Received: 24 September 2022 Revised: 8 February 2023 Accepted: 21 February 2023

DOI: 10.1002/wsbm.1606

PERSPECTIVE

Potential role of exitron-containing homeobox

genes in cancer

Joice de Faria Poloni 1,2 | Bruno César Feltes 2

1
School of Life and Health Sciences,
Pontifical Catholic University of Rio Abstract
Grande do Sul, Porto Alegre, Rio Grande Homeobox genes are protagonists in developmental and cancer biology, mak-
do Sul, Brazil
ing comprehending their regulation pivotal in multiple molecular pathways.
2
Department of Theoretical Informatics,
Exitrons, also known as intronic exons, are new players in the transcriptional
Federal University of Rio Grande do Sul,
Porto Alegre, Rio Grande do Sul, Brazil organization, providing additional splicing variants whose functions are still
vastly unknown. Exitron splicing sites were identified in eight homeobox
Correspondence
Bruno César Feltes, Institute of
genes, which has not been yet debated in the scientific literature. Due to the
Informatics, Department of Theoretical intimate connection between homeobox genes and tumorigenesis, it is worth
Informatics, Universidade Federal do Rio investing more time in understanding how these less explored exitron-
Grande do Sul—UFRGS, Avenida Bento
Gonçalves 9500, Prédio 72, Porto Alegre, containing transcriptional isoforms could play a role in modulating the
RS 91501-970, Brazil. homeobox gene's biological functions. The perspectives devised in this article
Email: [email protected]
are meant to instigate fresh debates on how the transcriptional variants
Funding information retaining exitrons identified in the human homeobox genes HOXA1, HOXA9,
Conselho Nacional de Desenvolvimento HOXD8, NKX3.1, and DLX6 can be examined in the context of tumorigenesis.
Científico e Tecnol
ogico, Grant/Award
Numbers: 151591/2022-9, 465450/2014-8; This article is categorized under:
Coordenação de Aperfeiçoamento de
Cancer > Genetics/Genomics/Epigenetics
Pessoal de Nível Superior, Grant/Award
Number: 001; Fundação de Amparo à
KEYWORDS
Pesquisa do Estado do Rio Grande do Sul,
Grant/Award Number: 17/2551-0000520-1 cancer, exitron, homeobox, HOX, splicing

Edited by: Emily Frieben, Executive

Editor

1 | INTRODUCTION

Alternative splicing is a post-transcriptional regulation mechanism in eukaryotes, indispensable for transcriptomic and
proteomic diversity and modulating cell fate decisions by regulating gene expression. The splicing process is primarily
known for removing introns from pre-mRNA and combining different exons to form a mature mRNA. However, these
small pieces can be combined in various assortments, assembling alternative transcripts from the same gene, creating
new layers of gene expression control and proteomic diversity.
In humans, approximately 95% of genes undergo the splicing process producing at least two alternative isoforms,
which can be specific to tissues, developmental stages, or in response to environmental stimuli (Bonnal et al., 2020; De
Faria Poloni & Bonatto, 2018; Monteuuis et al., 2019). Splicing is carried out by the spliceosome, a dynamic complex
primarily composed of small nuclear ribonucleoproteins (snRNPs; U1, U2, U4, U5, and U6) associated with RNA-
binding proteins (RBPs) that recognize short consensus sequences within pre-mRNA during transcription elongation,
named splice sites (Figure 1; Bonnal et al., 2020; De Faria Poloni & Bonatto, 2018). Splice sites' recognition is influenced

WIREs Mech Dis. 2023;15:e1606. wires.wiley.com/mechdisease © 2023 Wiley Periodicals LLC. 1 of 10

https://doi.org/10.1002/wsbm.1606
2 of 10 de FARIA POLONI and FELTES

F I G U R E 1 (a) Sequences of 30 and 50 splicing sites define the boundaries between introns and exons, being recognized by the
spliceosome. This recognition occurs with the aid of splicing factors acting in trans together with elements acting in cis, which can inhibit or
promote the recognition of nearby splicing sites and affect the activity of the spliceosome. (b) Exitrons are cryptic introns that present both
splicing sites located within an exon. These have coding potential and lack stop codons, as it is found in many introns. Thus, during splicing,
exitrons may be retained in the final transcript (1) or may be removed, possibly altering the reading frame (2). Intronic retention events
(3) generally produce transcripts that are retained in the nucleus or are directed to nonsense mediated decay in the cytoplasm, or are
translated into truncated proteins. Figure adapted from (De Faria Poloni & Bonatto, 2018).

by cis-regulatory sequences, such as exonic or intronic silencers or enhancers, which are targeted by trans-acting
factors, such as serine/arginine-rich (SR) proteins and heterogeneous ribonucleoproteins (hnRNPs) (Figure 1).
Trans-acting factors associated with cis-acting elements either promote or prevent the recruitment of the spliceosome
components at adjacent sites (Cherry & Lynch, 2020). RBPs are required for efficient splice site recognition by their
binding along exons and introns, directing the catalytic complex to the appropriate splice sites (Cherry &
Lynch, 2020). This splicing machinery's functioning is crucial to recognizing the correct functional units that com-
pose the mature mRNA.
Perturbations of the splicing process, which could involve aberrant splicing per se or alternative splicing deregula-
tion, are a driving mechanism for several pathologies, and studies have observed differentially spliced transcripts in dif-
ferent diseases (Cooper et al., 2009; Lord & Baralle, 2021; Montes et al., 2019), most notably in cancer (Bonnal
et al., 2020; Climente-Gonzalez et al., 2017; De Faria Poloni & Bonatto, 2018; El Marabti & Younis, 2018; Tang
et al., 2020). The correct regulation of alternative splicing events is crucial to ensure the proper transcript production,
according to the cell demand. Among splicing events, intron retention is the most related to tumor-suppressor genes
inactivation (Jung et al., 2015). Intron retention event is assumed to have a regulatory function of gene expression by
leading to RNA decay since many introns show in-frame premature termination codons (Monteuuis et al., 2019). A
study evaluating splicing patterns across matched tumor and normal samples between 16 distinct cancer types found
that intron retention was more frequent in almost all cancer types, even in the absence of mutations in the components
of the splicing machinery (Dvinge & Bradley, 2015). Likewise, intron-containing mRNAs were present at higher levels
in the nucleus; however, a small fraction was detected in the cytoplasm, suggesting that they were stable and could con-
tribute, if translated, to the proteomic repertoire or directed to cytoplasmic degradation via nonsense-mediated decay
(Dvinge & Bradley, 2015; Kumari et al., 2022). Marquez et al. in 2012 and 2015 described that some retained intron
transcript introns have specific intronic and exonic characteristics and display both splice sites (30 and 50 ) within an
annotated exon, being called exitrons or exonic introns (Marquez et al., 2012, 2015). Exitrons are introns within a
protein-coding exon, and, differently from conventional intron retention, they keep the transcript coding potential
when retained in the mature mRNA (Marquez et al., 2015). In addition, exitrons could be present in parts or in whole
protein domains, overrepresented in disordered regions and short linear motifs, and enriched in phosphorylation and
ubiquitylation sites (Marquez et al., 2015). Exitrons regulation shows distinct characteristics from introns, such as the
absence of stop codons, high GC content, weaker splice sites, and prevalence of synonymous substitutions, which are
also frequently observed in exons (Marquez et al., 2015; Sibley et al., 2016). Nevertheless, the research on exitrons is still
in its initial stages, which constitute a fertile ground for new discussions and perspectives.
de FARIA POLONI and FELTES 3 of 10

In this sense, in the original data of Marquez et al. (2015), several homeobox genes were described as having
exitrons in their sequence, which is not yet commented in the scientific literature. Homeobox is a diverse superclass of
genes coding for proteins with a homeodomain, an evolutionarily conserved sequence characterized by a helix–loop–
helix–turn–helix motif, deeply connected to the developmental process and differentiation (Bürglin, 2011;
Holland, 2013; Figure 2). Additionally, the strong connection between homeobox genes and cancer biology has been a
matter of discussion for decades, where these genes have protagonistic roles in tumorigenesis (Abate-Shen, 2002; Cillo
et al., 1999; Samuel & Naora, 2005). More importantly, homeobox genes have dual roles in multiple cancer types, been
described as suppressors or promoters of tumorigenesis, which could be associated with their alternative splicing. Like-
wise, homeobox genes are also associated with multiple DNA repair genes, implying a role yet to be understood
(Feltes, 2019). In this perspective article, we discuss some of the homeobox genes described to have exitrons in their
sequence and debate their roles in cancer.

2 | POTENTIAL R OLE OF EXITRON- CONTAINING HOM EOBOX GENES I N

CANCER

The work of Marquez et al. (2015) revealed nine homeobox genes with exitrons splicing sites: DLX6, HOXA1, HOXA9,
HOXD8, NKX3.1, ZEB2, ONECUT1, SIX5, and HOMEZ. In agreement with this find, the authors discuss that intrinsi-
cally disordered regions (IDRs), which are long associated with homeodomain-containing proteins (Hsiao et al., 2014;
th-Petro
Robertson et al., 2018; To czy et al., 2009; Vuzman & Levy, 2012), are the most enriched by exitron splicing sites.
Although multiple layers of complexity surround the fine-tuning of gene expression, it is worth discussing the dual roles
in tumorigenesis of the most preeminent genes in that list. The discussion presented in this perspective article was tai-
lored as “food-for-thoughts” since the probability of the abnormal functions seen in different cancer types of these tar-
gets being associated with exitron splicing is still to be unequivocally proved.
We highlight that the work of Marques et al. was based on the GRCh37 rather than GRCh38. However, the authors
highlighted in the original work that this update did not influence their conclusions and results.

F I G U R E 2 Homeodomains of the homeobox genes discussed in this article. (a) The homeodomain of mice HoxA9 and Pdx1 bound to
DNA (PDB: 1PUF) (LaRonde-LeBlanc & Wolberger, 2003). (b) Surface view of the homeodomain of HoxA9 (PDB: 1PUF). A long coil portion
are inserted near the minor grove of the DNA molecule, while the α-helices make contact with the major grove. (c) The homeodomain of
NKX3.1 from Homo sapiens (PDB: 2L9R, unpublished); (d) The homeodomain of DLX6 is not resolved at the time of this work. The figure
depict the homeodomain of DLX3 bound to MEIS1 and the DNA molecules (PDB: 4XRS) (Jolma et al., 2015). Note that the folding and the
conformational preference of DLX3, NKX3.1, and HoxA9 are highly similar.
4 of 10 de FARIA POLONI and FELTES

2.1 | HOXA9

Composed of only two exons, HOXA9 produces an ample assortment of associated transcripts, which are frequently
conserved in multiple species in embryogenesis and various cancer types (Bhatlekar et al., 2014; Popovic et al., 2008;

F I G U R E 3 Representation of the genes described with their transcriptional variants and the position of the exitrons described by
Marquez et al., 2015, according to the annotation available in Ensembl GRCh37. The gene orientation is indicated by arrows. (a) HOXA9;
(b) HOXA1; (c) HOXD8; (d) NKX3-1; and (e) DLX6.

TABLE. 1 Summary of the homeobox proteins discussed in the work.

Gene Protein lengtha Chromosome No. of exons Exitron coordinate

HOXA1 335 7p15.2 2 27,134,974–27,135,178 (exon I)
HOXA9 272 7p15.2 2 27,204,586–27,204,760 (exon I)
HOXD8 290 2q31.1 2 176,995,211–176,995,524 (exon I)
176,996,187–176,996,266 (exon II)
NKX3.1 234 8p21.2 2 23,540,144–23,540,370 (exon I)
DLX6 175 7q21.3 3 96,635,384–96,635,469 (exon I)

Note: The exitron coordinates were mapped using GRCh37 (Marquez et al., 2015).
a
Based on the most common (canonical) isoform.
de FARIA POLONI and FELTES 5 of 10

Figures 2a,b and 3; Table 1). All transcriptional isoforms variants described in GRCh37 are still present in the GRCh38
genome version.
One of the most intriguing features of HOXA9 is its dual role as a tumor suppressor in carcinoma and an oncogene
in leukemia, especially in acute myeloid leukemia (AML) and mixed lineage leukemia (MLL) (He et al., 2012; J. D.
Wang et al., 2019). The homeodomain is located at exon II, but the exitron splicing region was identified at the end of
exon I (GRCh37, 7: 27,204,586–27,204,760). This region was previously described in murine models in 1998 by Fujimoto
et al., which demonstrated that this isoform (HOXA9T) was found: (i) in higher quantities in kidney and intestinal tis-
sues in adults; (ii) in the trunk region of 12.5 days embryos; and (iii) would produce a truncated protein with no
homeodomain (Fujimoto et al., 1998). This first study was not conducted in cancer models, suggesting that HOXA9T
has potential physiological roles. However, J. D. Wang et al. (2019) argued that in humans and mice, this isoform could
produce an alternate protein starting at an out-of-frame start codon at the end of the exitron splicing site (7: 27,204,586)
and the full homeodomain C-terminal. Under this model, conserving an intact homeodomain implies that potential
new roles would not necessarily be associated with DNA binding but with other potential protein–protein (PPI) binding
sites. For example, this region was described as binding Pbx1 in mice and required to recognize HOX DNA-binding sites
(LaRonde-LeBlanc & Wolberger, 2003).
Other works discuss that HOXA9T could produce different transcriptional variants with roles in leukemogenesis,
but none could safely define the possibility of this new variant (He et al., 2012; Stadler et al., 2014). This is an interest-
ing debate since the conservation of the HOXA9T in mice and humans in healthy tissues and leukemia suggests that it
has biological roles that are yet to be elucidated. Although the importance of this isoform has been primarily debated in
leukemia, studying it in the context of solid tumors might deliver new clues to its biological consequence.
Another interesting debate regarding the dual roles of HOXA9 is that the action of its transcriptional isoforms could
explain its participation as a positive or negative regulator of DNA repair genes. In this sense, HOXA9 was observed to
be a positive transcriptional regulator of double-stand break repair genes, such as RAD51 and BRCA1 in leukemia; at
the same time, it was observed to be a negative regulator of mismatch repair genes in glioblastoma cells treated with
temozolomide, such as MSH2-3 and 6, PMS2, MLH1, and 3 (Feltes, 2019).

2.2 | HOXA1

HOXA1 showed an exitron identified in exon I (GRCh37, 7: 27,134,974–27,135,178), where, comparable to HOXA9, it is
located near the end of the exon (Figure 3). All transcriptional isoforms described in GRCh37 are still present in the
GRCh38 genome version.
One intriguing aspect of HOXA1 is that it is related to a distinct autosomal recessive phenotype, the Bosley–Salih–
Alorainy syndrome (BSAS), characterized by cerebrovascular and cardiac anomalies, deafness, and, on rare occasions,
autism (Bosley et al., 2007). Most HOXA1 mutations were described in exon I, thus, hindering the final protein of the
homeodomain (Bosley et al., 2008). Still, a mutation in exon II (:c.669C>A, p. Tyr223Stop) was also reported (Patil
et al., 2020). HOXA1 is a known oncogene in multiple cancers, such as breast (Belpaire et al., 2022), prostate (H. Wang
et al., 2015), and liver (T.-Z. Zha et al., 2012), among others. The amino acid sequence encoded near the exitron region
display residues which are conserved from fly to humans (Singh et al., 2020) and were also associated with a PPI with
the O-linked N-acetylglucosamine transferase OGT (Draime et al., 2018). OGT-associated post-translational modifica-
tion (PTM) can modulate intracellular translocation, DNA binding capacity, PPI, and even stability (Özcan et al., 2010).
Draime et al. demonstrated that the portion comprising the 79–199 amino acid region of HOXA1, which is encoded by
exon I, was required for PPI with OGT and that it could be O-GlcNAcylated on Tyr149. Likewise, the authors showed
that this PPI occurs in the DNA rather than in the cytoplasm. Despite the PPI and the PTM, OGT did not influence the
stability, localization, or transcriptional activity of HOXA1, suggesting that it probably exerts such influence in the con-
text of complex formation (i.e., together with other proteins), such as with Hoxb1, which is not only a known HOXA1
target in neurodevelopment (Studer et al., 1998) but also an OGT-interacting protein (Vella et al., 2013). Another
hypothesis was that HOXA1 could have nontranscriptional roles, thus, influencing OGT rather than being
affected by it.
OGT is a known protagonist in cancer cells (Ferrer et al., 2014; Itkonen et al., 2019; X. Li et al., 2021) but was also
described to play a role in mice neurogenesis by affecting Notch signaling (Chen et al., 2021). Remarkably, in chick
embryos, the expression of Hoxa1, Hoxb1, and Hoxb2 can induce a Notch cascade during neural crest formation (Gouti
et al., 2011). Thus, OGT appears as a protagonist during neurodevelopment in embryogenesis, as well as in
6 of 10 de FARIA POLONI and FELTES

tumorigenesis. The effects of the HOXA1 transcriptional variant (GRCh37, 7: 27,134,974–27,135,178) are not yet studied
in the scientific literature. Considering the strong association between HOXA1 and OGT and their dual roles in
embryogenesis and disease, studies focusing on their relationship in different cancers could shed light on new roles for
these proteins. Likewise, investigating the effects of the new variant, like in the work of (J. D. Wang et al., 2019), could
provide additional clues on how HOXA1 acts as an oncogene.

2.3 | HOXD8

The work of (Marquez et al., 2015) identified two exitron-containing transcriptional isoforms in HOXD8, one in exon I
(GRCh37, 2: 176,995,211–176,995,524) and one in exon II (GRCh37, 2: 176,996,187–176,996,266) (Figure 3). PFAM anal-
ysis showed that the second variant is partially located in the homeodomain (residues 199–255), between the region
240–266. The new version of the human genome depicts one less variant than in GRCh37 (ENST00000548663.1), which
was found to be part of a different transcript (GRCh38, ENST00000544999.2). All other four transcriptional variants
described for HOXD8 are protein-coding.
Among the HOX genes discussed so far, HOXD8 has the most contradictory roles. Through different mechanisms,
HOXD8 was a tumor suppressor in breast (Wen et al., 2021; Zhang et al., 2021), neuroblastoma (Y. Zha et al., 2012),
and colorectal cancer (Mansour & Senga, 2017). However, HOXD8 was found to be recurrently overexpressed in lung
squamous carcinoma (Bao et al., 2016) and cisplatin chemoresistant ovarian cancer (Sun et al., 2018). While the differ-
ent roles of HOXD8 imply that it acts as a suppressor or oncogene through different molecular mechanisms, the scien-
tific literature has no reports on the biological functions of any transcriptional variant, creating a fertile ground for
discoveries in the field of cancer biology. In fact, other HOXD genes have known splicing variants, such as
HOXD11-NUP98 in AML (Taketani et al., 2002), a 758-2delA in HOXD13, which leads to foot malformations, and
HOXD-AS2 reported to confer temozolomide resistance in glioblastoma (Nie et al., 2021).

2.4 | NKX3.1

NKX3.1 is a homeobox whose protein is highly present during the formation of the urogenital system (Padmanabhan
et al., 2016) (Figure 2c). It has two exons, where the exitron (GRCh37, 8: 23,540,144–23,540,370) lies in exon I
(Figure 3). All variants described in GRCh37 are still present in the GRCh38 genome version.
NKX3.1 was described as both suppressor and oncogene in prostate cancer (Antao et al., 2021; Gurel et al., 2010). In
terms of transcriptional variants, Korkmaz et al. in 2000 identified three cDNA originated from small in-frame deletions
in prostate cancer cells, which were highly similar to mice. Still, no additional studies were performed to understand
their biological functions (if any). However, the impact of the exitron-containing transcriptional variant might not nec-
essarily relate to DNA-binding but PTM. Exon I encodes for the N-terminal of NKX3.1, which has three known phos-
phorylation sites associated with its DNA binding and protein stability (Padmanabhan et al., 2016). In this sense,
NKX3.1 is phosphorylated at Thr89 and Thr93 by a CDK2 isoform, protecting it from degradation in prostate cancer
cells (X. Li et al., 2006). The authors argue that the PTM might be one of the defining variables that modulate NKX3.1
role as a tumor suppressor since NKX3.1 down-regulation is a common phenomenon in aggressive types of prostate
cancer.
Two layers of complexity arise from this observation. The first is that the exitron-containing transcriptional variant
could display an altered N-terminal, hindering the possibility of NKX3.1 being protected from degradation in prostate
cancer cells, thus, exerting an oncogenic role. The other is that this PTM is not the only defining factor, and the other
seven phosphorylation sites in NKX3.1 are associated with fine-tuning this protein in cancer cells. Nevertheless, com-
prehensive knowledge of this protein splicing variants would help elucidate the dual roles of NKX3.1.

2.5 | DLX6

DLX6 is composed of three exons, where the exitron splicing site is located at exon I (GRCh37, 7: 96,635,384–
96,635,469) (Figures 2d and 3). The new version of the human genome depicts one less variant than in GRCh37
(ENST00000007660.5); all other three transcriptional variants remain the same.
de FARIA POLONI and FELTES 7 of 10

BOX 1 Homeobox genes, exitrons, and cancer.

Homeobox genes are protagonists in developmental and cancer biology, making comprehending their regula-
tion pivotal in multiple molecular pathways. Exitrons, also known as intronic exons, are new players in the
transcriptional organization, providing additional splicing variants whose functions are still vastly unknown.
The perspectives devised in this article are meant to instigate fresh debates on how the exitron-containing tran-
scriptional variants identified in human homeobox genes can be examined in the context of tumorigenesis. Fur-
ther studies benefit an interdisciplinary audience since the discussion has ramifications in genomic,
transcriptomic, and protein structure research.

DLX6 promotes proliferation and the inhibition of apoptosis in oral squamous cell carcinoma (Liang et al., 2022). A
long noncoding RNA arising from a conserved intergenic region between DLX5 and DLX6, named DLX6-AS1 (Feng
et al., 2006; Zheng et al., 2021), was found to positively regulate DLX6 in endometrial cancer, also promoting prolifera-
tion apoptosis inhibition (Zhao & Xu, 2020). Likewise, downregulation of DLX6-AS1 lower both DLX6 mRNA and pro-
tein levels in lung adenocarcinoma (J. Li et al., 2015).
Reports demonstrate that DLX6 has implications in tumorigenesis, but the scientific literature lacks in-depth knowl-
edge of this gene in the tumoral process. Likewise, little is known about the DLX6 protein structure to allow some level
of inference of potential impacts of an alternative transcript in the final protein. While this reality hinders further
detailed discussion of DLX6 in cancer, it is also an excellent opportunity for new genomic, transcriptomic, and struc-
tural biology research to target DLX6 for further investigation.

3 | C ON C L U S I ON

The intimate relationship of homeobox genes with tumorigenesis is unequivocal. Comprehensive research also continu-
ously proves how the splicing process plays a pivotal role in modulating the unique molecular profile of different cancer
types. Considering the newly found exitron splicing sites in homeobox genes, distinct molecular behaviors of these
genes in the tumoral tissue may be associated with these variants. Either due to the “Jekyll and Hyde” roles of HOXD8
in cancer, the numerous variants of HOXA9 in leukemia, or the potential loss of protein function in HOXA1 and
NKX3.1, which could lead to tumor progression, the mechanisms behind their exitronic mRNA are still to be eluci-
dated. Some homeobox genes, such as DLX6, offer a fertile ground for new works since we have multiple knowledge
gaps regarding its potential variants and protein structure information. We argue that, under a given light, it is possible
that previous splicing events classified as intron retention could be exitrons; the best example is HOXA9T. When fist
described HOXA9T was considered a transcriptional variant with absent homeodomain. However, the work of
(Marquez et al., 2015) showed that the identified region was an exitron, opening new mRNA processing possibilities, a
perspective discussed later by (J. D. Wang et al., 2019). Finally, the work of (T. Y. Wang et al., 2021) described other
exitron-spliced HOX genes in TCGA cohorts, such as HOXD4, HOXB2, and NKX2.1 that could also be further explored
in the same manner (Box 1).

A U T H O R C ON T R I B U T I O NS
Bruno César Feltes: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition
(equal); investigation (equal); methodology (equal); project administration (equal); supervision (equal); writing – origi-
nal draft (equal); writing – review and editing (equal). Joice de Faria Poloni: Conceptualization (equal); data curation
(equal); formal analysis (equal); funding acquisition (equal); investigation (equal); methodology (equal); project admin-
istration (equal); supervision (equal); writing – original draft (equal); writing – review and editing (equal).

FUNDING INFORMATION
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul [17/
2551-0000520-1] and from the Conselho Nacional de Desenvolvimento Científico e Tecnol
ogico [151591/2022-9 and
465450/2014-8]. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior—Brasil (CAPES)—Finance Code 001.
8 of 10 de FARIA POLONI and FELTES

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

ORCID
Joice de Faria Poloni https://orcid.org/0000-0002-3896-6404
Bruno César Feltes https://orcid.org/0000-0002-2825-8295

R EF E RE N C E S
Abate-Shen, C. (2002). Deregulated homeobox gene expression in cancer: Cause or consequence? Nature Reviews Cancer, 2(10), 777–785.
https://doi.org/10.1038/nrc907
Antao, A. M., Ramakrishna, S., & Kim, K.-S. (2021). The role of Nkx3.1 in cancers and stemness. International Journal of Stem Cells, 14, 168–
179. https://doi.org/10.15283/ijsc20121
Bao, L., Zhang, Y., Wang, J., Wang, H., Dong, N., Su, X., Xu, M., & Wang, X. (2016). Variations of chromosome 2 gene expressions among
patients with lung cancer or non-cancer. Cell Biology and Toxicology, 32(5), 419–435. https://doi.org/10.1007/s10565-016-9343-z
Belpaire, M., Taminiau, A., Geerts, D., & Rezsohazy, R. (2022). HOXA1, a breast cancer oncogene. Biochimica et Biophysica Acta (BBA) -
Reviews on Cancer, 1877(4), 188747. https://doi.org/10.1016/j.bbcan.2022.188747
Bhatlekar, S., Fields, J. Z., & Boman, B. M. (2014). HOX genes and their role in the development of human cancers. Journal of Molecular
Medicine, 92(8), 811–823. https://doi.org/10.1007/s00109-014-1181-y
Bonnal, S. C., L opez-Oreja, I., & Valcarcel, J. (2020). Roles and mechanisms of alternative splicing in cancer—Implications for care. Nature
Reviews Clinical Oncology, 17(8), 457–474. https://doi.org/10.1038/s41571-020-0350-x
Bosley, T. M., Alorainy, I. A., Salih, M. A., Aldhalaan, H. M., Abu-Amero, K. K., Oystreck, D. T., Tischfield, M. A., Engle, E. C., &
Erickson, R. P. (2008). The clinical spectrum of homozygousHOXA1 mutations. American Journal of Medical Genetics Part A, 146A(10),
1235–1240. https://doi.org/10.1002/ajmg.a.32262
Bosley, T. M., Salih, M. A., Alorainy, I. A., Oystreck, D. T., Nester, M., Abu-Amero, K. K., Tischfield, M. A., & Engle, E. C. (2007). Clinical
characterization of the HOXA1 syndrome BSAS variant. Neurology, 69(12), 1245–1253. https://doi.org/10.1212/01.wnl.0000276947.
59704.cf
Bürglin, T. R. (2011). Homeodomain subtypes and functional diversity. Sub-Cellular Biochemistry, 52, 95–122. https://doi.org/10.1007/978-90-
481-9069-0_5
Chen, J., Dong, X., Cheng, X., Zhu, Q., Zhang, J., Li, Q., Huang, X., Wang, M., Li, L., Guo, W., Sun, B., Shu, Q., Yi, W., & Li, X. (2021). Ogt
controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling. Cell Reports, 34(13), 108905.
https://doi.org/10.1016/j.celrep.2021.108905
Cherry, S., & Lynch, K. W. (2020). Alternative splicing and cancer: Insights, opportunities, and challenges from an expanding view of the
transcriptome. Genes and Development, 34(15–16), 1005–1016. https://doi.org/10.1101/GAD.338962.120
Cillo, C., Faiella, A., Cantile, M., & Boncinelli, E. (1999). Homeobox genes and cancer. Experimental Cell Research, 248(1), 1–9. https://doi.
org/10.1006/excr.1999.4451
Climente-Gonzalez, H., Porta-Pardo, E., Godzik, A., & Eyras, E. (2017). The functional impact of alternative splicing in cancer. Cell Reports,
20(9), 2215–2226. https://doi.org/10.1016/j.celrep.2017.08.012
Cooper, T. A., Wan, L., & Dreyfuss, G. (2009). RNA and disease. Cell, 136(4), 777–793. https://doi.org/10.1016/j.cell.2009.02.011
De Faria Poloni, J., & Bonatto, D. (2018). Influence of transcriptional variants on metastasis. RNA Biology, 15(8), 1006–1024. https://doi.org/
10.1080/15476286.2018.1493328
Draime, A., Bridoux, L., Belpaire, M., Pringels, T., Degand, H., Morsomme, P., & Rezsohazy, R. (2018). The O-GlcNAc transferase OGT inter-
acts with and post-translationally modifies the transcription factor HOXA1. FEBS Letters, 592(7), 1185–1201. https://doi.org/10.1002/
1873-3468.13015
Dvinge, H., & Bradley, R. K. (2015). Widespread intron retention diversifies most cancer transcriptomes. Genome Medicine, 7(1), 1–13.
https://doi.org/10.1186/s13073-015-0168-9
El Marabti, E., & Younis, I. (2018). The cancer spliceome: Reprograming of alternative splicing in cancer. Frontiers in Molecular Biosciences,
5(SEP), 1–11. https://doi.org/10.3389/fmolb.2018.00080
Feltes, B. C. (2019). Architects meets repairers: The interplay between homeobox genes and DNA repair. DNA Repair, 73, 34–48. https://doi.
org/10.1016/j.dnarep.2018.10.007
Feng, J., Bi, C., Clark, B. S., Mady, R., Shah, P., & Kohtz, J. D. (2006). The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultra-
conserved region and functions as a Dlx-2 transcriptional coactivator. Genes & Development, 20(11), 1470–1484. https://doi.org/10.1101/
gad.1416106
Ferrer, C. M., Lynch, T. P., Sodi, V. L., Falcone, J. N., Schwab, L. P., Peacock, D. L., Vocadlo, D. J., Seagroves, T. N., & Reginato, M. J. (2014).
O-GlcNAcylation regulates cancer metabolism and survival stress signaling via regulation of the HIF-1 pathway. Molecular Cell, 54(5),
820–831. https://doi.org/10.1016/j.molcel.2014.04.026
de FARIA POLONI and FELTES 9 of 10

Fujimoto, S., Araki, K., Chisaka, O., Araki, M., Takagi, K., & Yamamura, K. I. (1998). Analysis of the murine Hoxa-9 cDNA: An alternatively
spliced transcript encodes a truncated protein lacking the homeodomain. Gene, 209(1–2), 77–85. https://doi.org/10.1016/S0378-1119(98)
00014-6
Gouti, M., Briscoe, J., & Gavalas, A. (2011). Anterior Hox genes interact with components of the neural crest specification network to induce
neural crest fates. Stem Cells, 29(5), 858–870. https://doi.org/10.1002/stem.630
Gurel, B., Ali, T. Z., Montgomery, E. A., Begum, S., Hicks, J., Goggins, M., Eberhart, C. G., Clark, D. P., Bieberich, C. J., Epstein, J. I., & De
Marzo, A. M. (2010). NKX3.1 as a marker of prostatic origin in metastatic tumors. American Journal of Surgical Pathology, 34(8), 1097–
1105. https://doi.org/10.1097/PAS.0b013e3181e6cbf3
He, M., Chen, P., Arnovitz, S., Li, Y., Huang, H., Neilly, M. B., Wei, M., Rowley, J. D., Chen, J., & Li, Z. (2012). Two isoforms of HOXA9 func-
tion differently but work synergistically in human MLL-rearranged leukemia. Blood Cells, Molecules, and Diseases, 49(2), 102–106.
https://doi.org/10.1016/j.bcmd.2012.05.003
Holland, P. W. H. (2013). Evolution of homeobox genes. Wiley Interdisciplinary Reviews: Developmental Biology, 2(1), 31–45. https://doi.org/
10.1002/wdev.78
Hsiao, H. C., Gonzalez, K. L., Catanese, D. J., Jordy, K. E., Matthews, K. S., & Bondos, S. E. (2014). The intrinsically disordered regions of the
Drosophila melanogaster hox protein ultrabithorax select interacting proteins based on partner topology. PLoS One, 9(10), e108217.
https://doi.org/10.1371/journal.pone.0108217
Itkonen, H. M., Urbanucci, A., Martin, S. E., Khan, A., Mathelier, A., Thiede, B., Walker, S., & Mills, I. G. (2019). High OGT activity is essen-
tial for MYC-driven proliferation of prostate cancer cells. Theranostics, 9(8), 2183–2197. https://doi.org/10.7150/thno.30834
Jolma, A., Yin, Y., Nitta, K. R., Dave, K., Popov, A., Taipale, M., Enge, M., Kivioja, T., Morgunova, E., & Taipale, J. (2015). DNA-dependent
formation of transcription factor pairs alters their binding specificity. Nature, 527(7578), 384–388. https://doi.org/10.1038/nature15518
Jung, H., Lee, D., Lee, J., Park, D., Kim, Y. J., Park, W. Y., Hong, D., Park, P. J., & Lee, E. (2015). Intron retention is a widespread mechanism
of tumor-suppressor inactivation. Nature Genetics, 47(11), 1242–1248. https://doi.org/10.1038/ng.3414
Korkmaz, K. S., Korkmaz, C. G., Ragnhildstveit, E., Kizildag, S., Pretlow, T. G., & Saatcioglu, F. (2000). Full-length cDNA sequence and
genomic organization of human NKX3A—alternative forms and regulation by both androgens and estrogens. Gene, 260(1-2), 25-36.
Kumari, A., Sedehizadeh, S., Brook, J. D., Kozlowski, P., & Wojciechowska, M. (2022). Differential fates of introns in gene expression due to
global alternative splicing. Human Genetics, 141(1), 31–47. https://doi.org/10.1007/s00439-021-02409-6
LaRonde-LeBlanc, N. A., & Wolberger, C. (2003). Structure of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide and DNA recognition ante-
rior to posterior. Genes and Development, 17(16), 2060–2072. https://doi.org/10.1101/gad.1103303
Li, J., Li, P., Zhao, W., Yang, R., Chen, S., Bai, Y., Dun, S., Chen, X., Du, Y., Wang, Y., Zang, W., Zhao, G., & Zhang, G. (2015). Expression of
long non-coding RNA DLX6-AS1 in lung adenocarcinoma. Cancer Cell International, 15(1), 48. https://doi.org/10.1186/s12935-015-
0201-5
Li, X., Guan, B., Maghami, S., & Bieberich, C. J. (2006). NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells. Molecular and
Cellular Biology, 26(8), 3008–3017. https://doi.org/10.1128/MCB.26.8.3008-3017.2006
Li, X., Wu, Z., He, J., Jin, Y., Chu, C., Cao, Y., Gu, F., Wang, H., Hou, C., Liu, X., & Zou, Q. (2021). OGT regulated O-GlcNAcylation pro-
motes papillary thyroid cancer malignancy via activating YAP. Oncogene, 40(30), 4859–4871. https://doi.org/10.1038/s41388-021-01901-7
Liang, J., Liu, J., Deng, Z., Liu, Z., & Liang, L. (2022). DLX6 promotes cell proliferation and survival in oral squamous cell carcinoma. Oral
Diseases, 28(1), 87–96. https://doi.org/10.1111/odi.13728
Lord, J., & Baralle, D. (2021). Splicing in the diagnosis of rare disease: Advances and challenges. Frontiers in Genetics, 12(July), 689892.
https://doi.org/10.3389/fgene.2021.689892
Mansour, M. A., & Senga, T. (2017). HOXD8 exerts a tumor-suppressing role in colorectal cancer as an apoptotic inducer. The International
Journal of Biochemistry & Cell Biology, 88, 1–13. https://doi.org/10.1016/j.biocel.2017.04.011
Marquez, Y., Brown, J. W. S., Simpson, C., Barta, A., & Kalyna, M. (2012). Transcriptome survey reveals increased complexity of the alterna-
tive splicing landscape in Arabidopsis. Genome Research, 22(6), 1184–1195. https://doi.org/10.1101/gr.134106.111
Marquez, Y., Höpfler, M., Ayatollahi, Z., Barta, A., & Kalyna, M. (2015). Unmasking alternative splicing inside protein-coding exons defines
exitrons and their role in proteome plasticity. Genome Research, 25(7), 995–1007. https://doi.org/10.1101/gr.186585.114
Montes, M., Sanford, B. L., Comiskey, D. F., & Chandler, D. S. (2019). RNA splicing and disease: Animal models to therapies. Trends in
Genetics, 35(1), 68–87. https://doi.org/10.1016/j.tig.2018.10.002
Monteuuis, G., Wong, J. J. L., Bailey, C. G., Schmitz, U., & Rasko, J. E. J. (2019). The changing paradigm of intron retention: Regulation,
ramifications and recipes. Nucleic Acids Research, 47(22), 11497–11513. https://doi.org/10.1093/nar/gkz1068
Nie, E., Jin, X., Miao, F., Yu, T., Zhi, T., Shi, Z., Wang, Y., Zhang, J., Xie, M., & You, Y. (2021). TGF-β1 modulates temozolomide resistance
in glioblastoma via altered microRNA processing and elevated MGMT. Neuro-Oncology, 23(3), 435–446. https://doi.org/10.1093/neuonc/
noaa198
Özcan, S., Andrali, S. S., & Cantrell, J. E. L. (2010). Modulation of transcription factor function by O-GlcNAc modification. Biochimica et Bio-
physica Acta (BBA) - Gene Regulatory Mechanisms, 1799(5–6), 353–364. https://doi.org/10.1016/j.bbagrm.2010.02.005
Padmanabhan, A., Rao, V., De Marzo, A. M., & Bieberich, C. J. (2016). Regulating NKX3.1 stability and function: Post-translational modifica-
tions and structural determinants. The Prostate, 76(6), 523–533. https://doi.org/10.1002/pros.23144
Patil, S. J., Karthik, G. A., Bhavani, G. S. L., Bhat, V., Matalia, J., Shah, J., Shukla, A., & Girisha, K. M. (2020). Bosley–Salih–Alorainy syn-
drome in patients from India. American Journal of Medical Genetics, Part A, 182(11), 2699–2703. https://doi.org/10.1002/ajmg.a.61809
Popovic, R., Erfurth, F., & Zeleznik-Le, N. (2008). Transcriptional complexity of the HOXA9 locus. Blood Cells, Molecules, and Diseases,
40(2), 156–159. https://doi.org/10.1016/j.bcmd.2007.07.016
10 of 10 de FARIA POLONI and FELTES

Robertson, N. O., Smith, N. C., Manakas, A., Mahjoub, M., McDonald, G., Kwan, A. H., & Matthews, J. M. (2018). Disparate binding kinetics
by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation. Proceedings of the National
Academy of Sciences, 115(18), 4643–4648. https://doi.org/10.1073/pnas.1714646115
Samuel, S., & Naora, H. (2005). Homeobox gene expression in cancer: Insights from developmental regulation and deregulation. European
Journal of Cancer, 41(16), 2428–2437. https://doi.org/10.1016/j.ejca.2005.08.014
Sibley, C. R., Blazquez, L., & Ule, J. (2016). Lessons from non-canonical splicing. Nature Reviews Genetics, 17(7), 407–421. https://doi.org/10.
1038/nrg.2016.46
Singh, N. P., de Kumar, B., Paulson, A., Parrish, M. E., Zhang, Y., Florens, L., Conaway, J. W., Si, K., & Krumlauf, R. (2020). A six-amino-
acid motif is a major determinant in functional evolution of HOX1 proteins. Genes and Development, 34(23–24), 1680–1696. https://doi.
org/10.1101/gad.342329.120
Stadler, C. R., Vegi, N., Mulaw, M. A., Edmaier, K. E., Rawat, V. P. S., Dolnik, A., Bullinger, L., Heilmeier, B., Quintanilla-Fend, L.,
Spiekermann, K., Hiddemann, W., Döhner, K., Döhner, H., Feuring-Buske, M., & Buske, C. (2014). The leukemogenicity of Hoxa9
depends on alternative splicing. Leukemia, 28(9), 1838–1843. https://doi.org/10.1038/leu.2014.74
Studer, M., Gavalas, A., Marshall, H., Ariza-McNaughton, L., Rijli, F. M., Chambon, P., & Krumlauf, R. (1998). Genetic interactions between
Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning. Development, 125(6), 1025–1036. https://doi.org/10.1242/
dev.125.6.1025
Sun, P., Song, Y., Liu, D., Liu, G., Mao, X., Dong, B., Braicu, E. I., & Sehouli, J. (2018). Potential role of the HOXD8 transcription factor in cis-
platin resistance and tumour metastasis in advanced epithelial ovarian cancer. Scientific Reports, 8(1), 13483. https://doi.org/10.1038/
s41598-018-31030-3
Taketani, T., Taki, T., Shibuya, N., Ito, E., Kitazawa, J., Terui, K., & Hayashi, Y. (2002). The HOXD11 gene is fused to the NUP98 gene in
acute myeloid leukemia with t(2;11)(q31;p15). Cancer Research, 62(1), 33–37.
Tang, S. J., Shen, H., An, O., Hong, H. Q., Li, J., Song, Y., Han, J., Tay, D. J. T., Ng, V. H. E., Bellido Molias, F., Leong, K. W.,
Pitcheshwar, P., Yang, H., & Chen, L. (2020). Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer
development. Nature Communications, 11(1), 1–17. https://doi.org/10.1038/s41467-020-14621-5
T
oth-Petr oczy, Á., Simon, I., Fuxreiter, M., & Levy, Y. (2009). Disordered tails of homeodomains facilitate DNA recognition by providing a
trade-off between folding and specific binding. Journal of the American Chemical Society, 131(42), 15084–15085. https://doi.org/10.1021/
ja9052784
Vella, P., Scelfo, A., Jammula, S., Chiacchiera, F., Williams, K., Cuomo, A., Roberto, A., Christensen, J., Bonaldi, T., Helin, K., & Pasini, D.
(2013). Tet proteins connect the O-linked N-acetylglucosamine transferase Ogt to chromatin in embryonic stem cells. Molecular Cell,
49(4), 645–656. https://doi.org/10.1016/j.molcel.2012.12.019
Vuzman, D., & Levy, Y. (2012). Intrinsically disordered regions as affinity tuners in protein–DNA interactions. Molecular BioSystems, 8(1),
47–57. https://doi.org/10.1039/C1MB05273J
Wang, H., Liu, G., Shen, D., Ye, H., Huang, J., Jiao, L., & Sun, Y. (2015). HOXA1 enhances the cell proliferation, invasion and metastasis of
prostate cancer cells. Oncology Reports, 34(3), 1203–1210. https://doi.org/10.3892/or.2015.4085
Wang, J. D., Gross, G. L., Andrew, E. R., Padovan, A., & Fahrer, A. M. (2019). A predicted novel protein isoform of HOXA9. Leukemia
Research, 82(May), 7–10. https://doi.org/10.1016/j.leukres.2019.05.002
Wang, T. Y., Liu, Q., Ren, Y., Alam, S. K., Wang, L., Zhu, Z., Hoeppner, L. H., Dehm, S. M., Cao, Q., & Yang, R. (2021). A pan-cancer trans-
criptome analysis of exitron splicing identifies novel cancer driver genes and neoepitopes. Molecular Cell, 81(10), 2246–2260.e12. https://
doi.org/10.1016/j.molcel.2021.03.028
Wen, X., Chen, Y., & Fang, X. (2021). Overexpression of HOXD8 inhibits the proliferation, migration and invasion of breast cancer cells by
downregulating ILP2 expression. Experimental and Therapeutic Medicine, 22(3), 1006. https://doi.org/10.3892/etm.2021.10439
Zha, T.-Z., Hu, B.-S., Yu, H.-F., Tan, Y.-F., Zhang, Y., & Zhang, K. (2012). Overexpression of HOXA1 correlates with poor prognosis in
patients with hepatocellular carcinoma. Tumor Biology, 33(6), 2125–2134. https://doi.org/10.1007/s13277-012-0472-6
Zha, Y., Ding, E., Yang, L., Mao, L., Wang, X., McCarthy, B. A., Huang, S., & Ding, H.-F. (2012). Functional dissection of HOXD cluster genes
in regulation of neuroblastoma cell proliferation and differentiation. PLoS One, 7(8), e40728. https://doi.org/10.1371/journal.pone.
0040728
Zhang, Y., Yu, Y., Su, X., & Lu, Y. (2021). HOXD8 inhibits the proliferation and migration of triple-negative breast cancer cells and induces
apoptosis in them through regulation of AKT/mTOR pathway. Reproductive Biology, 21(4), 100544. https://doi.org/10.1016/j.repbio.2021.
100544
Zhao, H., & Xu, Q. (2020). Long non-coding RNA DLX6-AS1 mediates proliferation, invasion and apoptosis of endometrial cancer cells by
recruiting p300/E2F1 in DLX6 promoter region. Journal of Cellular and Molecular Medicine, 24(21), 12572–12584. https://doi.org/10.
1111/jcmm.15810
Zheng, Q., Gu, X., Yang, Q., Chu, Q., Dai, Y., & Chen, Z. (2021). DLX6-AS1 is a potential biomarker and therapeutic target in cancer initia-
tion and progression. Clinica Chimica Acta, 517(79), 1–8. https://doi.org/10.1016/j.cca.2021.02.006

How to cite this article: de Faria Poloni, J., & Feltes, B. C. (2023). Potential role of exitron-containing
homeobox genes in cancer. WIREs Mechanisms of Disease, 15(3), e1606. https://doi.org/10.1002/wsbm.1606