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Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Irma Rangel Alarcón, UCSF Helen Diller Family Comprehensive Cancer
Center and Cancer Research Institute, San Francisco, California 94158,
USA (1)
Ann F. Chambers, Department of Pathology; Department of Medical
Biophysics; and London Regional Cancer Program, London Health
Sciences Centre, and Department of Oncology, Schulich School of
Medicine and Dentistry, University of Western Ontario, London, Ontario,
Canada (67)
Benjamin D. Hedley, Division of Hematology, London Health Sciences
Centre, London, Ontario, Canada (67)
Bing-Hua Jiang, Department of Pathology, Cancer Center, Nanjing Medical
University, Nanjing 210029, Jiangsu, China; Mary Babb Randolph
Cancer Center and Department of Microbiology, Immunology and
Cell Biology, West Virginia University, Morgantown, West Virginia
26506, USA (19)
C. Christian Johansson, Department of Oncology and Pathology, Cancer
Center Karolinska, Karolinska Institutet, Stockholm 17176, Sweden (197)
Sotirios C. Kampranis, Molecular Oncology Research Institute, Tufts
Medical Center, Boston, Massachusetts 02111, USA (103)
Rolf Kiessling, Department of Oncology and Pathology, Cancer Center
Karolinska, Karolinska Institutet, Stockholm 17176, Sweden (197)
Sonia Lain, Department of Surgery and Molecular Oncology, Ninewells
Hospital, University of Dundee, Dundee DD1 9SY, Scotland, United
Kingdom; and Department of Microbiology, Tumor and Cell Biology,
Karolinska Institute, Nobels väg 16, 171 77 Stockholm, Sweden (171)
Ingeborg van Leeuwen, Department of Surgery and Molecular Oncology,
Ninewells Hospital, University of Dundee, Dundee DD1 9SY, Scotland,
United Kingdom; and Department of Microbiology, Tumorand CellBiology,
Karolinska Institute, Nobels väg 16, 171 77 Stockholm, Sweden (171)
Ling-Zhi Liu, Mary Babb Randolph Cancer Center and Department of
Microbiology, Immunology and Cell Biology, West Virginia University,
Morgantown, West Virginia 26506, USA (19)
ix

Jesse Lyons, UCSF Helen Diller Family Comprehensive Cancer Center and
Cancer Research Institute, San Francisco, California 94158, USA (1)
Frank McCormick, UCSF Helen Diller Family Comprehensive Cancer
Center and Cancer Research Institute, San Francisco, California 94158,
USA (1)
Shikhar Mehrotra, Department of Surgery, Medical University of South
Carolina, Charleston, South Carolina 29425, USA (197)
Abigail L. Miller, UCSF Helen Diller Family Comprehensive Cancer Center
and Cancer Research Institute, San Francisco, California 94158, USA (1)
Dimitrios Mougiakakos, Department of Oncology and Pathology, Cancer
Center Karolinska, Karolinska Institutet, Stockholm 17176, Sweden (197)
Vernon T. Phan, UCSF Helen Diller Family Comprehensive Cancer Center
and Cancer Research Institute, San Francisco, California 94158, USA (1)
Philip N. Tsichlis, Molecular Oncology Research Institute, Tufts Medical
Center, Boston, Massachusetts 02111, USA (103)
Christina Voelkel-Johnson, Department of Microbiology and Immunology,
Medical University of South Carolina, Charleston, South Carolina 29425,
USA (197)
Amy Young, UCSF Helen Diller Family Comprehensive Cancer Center and
Cancer Research Institute, San Francisco, California 94158, USA (1)
x Contributors

Ras Signaling and Therapies
Amy Young, Jesse Lyons, Abigail L. Miller, Vernon T. Phan,
Irma Rangel Alarcón, and Frank McCormick
UCSF Helen Diller Family Comprehensive Cancer Center and Cancer Research
Institute, San Francisco, California 94158, USA
I. Introduction: The Ras Superfamily and Their Regulators
II. The Raf/MAPK Pathway
III. Ras and the PI3K Pathway
IV. Cellular Signals that Block Ras Pathways
A. EphA2
B. Sprouty and Spred
V. Ras and Neurofibromatosis
VI. Therapeutic Opportunities
References
More than 25 years have passed since activating mutations in Ras genes were identified
in DNA from human tumors. In this time, it has been established beyond doubt that these
mutations play a direct role in causing cancer, and do so in collaboration with a number of
other oncogenes and tumor suppressors. Oncogenic mutant Ras proteins are resistant to
downregulation by GAP‐mediated hydrolysis of bound GTP, and therefore signal persis-
tently. Efforts to develop therapies that block Ras oncoprotein function directly have
failed. The high affinity of Ras proteins for GTP has discouraged attempts to identify
GTP‐analogs. Ras processing enzymes have been targeted, but unfortunately, K‐Ras, the
Ras protein that plays the major role in human cancer, has proven refractory to these
approaches. Further progress has been made with drugs that block downstream signaling:
the approved drug Sorafenib inhibits Raf kinase, and its clinical benefits in liver cancer are
greatest in patients in which the mitogen activated protein kinase (MAPK) signaling
pathway is hyperactive. Other Raf kinase inhibitors, as well as drugs that block mitogen-
activated protein kinase / extracellular signal-regulated kinase kinase (MEK) and various
steps in the PI 30
kinase pathway, are under development. Here we will discuss the
complexities of Ras signaling and their effects on targeting the Ras pathway in the future.
# 2009 Elsevier Inc.
I. INTRODUCTION: THE Ras SUPERFAMILY
AND THEIR REGULATORS
The tumor oncoproteins H‐Ras, K‐Ras, and N‐Ras are founding members
of a larger superfamily of proteins that bind GDP and GTP with high affinity
and can hydrolyze bound GTP to GDP. This Ras superfamily is comprised
of more than 150 proteins and can be grouped into at least five subfamilies:
Advances in CANCER RESEARCH 0065-230X/09 $35.00
Copyright 2009, Elsevier Inc. All rights reserved. DOI: 10.1016/S0065-230X(09)02001-6
1

the Ras, Rho, Rab, Arf, and Ran families (Rajalingam et al., 2007). The Ras
subfamily itself now includes at least 21 members: H‐Ras, K‐Ras (A and B),
N‐Ras, R‐Ras, TC21/R‐Ras2, M‐Ras/R‐Ras3, Rap1a, Rap1b, Rap2a, Rap2b,
Rap2c, Rit, Rin, Rheb, Noey2, DiRas1/Rig, DiRas2, ERas, RalA, RalB,
DexRas/RasD1, and RasD2/Rhes. Many of these Ras family proteins have
yet to be fully characterized. However, several appear to share some of the
properties and the functions of the canonical Ras proteins (Rodriguez‐Viciana
et al., 2004; Takai et al., 2001).
Given the quantity and functional promiscuity of these proteins, it is
important to understand their specificity, both in terms of their downstream
effector functions, and how they are regulated. Recent work in our laboratory
attempted to understand effector specificity by comprehensively examining
the ability of a panel of Ras superfamily proteins to interact with and directly
activate different isoforms of three well characterized effector families: Raf,
PI3K, and RalGEF. This work led to a model in which signaling specificity is
achieved by each family member selectively interacting with distinct sets of
effectors in a combinatorial fashion, as well as by selective interactions
between isoforms of the same effector families (Rodriguez‐Viciana et al.,
2004). For example, H‐Ras, K‐Ras, and N‐Ras appear to be stronger binders
and activators of Raf kinases than TC21, M‐Ras, or Rit. On the other hand,
all members of this branch of the Ras superfamily interact with similar
intensity with RalGEFs, but interaction does not correlate with the induced
enzymatic activity of the RalGEF isoform (Rodriguez‐Viciana et al., 2004).
Ras proteins show significant selectivity in binding to the Class I PI3K
isoforms, another major, well established effector. H‐Ras, K‐Ras, N‐Ras,
R‐Ras, TC21, and M‐Ras are able to activate the p110 and p110
isoforms. However, only R‐Ras and TC21 are able to activate the p110
isoform (Rodriguez‐Viciana et al., 2004). The specificity of Ras for particu-
lar p110 isoforms suggests that the expression profile of p110 in different
cell types may determine the consequences of Ras activation. This specificity
also highlights the importance of finding isoform‐specific inhibitors to block
the PI3K pathway in cells harboring oncogenic Ras mutations.
While it is well established that the canonical Ras family members play a
major role in human cancer, the contribution of other family members is less
understood. To better understand their biological functions, it is important
to determine which effector families and downstream pathways they are
able to regulate. Dissecting the complexity of signaling specificity among the
families will further our understanding of how different Ras proteins
promote differing cellular functions as well as aid in designing strategies
for targeting diseases where these pathways are frequently deregulated. It
remains striking that no member of the Ras family, except for the prototypic
H‐Ras, K‐Ras, and N‐Ras, has been found mutated recurrently in human
cancer, even though some appear capable of activating effector pathways
2 Amy Young et al.

that are thought to be causally involved in cancer. On the other hand,
inactivating mutations in neurofibromin, a negative regulator of Ras pro-
teins, have been detected in many types of solid tumors, such as glioblastoma
and prostate cancer, and in many leukemias as well as malignant tumors
associated with neurofibromatosis type I, and more recently, serous tumors
of the ovary (Sangha et al., 2008). Loss of expression of Sprouty family
proteins also occurs in some cancers, such as liver cancer (Lee et al., 2008).
In each of these cases, Ras activity is expected to increase. However, it is
not clear which Ras family member is most strongly affected and which
downstream effectors are therefore activated.
II. THE Raf/MAPK PATHWAY
Of all of the Ras effector pathways, the Raf/MAPK pathway is one of the
best studied both in terms of its biochemistry and signaling and its role
in disease (Gollob et al., 2006). The Raf proteins are a family of serine/
threonine kinases (A‐Raf, B‐Raf, and C‐Raf/Raf‐1) which are conserved
from Drosophila melanogaster and Caenorhabditis elegans to vertebrates
(Wellbrock et al., 2004). Raf binds to and is activated by GTP‐bound Ras.
Raf activation results in activation of the MAPK cascade through phos-
phorylation of MEK which, in turn, phosphorylates extracellular signal-regu-
lated kinase (ERK). Following phosphorylation, ERK translocates to the
nucleus where it activates various transcription factors and cell cycle regulato-
ry proteins (Downward, 2003; Garnett and Marais, 2004; Wellbrock et al.,
2004). The effects of MAPK cascade activation range from proliferation and
survival to differentiation depending on the cellular context. The critical
biological role of Raf and the MAPK cascade in Ras signaling has been
described thoroughly in cell culture and mouse models (Wellbrock et al.,
2004). In addition, the key role for the Raf arm of Ras signaling has been
confirmed in human pathologies involving abnormal Ras signaling. The first
reports of B‐Raf mutations in cancer emerged in 2002 (Davies et al., 2002;
Dhomen and Marais, 2007). B‐Raf has been found to be mutated in many
types of cancer and is commonly found in tumors such as melanoma (66%)
and colorectal cancer (15%). It is interesting to note that the tumor types that
have the highest rates of B‐Raf mutation also have frequent Ras family muta-
tions. These mutations, however, are almost always mutually exclusive within
a single tumor (Garnett and Marais, 2004; Tsao et al., 2004). This suggests that
Raf itself can serve the same role as Ras in tumor formation, although activa-
tion of other pathways such as PI3K are often also required.
Dr. Kate Rauen, in collaboration with our laboratory, recently provided
an additional example of the similar effects of Ras, Raf, and MAPK
Ras Signaling and Therapies 3

activation in human disease (Rodriguez‐Viciana et al., 2006b). Costello,
Noonan, and Cardio‐Facio‐Cutaneous (CFC) syndromes are a group of
developmental disorders that show an overlapping range of symptoms,
particularly cardiac defects and short stature (Aoki et al., 2008; Schubbert
et al., 2007a,b; Tidyman and Rauen, 2008). Based on the fact that Costello
syndrome patients have been shown to have germ‐line mutations in H‐Ras,
the Ras proteins were sequenced in CFC patients, but found to be wild type.
However, sequencing of downstream targets of Ras signaling showed germ‐
line mutations in BRAF in 78% (18 of 23) of patients and mutations in the
MAPK pathway (MEK1 or MEK2) in another 13% of patients (Rodriguez‐
Viciana et al., 2006b). Furthermore, a recent paper from Schubbert and
colleagues reports that mutations in KRAS are found in patients with Noo-
nan syndrome (Schubbert et al., 2006). The finding that germ‐line mutations
in Ras, Raf, and MEK result in syndromes with a similar spectrum of
symptoms underscores the critical role that Ras signaling plays in these
developmental disorders (Aoki et al., 2008; Schubbert et al., 2007a,b;
Tidyman and Rauen, 2008).
While the linear pathway from Ras to Raf to MAPK has been clear for
some time, the precise mechanisms of signal transduction from Ras to Raf is
complex and not fully understood. It is known that Raf activation requires
its recruitment to the membrane by GTP‐bound Ras (Marais et al., 1995).
Additionally, there are a number of phosphorylation and dephosphoryla-
tion events that regulate Raf’s interactions with its own autoinhibitory
domains as well as other proteins (Light et al., 2002). Serine‐259 is a key
regulatory site on Raf which must be dephosphorylated for full activation
of Raf (Kubicek et al., 2002). In a recent paper by Rodriguez‐Viciana and
colleagues, it was shown that a newly identified Ras effector, Shoc2, can
mediate dephosphorylation of this site via an interaction with activated
M‐Ras and protein phosphatase 1C (PP1C) (Rodriguez‐Viciana et al.,
2006a). Shoc2, which is comprised almost entirely of leucine‐rich repeats,
is an adaptor protein which serves as a regulatory domain for PP1C,
directing it to Raf. One of the most important aspects of this discovery is
the fact that M‐Ras, Shoc2, and PP1C can dephosphorylate S259 on Raf
molecules attached not only to M‐Ras but to other Ras family members as
well (Fig. 1). This was proven in several tumor lines with activated Ras in
which Shoc2 inhibition by siRNA decreased the basal levels of Raf signaling
and ERK phosphorylation. This work adds a new layer to our understand-
ing of the role played by Ras family members in Raf and ERK activation
and suggests that there may be important roles for the less studied Ras
family members in normal Ras signaling and in cancer. Additionally, this
work identifies Shoc2 as a potential target for inhibition in Ras mutant
cancers.
4 Amy Young et al.

III. Ras AND THE PI3K PATHWAY
Another well characterized effector pathway of Ras family GTPases is the
PI3K pathway. As described earlier, multiple Ras family members directly
bind to and activate the p110 catalytic subunit of the Class I PI3Ks
(Rodriguez‐Viciana and Downward, 2001; Rodriguez‐Viciana et al., 1994,
1996, 2004). These heterodimeric PI3K enzymes convert the membrane
lipids PI‐4,5‐P2 (PIP2) into PI‐3,4,5‐P3 (PIP3), which serve as secondary
messengers that trigger a host of cellular responses. Primary effectors of
these lipids, such as the serine/threonine kinase Akt, are recruited to the
membrane and stimulate cell cycle entry, cell survival, glucose transport,
migration, and protein synthesis (Jiang and Liu, 2008). Under normal con-
ditions, the PI3K pathway is tightly regulated by the lipid phosphatases
PTEN, SHIP1, and SHIP2 (Fig. 2). However, in tumor cells, frequent muta-
tions in the PIK3CA and PTEN genes upregulate PI3K signaling, indicating
the importance of this pathway in tumorigenesis (Yuan and Cantley, 2008;
Zhao and Vogt, 2008).
H-Ras
Raf
S259-P
P-S259
Raf
M-Ras
Shoc2
PP1C
MEK
ERK
M-Ras
Fig. 1 Raf is dephosphorylated and activated by a novel M‐Ras/Shoc2/PP1C complex. Acti-
vated M‐Ras recruits a complex of Shoc2 and PP1C to the membrane where it dephosphorylates
Serine‐259 of Raf molecules bound to both M‐Ras and other canonical Ras family members
such as H‐Ras, K‐Ras, and N‐Ras. This dephosphorylation event contributes to activation of
Raf and the MAPK cascade.

The high incidence of PI3K pathway mutations described above indicates
that Ras mutant tumors may be dependent on the activation of the PI3K
pathway. Several lines of experimental evidence support this hypothesis.
For example, dominant negative PI3K inhibits Ras transformation of
NIH3T3 cells (Rodriguez‐Viciana et al., 1997). Additionally, a mutant
form of p110 that fails to bind Ras inhibits fibroblast transformation.
More importantly, mice generated with this p110 mutation are highly
resistant to K‐Ras driven lung adenocarcinomas and skin carcinomas devel-
oped in a two‐stage chemical carcinogenesis model, in which the mutagen
Growth factors
RTKs
E4-ORF1
PI-4,5-P2
PI-3,4,5-P3
AKT
PI-3,4-P2
GEFs
GAPs
neurofibromin
Spred
Stimulation of
EphA2 with
ephrin-A1
Ras-GDP Ras-GTP
Raf
MEK
ERK
EphA2
PI3K PTEN
SHIP1/2
Fig. 2 The Raf/MAPK and PI3K signaling pathways. Receptor tyrosine kinases (RTKs)
integrate signals from extracellular growth factors to recruit guanine nucleotide exchange
factors (GEFs), which promote the exchange of GDP for GTP on Ras. In its GTP‐bound state,
Ras activates downstream effector pathways, including the Raf/MAPK and PI3K pathways.
GTPase activating proteins (GAPs), such as neurofibromin, promote the hydrolysis of Ras‐GTP
to Ras‐GDP, thereby downregulating both Raf/MAPK and PI3K signaling. Spred proteins have
been reported to negatively regulate Raf/MAPK signaling through either direct interaction with
Ras or Raf. High levels of Raf/MAPK signaling induce expression of the receptor tyrosine kinase
EphA2, which can form a negative feedback loop to inhibit MAPK and PI3K signaling when
stimulated with its ligand, ephrin‐A1. While PI3K can be activated by GTP‐bound Ras, it can
also be activated by growth factor‐stimulated RTKs, or by the adenoviral protein E4‐ORF1.
The PI3K pathway is negatively regulated by phosphatases such as PTEN, SHIP1, and SHIP2.
6 Amy Young et al.

7,12-dimethylbenzanthracene (DMBA) causes activating mutations in
H‐Ras (Gupta et al., 2007). Unlike previous Ras effector studies, these
experiments were performed in vivo with endogenous levels of mutated
p110 and Ras, giving a more accurate representation of naturally occurring
tumors than can be obtained through tissue culture models with ectopic
expression. Furthermore, Gupta et al. (2007) found that adult mice with this
p110 mutation are healthy, indicating that targeting the Ras‐PI3K interac-
tion would be viable therapeutically.
Ras driven tumors exploit the functions of both the MAPK and PI3K
pathways in mitosis, apoptosis, motility, proliferation, and differentiation.
In a similar fashion, viruses also target these pathways to drive viral replica-
tion, inhibit apoptosis during infection, and evade the host immune response.
In fact, mutant Ras was discovered as a viral oncogene from the rat sarcoma
virus, while PI3K was uncovered as an activity associated with the polyoma
virus middle T antigen (Jiang and Liu, 2008; Whitman et al., 1985). Since
then a number of viruses have been found to upregulate these signaling
pathways, PI3K in particular. One of the more recent discoveries involves
human adenovirus. The early adenoviral protein E4‐ORF1 potently acti-
vates PI3K, producing high PIP3 levels and activation of its downstream
effector Akt. Pharmacologic studies show that Class I PI3Ks are activated
and that E4‐ORF1’s carboxy‐terminal PDZ binding motif is required for
activation (Frese et al., 2003; O’Shea et al., 2005). Our laboratory deter-
mined that E4‐ORF1 did not act through several known mechanisms of
PI3K activation, including directly binding and activating PI3K, inactivating
PTEN, or increasing Ras activity. To get a better understanding of E4‐ORF1
function, we used tandem affinity purification to identify E4‐ORF1 binding
partners. We are now studying various PDZ containing protein complexes
and determining their role in PI3K signaling (unpublished). This may indi-
cate a novel mechanism of regulating the PI3K pathway, and thus may
impact the study and treatment of cancers in which PI3K, Ras, or upstream
receptor tyrosine kinases (RTKs) are mutated.
IV. CELLULAR SIGNALS THAT BLOCK Ras PATHWAYS
It has become increasingly clear that in addition to the core components of
the Raf/MAPK cascade, there are a number of negative modulators that act
to regulate the intensity and duration of signaling downstream of Ras
(Dhillon et al., 2007; Kolch, 2005). Several endogenous antagonists of
Ras‐mediated signaling have been identified, and below we present two
examples—the EphA2 receptor tyrosine kinase and the Sprouty and Spred
proteins.

A. EphA2
The Raf/MAPK pathway activates the Ets family of transcription factors,
which in turn regulate the activity of several genes (Coffer et al., 1994). One
gene regulated by this pathway encodes an Eph receptor tyrosine kinase
called EphA2. Here, we discuss recent studies from our laboratory that
identify a negative feedback loop involving the Raf/MAPK target EphA2,
in which stimulation of EphA2 with its ligand ephrin‐A1 inhibits MAPK
signaling (Macrae et al., 2005).
The Eph family of RTKs comprises the largest family of tyrosine kinases in
the human genome, with 14 Eph receptors and 8 membrane associated
ephrin ligands currently described (Edwards and Mundy, 2008). Eph recep-
tors are divided into two classes based on sequence similarity and ligand
specificity: EphA receptors interact with ephrin‐A ligands, which are teth-
ered to the cell membrane by GPI‐linkage, whereas EphB receptors interact
with ephrin‐B ligands, which are transmembrane proteins (Kullander and
Klein, 2002; Pasquale, 2005). The interaction between an Eph receptor and
its membrane associated ephrin ligand occurs between two neighboring cells
at sites of cell–cell contact, providing a conduit through which one cell can
affect a neighboring cell’s signaling program. Through these interactions,
Eph receptors and ligands mediate cell adhesion, motility, and migration in a
variety of biological settings, including tissue patterning, neuronal targeting,
and vascular development (Ireton and Chen, 2005; Kullander and Klein,
2002; Pasquale, 2005). Eph receptors and ligands are misexpressed or over-
expressed in several human cancers, and are often associated with the most
aggressive and metastatic tumors (Surawska et al., 2004). This underscores
the importance of understanding the regulation of Eph receptor and ephrin
ligand expression.
Expression of the Eph receptor tyrosine kinase EphA2 is frequently ele-
vated in several different types of cancer, and its expression correlates with
poor clinical outcome (Ireton and Chen, 2005). Reports indicate that EphA2
is overexpressed in 40% of breast cancers (Miyazaki et al., 2003). Our
laboratory recently demonstrated that expression of EphA2 and its ligand
ephrin‐A1 is mutually exclusive in a panel of 28 human breast cancer cell
lines (Macrae et al., 2005). Interestingly, the eight cell lines that express the
EphA2 receptor have a distinct phenotype from those that express the
ephrin‐A1 ligand. While the EphA2‐expressing breast cancer cell lines
express several markers that are characteristic of a mesenchyme‐like pheno-
type, those that express the highest levels of ephrin‐A1 ligand maintain an
epithelial phenotype (Macrae et al., 2005). These findings prompted further
studies to investigate the regulation of EphA2 and ephrin‐A1 expression in
breast cancer cells.
8 Amy Young et al.

Through the utilization of microarray analysis, EPHA2 was identified as a
direct transcriptional target of the Raf/MAPK pathway. However, while the
Raf/MAPK pathway activates transcription of EPHA2, it also downregu-
lates the expression of its ligand, ephrin‐A1 (Macrae et al., 2005). In both
mouse embryonic fibroblasts and human breast cancer cell lines, activation
of the Raf/MAPK pathway induced expression of the EphA2 receptor and
downregulated expression of the ephrin‐A1 ligand. Similarly, treatment of a
breast epithelial cell line with MEK inhibitors reduced EphA2 protein levels
but induced ephrin‐A1 expression, further confirming that the MAPK path-
way is critical in regulating both EphA2 receptor and ephrin‐A1 ligand
expression. Therefore, the reciprocal pattern of EphA2 receptor and ephrin‐
A1 ligand expression in the panel of breast cancer cell lines may result in part
to differing MAPK pathway activation levels. These findings also suggest
that high MAPK activity may contribute to the high EphA2 levels observed
in many cancers.
The interaction between an Eph receptor and ephrin ligand occurs between
two neighboring cells, resulting in Eph receptor phosphorylation and activa-
tion. In cell culture, this can be mimicked by stimulating EphA2‐expressing
cells with a soluble form of dimerized ephrin‐A1 ligand (ephrin‐A1/Fc).
When EphA2‐expressing breast cancer cells are stimulated with ephrin‐A1/Fc,
MAPK signaling is significantly attenuated. These results indicate that the
interplay between MAPK signaling and EphA2 signaling forms a conditional
feedback loop to regulate MAPK activity in a ligand‐dependent manner:
high MAPK activity stimulates EphA2 expression, and activation of EphA2
by its ligand ephrin‐A1 then inhibits MAPK signaling (Fig. 2). It is of note
that none of the 28 breast cancer cell lines examined appears to express both
EphA2 receptor and ephrin‐A1 ligand, and therefore none maintain the
negative feedback of MAPK signaling through EphA2 activation. An escape
from this negative feedback loop—by loss of receptor or ligand expression—
may serve as a mechanism to escape MAPK pathway suppression and may
therefore be important in the development of cancer. In unpublished work,
our laboratory has found that stimulation of EphA2‐expressing cells with
ephrin‐A1/Fc attenuates Akt activation, suggesting that activation of EphA2
additionally has negative effects on another important signaling pathway
downstream of Ras, the PI3K pathway. These findings present a new poten-
tial therapeutic strategy for breast cancer: because both the MAPK and PI3K
pathways are commonly deregulated in cancer, stimulating the EphA2 re-
ceptor with its ligand ephrin‐A1 may present a viable method to suppress the
tumorigenicity of breast cancer cells by attenuating the downstream signal-
ing events and biological activities associated with these pathways. Current
work in our laboratory aims to define the molecular mechanism by which
EphA2 attenuates MAPK and PI3K signaling.

B. Sprouty and Spred
Sprouty and Spred (Sprouty‐related with an EVH1 domain) proteins are
members of another family of proteins involved in negatively regulating Ras
signaling (Kim and Bar‐Sagi, 2004). A key feature of the Sprouty family of
proteins is the conserved C‐terminal cysteine‐rich SPR domain, which is
thought to be necessary for plasma membrane localization (Bundschu
et al., 2006, 2007; Wakioka et al., 2001). D. melanogaster SPRY was the
first member of this family to be identified. This protein regulates tracheal
branching in response to fibroblast growth factor (Hacohen et al., 1998). In
mammals, four Sprouty isoforms have been identified and shown to regulate
growth factor‐mediated actions. At the cellular level, overexpression of
Sprouty proteins inhibits migration and proliferation of a variety of cell
types in response to serum and growth factors, though the precise mecha-
nism is not clear. Direct association with Ras and Raf proteins has been
reported, as well as indirect effects mediated through organization of signal-
ing complexes at the plasma membrane (Kim and Bar‐Sagi, 2004; Kim et al.,
2007). Interestingly, EGF or FGF stimulation induces the expression of
several Sprouty isoforms, suggesting the Sprouty proteins may play an
important role in negative feedback control of receptor tyrosine kinase
signaling (Dhillon et al., 2007; Kim and Bar‐Sagi, 2004).
Spred family members are characterized by an N‐terminal Enabled/VASP
homology 1 (EVH1) domain and the C‐terminal SPR domain. Thus far,
three Spred isoforms have been identified in humans. Spred proteins have
been reported to negatively regulate Raf/MAPK signaling through either
direct interaction with Ras or Raf (Fig. 2). Recently, germ‐line loss‐of‐
function mutations in SPRED1 were reported in five families that showed
some hallmarks of neurofibromatosis type 1 (NF1; below), suggesting that
loss of Spred‐1 upregulates Ras signaling and so phenocopies some features
of NF1 (Brems et al., 2007). This provides yet another example of a neuro‐
cardio‐facial‐cutaneous syndrome caused by germ‐line mutation in a gene of
the Ras/Raf/MAPK cascade (Denayer et al., 2008).
Recent studies have shown that Sprouty and Spred expression is deregu-
lated in various types of cancer. Spred‐1 and Spred‐2 expression has been
shown to be reduced in hepatocellular carcinoma (HCC), while loss of
expression of Sprouty family proteins has been reported to occur in liver,
breast, prostate, and skin cancers (Bundschu et al., 2007; Lee et al., 2008;
Lo et al., 2006). Elucidating the precise molecular mechanism by which
Sprouty and Spred proteins negatively regulate Ras signaling may be an
important first step in understanding the implications of downregulating
these proteins in cancer.
10 Amy Young et al.

V. Ras AND NEUROFIBROMATOSIS
NF1 was first described by Fredrich von Recklinghausen in 1882, and the
NF1 gene was cloned in 1990 by the laboratories of Collins and White
(Ballester et al., 1990; Cawthon et al., 1990). Single copy germ‐line muta-
tions in the NF1 gene cause NF1, which is one of the most common familial
genetic disorders, affecting approximately 1 in 3500 individuals. One of the
hallmarks of NF1 is the development of benign neurofibromas, as well as the
predisposition to the development of malignant tumors of the nervous
system. Nontumor manifestations of the disorder include visual anomalies,
skeletal deformities, abnormal skin pigmentation, and learning disabilities
(McClatchey, 2007). Neurofibromin, the NF1 gene product, contains a GAP
domain with function similar to the catalytic domain of p120 RasGAP,
which accelerates the hydrolysis of active Ras‐GTP to inactive Ras‐GDP
(Fig. 2). By promoting the conversion of Ras‐GTP to Ras‐GDP, neurofibro-
min negatively regulates Ras, MAPK, and PI3K signaling—all of which are
critical in the development of human cancer.
Importantly, NF1‐associated tumors display a loss or mutation of the
wild‐type NF1 allele, suggesting that NF1 acts as a tumor suppressor gene
(Perry et al., 2001; Side and Shannon, 1997). Additionally, cells derived
from NF1‐associated tumors have elevated levels of Ras activity. For exam-
ple, Schwann cells derived from NF1 tumors have hyperactive Ras signaling
as well as activation of the MAPK and PI3K pathways (Sherman et al.,
2000). Recently, the mTOR pathway, a downstream effector of the Ras
and PI3K pathways, has also been found to be activated in NF1‐associated
tumors, further supporting the role of neurofibromin as a Ras‐GAP
(Johannessen et al., 2005, 2008).
The use of model organisms has helped to confirm the role of neurofibro-
min as a Ras‐GAP protein. The NF1 gene is highly conserved from yeast to
mammals (McClatchey, 2007). Early work in the yeast Saccharomyces
cerevisae demonstrated that loss of the neurofibromin‐like proteins Ira1
and Ira2 results in hyperactivation of the Ras pathway. While it is clear
that neurofibromin plays a critical role as a Ras‐GAP, little else is known
about neurofibromin signaling and regulation. The GAP domain represents
approximately 10% of the neurofibromin protein, suggesting that neurofi-
bromin may have other, uncharacterized functions. Additionally, exactly
how neurofibromin activity is regulated is poorly understood. Identifying
the physiological signals that regulate neurofibromin could lead to strategies
for regulating Ras in NF1 patients that still retain a normal copy of the NF1
allele. To identify the biological signals that regulate neurofibromin, our
laboratory has utilized unbiased genetic and proteomic approaches to

identify novel protein associations with neurofibromin. We hope to further
our understanding of neurofibromin signaling by determining what proteins
directly bind to neurofibromin. Our preliminary data reveal both previously
reported and unknown binding partners that interact with neurofibromin.
We are currently dissecting how these interacting proteins regulate neurofi-
bromin signaling. This approach should reveal critical biological functions
of neurofibromin and identify new protein candidates as potential therapeu-
tic targets for the treatment of NF1.
VI. THERAPEUTIC OPPORTUNITIES
Current efforts to block oncogenic Ras activity are focused on down-
stream pathways, in which a number of protein and lipid kinases present
suitable targets for drug development. The first pathway described down-
stream of Ras was the Raf/MAPK pathway, and efforts to develop drugs
blocking proteins in this pathway were launched in the early 1990s. At
ONYX Pharmaceuticals, Raf‐1 kinase was targeted, and indeed, in 2000
Sorafenib became the first drug to enter clinical trials based on its expected
effects on the Ras pathway. In 2004, Sorafenib was approved for treatment
of renal cancer, based initially on delayed time to progression in patients
suffering from this disease. However, in this indication it is more likely that
VEGFR2 is the major target, rather than Raf kinases themselves (Wilhelm
et al., 2004). This assumption is based on the relatively high potency of
Sorafenib against VEGFR2 (an unexpected property, as Sorafenib was de-
veloped based on its activity against the serine/threonine kinase Raf‐1), the
known dependence of renal cancer on VEGF signaling, and the observation
that another VEGFR2 inhibitor, Sutent, is also active in renal cancer even
though it does not affect Raf kinase activity.
In liver cancer, an indication in which Sorafenib has also been approved,
Raf kinase inhibition may be involved in clinical activity, since responses
correlate with high MAPK activity (Abou‐Alfa et al., 2006). Furthermore, in
liver cancer, the Raf/MAPK pathway is frequently active through loss of
negative regulators, such as Sprouty, as described above. However, it is clear
that many opportunities exist for development of more potent and effective
Raf kinase inhibitors. A compound with a similar spectrum of activities as
Sorafenib, but with much higher potency, Novartis‐265, and compounds
that show selectivity for V600E BRAF from Plexxicon, Exelixis, and others,
are under clinical and preclinical development. In parallel to these attempts
to block Raf kinase, drugs that inhibit MEK are under clinical investigation
(Downward, 2008).
The PI 30 kinase pathway has also been extensively targeted more recently.
Multiple inhibitors of PI 30 kinase, Akt, and mTOR are under clinical
12 Amy Young et al.

development. In the latter group, an mTOR inhibitor, Temsirolimus, has
already been approved, interestingly also for treatment of renal cancer
(Downward, 2008). Efforts to target this arm of the Ras effector pathway
have been encouraged by the high frequency of mutations in cancer, as
discussed above, and by the demonstration that efficient Ras transformation
requires direct interaction of activated Ras with PI 30 kinase. This interaction
appears less important in normal cell signaling, suggesting that therapeutic
selectivity could be obtained by preventing this interaction.
We and others demonstrated that full oncogenic transformation requires
activation of both major arms of the Ras effector pathways: effective inhibi-
tion might also require simultaneous inhibition of both arms. Recently, it
was demonstrated that effective treatment of murine lung cancers driven by
mutant K‐Ras could be achieved by inhibiting PI3K and MEK pathways, but
not by either alone (Engelman et al., 2008). Clinical trials involving inhibi-
tors of both effector arms will soon be launched to test this hypothesis in
cancer patients (Downward, 2008).
The success of inhibitors targeting a pathway of such central importance
to growth and survival of normal cells is likely to depend on two major
principles: oncogene addiction and signaling redundancy. Oncogene addic-
tion refers to the empirical observation that cells transformed by mutant
oncoproteins are far more dependent on these proteins and the pathways
they control for their survival than their normal counterparts. For example,
cells transformed by mutant BRAF are far more sensitive to MEK inhibitors
than wild‐type cells, despite similar levels of signaling through the MEK/
MAPK pathway. The precise basis of this phenomenon is not yet clear,
though several interpretations have been proposed. One outcome of the
principle of oncogene addiction is the unexpected discovery that more
advanced cancers respond better to targeted therapies, since they are more
dependent on a small number of driver pathways. This suggests that drugs
that target the Ras pathway may be less effective against a benign state such
as NF1, than a fully transformed Ras driven tumor cell.
Finally, it has become clear that signaling pathways are altered in Ras
transformed cells in ways that we do not yet understand. This has been
underscored by the recent discovery that EGF‐receptor inhibitors appear to
promote tumor progression in tumors with mutant Ras, suggesting that EGF
is a negative growth factor for these cells, in contrast to its clear role in
promoting growth of tumor cells in which its receptor is amplified or
activated (Eberhard et al., 2005). We, therefore, believe that more extensive
analysis of the Ras pathway in human cancer will guide future development
of therapies that impact the pathway and thus benefit patients suffering from
tumors in which the pathway is deregulated.

ACKNOWLEDGMENTS
The authors gratefully acknowledge Pablo Rodriguez‐Viciana for thoughtful discussions,
Jessie Castillo for assistance in preparing the manuscript, and Daiichi‐Sankyo Pharmaceutical
Co., Ltd. for support.
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PI3K/PTEN Signaling in
Angiogenesis and Tumorigenesis
Bing‐Hua Jiang*,{
and Ling‐Zhi Liu*
*Mary Babb Randolph Cancer Center and Department of Microbiology,
Immunology and Cell Biology, West Virginia University, Morgantown,
West Virginia 26506, USA
{
Department of Pathology, Cancer Center, Nanjing Medical University, Nanjing
210029, Jiangsu, China
I. Introduction of PI3K/PTEN Signaling Pathway
II. Angiogenesis Regulated by VEGF, Angiopoietins, and PI3K Activation
III. Genetic Aberrations of PI3K, PTEN, and AKT in Cancer
IV. Roles of PI3K and AKT in Regulating Angiogenesis
V. PI3K/PTEN Controls Angiogenesis through Increasing HIF‐1 and VEGF Expression
VI. The Downstream Signaling Molecules Mediated by PI3K/PTEN in Regulating Tumor
Growth and Angiogenesis
A. Tumor Growth
B. Tumor Metastasis
C. Tumor Angiogenesis
VII. Inhibition of PI3K Signaling Pathway for Cancer Treatment and Prevention
A. PI3K Inhibitors
B. AKT Inhibitors
C. mTOR Inhibitors
VIII. Concluding Remarks
References
Phosphatidylinositol 3‐kinase (PI3K) and phosphatase and tensin homolog deleted on
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functions such as cell metabolism, proliferation, cell‐cycle progression, and survival.
PI3K is activated by growth factors and angiogenesis inducers such as vascular endothe-
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trials for cancer treatment. # 2009 Elsevier Inc.
Advances in CANCER RESEARCH 0065-230X/09 $35.00
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19

I. INTRODUCTION OF PI3K/PTEN
SIGNALING PATHWAY
The phosphatidylinositol 3‐kinases (PI3Ks) in mammalian cells form a
family that can be divided into three classes, class I, II, and III, based on their
structure, substrate, distribution, mechanism of activation, and functions
(Domin and Waterfield, 1997; Walker et al., 1999). Among these classes,
class I PI3Ks are the best understood to play vital roles in regulating cell
proliferation, growth, and survival initiated by many growth and survival
factors (Cantley, 2002; Fruman et al., 1999; Morita et al., 1999). Based on
different associated adaptors, class I PI3Ks are divided into class IA and IB
PI3Ks. Class IA PI3Ks are activated by receptor tyrosine kinases (RTKs),
while class IB PI3Ks are activated by G‐protein‐coupled receptors (GPCRs)
(Engelman et al., 2006; Vanhaesebroeck et al., 1997). Class IA PI3Ks consist
of the heterodimers of a p110 catalytic subunit and a p85 regulatory subunit,
and use phosphatidylinositol, phosphatidylinositol‐4‐phosphate (PIP), and
phosphatidylinositol‐4,5‐bisphosphate (PIP2) as substrates. Three isoforms
of p110, p110 , p110 , and p110 are encoded by PIK3CA, PIK3CB, and
PIK3CD, respectively. There are also three isoforms of p85 subunit: p85 ,
p85 , and p85 that are encoded by PIK3R1, PIK3R2, and PIK3R3,
respectively. Class IB PI3Ks are composed of the heterodimers of a p110
catalytic subunit and a p101 regulatory subunit or its homologues p84 or
p87PIKAP (PI3K adaptor protein of 87 kDa). Class II PI3Ks include
PIK3C2 , PIK3C2 , and PIK3C2 , all of them are characterized by contain-
ing a common C2 domain at the C‐terminus. Class II PI3Ks can also be
activated by RTKs, cytokine recepors and integrins, and use phosphatidyli-
nositol and PIP as substrates (Arcaro et al., 2000; Falasca and Maffucci,
2007; MacDougall et al., 2004; Wheeler and Domin, 2001). But the specific
functions of class II PI3Ks in response to these activators are poorly under-
stood. Class III PI3Ks are composed of the heterodimers of catalytic and
adaptor subunits. This class of PI3Ks only uses phosphatidylinositol as a
substrate (e.g., mammalian PI3K and yeast Vps34p). The structure of PI3K
family is shown in Box 1. It has been indicated that class III PI3Ks are
involved in the regulation of mammalian target of rapamycin (mTOR)
activity in response to amino acid levels, and the regulation of autophagy
in response to cellular stress (Gulati et al., 2008; Tassa et al., 2003). The
class III PI3K Vps34 is present in all eukaryotic organisms, while both class I
and II PI3Ks only exist in multicellular organisms.
The two subfamilies of class IA and IB PI3Ks have evolved in mammals.
Class I, especially class IA PI3Ks, are the most extensively investigated in
regulating cellular functions such as cell proliferation, growth, and survival.
Class I PI3Ks catalyze the conversion of PIP2 at the D‐3 position to
20 Bing‐Hua Jiang and Ling‐Zhi Liu

Box 1 PI3K Family and Its Cellular Function
PI3K composes of three classes based on the substrate, structure, distribution, mechanism of
activation, and function. The structure of class I, II, and III PI3Ks is shown as below.
p85a
p55a
p50a
p85b
p55g
p110a
p110b
p110d
Class IA
SH3 BCR P
P SH2 Inter SH2 SH2
Regulatory subunits
P
Catalytic subunits
p85
binding
Ras
binding C2 PIK
Kinase
domain
Class II
PX C2
Catalytic (PIK3C2a,
PIK3C2b, PIK3C2g)
Homology I
Homology I Homology II
Class IB Regulatory subunits
Homology II
p101
Catalytic subunits
Ras
binding C2 PIK
Kinase
domain
Ras
binding C2 PIK
Kinase
domain
p110g
p84
Continued
PI3K/PTEN Signaling in Angiogenesis and Tumorigenesis 21

Box 1—Continued
Class III Regulatory subunits
Heat domain WD40 domain
p150
Catalytic subunits
C2 PIK
Kinase
domain
hVPS34
Kinase
domain
PI3K exerts various cellular functions through its downstream target AKT.
Cell metabolism. AKT promotes glucose uptake in muscle and fat cells by stimulating the
glucose transporter, GLUT4, to cell membrane. AKT increases glycogen synthesis by inhi-
biting glycogen synthase kinase 3 (GSK‐3) (Cohen and Frame, 2001). AKT also regulates
fatty‐acid synthesis by activating ATP citrate lyase (Berwick et al., 2002). Moreover, AKT
inhibits gluconeogenesis by blocking forkhead (FOXO)‐mediated transcription of gluconeo-
genic enzymes and regulates insulin metabolism in the liver (Engelman et al., 2006).
Abnormality of AKT is related with diabetes. AKT2‐deficient mice exhibit a diabetes‐like
syndrome with an elevated fasting plasma glucose level, elevated hepatic glucose output, and
peripheral insulin resistance (Cho et al., 2001a; Garofalo et al., 2003).
Initiation of translation and protein synthesis. AKT inhibits the GTPase‐activating protein
(GAP) activity of the tuberous sclerosis complex 1 (TSC1)‐TSC2 complex by phosphorylat-
ing TSC2 tuberin protein, leading to the accumulation and activation of the mTOR‐raptor
kinase complex. mTOR mediates the phosphorylation of the ribosomal protein S6 kinases
(p70S6K) and eukaryotic translation initiation factor 4E‐binding protein 1 (4E‐BP1) leading
to the release of the translation initiation factor eIF4E (Hennessy et al., 2005; Schmelzle and
Hall, 2000). However, there are complicated interactions and feedback loops in this signal-
ing pathway since TSC/mTOR/S6K cascade also inhibits PI3K/AKT pathway by down-
regulating insulin receptor substrate (IRS) 1/2 and PDGFR (Harrington et al., 2004; Zhang
et al., 2003).
Cell survival/inhibition of apoptosis. One of the important downstream targets of AKT is
FOXO family of transcription factors. AKT inactivates FOXO proteins by phosphorylation.
Some other important targets of AKT are GSK‐3, BAD (Bcl2‐antagonist of cell death),
IkappaB kinase (IKK), and MDM2. AKT blocks FOXO‐mediated transcription of some
proapoptotic proteins such as Fas‐ligand (FasL) and Bim, directly phosphorylates the
proapoptotic protein BAD, thus repressing the prosurvival molecule Bcl‐XL. The phosphor-
ylation of IKK results in phosphorylating IB (inhibitor of NF‐B), leading to its proteaso-
mal degradation and NF‐B nuclear localization. On the other hand, the phosphorylation of
MDM2 leads to the degradation of p53, exhibiting the antiapoptotic effect (Brazil et al.,
2002). In addition, eIF4E also has antiapoptotic activity in vitro and in vivo (Contreras
et al., 2008; Yamaguchi et al., 2008).
Cell cycle. AKT promotes G1‐S phase transition by blocking FOXO‐mediated trans-
cription of cell‐cycle inhibitors including p27Kip1 (Chandramohan et al., 2004;
Continued

phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) via its regulatory subunit
p85 linking to upstream receptors that are activated by growth factors or
hormones (Cantley, 2002; Luo et al., 2006; Zhao et al., 2006). RTKs, such
as epidermal growth factor receptor (EGFR), platelet‐derived growth factor
receptor (PDGFR), fibroblast growth factor receptor (FGFR), and insulin‐
like growth factor 1 receptor (IGF‐1R), can interact with the p85 regulatory
subunit to activate PI3K (Hu et al., 1992; McGlade et al., 1992; Zhu et al.,
1992), while Ras protein directly interacts with the p110 catalytic subunit of
PI3K in a GTP‐dependent manner (Peyssonnaux et al., 2000; Rodriguez‐
Viciana et al., 1996). In addition, p85 subunit also binds to the intracellular
proteins such as protein kinase C, SHP1, Rac, Rho, hormonal receptors,
mutated Ras and Src, providing an integration point for p110 activation
(Hennessy et al., 2005). It has been demonstrated that PI3K can be regulated
by the molecular switch, which is formed by a GTPase‐responsive domain
and an inhibitory domain on p85 regulatory subunit of PI3K. H‐Ras and
Rac1 activate PI3K by targeting the GTPase‐responsive domain and the
stimulatory effects can be blocked by the inhibitory domain, which func-
tions by binding to tyrosine‐phosphorylated molecules (Chan et al., 2002).
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN),
which is also known as MMAC1 or TEP1, was named due to its sequence
homology with phosphatases and the cytoskeletal protein tensin (Dahia
et al., 1997; Li et al., 1997b; Maehama and Dixon, 1998). PTEN is a
tumor suppressor commonly mutated in many human cancers (Salmena
et al., 2008). PTEN locates on 10q23.3, which encodes a 403‐residue
dual‐specificity phosphatase that has protein phosphatase activity, and
lipid phosphatase activity that antagonizes PI3K activity (Maehama and
Dixon, 1998). Since the product of p110 , PIP3, is a second messenger for
promoting cell proliferation, growth, metabolism, and survival, PTEN
hydrolyzes the 3‐phosphate on PIP3 to generate PIP2, and negatively reg-
ulates PIP3‐mediated signaling pathways. Thus, PTEN plays an important
role in phosphatidylinositol homeostasis (Maehama and Dixon, 1998).
It has been demonstrated that PTEN can be upregulated by early growth
regulated transcription factor 1 (EGR1) through direct binding to the PTEN
promoter. In addition, peroxisome proliferator activated receptor
Box 1—Continued
Schmidt et al., 2002). AKT also indirectly stabilizes the cell‐cycle protein c‐Myc and cyclin
D1 by inhibiting GSK‐3 (Diehl et al., 1998; Engelman et al., 2006; Gregory et al., 2003).
In addition, PI3K plays a role in regulating cell polarity and motility (Engelman
et al., 2006).

(PPAR ), p53, and activating transcription factor 2 (ATF2) can also tran-
scriptionally upregulate PTEN by binding to its promoter (Patel et al., 2001;
Shen et al., 2006; Stambolic et al., 2001), while transforming growth factor
(TGF)‐ , nuclear factor kappaB (NF‐B), and Jun negatively regulate PTEN
mRNA expression (Hettinger et al., 2007; Mahimainathan et al., 2006; Xia
etal., 2007).Recently,ithasbeenfound thatsomemicroRNAssuchasmiR‐21,
miR‐19a, and miR‐214 inhibit PTEN through targeting the 30‐untranslated
region (UTR) of PTEN, leading to inhibition of PTEN translation (Meng
et al., 2007; Pezzolesi et al., 2008; Yang et al., 2008). PTEN activity can also
be regulated by the posttranslational regulation including phosphorylation,
acetylation, oxidation, and control of its localization (Gericke et al., 2006;
Ikenoue et al., 2008; Leslie, 2006; Planchon et al., 2008; Tamguney and
Stokoe, 2007).
Serine–threonine protein kinase AKT (also known as protein kinase B) is
initially found to be the cellular homolog of AKT8 retroviral oncogene
(Bellacosa et al., 1991). AKT is one of the most important downstream
targets of PI3K. Human AKT has three isoforms: AKT1, AKT2, and
AKT3 (also known as PKB , PKB , and PKB , respectively). The product
of PI3K, PIP3, binds to AKTand leads to the membrane recruitment of AKT,
and also binds to phosphoinositide‐dependent kinase 1 (PDK1) via their
plekstrin homology (PH) domains (Downward, 1998; Engelman et al.,
2006), then PDK1 phosphorylates AKT in the kinase domain (Thr 308 in
AKT1). For the full activation of AKT, the phosphorylation within the
carboxyl‐terminal hydrophobic motif (Ser 473 in AKT1) of AKT by PDK2
is required (Hresko et al., 2003; Sarbassov et al., 2005; Stokoe et al., 1997).
Once activated, AKT moves to the cytoplasm and nucleus, where it phos-
phorylates, activates, or inhibits many downstream targets to regulate vari-
ous cellular functions including cell metabolism, protein synthesis, cell
survival/inhibition of apoptosis, and cell‐cycle progression (Box 1). In this
review, we will focus on the roles of class IA PI3Ks, PTEN, and AKT in
tumor growth and angiogenesis.
II. ANGIOGENESIS REGULATED BY VEGF,
ANGIOPOIETINS, AND PI3K ACTIVATION
Angiogenesis is the process by which new blood capillaries are generated
from the preexisting vasculature. It is essential for the embryo development,
female reproduction, tissue repair, inflammatory diseases, tumor growth,
and metastasis. Tumor angiogenesis occurs by sprouting the new
vessels from preexisting blood vessels or by inserting interstitial tissue
columns into the lumen of preexisting vessels (Carmeliet and Jain, 2000).

This process can be triggered by extracellular signals such as growth factors,
by genetic alterations such as activation of oncogenes including PI3K, and by
mutations of tumor suppressor genes such as PTEN and p53 (Carmeliet and
Jain, 2000; Folkman, 1995). Among all the proangiogenic factors, vascular
endothelial growth factor (VEGF) and angiopoietins (Ang) and their recep-
tors—VEGF and Tie [tyrosine kinase with immunoglobulin (Ig) and EGF
homology domains] receptors play important roles during tumor growth
and angiogenesis.
VEGFR family and the Tie receptor family are expressed specifically in
endothelium. The VEGF family members are secreted, dimeric glycopro-
teins. In mammals, VEGF family members consist of VEGF‐A, ‐B, ‐C, ‐D,
and placenta growth factor (PLGF) (Olsson et al., 2006). VEGF‐A plays a
key role in vasculogenesis and angiogenesis. Genetic studies have demon-
strated that VEGF‐A gene knockout mice either homozygotes or heterozy-
gotes die in the embryonic stage due to the defects in vasculature (Carmeliet
et al., 1996; Ferrara et al., 1996). There are five human isoforms of VEGF‐A:
VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Among them,
VEGF121, VEGF165, and VEGF189 are the dominant subtypes based on
the amount and biological activity (Olsson et al., 2006; Shibuya, 2008).
VEGF receptors have three family members: VEGFR1 (fms‐like tyrosine
kinase, Flt‐1), VEGFR2 (Flk‐1/KDR), and VEGFR3 (Flt‐4). All three VEGF
receptors contain tyrosine phosphorylation sites with regulatory and signaling
functions. These receptors play critical role in promoting vasculogenesis
during normal embryogenesis and pathologic angiogenesis. VEGF‐A binds
to both VEGFR1 and VEGFR2 to regulate tumorigenesis and angiogenesis,
while VEGF‐B and PLGF bind to VEGFR1. Under pathological conditions,
the increased PLGF and VEGF‐A can recruit monocytes/macrophages via
VEGFR1 to cancer tissues or inflammatory lesions, and significantly induce
angiogenesis (Brown et al., 2001; Murakami et al., 2008). VEGF‐C and ‐D
mainly bind to VEGFR3, and stimulate lymphangiogenesis.
VEGFR1 binds to the p85 regulatory subunit of PI3K on Tyr1213 and
1333 and has crosstalk with VEGFR2 in controlling cell migration, differ-
entiation, and angiogenesis (Autiero et al., 2003; Cunningham et al., 1995).
VEGFR2 is the predominant receptor in angiogenic signaling since it reg-
ulates endothelial cell migration, proliferation, differentiation and survival,
as well as vessel permeability and dilation (Cebe‐Suarez et al., 2006). It has
been demonstrated that tyrosines 799 and 1173 of VEGFR2 are binding
sites for the p85 subunit, and that activation of PI3K is responsible for
endothelial cell proliferation (Dayanir et al., 2001). Previous study showed
that VEGFR2 was associated with p85 regulatory subunit of PI3K to phos-
phorylate p85 subunit, resulting in increased PI3K and AKT activities
in vitro (Gerber et al., 1998). Grb2‐adapter binder 1 (Gab1) PH domain
serves as a primary actor in coupling VEGFR2 to PI3K through an

amplification loop involving PIP3 and its PH domain (Dance et al., 2006;
Laramee et al., 2007). VEGF‐induced endothelial cell survival was blocked
by PI3K inhibitors, wortmannin and LY294002, and by overexpression
of a dominant‐negative form of AKT (AKT‐DN) (Gerber et al., 1998).
VEGFR3 is expressed in developing veins and lymphatics, in blood vessels
in the vicinity of tumors, and in several benign and malignant tumor cells
(Cebe‐Suarez et al., 2006). VEGFR3 can promote cell migration and survival
in lymphatic endothelial cells by activating PI3K and mitogen‐activated pro-
tein kinase (MAPK) pathways (Lin et al., 2005; Makinen et al., 2001).
The angiopoietins are a family of secreted proteins including three human
angiopoietins (Ang‐1, Ang‐2, and Ang‐4), and one mouse angiopoietin, Ang‐3.
Ang‐1 is an angiogenic growth factor with a central role in promotion of
structural integrity in the vasculature. Both Ang‐1 and Ang‐2 can bind to
Tie2 receptor. Ang‐1 is a Tie2 agonist, while Ang‐2 could act as either a
context‐dependent competitive antagonist or an agonist depending on cell
type and microenvironmental conditions (Davis et al., 1996; Maisonpierre
et al., 1997). Transgenic overexpression of both Ang‐1 and Ang‐2 led to
vascular defects (Sato et al., 1995). Ang‐3 is moderately expressed in multi-
ple mouse tissues, and functions as a Tie2 activator or as a Tie2 antagonist.
Ang‐4 mRNA is abundantly expressed in human lungs, and functions as a
Tie2 agonist (Jones et al., 2001; Makinde and Agrawal, 2008). The Tie
receptor family is comprised of Tie1 and Tie2/Tek. Ang‐1, 2, 3, and 4 are
specific ligands for Tie2. The specific ligand for Tie1 is unknown. The
phosphorylation of Tie1 is dependent on Tie2 activation, suggesting that
Tie2 tyrosine kinase domain may be responsible for phosphorylating Tie1 as
a result of heterodimerization (Yuan et al., 2007). Tie2 is expressed not only
in vascular cells, but also in cancer cells. Several tumor cells express high
levels of Ang‐1, indicating an autocrine/paracrine loop of Ang‐1‐Tie2 sig-
naling in the tumor. Genetic studies have showed that deletion of Ang‐1 or
Tie2 genes led to severe defects in the vasculature and subsequent lethality,
suggesting that Ang‐1/Tie2 signaling pathway is required in microvascular
development (Makinde and Agrawal, 2008). There are several lines of
evidence suggesting that PI3K/AKT signaling plays a major role in Ang‐1‐
mediated cell migration, survival, and angiogenesis: (1) Ang‐1 was shown to
induce phosphorylation of Tie2, then recruited and interacted with p85
subunit of PI3K in a phosphotyrosine‐dependent manner through their Src
homology 2 (SH2) domains, resulting in the induction of PI3K activities and
activation of AKT (Jones et al., 1999); (2) Ang‐1 induced survival, migra-
tion, and sprouting of endothelial cells through PI3K and AKT activation
(Jones et al., 1999; Kanda et al., 2005; Kim et al., 2000); (3) In vivo studies
also showed that Ang‐1 induced angiogenesis through increasing AKT
phosphorylation and PI3K‐mediated endothelial nitric oxide synthase
(eNOS) activation (Babaei et al., 2003; Cho et al., 2004).

III. GENETIC ABERRATIONS OF PI3K, PTEN,
AND AKT IN CANCER
PI3K activation is implicated to be involved in oncogenesis by the obser-
vation that PI3K is associated with the Src and the middle T oncoproteins
(Sugimoto et al., 1984; Whitman et al., 1985). The activation of PI3K is
through the interaction with p85 regulatory subunit of PI3K, which contains
SH2 domains that bind to phosphotyrosines, and localize PI3K to the
plasma membrane (Otsu et al., 1991). The p110 catalytic subunit of
PI3K was initially identified as an oncogene from the spontaneous chicken
tumor (Chang et al., 1997). The expression of active PI3K by avian retrovi-
rus induced the transformation of chick embryo fibroblasts in vitro, and
induced tumor in chicken (Chang et al., 1997). Abnormalities of PI3K
upstream molecules are common in cancer and this cascade has a role in
tumorigenesis and neoplastic transformation. PI3K is also frequently mutat-
ed in various kinds of human cancers such as ovarian, breast, gastric, bowel,
brain, colon, and hepatocellular carcinomas (Engelman et al., 2006;
Hennessy et al., 2005; Jiang and Liu, 2008). The amplification of
PIK3CA, the gene encoding p110 catalytic subunit of PI3K, was observed
in ovarian, cervical, gastric, and breast cancers (Engelman et al., 2006;
Hennessy et al., 2005; Jiang and Liu, 2008). In addition, the somatic
missense mutations of PIK3CA are the most frequently genetic aberrations
in breast cancer, especially in HER2‐amplified and hormone‐receptor‐posi-
tive breast cancers (Paradiso et al., 2007). The mutations of PIK3CA were
also found in colorectal, gastric, lung, ovarian, hepatocellular, thyroid,
endometrial cancers, glioblastomas, acute leukemia, as well as in malignan-
cies of the central nervous system (Campbell et al., 2004; Jiang and Liu,
2008; Samuels et al., 2004). The p85 regulatory subunit dimerizes with p110
catalytic subunit, and inhibits PI3K activity in normal cells. The deletion of
p85 protein that lacks the inhibitory domain, and loss of the autophosphor-
ylation site at the p85 inhibitory domain, commonly increases PI3K activity.
The deletion and somatic mutations of p85 regulatory subunit (PIK3R1)
were rare, and occurred in primary human glioblastoma, colon, ovarian
cancers, and lymphoma (Jucker et al., 2002; Philp et al., 2001). Recent
study has demonstrated that PIK3CA‐knockout mouse embryonic fibro-
blasts are deficient in cellular signaling in response to various growth factors,
unable to differentiate into adipocytes, and are resistant to oncogenic trans-
formation induced by RTKs (Zhao et al., 2006). Another genetic study
indicated that the kinase activity of p110 was required for GPCR signaling
triggered by lysophosphatidic acid and had a function in oncogenic
transformation.

PTEN was first discovered as the tumor suppressor on human chromo-
some 10q23 in 1997 (Li et al., 1997a; Steck et al., 1997). PTEN is highly
susceptible to deletion or mutation in many human malignancies including
brain, breast, kidney, and prostate cancers (Li et al., 1997a; Steck et al.,
1997). A serial of studies have shown that the tumor suppressor PTEN is
frequently mutated or lost in many kinds of human primary cancers includ-
ing glioblastomas, kidney and uterine endometrioid carcinomas, breast
cancer, lung cancer, colon cancer, and melanoma (Jiang and Liu, 2008;
Salmena et al., 2008; Steck et al., 1997). In addition, the decreasing levels
of PTEN expression are correlated with the progressive outcome of solid
cancers, including ovarian, prostate, and cervical cancers (Harima et al.,
2001; Yoshimoto et al., 2007). PTEN germline mutations lead to a group of
autosomal dominant syndromes including Cowden syndrome, Lhermitte–
Duclos disease, Bannayan–Riley–Ruvalcaba syndrome, and Proteus and
Proteus‐like syndromes characterized by developmental disorders, neurolog-
ical deficits, multiple hamartomas, and an increased risk of breast, thyroid,
and endometrial cancers (Liaw et al., 1997; Marsh et al., 1997; Tsou et al.,
1997; Tsuchiya et al., 1998). Mice with PTEN deletion and mutation are
highly susceptible to tumor induction and conditional knockout of PTEN
leads to neoplasia in multiple organs such as the mammary gland, skin, and
prostate (Backman et al., 2004; Li et al., 2002; Suzuki et al., 1998). In an
animal model of prostate tumor induced by PTEN loss, ablation of p110
impeded tumorigenesis with a concomitant diminution of AKT phosphory-
lation (Jia et al., 2008), indicating the important role of p110 in cell
transformation and tumorigenesis. These studies demonstrate the key roles
of PI3K and PTEN in cancer development. The transgenic ablation models
of PI3K and PTEN in tumorigenesis are summarized in Table I.
IV. ROLES OF PI3K AND AKT IN
REGULATING ANGIOGENESIS
PI3K/AKT signaling pathway also plays an important role in regulating
the vasculature and angiogenesis. In zebrafish, K‐ras/PI3K/AKT signaling is
essential for hematopoiesis and angiogenesis (Liu et al., 2008a). The direct
evidence of PI3K and AKT involvement in regulating angiogenesis in vivo
was initially observed by the forced expression of PI3K and AKT using
RCAS retroviral vector system (Jiang et al., 2000). Overexpression of PI3K
or AKT induced angiogenesis, while overexpression of PTEN or of
dominant‐negative constructs of PI3K inhibited angiogenesis in chicken
embryos, suggesting that PI3K signaling is required for normal embryonal
angiogenesis (Jiang et al., 2000). Mice deficient in the p110 catalytic

Table I Transgenic Ablation Models of PI3K/PTEN/AKT Signaling Pathway Related to Carcinogenesis, Vasculature, and Angiogenesis
Targeted subunit Genetic alteration Comments
p110 p110
/
Embryonic lethality, multiple vascular defects, lower Tie2 protein
levels (Lelievre et al., 2005)
Endothelial cell‐specific‐p110
/
Embryonic lethality at mid‐gestation because of severe defects in an-
giogenic sprouting and vascular remodeling (Graupera et al., 2008)
p110 p110
/
The significantly diminished vascular permeability in response to both
Ras and VEGF (Serban et al., 2008)
p85 /p55 /p50 (pan‐p85 ) pan‐p85
/
Embryonic lethality, subepidermal blebs flanking the neural tube
and bleeding into the blebs during the turning process (Brachmann
et al., 2005)
p85 subunits Muscle‐specific pan‐p85
/
p85
/
Viable, exhibit attenuated AKT signaling in the heart, reduced heart
size, and altered cardiac gene expression (Luo et al., 2005)
PTEN PTEN
/
Early embryonic lethality
PTEN
þ/
Showed neoplasms in multiple organs including prostate, skin and
endometrium, liver, colon, gastrointestinal tract, and thymus,
spontaneously developed germ cell, gonadostromal, breast, thyroid
tumors, and lymphomas (Di Cristofano et al., 1998; Podsypanina
et al., 1999; Stambolic et al., 2000; Suzuki et al., 1998)
PTEN
/
in smooth muscle cells Died before 6 weeks, increase in phosphorylated AKT in major vessels,
hearts, and lungs, pathological vascular remodeling and vascular
recruitment of progenitor cells, widespread smooth muscle cell
hyperplasia and abdominal leiomyosarcomas (Hernando et al.,
2007; Nemenoff et al., 2008)
Bronchioalveolar epithelium‐specific
PTEN
/
90% of SOPten
flox/flox
(E10–E16) mice died within 2 h of birth,
surviving mice developed spontaneous lung adenocarcinomas with
hyperplasia of bronchioalveolar epithelial cells and myofibroblast
precursors, enlarged alveolar epithelial cells, and impaired produc-
tion of surfactant proteins. K‐ras was frequently mutated in adeno-
carcinomas (Yanagi et al., 2007)
(continues)

Table I (continued)
Targeted subunit Genetic alteration Comments
Hepatocyte‐specific PTEN
/
Massive hepatomegaly and steatohepatitis with triglyceride accumu-
lation followed by liver fibrosis and hepatocellular carcinoma (Horie
et al., 2004; Watanabe et al., 2007)
PTEN
/
in endothelial cells Embryonic lethality due to endothelial cell hyperproliferation and
impaired vascular remodeling (Suzuki et al., 2007)
PTEN
þ/
in endothelial cells Enhances postnatal neovascularization, including tumor angiogenesis
necessary for tumor growth (Suzuki et al., 2007)
Urothelium‐specific PTEN
/
Exhibited urothelial hyperplasia, 10% of mice spontaneously devel-
oped pedicellate papillary transitional cell carcinomas (Tsuruta
et al., 2006)
Pancreas‐specific PTEN
/
Progressive replacement of the acinar pancreas with highly prolifera-
tive ductal structures, a fraction of these mice develop ductal ma-
lignancy (Stanger et al., 2005)
Prostate‐targeted PTEN
/
Hyperproliferation and neoplastic changes in prostate (Backman et al.,
2004; Ma et al., 2005; Trotman et al., 2003; Wang et al., 2003,
2006)
Astrocytes‐specific PTEN
/
Hypertrophy and increased proliferation of astrocytes in vivo (Fraser
et al., 2004)
Skin‐specific PTEN
/
Hyperproliferation and spontaneous tumorigenesis of the skin kerati-
nocytes (Komazawa et al., 2004)
PTEN
þ/
in primordial germ cells Testicular teratoma and enhanced embryonic germ cell production
(Kimura et al., 2003)
Mammary‐specific PTEN
/
Precocious development and neoplasia in the mammary gland
(Li et al., 2002)
30

AKT1 AKT1
/
Smaller litter sizes, reduced fetal weight, and a higher fetal mortality
due to the impaired extraembryonic vascularization and placental
hypotrophy (Chen et al., 2001; Cho et al., 2001b; Yang et al., 2003)
Impairment of blood vessel maturation and increased vascular perme-
ability, reduced activation of eNOS, and reduced expression of
thrombospondins 1 (TSP‐1) and TSP‐2 (Chen et al., 2005)
Defective ischemia‐ and VEGF‐induced angiogenesis and severe pe-
ripheral vascular disease (Ackah et al., 2005)
Abrogated polarity, migratory directionality, and breast cancer onset of
mammary epithelial cells with ErbB2 overexpression (Ju et al., 2007)
Resistant to tumors and skin carcinogenesis induced MMTV‐v‐H‐Ras‐
induced (Skeen et al., 2006)
AKT2 AKT2
/
Displayed normal cardiac growth in responses to provocative stimu-
lation, and sensitized to cardiomyocyte apoptosis in response to
ischemic injury (DeBosch et al., 2006)
31

subunit of PI3K displayed multiple vascular defects, including dilated vessels
in the head, reduced branching morphogenesis in the endocardium, lack of
hierarchical order of large and small branches in the yolk sac, impaired
development of anterior cardinal veins, and significant decrease of Tie2
protein level (Lelievre et al., 2005). In mice deficient in p110 , the vascular
permeability response to both Ras and VEGF was significantly diminished,
suggesting that PI3K is necessary and sufficient for vascular permeability
(Serban et al., 2008). Endothelial cell‐specific‐p110 /
led to embryonic
lethality at mid‐gestation due to severe defects in angiogenic sprouting and
vascular remodeling (Graupera et al., 2008). Knockout of p85 /p55 /p50
caused perinatal lethality with bleeding into the blebs during the turning
process (Brachmann et al., 2005). Muscle‐specific pan‐p85 /
p85 /
mice exhibited reduced heart size and altered cardiac gene expression (Luo
et al., 2005). Mutated p110 proteins show a gain of enzymatic function
in vitro. Recent studies show that three prevalent mutants of p110 , E542K,
E545K, and H1047R, are oncogenic in vivo (Bader et al., 2006). These
tumors are marked by increased angiogenesis and the activation of AKT
pathway (Bader et al., 2006).
AKT was initially found to be the homolog of a viral oncogene (Bellacosa
et al., 1991). In various kinds of tumors, AKT is also overexpressed or
amplified, with elevated level of AKT phosphorylation (Hennessy et al.,
2005; Jiang and Liu, 2008). There are several reports showing the genetic
amplification of AKT isoforms. AKT1 amplification has been observed in
gastric adenocarcinoma, glioblastoma, gliosarcoma, and high‐grade gliomas
(Jiang and Liu, 2008; Liaw et al., 1997; Sasaki et al., 2003; Staal, 1987).
AKT2 amplification or mutations are found in head and neck squamous cell
carcinoma, pancreatic, ovarian, breast, and colorectal cancers (Hennessy
et al., 2005; Jiang and Liu, 2008). Increased AKT3 mRNA level is correlated
to breast and prostate cancers (Nakatani et al., 1999). Recent studies have
shown that AKT1/
mice are resistant to ErbB2‐ or MMTV‐v‐H‐Ras‐
induced carcinogenesis, indicating the key role of AKT1 in oncogenesis
(Ju et al., 2007; Skeen et al., 2006). Among three isoforms of AKT, AKT1
shows closely related with vasculature during animal development and
pathological angiogenesis. AKT1/
mice have defects in both fetal and
postnatal growth into adulthood with smaller litter sizes and reduced fetal
weight (Chen et al., 2001; Cho et al., 2001b). Since AKT1 is widely
expressed in placenta including all types of trophoblast and vascular endo-
thelial cells, AKT1/
mice exhibited a higher fetal mortality due to the
impaired extraembryonic vascularization and placental hypotrophy, indicat-
ing the significant role of AKT1 in fetal development and vascularization
(Yang et al., 2003). AKT1 is the predominant isoform in vascular cells.
AKT1/
mice showed impaired vascular maturation due to reduced activa-
tion of eNOS and the major phenotypic changes in vascular permeability

and angiogenesis with decreased expression of thrombospondins 1 and 2
(TSP‐1 and TSP‐2) (Chen et al., 2005). AKT1 is critical for ischemic‐ and
VEGF‐induced angiogenesis. AKT1/
mice exhibited defective ischemia‐
and VEGF‐induced angiogenesis and showed severe peripheral vascular dis-
ease. In response to ischemia, AKT1/
mice had much less endothelial
progenitor cell (EPC) mobilization. Intravenous administration of EPCs
from wild‐type AKT1 mice, but not EPCs isolated from AKT1/
mice, into
mice improved limb blood flow, increased the migration of fibroblasts and
endothelial cells after femoral ligation. These results indicate that AKT1 is
sufficient and essential for regulating ischemia‐induced angiogenesis (Ackah
et al., 2005). AKT2/
mice displayed normal cardiac growth in response to
provocative stimulation, and were sensitized to cardiomyocyte apoptosis in
response to ischemic injury (DeBosch et al., 2006). The studies on transgenic
models related to vasculature and angiogenesis are summarized in Table I.
V. PI3K/PTEN CONTROLS ANGIOGENESIS THROUGH
INCREASING HIF‐1 AND VEGF EXPRESSION
Hypoxia is an integral characteristic of the tumor microenvironment,
associated with accelerated neoplastic growth. Hypoxia‐inducible factor 1
(HIF‐1) is a heterodimer consisting of HIF‐1 and HIF‐1 [also known as
the aryl hydrocarbon nuclear translocator (ARNT)] subunits, and acts as a
mediator of transcriptional activation in responses to hypoxia (Wang et al.,
1995). HIF‐1 is rapidly degraded under normoxic conditions by hydroxyl-
ation at several proline residues, and acetylation at lysine 5328 (Jeong et al.,
2002; Semenza, 2000). The von Hippel‐Lindau tumor suppressor gene
product, pVHL, functions as the substrate recognition component of an
E3‐ubiquitin ligase, which targets the oxygen‐sensitive HIF alpha‐subunit
for rapid proteasomal degradation under normoxic conditions and as such
plays a central role in oxygen sensing (Maxwell et al., 1999). Hypoxia or
lossofpVHLinhibitsprolyl‐hydroxylation,leadingtoaccumulationofHIF‐1
protein in the cytoplasm (Kapitsinou and Haase, 2008). Growth factors,
cytokines, and other signaling molecules stimulate HIF‐1 synthesis via
activation of PI3K or MAPK pathways (Mazure et al., 1997; Zhong et al.,
2000). HIF‐1 regulates VEGF expression by binding to the hypoxia respon-
sive element (HRE) of VEGF promoter (Levy et al., 1995; Wang et al.,
1995). HIF‐1 can activate more than 60 known genes, which are related
to cell proliferation, survival, apoptosis, cell mortality, adhesion, erythro-
poiesis, cytoskeletal structure, pH regulation, epithelial homeostasis, drug
resistance, iron, nucleotide, glucose, energy, amino acid, and extracellular‐
matrix metabolisms, vascular tone, and angiogenesis (Semenza, 2003).

HIF1 is upregulated in many human cancers. Among all the angiogenic
factors, VEGF is the most potent one in physiological and pathological
angiogenesis.
HIF‐1 expression is regulated by PI3K activation in response to growth
factors. Insulin and EGF induced expression of HIF‐1 and VEGF by PI3K
signaling pathway (Jiang et al., 2001). Cobalt and hypoxia induced HIF‐1
expression through PI3K‐dependent mechanism in airway smooth muscle
and pulmonary artery smooth muscle cells (Belaiba et al., 2007; Chachami
et al., 2004). HIF‐1‐dependent gene transcription was blocked by AKT‐DN
or PI3K, and by wild‐type PTEN, whereas transcription was stimulated by
constitutively active form of AKT. PI3K inhibitor LY294002 and mTOR
inhibitor rapamycin also inhibited growth factor‐ and mitogen‐induced
secretion of VEGF, which may provide the connection of PI3K/PTEN/AKT
to mTOR, HIF‐1, and tumor angiogenesis (Jiang et al., 2001; Zhong et al.,
2000). On the other hand, overexpression of PI3K or AKT elevated the
mRNA levels of VEGF. LY294002 suppressed VEGF mRNA expression,
while this inhibition was restored by overexpression of PI3K or AKT (Jiang
et al., 2000). These results indicate that PI3K is sufficient to induce angio-
genesis, and the effect may be partially through increasing HIF‐1 and VEGF
expression. Similarly, VEGF transcriptional activation in ovarian cancer
cells was regulated by PI3K/AKT through HIF‐1 expression (Skinner
et al., 2004). A number of studies have demonstrated that PI3K/PTEN/
AKT signaling regulates HIF‐1 and VEGF expression in different types of
cancer cells, Ras‐transformed cells, airway smooth muscle cells, pulmonary
artery smooth muscle cells, osteoblasts, pulmonary vascular endothelial
cells, and mast cells (Belaiba et al., 2007; Carver et al., 2007; Chachami
et al., 2004; Jiang et al., 2001; Lee et al., 2008; Mazure et al., 1997;
Trisciuoglio et al., 2005; Yen et al., 2005; Zhong et al., 2000). Mast cells
mediated VEGF expression by HIF‐1 activation through PI3K‐HIF‐1
pathway in mice with allergic airway disease, resulting in the increase of
vascular permeability (Lee et al., 2008). Hypoxia exposure of melanoma
cells overexpressing bcl‐2 activated phosphorylation of AKT and extracellu-
lar signal‐regulated kinase (ERK)1/2 proteins, induced VEGF and HIF‐1
expression, which can be suppressed by PI3K and MAPK inhibitors, suggest-
ing that bcl‐2 synergizes with hypoxia to promote expression of angiogenesis
factors in melanoma cells through both PI3K and ERK pathways
(Trisciuoglio et al., 2005).
Consistent with those results in vitro, in vivo studies showed that
LY294002 significantly decreased the tumor burden of mice and inhibited
peritoneal and tumor vascularization, which resulted in numerous leaky,
irregular, tortuous vessels in scant, straight, relatively impermeable vessels,
demonstrating the role of PI3K in mediating angiogenesis and vas-
cular permeability associated with ovarian carcinoma (Hu et al., 2005).

Specific downregulation of p110 expression in ovarian cancer cells using
small interfering RNA (siRNA) showed that p110 knockdown greatly
decreased ovarian tumor growth and angiogenesis, inhibited VEGF expres-
sion through decreasing HIF‐1 expression in both ovarian cancer cells and
tumor tissues. Moreover, AKT1 is a major downstream mediator for reg-
ulating tumor growth, angiogenesis, and VEGF expression, suggesting that
p110 and AKT1 play an important role in tumor growth by inducing
angiogenesis and by increasing HIF‐1 and VEGF expression (Xia et al.,
2006). Inhibition of PI3K activity by LY294002 decreased cancer cell‐
induced angiogenesis (Fang et al., 2007). Reconstitution of PTEN or over-
expression of AKT dominant negative also inhibited angiogenesis and tumor
growth associated with the decrease of HIF‐1 and VEGF expression in the
tumor xenographs (Fang et al., 2007). These results suggest that PI3K and
AKT may regulate tumorigenesis and angiogenesis through HIF‐1 and
VEGF expression in cancer cells.
VI. THE DOWNSTREAM SIGNALING MOLECULES
MEDIATED BY PI3K/PTEN IN REGULATING TUMOR
GROWTH AND ANGIOGENESIS
Overexpression and activation of AKT play an important role in carcino-
genesis (Engelman et al., 2006; Hennessy et al., 2005; Jiang and Liu, 2008).
The mutations or deletions of PTEN are presented in many kinds of solid
tumors. As shown in Fig. 1, upon the stimulation of VEGF and other growth
factors, RTKs can activate PI3K which exerts its effect through AKT and
other downstream targets (Engelman et al., 2006; Jiang and Liu, 2008).
GSK‐3 , the downstream target of AKT, together with the adenomatous
polyposis coli (APC) protein and axin, forms a multiprotein complex which
phosphorylates ‐catenin making it for subsequent ubiquitination and deg-
radation (Liu et al., 2005; Rubinfeld et al., 1996). Thus, the reduced expres-
sion of GSK‐3 can cause the increase of ‐catenin activity. On the other
hand, PI3K may indirectly activate ERK and p38 MAPK signaling pathways
through Rho GTPases (Mizukami et al., 2006; Xue et al., 2006). Recent
study has demonstrated that in addition to suppress AKT activation, PTEN
also controls the activity of Jun N‐terminal kinase (JNK) (Vivanco et al.,
2007). Both AKT and ERK can activate NF‐B pathway, performing a
complicated network in regulating tumor growth, metastasis, and angiogen-
esis (Fig. 1). The downstream signaling molecules related to tumorigenesis
and angiogenesis are outlined in Fig. 1, and briefly described below.

A. Tumor Growth
PI3K may transmit oncogenic signals to AKT for regulating tumorigenesis
through several downstream targets. AKT can directly phosphorylate
human double minute 2 (HDM2) and regulate HDM2 through p70S6K1
activation (Fang et al., 2005; Mayo and Donner, 2001; Skinner et al., 2004).
HDM2 regulates tumor suppressor p53 by promoting its proteasome‐
mediated degradation (Fang et al., 2006; Skinner et al., 2004). p53 plays a
key role in carcinogenesis and cellular apoptosis. AKT activates NF‐B
pathway by the phosphorylation of I kappaB kinase (IKK) / (Hurt et al.,
2002; Lu and Wahl, 2005; Ozes et al., 1999; Tanaka et al., 2005). Activated
AKT pathway also exhibits the antiapoptotic effect through the activation of
nitric oxide synthase (NOS), the inhibition of FOXO‐mediated transcription
of proapoptotic proteins, and the inactivation of proapoptotic protein BAD
by phosphorylation to activate survival signals. In addition, AKT regulates
cell proliferation and tumor growth by increasing the cell‐cycle progression.
AKT blocks FOXO‐mediated transcription of cell‐cycle inhibitors, and pro-
motes G1 to S phase transition. AKT stabilizes c‐Myc and cyclin D1 through
eIF4E CXCL-8,
CXCL-1,
VCAM,
ICAM,
COX-2
AKT
VEGF and other growth factors
VEGF
HIF-1a
RTKs
PI3K
PTEN
IKK
NF-kB
GSK-3b
b-catenin
NOS
FOXO
TNF, IL-
1, IL-6
MMPs
Metastasis
c-myc,
cyclin-D1
HDM2
p53
Tumor growth
TSC1-TSC2
mTOR
S6K
4EBP
S6
Tumor angiogenesis
Rho GTPases
ERK/
p38MAPK
Hypoxia
TSP1
Protein synthesis
Anti-apoptosis
BAD
JNK
Fig. 1 Targets of PI3K and PTEN in regulating tumor growth, metastasis, and angiogenesis.
(See Page 1 in Color Section at the back of the book.)

the activation of NF‐B pathway and GSK‐3 / ‐catenin‐signaling axis. Cell
proliferation, size, and growth are tightly regulated by the activation of
mTOR through PI3K/AKT and MAPK pathways. AKT and MAPK can
regulate mTOR to control protein synthesis and cell proliferation, which
are associated with carcinogenesis. The regulation of cell survival and cell
cycle is associated with the increased cell number in tumors.
B. Tumor Metastasis
The basement membrane forms a cellular support for tumors, and is
made up of a complex mix of extracellular matrix (ECM) proteins. The
proteolytic enzymes including matrix metalloproteinases (MMPs) can de-
grade ECM (Orlichenko and Radisky, 2008). PI3K activates MMP‐2,
MMP‐9, and Urokinase‐type plasminogen activator (uPA), leading to de-
struction of ECM (Ispanovic and Haas, 2006; Shukla et al., 2007). PI3K
activity is shown to be higher in metastatic cells when compared to non-
metastatic cancer cells. Increased levels of MMPs are also due to the
activation of AKT/IKK/NF‐B pathway and AKT/GSK‐3 / ‐catenin axis
(Agarwal et al., 2005; Amiri and Richmond, 2005; Ispanovic and Haas,
2006; Kim et al., 2005). PI3K signaling also regulates chemokine (C‐X‐C
motif) ligand 1 (CXCL‐1), cyclooxygenase‐2 (COX‐2), and interleukin‐
8 (CXCL‐8) that enhance tumor metastasis. PI3K and AKT regulate
epithelial–mesenchymal transition (EMT), which is a change thought to
herald tissue invasion and prophesize metastatic potential (Cheng et al.,
2008; Onoue et al., 2006). NF‐B plays a key role in EMT by the activa-
tion of mesenchymal program (involving genes such as MMP2/9, VCAM‐
1, ICAM‐1, and Cathepsins B and Z) (Huber et al., 2004) and the repres-
sion of E‐cadherin, a metastasis suppressor protein, by activating bcl‐2 and
TWIST (Naugler and Karin, 2008). E‐cadherin is a key marker of EMT and
loss of E‐cadherin disrupts not only cell–cell junctions, but also allows for
loss of the normal organ architecture. ‐Catenin plays an important role in
downregulating E‐cadherin expression (Brabletz et al., 2005; Lu et al.,
2003). PI3K and AKT also increase invasiveness and downregulate
E‐cadherin expression (Grille et al., 2003; Larue and Bellacosa, 2005;
Schramek et al., 2003; Thiery and Sleeman, 2006). Cell motility is a
fundamental process during tumor metastasis. PI3K in combination with
the small GTPase Rac and Cdc42 regulates cell motility by controlling
actin dynamics in motile cells (Engelman et al., 2006). ERK pathway is
also involved in regulating the expression of MMPs, cell migration, and
EMT (Reddy et al., 2003).

C. Tumor Angiogenesis
First, PI3K and AKT may regulate tumor angiogenesis by several down-
stream targets such as mTOR/p70S6K1 signaling axis, the inhibition of
FOXO, the induction of NOS (Emerling et al., 2008; Engelman et al.,
2006; Quintero et al., 2006; Wang et al., 2004), and/or the inhibition of
GSK‐3 . These targets commonly increase HIF‐1 expression which induces
VEGF transcriptional activation. Inhibition of GSK‐3 by the activation of
PI3K/AKT can upregulate HIF‐1 expression, and increases ‐catenin ac-
tivity, which can enhance HIF‐1‐mediated transcription through the ‐cate-
nin‐HIF‐1 interaction at the promoter region of HIF‐1 target genes (Kaidi
et al., 2007; Mottet et al., 2003). In addition, hypoxia is a hallmark of the
tumor microenvironment in the fast growth tumor. Hypoxia induces HIF‐1
production through the increase of its stability and the activation of ERK1/2
pathway. In some kinds of cancer cells, hypoxia stimulates multiple K‐ras
effectors and PI3K, which induces VEGF expression in a HIF‐1‐dependent
manner or via PI3K/Rho/ROCK/c‐myc pathway (Mizukami et al., 2006; Xue
et al., 2006). PI3K can induce VEGF expression through HIF‐1, ERK1/2, and
NF‐B activation to induce tumor angiogenesis. NF‐B can also stimulate
tumor necrosis factor (TNF), CXCL‐8, IL‐1, and IL‐6 to induce VEGF
(Amiri and Richmond, 2005; Sparmann and Bar‐Sagi, 2004). Growing
evidence has shown the key roles of PI3K, AKT, mTOR, and their effectors
HIF‐1 and VEGF in regulating cancer cell‐induced angiogenesis (Fang
et al., 2007; Hu et al., 2005; Xia et al., 2006).
Next, the angiogenesis and vasculature are regulated though the change of
balance between the collective actions of proangiogenic factors (e.g., VEGF)
and angiogenic inhibitors (e.g., TSP‐1). PI3K/AKT can increase VEGF expres-
sion and suppress TSP‐1, the endogenous antiangiogenic molecule, in both
cancer cells and endothelial cells (Niu et al., 2004; Wen et al., 2001). Further-
more, AKT1/
mice showed impaired vascular maturation with decreased
expression of TSP‐1 and TSP‐2, while reexpression of TSP‐1 and TSP‐2 in
mice transplanted with wild‐type bone marrow is associated with the angio-
genic abnormalities in AKT1/
mice (Chen et al., 2005). Thus, PI3K/AKT
signaling pathway induces tumor growth through the overexpression of an-
giogenic factors and the inhibition of antiangiogenic molecules.
Third, tumor angiogenesis is regulated by the tumor microenvironments
composed of tumor cells, vascular endothelial cells, and stromal cells. In
addition to cancer cells, the microvascular endothelial cells recruited by the
tumor are important for cancer development (Carmeliet and Jain, 2000;
Stoeltzing et al., 2006). PI3K/AKT pathway also controls tumor microenvir-
onments, including endothelial cells (Phung et al., 2006; Yuan et al., 2007).
PI3K can regulate endothelial migration, proliferation, and survival through
the effect of its downstream targets such as NOS, p70S6K1, and FOXO to

regulate tumor angiogenesis (Fosbrink et al., 2006; Nakao et al., 2007;
Zheng et al., 2008). Class IA PI3Ks regulate vessel integrity during develop-
ment and tumorigenesis (Yuan et al., 2008). Further analysis of p110 iso-
forms has demonstrated that p110 is required to control endothelial cell
migration and angiogenesis, and p110 /
endothelial cells lead to embry-
onic lethality with severe defects in angiogenic sprouting and vascular
remodeling (Graupera et al., 2008; Suzuki et al., 2007). PTEN/
endothe-
lial cells cause embryonic lethality due to endothelial cell hyperproliferation
and impaired vascular remodeling; PTENþ/
endothelial cells enhance post-
natal neovascularization and tumor angiogenesis to increase tumor growth
(Suzuki et al., 2007). Transgenic expression of Myr‐AKT1 in endothelial
cells is sufficient to recapitulate the abnormal structural and functional
features of tumor blood vessels innontumortissues,likelydue tothe induction
of VEGF‐A (Jiang et al., 2000; Phung et al., 2006). Sustained endothelial
AKT activation causes enlarged and hyperpermeable blood vessels and its
effect can be completely reversed by AKT inhibition or by rapamycin
treatment (Phung et al., 2006). Our studies using chimeric tumor model
found that overexpression of p70S6K1 in human dermal microvascular endo-
thelial cells (HDMECs) enhanced tumor growth and angiogenesis, while over-
expression of p70S6K1‐kinase mutant, or of HIF‐1 siRNA significantly
inhibited tumor growth and angiogenesis, suggesting that endothelial
p70S6K1 controls tumor angiogenesis through HIF‐1 and VEGF expression
(Liu et al., 2008b).
The interaction of cancer cells and vascular endothelial cells in the tumor
microenvironment affects angiogenesis. In cancer cells, stimuli such as growth
factors, insulin, and other hormones activate PI3K/AKT/mTOR/ HIF‐1 axis,
and induce the production of VEGF, which switches angiogenic response and
causes endothelial cell activation and permeability increased by PI3K pathway
(Nyberg et al., 2008; Stoeltzing et al., 2006). Thus, inhibition of PI3K/AKT/
mTOR pathway is one of the choices in cancer treatment, which is going on
under the preclinical and clinical trials. The signaling pathway of PI3K related
to tumor growth, metastasis, and angiogenesis is shown in Fig. 1.
VII. INHIBITION OF PI3K SIGNALING PATHWAY
FOR CANCER TREATMENT AND PREVENTION
Given the important role of PI3K signaling pathway in regulating tumor
growth and angiogenesis, development of therapeutic drugs using PI3K,
AKT, and mTOR inhibitors becomes important for cancer treatment.
Here, we introduce the inhibitors of PI3K, AKT, and mTOR.

A. PI3K Inhibitors
PI3K inhibitors, wortmannin, and LY294002, are commonly used to inhibit
cancer cell proliferation and tumor growth, and sensitize tumor cells to the
treatment of chemotherapeutic drugs and radiation (Granville et al., 2006).
Wortmannin is a fungal product isolated from Penicillium wortmanni in
1957, which exerts its effect by the covalent interaction to the conserved
Lys802 of the PI3K catalytic subunit and Lys833 in PI3K (Walker et al.,
2000; Wymann et al., 1996). The pan‐PI3K inhibitor LY294002 was synthe-
sized in the early nineties. Both wortmannin and LY294002 also cross‐react
with PI3K‐related kinases such as mTOR and DNA‐dependent protein
kinases (DNA‐PKs). These PI3K inhibitors have poor solubility and high
toxicity because they target a broad range of PI3K‐related enzymes, which
limits their clinical application (Marone et al., 2008). To overcome these
shortcomings, many derivatives of wortmannin and LY294002 are being
developed (Marone et al., 2008). In addition, inositol(1,3,4,5,6) pentakispho-
sphate [Ins(1,3,4,5,6)P5], the PI3K/AKT inhibitor, inhibits tumor growth and
angiogenesis in vitro and in vivo (Maffucci et al., 2005). PWT‐458, a novel
pegylated 17‐hydroxywortmannin, is water‐soluble and has shown significant
improvements in drug stability as well as in vivo pharmacokinetic parameters.
It inhibits PI3K signaling and suppresses growth of solid tumors in nude mice
(Yu et al., 2005). SF1126, a small molecule conjugate containing a pan‐PI3K
inhibitor, suppresses PI3K class IA isoforms and other key members of the
PI3K superfamily including DNA‐PK. In preclinical studies, it has been shown
to inhibit tumor growth, dissemination, and angiogenesis (Garlich et al.,
2008). The other two pan‐PI3K inhibitors, PI‐103 and ZSTK474 share the
arylmorpholine structure of LY294002. PI‐103 is a dual PI3K IA/mTOR
inhibitor, while ZSTK474 inhibits the activity of all class I PI3Ks. Both of
these drugs exhibit antitumor effect on various kinds of cancers (Chaisuparat
et al., 2008; Fan et al., 2006; Kong and Yamori, 2007; Yaguchi et al., 2006;
Yuan and Cantley, 2008). IC486068, a p110 specific inhibitor, enhances
radiation‐induced tumor vascular destruction (Geng et al., 2004). NVP‐
BEZ235, an orally administered inhibitor of dual pan‐class I PI3K and
mTOR kinase, inhibits the growth of breast and prostate cancer cells with
active mutations of PI3K, and decreases tumor vasculature (Maira et al.,
2008; Schnell et al., 2008; Serra et al., 2008). Recent study has shown
that the dual PI3K/PDK‐1 inhibitor, BAG956, has inhibitory effect on BCR‐
ABL‐ and mutant FLT3‐expressing cells both in vitro and in vivo (Weisberg
et al., 2008).
Several PI3K inhibitors are used in clinical trials now. For example, XL147
and XL765, the exelixis compounds, are in phase I trials for the treatment of
solid tumors. NVP‐BEZ235 and another Novartis compound, BGT226, are

in ongoing trials for breast and other solid tumors with some promising
results (Yuan and Cantley, 2008).
B. AKT Inhibitors
AKT is a major downstream target of PI3K for regulating tumor growth
and angiogenesis. The first developed group of AKT inhibitors were lipid‐
based inhibitors that include perifosine, phosphatidylinositol ether lipid
analogs (PIAs), and D‐3‐deoxy‐phosphatidylmyoinositol‐1‐[(R)‐2‐methoxy‐
3‐octadecyloxyropyl hydrogen phosphate] (PX‐316), which showed antitu-
mor effects in vitro and in vivo (Gills et al., 2006; Granville et al., 2006;
Jiang and Liu, 2008; Meuillet et al., 2004). Several other AKT antagonists
such as 9‐methoxy‐2‐methylellipticinium acetate (API‐59‐OMe), indazole‐
pyridine A‐443654, and isoform‐specific canthine alkaloid analogs have
been identified using high‐throughput screening of the chemical libraries
and shown to inhibit human cancer cell growth and induce apoptosis
(Granville et al., 2006; Liu et al., 2008c; Shi et al., 2005). Other kinds of
AKT inhibitors being developed include peptide‐based inhibitors of AKT (e.
g., KP372‐1), pseudopeptide substrates of AKT, a single‐chain antibody
(scFv) against AKT, an inhibitory form of AKT expressed by adenovirus
virus system, and siRNA against AKT (Granville et al., 2006; Jiang and Liu,
2008; Litman et al., 2007; Mandal et al., 2006; Xia et al., 2006).
Perifosine is one of the best‐characterized AKT inhibitors, which inhibits
the translocation of AKT to the cell membrane. Perifosine inhibits tumor
growth in several different kinds of solid tumors. It has been used for clinical
trials for the treatment of prostate, breast, gastrointestinal stromal tumors,
melanoma, and soft tissue sarcoma, but the clinical outcomes were not
satisfied (Table II).
C. mTOR Inhibitors
The mTOR inhibitor, rapamycin (sirolimus) and its analogs CCI‐779
(temsirolimus), RAD001 (everolimus), and AP‐23573 (deforolimus) inhibit
mTOR activation by binding to FK506‐binding protein‐12 (Hennessy et al.,
2005). These drugs are currently under the clinical trials for cancer treat-
ment. Preclinical studies with these compounds indicated that these com-
pounds have synergistic effects for inhibiting tumor growth when they
are used with conventional chemotherapy agent or radiation treatment.
In clinical studies, these compounds have been shown to be effective against
many types of cancers (Easton and Houghton, 2006; Faivre et al., 2006).
In phase I trials, rapamycin has shown anticancer activity in recurrent

Table II Clinical Trials of PI3K/AKT/mTOR Pathway Inhibitors for Cancer Therapy
Targets Drug name Phase Tumor types Comments and references
AKT Perifosine I Incurable solid
malignancies
In order to get suitable dose, pharmacokinetic data,
and side effects (Crul et al., 2002; Van Ummersen et al., 2004)
I Advanced solid tumors Pharmacokinetic study showed that perifosine can be safely
combined with fractionated radiotherapy (Vink et al., 2006)
I/II Gastrointestinal stromal
tumor in combination
with imatinib
Ocular toxicity and ulcerative keratitis were associated with
Perifosine (Shome et al., 2008)
II Advanced breast cancer No objective responses were seen in this group of pretreated
metastatic breast cancer patients (Leighl et al., 2008)
II Androgen independent
prostate cancer
No significant clinical activity against prostate cancer was
observed in this population (Posadas et al., 2005)
II Advanced soft tissue
sarcoma
Optimism remains for this agent in STS patients (Bailey
et al., 2006)
No significant response was seen (Knowling et al., 2006)
II Recurrent, hormone‐
sensitive prostate
cancer
Modest single‐agent clinical activity (Chee et al., 2007)
II Pancreatic
adenocarcinoma
Perifosine did not appear to be worthy of further study in this
group of patients (Marsh et al., 2007)
II Recurrent or metastatic
head and neck cancer
(SCCHN)
Perifosine in the doses and schedule used lacked single‐agent
activity in SCCHN (Argiris et al., 2006)
mTOR Rapamycin
(sirolimus)
Hepatocellular and cho-
langiocellular cancer
A temporary disease‐control rate was identified and the toxicity
was acceptable (Rizell et al., 2008)
Chronic myeloid leukae-
mia (CML)
Rapamycin showed antileukemic effects in imatinib‐resistant
CML (Sillaber et al., 2008)
42

I Nonsmall cell lung cancer
(NSCLC)
Combination therapy with sirolimus, radiation, and cisplatin
was well tolerated in patients (Sarkaria et al., 2007)
I Recurrent PTEN‐deficient
glioblastoma
Rapamycin had anticancer activity in PTEN‐deficient
glioblastoma and warrants further clinical study alone
or in combination with PI3K pathway inhibitors
(Cloughesy et al., 2008)
I Recurrent malignant
glioma
Gefitinib plus sirolimus was safely coadministered on a
continuous, daily dosing schedule (Reardon et al., 2006)
CCI‐779
(temsirolimus)
Solid tumor, recurrent
malignant glioma, ad-
vanced renal cancer
To establish the safety, tolerability, and pharmacokinetic
parameters of CCI‐779 (Kuhn et al., 2007; Peralba et al.,
2003; Raymond et al., 2004)
I Solid tumors or
lymphomas
Antitumor efficacy was observed and CCI‐779 was generally
well tolerated on this intermittent schedule (Hidalgo et al.,
2006)
I Advanced solid tumors The administration of CCI‐779 and 5‐FU/LV at these doses
and schedule resulted in unacceptable toxicity and
therefore it is not recommended (Punt et al., 2003)
I Recurrent malignant
glioma
The recommended dose of CCI‐779 for patients on enzyme‐
inducing antiepileptic drugs was 250 mg IV weekly
(Chang et al., 2004)
I/II Advanced renal‐cell
carcinoma
The combination of CCI‐779 and IFN had an acceptable
safety profile and displays antitumor activity in patients
with advanced RCC (Motzer et al., 2007)
II Advanced breast cancer CCI‐779 showed antitumor activity and a generally tolerable
safety profile (Chan et al., 2005)
II Recurrent glioblastoma
multiforme
CCI‐779 was well tolerated in recurrent GBM patients. No
response or radiographic improvement was observed in
36% of CCI‐779 treated patients (Chang et al., 2005; Galanis
et al., 2005)
(continues)
43

Table II (continued)
Targets Drug name Phase Tumor types Comments and references
II Advanced neuroendocrine
carcinomas
CCI‐779 appeared to have little activity and does not warrant
further single‐agent evaluation in advanced NEC (Duran
et al., 2006)
II Extensive‐stage small‐cell
lung cancer
CCI‐779 seemed not to increase the progression‐free survival
in this patient population (Pandya et al., 2007)
II Metastatic melanoma CCI‐779 was not sufficiently active in this patient population
(Margolin et al., 2005)
II Advanced refractory
renal‐cell carcinoma
In patients with advanced RCC, CCI‐779 showed antitumor
activity and encouraging survival (Atkins et al., 2004)
III Advanced renal‐cell
carcinoma
CCI‐779 increased the effect of interferon alpha, improved
overall survival among patients with metastatic renal‐cell
carcinoma and a poor prognosis (Hudes et al., 2007)
RAD001
(everolimus)
I Refractory solid tumors in
children
Continuous, orally administered RAD001 was well tolerated
in children with recurrent or refractory solid tumors and
significantly inhibited the mTOR signaling pathway (Fouladi
et al., 2007)
I Advanced solid tumors RAD001 was satisfactorily tolerated at dosages up to 70 mg/
week and 10 mg/day, a dosage of 10 mg/day or 50 mg/week
was recommended for further development (O’Donnell et al.,
2008; Tabernero et al., 2008)
I Advanced NSCLC A dose of 5 mg daily in combination with daily gefitinib
250 mg was recommended. The two patients with radio-
graphic responses identified were encouraging (Milton
et al., 2007)
I Advanced breast cancer Daily therapy with RAD001 plus letrozole was promising
and a daily dose of RAD001 10 mg was recommended
for further trials (Awada et al., 2008)
44

I/II Relapsed or refractory he-
matologic malignancies
RAD001 was well tolerated at a daily dose of 10 mg daily
and was effective in patients with myelodysplastic
syndrome (Yee et al., 2006)
II Relapsed chronic lympho-
cytic leukemia
Although the patient initially responded to therapy, the
patient subsequently developed a rapidly fatal
Epstein–Barr‐virus‐associated lymphoproliferative
disorder (Gotze et al., 2007)
II Low‐ to intermediate‐
grade neuroendocrine
tumors
RAD001 at 5 or 10 mg/d was well tolerated in combination
with octreotide with promising antitumor activity
(Yao et al., 2008)
III Advanced renal‐cell
carcinoma
Treatment with everolimus prolonged progression‐free
survival relative to placebo in patients with metastatic
renal‐cell carcinoma that had progressed on other
targeted therapies (Motzer et al., 2008)
AP23573
(deforolimus)
I Advanced malignancies Deforolimus was well tolerated with encouraging antitumor
activity across a broad range of malignancies (Mita et al.,
2008)
II Relapsed or refractory he-
matologic malignancies
Deforolimus was well tolerated in patients with heavily
pretreated hematologic malignancies, and antitumor
activity was observed (Rizzieri et al., 2008)
45

glioblastoma and gefitinib plus rapamycin can be safely coadministered on a
continuous, daily dosing schedule (Cloughesy et al., 2008; Reardon et al.,
2006). In phase II and III clinical studies, CCI‐779 has been shown to have
effects for treating patients with advanced breast cancer and advanced
refractory renal‐cell carcinoma (Atkins et al., 2004; Chan et al., 2005).
Moreover, CCI‐779 increased the effect of interferon alpha, improved over-
all survival among patients with metastatic renal‐cell carcinoma, and a poor
prognosis (Hudes et al., 2007; Motzer et al., 2007). RAD001 is administered
orally for clinical application. The phase II clinical studies have shown that
RAD001 treatment enhances the effect of gefitinib in advanced nonsmall cell
lung cancer patients, increased the effect of lerozole in advanced breast
cancer patients. It is also shown benefits for treating low‐ to intermediate‐
grade neuroendocrine tumor combination with octreotide (Awada et al.,
2008; Milton et al., 2007; Yao et al., 2008). A recent study has shown that
treatment with RAD001 prolongs progression‐free patient survival when
compared to placebo treated patients with metastatic renal‐cell carcinoma
that has progressed on other targeted therapies (Motzer et al., 2008). AP‐
23573 is a phosphorus‐containing derivative of rapamycin, and developed
in both intravenous and oral formulations for clinical trials. Recent clinical
trials have demonstrated that it was well tolerated and showed encouraging
activity across a broad range of malignancies, and antitumor activity was
observed in patients with heavily hemotologic malignancies (Mita et al.,
2008; Rizzieri et al., 2008). The published results in the clinical trials were
summarized in Table II.
VIII. CONCLUDING REMARKS
PI3K/PTEN signaling pathway plays a central role in regulating various
kinds of cellular functions in response to growth factors, insulin, and other
hormones. The intensive interests are on the study of PI3K and PTEN in
tumorigenesis. Recent studies have shown that the active form of PI3K is an
oncogene, and that amplifications and mutations of PI3K are commonly
found in many kinds of human cancers. PTEN, as the tumor suppressor and
antagonist of PI3K, is frequently mutated or lost in a number of human
cancers. PI3K/PTEN signaling regulates angiogenesis through the interac-
tion of cancer cells and tumor microenvironments, especially endothelial
cells. Angiogenesis inducers such as VEGF and angiopoietins activate PI3K
signaling for inducing angiogenesis. Forced expression of PI3K alone is
sufficient to increase angiogenesis. Genetic alterations of PI3K lead to dys-
function of vasculature and angiogenesis. Mutations of RTKs regulate
tumor growth and angiogenesis through PI3K/PTEN signaling. PI3K in

turn regulates tumor growth and angiogenesis through downstream targets
AKT, mTOR, and p70S6K1; and through effectors, HIF‐1 and VEGF.
A growing list of evidence shows that PI3K, PTEN, and their upstream
and downstream molecules are commonly altered in human cancers; and
play an important role in tumorigenesis and angiogenesis. The inhibitors to
this signaling pathway, including PI3K, AKT, and mTOR inhibitors, are
currently in clinical trials with promising outcomes.
Pan‐PI3K inhibitors were initially discovered, and some recently devel-
oped versions of pan‐PI3K inhibitors broadly target the class IA PI3Ks
(p110 , p110 , and p110), and the catalytic site of mTOR. Isoform‐
specific PI3K inhibitors have less toxicity to the cells than those pan‐PI3K
inhibitors, which could be used to specifically target PI3K activation in
certain cancer cells. Clinical data indicates that mTOR inhibitors have
stronger effect and more promising results than PI3K and AKT inhibitors.
However, there is a feedback loop because p70S6K1 negatively regulates
IRS and PDGFR. Rapamycin or its analogs can activate upstream mole-
cules including AKT due to the loss of feedback inhibition. Thus, it is
important to exploit the potential benefits of the targeted therapies and
optimal treatment with these inhibitors. PI3K pathway inhibitors are likely
more effective in patients with active PI3K/AKT pathway, such as PIK3CA
mutations or PTEN mutations. In addition, PI3K/AKT signaling is
involved in resistance to both chemotherapeutic and radiotherapeutic
treatments. Therefore, it would be beneficial to combine these therapeutic
agents with PI3K inhibitors. We anticipate that the therapeutic methods
targeting PI3K pathway would represent the promising cancer therapy in
the near future.
ACKNOWLEDGMENTS
This work was supported in part by Grants CA109460, ES017237, and HL091456 from
National Institutes of Health by the National Basic Research Program of China Grant
2007CB947002.
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teak tree; Netām, the dog; Irpāchi, the mahua tree; Tumrāchi, the tendu tree;
Warkara, the wild cat, and so on. Generally the members of a sept do not
kill or injure their totem animals, but the rule is not always observed, and in
some cases they now have some other object of veneration, possibly
because they have forgotten the meaning of the sept name, or the object
after which it is named has ceased to be sacred. Thus the Markām sept,
though named after the mango, now venerate the tortoise, and this is also
the case with the Netām sept in Bastar, though named after the dog. In
Bastar a man revering the tortoise, though he will not catch the animal
himself, will get one of his friends to catch it, and one revering the goat, if
he wishes to kill a goat for a feast, will kill it not at his own house but at a
friend’s. The meaning of the important sept names Marābi, Dhurwa and
Uika has not been ascertained, and the members of the sept do not know it.
In Mandla the Marābi sept are divided into the Eti Marābi and Padi Marābi,
named after the goat and pig. The Eti or goat Marābi will not touch a goat
nor sacrifice one to Bura Deo. They say that once their ancestors stole a
goat and were caught by the owner, when they put a basket over it and
prayed Bura Deo to change it into a pig, which he did. Therefore they
sacrifice only pigs to Bura Deo, but apparently the Padi Marābi also both
sacrifice and eat pigs. The Dhurwa sept are divided into the Tumrāchi and
Nābalia Dhurwa, named after the tendu tree and the dwarf date-palm. The
Nābalia Dhurwas will not cut a dwarf date-palm nor eat its fruit. They
worship Bura Deo in this tree instead of in the sāj tree, making an iron doll
to represent him and covering it with palm-leaves. The Uika sept in Mandla
say that they revere no animal or plant, and can eat any animal or cut down
any plant except the sāj tree,21 the tree of Bura Deo; but in Betūl they are
divided into several subsepts, each of which has a totem. The Parteti sept
revere the crocodile. When a marriage is finished they make a sacrifice to
the crocodile, and if they see one lying dead they break their earthen pots in
token of mourning. The Warkara sept revere the wild cat; they also will not
touch a village cat nor keep one in their house, and if a cat comes in they
drive it out at once. The Kunjām sept revere the rat and do not kill it.

14. Connection of totemism with the gods.
In Betūl the Gonds explain the totemistic names of their septs by saying that
some incident connected with the animal, tree or other object occurred to
the ancestor or priest of the sept while they were worshipping at the Deo-
khulla or god’s place or threshing-floor. Mr. Ganga Prasād Khatri has made
an interesting collection of these. The reason why these stories have been
devised may be that the totem animals or plants have ceased to be revered
on their own merits as ancestors or kinsmen of the sept, and it was therefore
felt necessary to explain the sept name or sanctity attaching to the totem by
associating it with the gods. If this were correct the process would be
analogous to that by which an animal or plant is first held sacred of itself,
and, when this feeling begins to decay with some recognition of its true
nature, it is associated with an anthropomorphic god in order to preserve its
sanctity. The following are some examples recorded by Mr. Ganga Prasād
Khatri. Some of the examples are not associated with the gods.
Gajjāmi, subsept of Dhurwa sept. From gaj, an arrow. Their first ancestor
killed a tiger with an arrow.
Gouribans Dhurwa. Their first ancestor worshipped his gods in a bamboo
clump.
Kusadya Dhurwa. (Kosa, tasar silk cocoon.) The first ancestor found a silk
cocoon on the tree in which he worshipped his gods.
Kohkapath. Kohka is the fruit of the bhilawa22 or marking-nut tree, and
path, a kid. The first ancestor worshipped his gods in a bhilawa tree and
offered a kid to them. Members of this sept do not eat the fruit or flowers of
the bhilawa tree.
Jaglya. One who keeps awake, or the awakener. The first ancestor stayed
awake the whole night in the Deo-khulla, or god’s threshing-floor.

Sariyām. (Sarri, a path.) The first ancestor swept the path to the Deo-khulla.
Guddām. Gudda is a place where a hen lays her eggs. The first ancestor’s
hen laid eggs in the Deo-khulla.
Irpāchi. The mahua tree. A mahua tree grew in the Deo-khulla or
worshipping-place of this sept.
Admachi. The dhaura tree.23 The first ancestor worshipped his gods under a
dhaura tree. Members of the sept do not cut this tree nor burn its wood.
Sarāti Dhurwa. (Sarāti, a whip.) The first ancestor whipped the priest of the
gods.
Suibadiwa. (Sui, a porcupine.) The first ancestor’s wife had a porcupine
which went and ate the crop of an old man’s field. He tried to catch it, but it
went back to her. He asked the name of her sept, and not being able to find
it out called it Suibadiwa.
Watka. (A stone.) Members of this sept worship five stones for their gods.
Some say that the first ancestors were young boys who forgot where the
Deo-khulla was and therefore set up five stones and offered a chicken to
them. As they did not offer the usual sacrifice of a goat, members of this
sept abstain from eating goats.
Tumrecha Uika. (The tendu tree.24) It is said that the original ancestor of
this sept was walking in the forest with his pregnant wife. She saw some
tendu fruit and longed for it and he gave it to her to eat. Perhaps the original
idea may have been that she conceived through swallowing a tendu fruit.
Members of this sept eat the fruit of the tendu tree, but do not cut the tree
nor make any use of its leaves or branches.
Tumdan Uika. Tumdan is a kind of pumpkin or gourd. They say that this
plant grows in their Deo-khulla. The members drink water out of this gourd
in the house, but do not carry it out of the house.

Kadfa-chor Uika. (Stealer of the kadfa.) Kadfa is the sheaf of grain left
standing in the field for the gods when the crop is cut. The first ancestor
stole the kadfa and offered it to his gods.
Gadhamār Uika. (Donkey-slayer.) Some say that the gods of the sept came
to the Deo-khulla riding on donkeys, and others that the first ancestor killed
a donkey in the Deo-khulla.
Eti-kumra. Eti is a goat. The ancestors of the sept used to sacrifice a
Brāhman boy to their gods. Once they were caught in the act by the parents
of the boy they had stolen, and they prayed to the gods to save them, and
the boy was turned into a goat. They do not kill a goat nor eat its flesh, nor
sacrifice it to the gods.
Ahke. This word means ‘on the other side of a river.’ They say that a man of
the Dhurwa sept abducted a girl of the Uika sept from the other side of a
river and founded this sept.
Tirgām. The word means fire. They say that their ancestor’s hand was burnt
in the Deo-khulla while cooking the sacrifice.
Tekām. (The teak tree.) The ancestor of the sept had his gods in this tree.
Members of the sept will not eat food off teak leaves, but they will use them
for thatching, and also cut the tree.
Manapa. In Gondi mani is a son and apa a father. They say that their
ancestors sacrificed a Brāhman father and son to their gods and were saved
by their being turned into goats like the Eti-kumra sept. Members of the
sept do not kill or eat a goat.
Korpachi. The droppings of a hen. The ancestors of the sept offered these to
his gods.
Mandani. The female organ of generation. The ancestor of the sept slept
with his wife in the Deo-khulla.

Paiyām. Paiya is a heifer which has not borne a calf, such as is offered to
the gods. Other Gonds say that the people of this sept have no gods. They
are said not only to marry a girl from any other subsept of the Dhurwas and
Uikas, but from their own sept and even their own sisters, though this is
probably no longer true. They are held to be the lowest of the Gonds.
Except in this instance, as already seen, the subsepts of the Dhurwa and
Uika septs do not intermarry with each other.

(c) Marriage Customs
15. Prohibitions on intermarriage, and unions of relations.
A man must not marry in his own sept, nor in one which worships the same
number of gods, in localities where the classification of septs according to
the number of gods worshipped obtains. Intermarriage between septs which
are bhaiband or brothers to each other is also prohibited. The marriage of
first cousins is considered especially suitable. Formerly, perhaps, the match
between a brother’s daughter and sister’s son was most common; this is
held to be a survival of the matriarchate, when a man’s sister’s son was his
heir. But the reason has now been generally forgotten, and the union of a
brother’s son to a sister’s daughter has also become customary, while, as
girls are scarce and have to be paid for, it is the boy’s father who puts
forward his claim. Thus in Mandla and Bastar a man thinks he has a right to
his sister’s daughter for his son on the ground that his family has given a
girl to her husband’s family, and therefore they should give one back. This
match is known as Dūdh lautāna or bringing back the milk; and if the
sister’s daughter marries any one else her maternal uncle sometimes claims

what is known as ‘milk money,’ which may be a sum of Rs. 5, in
compensation for the loss of the girl as a wife for his son. This custom has
perhaps developed out of the former match in changed conditions of
society, when the original relation between a brother and his sister’s son has
been forgotten and girls have become valuable. But it is said that the dūdh
or milk money is also payable if a brother refuses to give his daughter to his
sister’s son. In Mandla a man claims his sister’s daughter for his son and
sometimes even the daughter of a cousin, and considers that he has a
legitimate grievance if the girl is married to somebody else. Frequently, if
he has reason to apprehend this, he invites the girl to his house for some
ceremony or festival, and there marries her to his son without the consent of
her parents. As this usually constitutes the offence of kidnapping under the
Penal Code, a crop of criminal cases results, but the procedure of arrest
without warrant and the severe punishment imposed by the Code are
somewhat unsuitable for a case of this kind, which, according to Gond
ideas, is rather in the nature of a civil wrong, and a sufficient penalty would
often be the payment of an adequate compensation or bride-price for the
girl. The children of two sisters cannot, it is said, be married, and a man
cannot marry his wife’s elder sister, any aunt or niece, nor his mother-in-
law or her sister. But marriage is not prohibited between grandparents and
grandchildren. If an old man marries a young wife and dies, his grandson
will marry her if she is of proper age. In this there would be no blood-
relationship, but it is doubtful whether even the existence of such
relationship would prevent the match. It is said that even among Hindu
castes the grandfather will flirt with his granddaughter, and call her his wife
in jest, and the grandmother with her grandson. In Bastar a man can marry
his daughter’s daughter or maternal grandfather’s or grandmother’s sister.
He could not marry his son’s daughter or paternal grandfather’s sister,
because they belong to the same sept as himself.
16. Irregular marriages.

In the Māria country, if a girl is made pregnant by a man of the caste before
marriage, she simply goes to his house and becomes his wife. This is called
Paithu or entering. The man has to spend Rs. 2 or 3 on food for the caste
and pay the price for the girl to her parents. If a girl has grown up and no
match has been arranged for her to which she agrees, her parents will ask
her maternal uncle’s or paternal aunt’s son to seize her and take her away.
These two cousins have a kind of prescriptive claim to the girl, and
apparently it makes no difference whether the prospective husband is
already married or not. He and his friends lie in wait near her home and
carry her off, and her parents afterwards proceed to his house to console
their daughter and reconcile her to the match. Sometimes when a woman is
about to become what is known as a Paisamundi or kept woman, without
being married, the relations rub her and the man whose mistress she is with
oil and turmeric, put marriage crowns of palm-leaves on their heads, pour
water on them from the top of a post, and make them go seven times round
a mahua branch, so that they may be considered to be married. When a
couple are very poor they may simply go and live together without any
wedding, and perform the ceremony afterwards when they have means, or
they distribute little pieces of bread to the tribesmen in lieu of the marriage
feast.
17. Marriage. Arrangement of matches.
Marriage is generally adult. Among the wild Māria Gonds of Bastar the
consent of the girl is considered an essential preliminary to the union. She
gives it before a council of elders, and if necessary is allowed time to make
up her mind. The boy must also agree to the match. Elsewhere matches are
arranged by the parents, and a bride-price which amounts to a fairly
substantial sum in comparison with the means of the parties is usually paid.
But still the girls have a considerable amount of freedom. It is generally
considered that if a girl goes of her own accord and pours turmeric and
water over a man, it is a valid marriage and he can take her to live in his

house. Married women also sometimes do this to another man if they wish
to leave their husbands.
18. The marriage ceremony.
The most distinctive feature of a Gond marriage is that the procession
usually starts from the bride’s house and the wedding is held at that of the
bridegroom, in contradistinction to the Hindu practice. It is supposed that
this is a survival of the custom of marriage by capture, when the bride was
carried off from her own house to the bridegroom’s, and any ceremony
which was requisite was necessarily held at the house of the latter. But the
Gonds say that since Dūlha Deo, the bridegroom god and one of the
commonest village deities, was carried off by a tiger on his way to his
wedding, it was decided that in future the bride must go to the bridegroom
to be married in order to obviate the recurrence of such a calamity. Any risk
incidental to the journey thus falls to the lady. Among the wilder Māria
Gonds of Bastar the ritual is very simple. The bride’s party arrive at the
bridegroom’s village and occupy some huts made ready for them. His father
sends them provisions, including a pig and fowls, and the day passes in
feasting. In the evening they go to the bridegroom’s house, and the night is
spent in dancing by the couple and the young people of the village. Next
morning the bride’s people go back again, and after another meal her
parents bring her to the bridegroom’s house and push her inside, asking the
boy’s father to take charge of her, and telling her that she now belongs to
her husband’s family and must not come back to them alone. The girl cries
a little for form’s sake and acquiesces, and the business is over, no proper
marriage rite being apparently performed at all. Among the more civilised
Mārias the couple are seated for the ceremony side by side under a green
shed, and water is poured on them through the shed in imitation of the
fertilising action of rain. Some elder of the village places his hands on them
and the wedding is over. But Hindu customs are gradually being adopted,
and the rubbing of powdered turmeric and water on the bodies of the bride
and bridegroom is generally essential to a proper wedding. The following

description is given of the Gonds of Kanker. On the day fixed for the
marriage the pair, accompanied by the Dosi or caste priest, proceed to a
river, in the bed of which two reeds five or six feet high are placed just so
far apart that a man can lie down between them, and tied together with a
thread at the top. The priest lies down between the reeds, and the bride and
bridegroom jump seven times over his body. After the last jump they go a
little way off, throw aside their wet clothes, and then run naked to a place
where their dry clothes are kept; they put them on and go home without
looking back. Among the Gonds in Khairāgarh the pair are placed in two
pans of a balance and covered with blankets. The caste priest lifts up the
bridegroom’s pan and her female relatives the bride’s, and walk round with
them seven times, touching the marriage-post at each time. After this they
are taken outside the village without being allowed to see each other. They
are placed standing at a little distance with a screen between them, and
liquor is spilt on the ground to make a line from one to the other. After a
time the bridegroom lifts up the screen, rushes on the bride, gives her a
blow on the back and puts the ring on her finger, at the same time making a
noise in imitation of the cry of a goat. All the village then indulge in
bacchanalian orgies, not sparing their own relations.
19. Wedding expenditure.
In Bastar it is said that the expenses of a wedding vary from Rs. 5 to Rs. 20
for the bride’s family and from Rs. 10 to Rs. 50 for the bridegroom’s,
according to their means.25 In a fairly well-to-do family the expenditure of
the bridegroom’s family is listed as follows: liquor Rs. 20, rice Rs. 12, salt
Rs. 2, two goats Rs. 2, chillies Rs. 2, ghī Rs. 4, turmeric Rs. 2, oil Rs. 3,
three cloths for the bride Rs. 8, two sheets and a loin-cloth for her relatives
Rs. 5, payment to the Kumhār for earthen pots Rs. 5, the bride-price Rs. 10,
present to the bride’s maternal uncle when she is not married to his son Rs.
2, and something for the drummers. The total of this is Rs. 76, and any
expenditure on ornaments which the family can afford may be added. In
wealthier localities the bride-price is Rs. 15 to 20 or more. Sometimes if the

girl has been married and dies before the bride-price has been paid, her
father will not allow her body to be buried until it is paid. The sum
expended on a wedding probably represents the whole income of the family
for at least six months, and often for a considerably longer period. In
Chānda26 the bride’s party on arrival at the bridegroom’s village receive the
Bara jawa or marriage greeting, every one present being served with a little
rice-water, an onion and a piece of tobacco. At the wedding the bridegroom
has a ring either of gold, silver or copper, lead not being permissible, and
places this on the bride’s finger. Often the bride resists and the bridegroom
has to force her fist open, or he plants his foot on hers in order to control
her while he gets the ring on to her finger. Elsewhere the couple hold each
other by the little fingers in walking round the marriage-post, and then each
places an iron ring on the other’s little finger. The couple then tie strings,
coloured yellow with turmeric, round each other’s right wrists. On the
second day they are purified with water and put on new clothes. On the
third day they go to worship the god, preceded by two men who carry a
chicken in a basket. This chicken is called the Dhendha or associate of the
bridal couple, and corresponds to the child which in Hindu marriages is
appointed as the associate of the bridegroom. Just before their arrival at the
temple the village jester snatches away the chicken, and pretends to eat it.
At the temple they worship the god, and deposit before him the strings
coloured with turmeric which had been tied on their wrists. In Chhindwāra
the bride is taken on a bullock to the bridegroom’s house. At the wedding
four people hold out a blanket in which juāri, lemons and eggs are placed,
and the couple walk round this seven times, as in the Hindu bhānwar
ceremony. They then go inside the house, where a chicken is torn asunder
and the blood sprinkled on their heads. At the same time the bride crushes a
chicken under her foot. In Mandla the bride on entering the marriage-shed
kills a chicken by cutting off its head either with an axe or a knife. Then all
the gods of her house enter into her and she is possessed by them, and for
each one she kills a chicken, cutting off its head in the same manner. The
chickens are eaten by all the members of the bride’s party who have come
with her, but none belonging to the bridegroom’s party may partake of
them. Here the marriage-post is made of the wood of the mahua tree, round
which a toran or string of mango leaves is twisted, and the couple walk

seven times round this. In Wardha the bride and bridegroom stand on the
heap of refuse behind the house and their heads are knocked together. In
Bhandāra two spears are placed on the heap of refuse and their ends are tied
together at the top with the entrails of a fowl. The bride and bridegroom
have to stand under the spears while water is poured over them, and then
run out. Before the bride starts the bridegroom must give her a blow on the
back, and if he can do this before she runs out from the spears it is thought
that the marriage will be lucky. The women of the bride’s and bridegroom’s
party also stand one at each end of a rope and have a competition in
singing. They sing against each other and see which can go on the longest.
Brāhmans are not employed at a Gond wedding. The man who officiates is
known as Dosi, and is the bridegroom’s brother-in-law, father’s sister’s
husband or some similar relative. A woman relative of the bride helps her to
perform her part and is known as Sawāsin. To the Dosi and Sawāsin the
bride and bridegroom’s parties present an earthen vessel full of kodon. The
donors mark the pots, take them home and sow them in their own fields,
and then give the crop to the Dosi and Sawāsin.
20. Special customs.
Some years ago in Bālāghāt the bride and bridegroom sat and ate food
together out of two leaf-plates. When they had finished the bride took the
leaf-plates, ran with them to the marriage-shed, and fixed them in the
woodwork so that they did not fall down. The bridegroom ran after her, and
if she did not put the plates away quickly, gave her one or two blows with
his fist. This apparently was a symbolical training of the bride to be diligent
and careful in her household work. Among the Rāj-Gonds of Saugor, if the
bridegroom could not come himself he was accustomed to send his sword
to represent him. The Sawāsin carried the sword seven times round the
marriage-post with the bride and placed a garland on her on its behalf, and
the bride put a garland over the sword. This was held to be a valid marriage.
In a rich Rāj-Gond or Khatola Gond family two or three girls would be
given with the bride, and they would accompany her and become the

concubines of the bridegroom. Among the Māria Gonds of Chānda the
wedded pair retire after the ceremony to a house allotted to them and spend
the night together. Their relatives and friends before leaving shout and make
merry round the house for a time, and throw all kinds of rubbish and dirt on
it. In the morning the couple have to get up early and clear all this off, and
clean up the house. A curious ceremony is reported from one part of
Mandla. When a Gond girl is leaving to be married, her father places inside
her litter a necklace of many strings of blue and yellow beads, with a
number of cowries at the end, and an iron ring attached to it. On her arrival
at the bridegroom’s house his father takes out the necklace and ring.
Sometimes it is said that he simply passes a stone through the ring, but
often he hangs it up in the centre of a room, and the bridegroom’s relatives
throw stones at it until one of them goes through the ring, or they throw
long bamboo sticks or shoot arrows at it, or even fire bullets from a gun. In
a recent case it is said that a man was trying to fire a bullet through the ring
and killed a girl. Until a stone, stick, arrow or bullet has been sent through
the ring the marriage cannot take place, nor can the bridegroom or his father
touch the bride, and they go on doing this all night until somebody
succeeds. When the feat has been done they pour a bottle of liquor over the
necklace and ring, and the bride’s relatives catch the liquor as it falls, and
drink it. The girl wears the necklace at her wedding, and thereafter so long
as her husband lives, and when he dies she tears the string to pieces and
throws it into the river. The iron ring must be made by a Gondi Lohār or
blacksmith, and he will not accept money in payment for it, but must be
given a cow, calf, or buffalo. The symbolical meaning of this rite does not
appear to require explanation.27 In many places the bride and bridegroom
go and bathe in a river or tank on the day after the wedding, and throw mud
and dirt over each other, or each throws the other down and rolls him or her
in the mud. This is called Chikhal-Mundi or playing in the mud. Afterwards
the bride has to wash the bridegroom’s muddy clothes, roll them up in a
blanket, and carry them on her head to the house. A see-saw is then placed
in the marriage-shed, and the bridegroom’s father sits on it. The bride
makes the see-saw move up and down, while her relations joke with her and
say, ‘Your child is crying.’ Elsewhere the bridegroom’s father sits in a
swing. The bride and bridegroom swing him, and the bystanders exclaim

that the old man is the child of the new bride. It seems possible that both
customs are meant to portray the rocking of a baby in a cradle or swinging
it in a swing, and hence it is thought that through performing them the bride
will soon rock or swing a real baby.
21. Taking omens.
In Bastar an omen is taken before the wedding. The village elders meet on
an auspicious day as Monday, Thursday or Friday, and after midnight they
cook and eat food, and go out into the forest. They look for a small black
bird called Usi, from which omens are commonly taken. When anybody
sees this bird, if it cries ‘Sun, Sun,’ on the right hand, it is thought that the
marriage will be lucky. If, however, it cries ‘Chi, Chi’ or ‘Fie, Fie,’ the
proposed match is held to be of evil omen, and is cancelled. The Koya
Gonds of Bastar distil mahua liquor before arranging for a match. If the
liquor is good they think the marriage will be lucky, and take the liquor with
them to cement the betrothal; but if it is bad they think the marriage will be
unlucky, and the proposal is dropped. Mondays, Wednesdays and Fridays
are held to be lucky days for marriages, and they are celebrated in the hot-
weather months of Baisākh, Jesth and Asār, or April, May and June, or in
Pūs (December), and rarely in Māgh (January). A wedding is only held in
Kārtik (October) if the bride and bridegroom have already had sexual
intercourse, and cannot take place in the rains.
22. Marriage by capture. Weeping and hiding.
Survivals of the custom of marriage by capture are to be found in many
localities. In Bastar the prospective bridegroom collects a party of his
friends and lies in wait for the girl, and they catch her when she comes out
and gets a little distance from her house. The girl cries out, and women of

the village come and rescue her and beat the boys with sticks till they have
crossed the boundary of the village. The boys neither resist nor retaliate on
the women, but simply make off with the girl. When they get home a new
cloth is given to her, and the boys have a carouse on rice-beer, and the
marriage is considered to be complete. The parents do not interfere, but as a
rule the affair is prearranged between the girl and her suitor, and if she
really objects to the match they let her go. A similar procedure occurs in
Chānda. Other customs which seem to preserve the idea that marriage was
once a forcible abduction are those of the bride weeping and hiding, which
are found in most Districts. In Bālāghāt the bride and one or two friends go
round to the houses of the village and to other villages, all of them crying,
and receive presents from their friends. In Wardha the bride is expected to
cry continuously for a day and a night before the wedding, to show her
unwillingness to leave her family. In Kanker it is said that before marriage
the bride is taught to weep in different notes, so that when that part of the
ceremony arrives in which weeping is required, she may have the proper
note at her command. In Chhindwāra the bridegroom’s party go and fetch
the bride for the wedding, and on the night before her departure she hides
herself in some house in the village. The bridegroom’s brother and other
men seek all through the village for her, and when they find her she runs
and clings to the post of the house. The bridegroom’s brother carries her off
by force, and she is taken on a bullock to the bridegroom’s house. In Seoni
the girl hides in the same manner, and calls out ‘Coo, coo,’ when they are
looking for her. After she is found, the bridegroom’s brother carries her
round on his back to the houses of his friends in the village, and she weeps
at each house. When the bride’s party arrive at the bridegroom’s village the
latter’s party meet them and stop them from proceeding further. After
waving sticks against each other in a threatening manner they fall on each
other’s necks and weep. Then two spears are planted to make an arch before
the door, and the bridegroom pushes the bride through these from behind,
hitting her to make her go through, while she hangs back and feigns
reluctance. In Mandla the bride sometimes rides to the wedding on the
shoulders of her sister’s husband, and it is supposed that she never gets
down all the way.

23. Serving for a wife.
The practice of Lamsena, or serving for a wife, is commonly adopted by
boys who cannot afford to buy one. The bridegroom serves his prospective
father-in-law for an agreed period, usually three to five or even six years,
and at its expiry he should be married to the girl without expense. During
this time he is not supposed to have access to the girl, but frequently they
become intimate, and if this happens the boy may either stay and serve his
unexpired term or take his wife away at once; in the latter case his parents
should pay the girl’s father Rs. 5 for each year of the bridegroom’s
unexpired service. The Lamsena custom does not work well as a rule, since
the girl’s parents can break their contract, and the Lamsena has no means of
redress. Sometimes if they are offered a good bride-price they will marry
the girl to another suitor when he has served the greater part of his term,
and all his work goes for nothing.
24. Widow remarriage.
The remarriage of widows is freely permitted. As a rule it is considered
suitable that she should marry her deceased husband’s younger brother, but
she may not marry his elder brother, and in the south of Bastar and Chānda
the union with the younger brother is also prohibited. In Mandla, if she will
not wed the younger brother, on the eleventh day after the husband’s death
he puts the tarkhi or palm-leaf earrings in her ears, and states that if she
marries anybody else he will claim dawa-bunda or compensation. Similarly
in Bastar, if an outsider marries the widow, he first goes through a joint
ceremony with the younger brother, by which the latter relinquishes his
right in favour of the former. The widow must not marry any man whom
she could not have taken as her first husband. After her husband’s death she

resides with her parents, and a price is usually paid to them by any outsider
who wishes to marry her. In Bastar there is a fixed sum of Rs. 24, half of
which goes to the first husband’s family and half to the caste panchāyat.
The payment to the panchāyat perhaps comes down from the period when
widows were considered the property of the state or the king, and sold by
auction for the benefit of the treasury. It is said that the descendants of the
Gond Rājas of Chānda still receive a fee of Rs. 1–8 from every Gond
widow who is remarried in the territories over which their jurisdiction
extended. In Bastar when a widow marries again she has to be transferred
from the gods of her first husband’s sept to those of her second husband.
For this two leaf-cups are filled with water and mahua liquor respectively,
and placed with a knife between them. The liquor and water are each
poured three times from one cup to the other and back until they are
thoroughly mixed, and the mixture is then poured over the heads of the
widow and her second husband. This symbolises her transfer to the god of
the new sept. In parts of Bastar when a man has been killed by a tiger and
his widow marries again, she goes through the ceremony not with her new
husband but with a lance, axe or sword, or with a dog. It is thought that the
tiger into which her first husband’s spirit has entered will try to kill her
second husband, but owing to the precaution taken he will either simply
carry off the dog or will himself get killed by an axe, sword or lance. In
most localities the ceremony of widow-marriage is simple. Turmeric is
rubbed on the bodies of the couple and they may exchange a pair of rings or
their clothes.
25. Divorce.
Divorce is freely allowed on various grounds, as for adultery on the wife’s
part, a quarrelsome disposition, carelessness in the management of
household affairs, or if a woman’s children continue to die, or she is
suspected of being a witch. Divorce is, however, very rare, for in order to
get a fresh wife the man would have to pay for another wedding, which few
Gonds can afford, and he would also have difficulty in getting a girl to

marry him. Therefore he will often overlook even adultery, though a wife’s
adultery not infrequently leads to murder among the Gonds. In order to
divorce his wife the husband sends for a few castemen, takes a piece of
straw, spits on it, breaks it in two and throws it away, saying that he has
renounced all further connection with his wife. If a woman is suspected of
being a witch she often has to leave the village and go to some place where
she is not known, and in that case her husband must either divorce her or go
with her. There is no regular procedure for a wife divorcing her husband,
but she can, if sufficiently young and attractive, take matters into her own
hands, and simply leave her husband’s house and go and live with some one
else. In such a case the man who takes her has to repay to the husband the
sum expended by the latter on his marriage, and the panchāyat may even
decree that he should pay double the amount. When a man divorces his wife
he has no liability for her maintenance, and often takes back any ornaments
he may have given her. And a man who marries a divorced woman may be
expected to pay her husband the expenses of his marriage. Instances are
known of a bride disappearing even during the wedding, if she dislikes her
partner; and Mr. Lampard of the Baihir Mission states that one night a Gond
wedding party came to his house and asked for the loan of a lantern to look
for the bride who had vanished.
26. Polygamy.
Polygamy is freely allowed, and the few Gonds who can afford the expense
are fond of taking a number of wives. Wives are very useful for cultivation
as they work better than hired servants, and to have several wives is a sign
of wealth and dignity. A man who has a number of wives will take them all
to the bazār in a body to display his importance. A Gond who had seven
wives in Bālāghāt was accustomed always to take them to the bazār like
this, walking in a line behind him.

(d) Birth and Pregnancy
27. Menstruation.
In parts of Mandla the first appearance of the signs of puberty in a girl is an
important occasion. She stays apart for four days, and during this time she
ties up one of her body-cloths to a beam in the house in the shape of a
cradle, and swings it for a quarter or half an hour every day in the name of
Jhulān Devi, the cradle goddess. On the fifth day she goes and bathes, and
the Baiga priest and his wife go with her. She gives the Baiga a hen and five
eggs and a bottle of wine, and he offers them to Jhulān Devi at her shrine.
To the Baigan she gives a hen and ten eggs and a bottle of liquor, and the
Baigan tattoos the image of Jhulān Devi on each side of her body. A black
hen with feathers spotted with white is usually chosen, as they say that this
hen’s blood is of a darker colour and that she lays more eggs. All this
ceremonial is clearly meant to induce fertility in the girl. The Gonds regard
a woman as impure for as long as the menstrual period lasts, and during this
time she cannot draw water nor cook food, nor go into a cowshed or touch
cowdung. In the wilder Māria tracts there is, or was till lately, a building out

of sight of the village to which women in this condition retired. Her
relatives brought her food and deposited it outside the hut, and when they
had gone away she came out and took it. It was considered that a great evil
would befall any one who looked on the face of a woman during the period
of this impurity. The Rāj-Gonds have the same rules as Hindus regarding
the menstrual periods of women.28
28. Superstitions about pregnancy and childbirth.
No special rites are observed during pregnancy, and the superstitions about
women in this condition resemble those of the Hindus.29 A pregnant woman
must not go near a horse or elephant, as they think that either of these
animals would be excited by her condition and would assault her. In cases
where labour is prolonged they give the woman water to drink from a
swiftly flowing stream, or they take pieces of wood from a tree struck by
lightning or by a thunder-bolt, and make a necklace of them and hang it
round her neck. In these instances the swiftness of the running water, or of
the lightning or thunder-bolt, is held to be communicated to the woman, and
thus she will obtain a quick delivery. Or else they ask the Gunia or sorcerer
to discover what ancestor will be reborn in the child, and when he has done
this he calls on the ancestor to come and be born quickly. If a woman is
childless they say that she should worship Bura Deo and fast continually,
and then on the termination of her monthly impurity, after she has bathed, if
she walks across the shadow of a man she will have a child. It is thus
supposed that the woman can be made fertile by the man’s shadow, which
will be the father of the child. Or she should go on a Sunday night naked to
a sāj tree30 and pray to it, and she may have a child. The sāj is the tree in
which Bura Deo resides, and was probably in the beginning itself the god.
Hence it is supposed that the woman is impregnated by the spirit of the tree,
as Hindu women think that they can be made fertile by the spirits of
unmarried Brāhman boys living in pīpal trees. Or she may have recourse to
the village priest, the Bhumka or the Baiga, who probably finds that her
barren condition is the work of an evil spirit and propitiates him. If a

woman dies in the condition of pregnancy they cut her belly open before
burial, so that the spirit of the child may escape. If she dies during or soon
after delivery they bury her in some remote jungle spot, from which her
spirit will find it difficult to return to the village. The spirit of such a woman
is supposed to become a Churel and to entice men, and especially drunken
men, to injury by causing them to fall into rivers or get shut up in hollow
trees. The only way they can escape her is to offer her the ornaments which
a married woman wears. Her enmity to men is due to the fact that she was
cut off when she had just had the supreme happiness of bearing a child, and
the present of these ornaments appeases her. The spirit of a woman whose
engagement for marriage has been broken off, or who has deserted her
husband’s house for another man’s, is also supposed to become a Churel. If
an abortion occurs, or a child is born dead or dies very shortly after birth,
they put the body in an earthen pot, and bury it under the heap of refuse
behind the house. They say that this is done to protect the body from the
witches, who if they get hold of it will raise the child’s spirit, and make it a
Bir or familiar spirit. Witches have special power over the spirits of such
children, and can make them enter the body of an owl, a cat, a dog, or a
headless man, and in this form cause any injury which the witch may desire
to inflict on a human being. The real reason for burying the bodies of such
children close to the house is probably, however, the belief that they will
thus be born again in the same family. If the woman is fat and well during
pregnancy they think a girl will be born, but if she is ailing and thin, that the
child will be a boy. If the nipples of her breasts are of a reddish colour they
think the birth of a boy is portended, but if of blackish colour, a girl. When
a birth occurs another woman carefully observes the knots or protuberances
on the navel-cord. It is supposed that the number of them indicates the
further number of children which will be born to the mother. A blackish
knot inclining downwards portends a boy, and a reddish one inclining
upwards a girl. It is supposed that an intelligent midwife can change the
order of these knots, and if a woman has only borne girl-children can
arrange that the next one shall be a boy.

29. Procedure at a birth.
Professional midwives are not usually employed at childbirth, and the
women look after each other. Among the Māria Gonds of Bastar the father
is impure for a month after the birth of a child and does not go to his work.
A Muria Gond father is impure until the navel-cord drops; he may reap his
crop, but cannot thresh or sow. This is perhaps a relic of the custom of the
Couvade. The rules for the treatment of the mother resemble those of the
Hindus, but they do not keep her so long without food. On some day from
the fifth to the twelfth after the birth the mother is purified and the child is
named. On this day its hair is shaved by the son-in-law or husband’s or
wife’s brother-in-law. The mother and child are washed and rubbed with oil
and turmeric, and the house is freshly whitewashed and cleaned with
cowdung. They procure a winnowing-fan full of kodon and lay the child on
it, and the mother ties this with a cloth under her arm. In the Nāgpur
country the impurity of the mother is said to last for a month, during which
time she is not allowed to cook food and no one touches her. Among the
poorer Gonds the mother often does not lie up at all after a birth, but eats
some pungent root as a tonic and next day goes on with her work.
30. Names.
On the Sor night, or that of purification, the women of the village assemble
and sing. The mother holds the child in her lap, and they each put a pice
(¼d.) in a dish as a present to it. A name is chosen, and an elderly woman
announces it. Names are now often Hindu words, and are selected very
much at random.31 If the child was born on a Tuesday, Wednesday, Friday
or Sunday the name of the day is often given, as Mangal, Budhu, Sukhiya,
Itwāri; or if born in the month of Māgh (January), Phāgun (February), Chait

(March), Baisākh (April), Jesth (May), or Pūs (December), the name may
be from the month, as Māhu, Phāgu, Chaitia, Baisākhu, Jetha and Puso. The
names of the other months are also given, but are less common. If any
Government official is in the village when the child is born it may be named
after his office, as Daroga, Havildar (head-constable), Vaccinator, Patwāri
(village surveyor), Jemadār (head process-server), or Munshi (clerk). If a
European officer is in the village the child may be called Gora (red) or
Bhura (brown). Other names are Zamīndār (landholder) or Kirsān (tenant).
Or the child may be named after any peculiarity, as Ghurman, fat, Kaluta,
black, Chatua, one who kicks, and so on. Or it may be given a bad name in
order to deceive the evil spirits as to its value, as Ghurha, a heap of
cowdung, Jhāru, sweepings, Dumre or Bhangi, a sweeper, Chamari, a
Chamār or tanner, and so on. If the mother has got the child after
propitiating a spirit, it may be called Bhūta, from bhūt, a spirit or ghost.
Nicknames are also given to people when they grow up, as Dariya, long-
footed, Bobdi, fat and sluggish, Putchi, having a tail or cat-like, Bera, an
idiot, and so on. Such names come into general use, and the bearers accept
and answer to them without objection. All the above names are Hindi.
Names taken from the Gond language are rare or non-existent, and it would
appear either that they have been completely forgotten, or else that the
Gonds had not advanced to the stage of giving every individual a personal
name prior to their contact with the Hindus.
31. Superstitions about children.
If a child is born feet first its feet are supposed to have special power, and
people suffering from pain in the back come and have their backs touched
by the toes of the child’s left foot. This power is believed to be retained in
later life. If a woman gets a child when the signs of menstruation have not
appeared, the child is called Lamka, and is held to be in danger of being
struck by lightning. In order to avert this fate an offering of a white cock is
made to the lightning during the month of Asārh (June) following the birth,
when thunderstorms are frequent, and prayer is made that it will accept this

sacrifice in lieu of the life of the child. They think that the ancestors who
have been mingled with Bura Deo may be born again. Sometimes such an
ancestor appears in a dream and intimates that he is coming back to earth.
Then if a newborn child will not drink its mother’s milk, they think it is
some important male ancestor, and that he is vexed at being in such a
dependent position to a woman over whom he formerly had authority. So
they call the Gunia or sorcerer, and he guesses what ancestor has been
reborn by measuring a stick. He says that if the length of the stick is an even
number of times the breadth of his hand, or more or less than half a hand-
breadth over, such and such an ancestor is reborn in the child. Then he
measures his hand along the stick breadthwise, and when the measurement
comes to that foretold for a particular ancestor he says that this one has
been reborn; or if they find any mark on the body of the child
corresponding to one they remember to have been borne by a particular
ancestor, they identify it with this ancestor. Then they wash the child’s feet
as a token of respect, and pass their hands over its head and say to it, ‘Drink
milk, and we will give you a ring and clothes and jewels.’ Sometimes they
think that an ancestor has been born again in a calf, and the Gunia
ascertains who he is in the same manner. Then this calf is not castrated if a
bull, nor put to the plough if it is a cow, and when it dies they will not take
off its hide for sale but bury it with the hide on.
It is believed that if a barren woman can get hold of the first hair of another
woman’s child or its navel-cord, she can transfer the mother’s fertility to
herself, so they dispose of these articles very carefully. If they wish the
child to grow fat, they bury the navel-cord in a manure-heap. The upper
milk teeth are thrown on to the roof, and the lower ones buried under a
water-pot. They say that the upper ones should be in a high place, and the
lower ones in a low place. The teeth thrown on the roof may be meant for
the rats, who in exchange for them will give the child strong white teeth like
their own, while those thrown under the water-pot will cause the new teeth
to grow large and quickly, like the grass under a water-pot. Diseases of
children are attributed to evil spirits. The illness called Sukhi, in which the
body and limbs grow weak and have a dried-up appearance, is very
common, and is probably caused by malnutrition. They attribute it to the
machinations of an owl which has heard the child’s name or obtained a

piece of its soiled clothing. If a stone or piece of wood is thrown at the owl
to scare it away, it will pick this up, and after wetting it in a stream, put it
out in the sun to dry. As the stone or wood dries up, so will the child’s body
dry up and wither. In order to cure this illness they use charms and amulets,
and also let the child wallow in a pig-sty so that it may become as fat as the
pigs. They say that they always beat a brass dish at a birth so that the noise
may penetrate the child’s ears, and this will remove any obstruction there
may be to its hearing. If the child appears to be deaf, they lay it several
times in a deep grain-bin for about half an hour at a time; when it cries the
noise echoes in the bin, and this is supposed to remove the obstruction to its
power of hearing. If they wish the boy to be a good dancer, they get a little
of the flesh of the kingfisher or hawk which hangs poised in the air over
water by the rapid vibration of its wings, on the look-out for a fish, and give
him this to eat. If they wish him to speak well, they touch his finger with
the tip of a razor, and think that he will become talkative like a barber. If
they want him to run fast, they look for a stone on which a hare has dropped
some dung and rub this on his legs, or they get a piece of a deer’s horn and
hang it round his neck as a charm. If a girl or boy is very dark-coloured,
they get the branches of a creeper called malkangni, and express the oil
from them, and rub it on the child’s face, and think it will make the face
reddish. Thus they apparently consider a black colour to be ugly.

(e) Funeral Rites
32. Disposal of the dead.
Burial of the dead has probably been the general custom of the Gonds in the
past, and the introduction of cremation may be ascribed to Hindu influence.
The latter method of disposal involves greater expense on account of the
fuel, and is an honour reserved for elders and important men, though in
proportion as the body of the tribe in any locality becomes well-to-do it
may be more generally adopted. The dead are usually buried with the feet
pointing to the north in opposition to the Hindu practice, and this fact has
been adduced in evidence of the Gond belief that their ancestors came from
the north. The Māria Gonds of Bastar, however, place the feet to the west in
the direction of the setting sun, and with the face upwards. In some places
the Hindu custom of placing the head to the north has been adopted.
Formerly it is said that the dead were buried in or near the house in which
they died, so that their spirits would thus the more easily be born again in
children, but this practice has now ceased. In most British Districts Hindu

ceremonial32 tends more and more to be adopted, but in Bastar State and
Chānda some interesting customs remain.
33. Funeral ceremony.
Among the Māria Gonds a drum is beaten to announce a death, and the
news is sent to relatives and friends in other villages. The funeral takes
place on the second or third day, when these have assembled. They bring
some pieces of cloth, and these, together with the deceased’s own clothes
and some money, are buried with him, so that they may accompany his
spirit to the other world. Sometimes the women will put a ring of iron on
the body. The body is borne on a hurdle to the burial- or burning-ground,
which is invariably to the east of the village, followed by all the men and
women of the place. Arrived there, the bearers with the body on their
shoulders face round to the west, and about ten yards in front of them are
placed three sāj leaves in a line with a space of a yard between each, the
first representing the supreme being, the second disembodied spirits, and
the third witchcraft. Sometimes a little rice is put on the leaves. An axe is
struck three times on the ground, and a villager now cries to the corpse to
disclose the cause of his death, and immediately the bearers, impelled, as
they believe, by the dead man, carry the body to one of the leaves. If they
halt before the first, then the death was in the course of nature; if before the
second, it arose from the anger of offended spirits; if before the third,
witchcraft was the cause. The ordeal may be thrice repeated, the
arrangement of the leaves being changed each time. If witchcraft is
indicated as the cause of death, and confirmed by the repeated tests, the
corpse is asked to point out the sorcerer or witch, and the body is carried
along until it halts before some one in the crowd, who is at once seized and
disposed of as a witch. Sometimes the corpse may be carried to the house of
a witch in another village to a distance of eight or ten miles. In Mandla in
such cases a Gunia or exorciser formerly called on the corpse to go forward
and point out the witch. The bearers then, impelled by the corpse, made one
step forward and stopped. The exorciser then again adjured the corpse, and

they made a step, and this was repeated again and again until they halted in
front of the supposed witch. All the beholders and the bearers themselves
thus thought that they were impelled by the corpse, and the episode is a
good illustration of the power of suggestion. Frequently the detected witch
was one of the deceased’s wives. In Mandla the cause of the man’s death
was determined in the digging of his grave. When piling in the earth
removed for the grave after burial, if it reached exactly to the surface of the
ground, they thought that the dead man had died after living the proper span
of his life. If the earth made a mound over the hole, they thought he had
lived beyond his allotted time and called him Sīgpur, that is a term for a
measure of grain heaped as high as it will stand above the brim. But if the
earth was insufficient and did not reach to the level of the ground, they held
that he had been prematurely cut off, and had been killed by an enemy or by
a witch through magic.
Children at breast are buried at the roots of a mahua tree, as it is thought
that they will suck liquor from them and be nourished as if by their
mother’s milk. The mahua is the tree from whose flowers spirits are
distilled. The body of an adult may also be burnt under a mahua tree so that
the tree may give him a supply of liquor in the next world. Sometimes the
corpse is bathed in water, sprinkled over with milk and then anointed with a
mixture of mahua oil, turmeric and charcoal, which will prevent it from
being reincarnated in a human body. In the case of a man killed by a tiger
the body is burned, and a bamboo image of a tiger is made and thrown
outside the village. None but the nearest relatives will touch the body of a
man killed by a tiger, and they only because they are obliged to do so. None
of the ornaments are removed from the corpse, and sometimes any other
ornaments possessed by the deceased are added to them, as it is thought that
otherwise the tiger into which his spirit passes will come back to look for
them and kill some other person in the house. In some localities any one
who touches the body of a man killed or even wounded by a tiger or panther
is put temporarily out of caste. Yet the Gonds will eat the flesh of tigers and
panthers, and also of animals killed and partly devoured by them. When a
man has been killed by a tiger, or when he has died of disease and before
death vermin have appeared in a wound, the whole family are temporarily
out of caste and have to be purified by an elaborate ceremony in which the

Bhumka or village priest officiates. The method of laying the spirit of a man
killed by a tiger resembles that described in the article on Baiga.
34. Mourning and offerings to the dead.
Mourning is usually observed for three days. The mourners abstain from
work and indulgence in luxuries, and the house is cleaned and washed. The
Gonds often take food on the spot after the burial or burning of a corpse and
they usually drink liquor. On the third day a feast is given. In Chhindwāra a
bullock or cow is slaughtered on the death of a male or female Gond
respectively. They tie it up by the horns to a tree so that its forelegs are in
the air, and a man slashes it across the head once or twice until it dies. The
head is buried under a platform outside the village in the name of the
deceased. Sometimes the spirit of the dead man is supposed to enter into
one of the persons present and inform the party how he died, whether from
witchcraft or by natural causes. He also points out the place where the
bullock’s or cow’s head is to be buried, and here they make a platform to
his spirit with a memorial stone. Red lead is applied to the stone and the
blood of a chicken poured over it, and the party then consume the bodies of
the cow and chicken. In Mandla the mourners are shaved at the grave nine
or ten days after the death by the brother-in-law or son-in-law of the
deceased, and they cook and eat food there and drink liquor. Then they
come home and put oil on the head of the heir and tie a piece of new cloth
round his head. They give the dead man’s clothes and also a cow or bullock
to the Pardhān priest, and offer a goat to the dead man, first feeding the
animal with rice, and saying to the dead man’s spirit, ‘Your son- or brother-
in-law has given you this.’ Sometimes the rule is that the priest should
receive all the ornaments worn on the right side of a man or the left side of
a woman, including those on the head, arm and leg. If they give him a cow
or bullock, they will choose the one which goes last when the animals are
let out to graze. Then they cook and eat it in the compound. They have no
regular anniversary ceremonies, but on the new moon of Kunwār
(September) they will throw some rice and pulse in front of the house and

pour water on it in honour of the dead. The widow breaks her glass bangles
when the funeral takes place, and if she is willing she may be married to the
dead man’s younger brother on the expiry of the period of mourning.
35. Memorial stones to the dead.
In Bastar, at some convenient time after the death, a stone is set up in
memory of any dead person who was an adult, usually by the roadside.
Families who have emigrated to other localities often return to their parent
village for setting up these stones. The stones vary according to the
importance of the deceased, those for prominent men being sometimes as
much as eight feet high. In some places a small stone seat is made in front,
and this is meant for the deceased to sit on, the memorial stone being his
house. After being placed in position the stone is anointed with turmeric,
curds, ghī and oil, and a cow or pig is offered to it. Afterwards irregular
offerings of liquor and tobacco are made to the dead man at the stone by the
family and also by strangers passing by. They believe that the memorial
stones sometimes grow and increase in size, and if this happens they think
that the dead man’s family will become extinct, as the stone and the family
cannot continue to grow together. Elsewhere a long heap of stones is made
in honour of a dead man, sometimes with a flat-topped post at the head.
This is especially done for men who have died from epidemic disease or by
an accident, and passers-by fling stones on the heap with the idea that the
dead man’s spirit will thereby be kept down and prevented from returning
to trouble the living. In connection with the custom of making a seat at the
deceased’s tomb for his spirit to sit upon, Mr. A. K. Smith writes: “It is well
known to every Gond that ghosts and devils cannot squat on the bare
ground like human beings, and must be given something to sit on. The
white man who requires a chair to sit on is thus plainly akin to the world of
demons, so one of the few effective ways of getting Gonds to open their
mouths and talk freely is to sit on the ground among them. Outside every
Gond house is placed a rough bench for the accommodation of any devils

that may be flitting about at night, so that they may not come indoors and
trouble the inmates.”
36. House abandoned after a death.
If one or two persons die in a house in one year, the family often leave it
and make another house. On quitting the old house they knock a hole in the
back wall to go out, so as to avoid going out by the front door. This is
usually done when the deaths have been due to an epidemic, and it is
presumably supposed that the dead men’s spirits will haunt the house and
cause others to die, from spite at their own untimely end. If an epidemic
visits a village, the Gonds will also frequently abandon it, and make a new
village on another site.
37. Bringing back the soul.
They believe that the spirits of ancestors are reincarnated in children or in
animals. Sometimes they make a mark with soot or vermilion on the body
of a dead man, and if some similar mark is subsequently found on any
newborn child it is held that the dead man’s spirit has been reborn in it. In
Bastar, on some selected day a short time after the death, they obtain two
small baskets and set them out at night, placing a chicken under one and
some flour of wheat or kutki under the other. The householder then says, “I
do the work of those old men who died. O spirits, I offer a chicken to you
to-day; be true and I will perform your funeral rites to-morrow.” On the
next morning the basket placed over the flour is lifted up, and if a mark
resembling a footprint of a man or any animal be found, they think that the
deceased has become incarnate in a human being or in that animal.
Subsequently they sacrifice a cow to the spirit as described. In other places
on the fifth day after death they perform the ceremony of bringing back the

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