BBA - Reviews on Cancer 1877 (2022) 188772

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BBA - Reviews on Cancer

journal homepage: www.elsevier.com/locate/bbacan

Review

Emerging roles of PIWI-interacting RNAs (piRNAs) and PIWI proteins in

head and neck cancer and their potential clinical implications
Trisha Chattopadhyay, Priyajit Biswal, Anthony Lalruatfela, Bibekanand Mallick *
RNAi and Functional Genomics Lab., Department of Life Science, National Institute of Technology, Rourkela 769008, Odisha, India

A R T I C L E I N F O A B S T R A C T

Keywords: Head and neck squamous cell carcinoma (HNSCC) are among the well-known neoplasms originating in the oral
piRNA cavity, pharynx, and larynx. Despite advancements in chemotherapy, radiotherapy, and surgery, the survival
PIWIL rates of the patients are low, which has posed a major therapeutic challenge. A growing number of non-coding
Gene regulation
RNAs (ncRNAs), for instance, microRNAs, have been identified whose abnormal expression patterns have been
Cancer
implicated in HNSCC. However, more recently, several seminal research has shown that piwi-interacting RNAs
Head and neck cancer
(piRNAs), a promising and young class of small ncRNA, are linked to the emergence and progression of cancer.
They can regulate transposable elements (TE) and gene expression through multiple mechanisms, making them
potentially more powerful regulators than miRNAs. Hence, they can be more promising ncRNAs candidates for
cancer therapeutic intervention. Here, we surveyed the roles and clinical implications of piRNAs and their PIWI
proteins partners in tumorigenesis and associated molecular processes of cancer, with a particular focus on
HNSCC, to offer a new avenue for diagnosis, prognosis, and therapeutic interventions for the malignancy,
improving patient's outcomes.

1. Introduction proteins are not only involved in piRNA biogenesis but also in piRNA-
induced silencing complex (piRISC) and modulate gene expression via
Non-coding RNAs (ncRNAs) are transcripts that belong to the non- chromatin remodelling [9]. The dysregulations of the PIWI proteins are
protein-coding fragment of the genome, which include the constitu­ observed among a variety of diseases, including neuroblastoma (NB),
tional RNAs, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), Alzheimer's disease, ovarian cancer (OC), fibrosarcoma, osteosarcoma
and ncRNAs with regulatory functions like microRNAs (miRNAs), piwi- (OS), and many more, implying they play roles in their pathogenesis
interacting RNAs (piRNAs), small interfering RNAs (siRNAs), and long [10–14]. The deregulated PIWI proteins also affect cell proliferation,
ncRNAs (lncRNAs), etc. The regulatory ncRNAs were initially known as apoptosis, stemness, maintenance of genomic integrity, invasion, and
transcriptional noise of genome [1–4], but not anymore. In the recent metastasis [15] in cancers.
past, new types of ncRNAs, namely circular RNAs (circRNAs) [5], In humans, more than 30,000 piRNAs have been reported [8]. piR­
promoter-associated RNAs (PARs) [1], enhancer RNAs (eRNAs) [1], and NAs modulate gene silencing pathways transcriptionally, post-
many more, have also been discovered. Among these ncRNAs, miRNAs transcriptionally, and epigenetically by binding to the PIWI protein
are well documented in fundamental biological phenomena. They con­ and forming the piRISC [8]. Studies have demonstrated that an imbal­
trol these processes by targeting mRNA transcripts at their 3'UTR, ance in the piRNA regulatory processes alters several layers of gene
5'UTRs, or even the coding region [6,7]. regulation that control DNA damage repair, chromatin organization,
Another young class of small ncRNAs is piRNAs, which are gaining and mitogenic signal response, among the others, propelling the cells
attention in the scientific community due to their recent role in somatic towards uncontrolled growth [13,14]. The expression aberration among
cells and their germline functions [8]. Numerous reports have suggested the piRNAs is reported in several cancers, such as those arising from
that the biogenesis of these piRNAs via PIWI proteins is linked to the gastric (GC), bladder (BLCA), breast (BC), colorectal (CRC), lung (LC),
presence of electron-dense cytoplasmic granules, which form a peri­ and Liver cancer [8,16].
nuclear organelle known as “Nuage” in eukaryotic germ cells. The PIWI Among the major cancer types, Head and Neck Squamous cell

* Corresponding author.
E-mail addresses: [email protected], [email protected] (B. Mallick).

https://doi.org/10.1016/j.bbcan.2022.188772
Received 12 June 2022; Received in revised form 29 July 2022; Accepted 29 July 2022
Available online 2 August 2022
0304-419X/© 2022 Elsevier B.V. All rights reserved.
T. Chattopadhyay et al. BBA - Reviews on Cancer 1877 (2022) 188772

Carcinomas (HNSCC), originating from the oral cavity, pharynx, and continuously being unfolded in cancer genomics [27–29]. The ncRNAs
larynx, ranked 6th worldwide [17–19]. HNSCC has an incidence rate of can solely be used or in combination with other conventional thera­
~8.7% and a death rate of 0.5 to 1.9 % [19]. Among all the HNSCC peutic strategies for effective treatment with minimal or no side effects.
subtypes, lip and oral cavity cancer account for the highest incidence Also, this RNAi therapy could function with no tumor relapse and side
and death rate [20]. The major etiological factors responsible for effects, making it more effective than other approaches.
developing HNSCC malignancies are smoking tobacco, intake of alcohol, Our review focuses on the role of piRNA and PIWILs in HNSCC,
and viral infection, especially due to the human papillomavirus (HPV) mainly focusing on their therapeutic potential. We have also talked
[21]. There has also been immense research in cancer treatment, about how they have progressed from molecular markers to key regu­
including surgical resection, radiation therapy, and chemotherapy lators in other cancers in recent times.
(Fig. 1) [22].
Although surgery is usually the preferred treatment for advanced- 2. Discovery of PIWI and piRNA
stage tumor, it has several drawbacks. Its success mainly depends on
the cancer stage and the patient’s health [23]. After surgery, radiation piRNAs are small single-stranded ncRNAs derived from specific
therapy is an effective treatment method, but atherosclerotic vessel genomic loci termed ‘piRNA clusters’, especially among various trans­
damage, functional mucositis, increased weight loss, and electrolyte or posable elements (TEs). The piwi gene was first discovered in 1997 in the
fluid loss are some of the significant side effects [24]. Aside from surgery terminal filament of the Drosophila ovary (Fig. 2), a somatic cell located
and radiation therapy, chemotherapy is another treatment modality for just next to germline stem cells (GSCs), which is required in their divi­
HNSCC. Cisplatin, 5-fluorouracil, docetaxel, methotrexate, and cetux­ sion. The name “P-element induced wimpy testis” (piwi) was given due
imab are the current FDA-approved chemotherapeutic drugs for HNSCC to P-element insertional mutations causing a severe defect in sper­
treatment, with cisplatin being the most used drug [25]. Unfortunately, matogenesis and male sterility [30]. Although, in the beginning, piwi
most HNSCC cancer types develop resistance against various chemo­ was primarily found in the germ cells of organisms [31–34], their
therapeutic drugs, resulting in poor treatment outcomes and cancer expression was also recorded in somatic cells of Drosophila [30]. Due to
progression [26]. their role in GSC maintenance [35], piwi drew the attention of re­
Hence, it is evident that a therapy that can mitigate the drawbacks of searchers long before its homologs in other species such as prg-1 and prg-
current therapies is urgently needed, and ncRNA-based therapy could be 2 (C. elegans) [30,36,37] and miwi and mili (mouse) [36] were found. In
a promising option in this regard. Because of its minimal or no side ef­ humans, HIWI (PIWIL1), HILI (PIWIL2), HIWI3 (PIWIL3), and HIWI2
fects and increased specificity in its course of action, ncRNA-based (PIWIL4) are the piwi paralogues found [33]. PIWI is an Argonaute
therapy is now emerging as an effective therapeutic option for treating protein, which is highly conserved among eukaryotes [38,39]. The
cancer. Among the ncRNAs, lncRNAs, miRNAs, and circRNAs have been family contains a PAZ domain with three subdomains- Piwi/Argonaute/
reported and studied in HNSCC, which may aid in developing RNAi Zwille (PAZ) [40], which is one of the binding pockets for several small
therapy for HNSCC treatment in addition to piRNAs that are RNAs [41–44]. Even before PIWI was fully established, Argonaute

Fig. 1. Various treatment strategies of HNSCC.

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Fig. 2. Timeline of discovery of piwil and piRNA.

family genes such as rde-1 in C. elegans, Argonaute1 (Ago1) in Arabi­ 3. Biogenesis of piRNA
dopsis, and quelling-defective2 (Qde2) in Neurospora were implicated in
post-transcriptional gene silencing (PTGS) [41–44]. Unsurprisingly, the Both piRNAs and PIWIL proteins have tissue and time-specific
RNAi role of PIWI came to light when anti-prg-1 injection in the germline expression in organisms as diverse as scorpions, honeybees, and mice
of C. elegans abolished the activity of the zygote and the maternal genes [62,63] [64]. The biogenesis of piRNAs involves several proteins, among
[45–47]. Later, a similar mechanism was also seen for other related which PIWI proteins are the leading players. PIWI is also involved in
protein families, namely rde1, rde2, rde3, and rde4 [36]. small RNA-mediated gene silencing by binding with piRNAs. The core of
Due to their ability to maintain stemness [30,36,37,48], PIWI pro­ this RNA silencing world lives within the PIWI proteins, which comprise
teins were thought to be cardinal in diseases like cancer, which also three prime domains: N-terminal positioned PAZ domain that binds to
display self-renewal. In 2005, Lee, Jae Ho et al. proved its oncogenic role the 3' end of guide RNA, MID domain that binds to the 5' end of guide
by demonstrating that Piwil2 was upregulated in testicular seminomas RNA, and the C-terminal positioned Piwi domain that has innate RNAse
and promoted cell proliferation by activating the expression of Bcl—XL, H activity [65] (Fig. 3A). The PAZ domain contains Tyr and His, aro­
an anti-apoptotic gene, its upstream regulators Stat3 and Akt and Cyclin matic residues forming the RNA binding pockets and are possibly
D1. Subsequently, this study also revealed the expression of the Piwil2 responsible for the 2'-O-methylated 3' end of piRNAs [66].
not only in tumors of germline cell origin but also in cancer of the GI
tract, prostate, ovary, and endometrium in humans, opening a whole 3.1. Origin of precursor piRNAs (pre-piRNAs)
new avenue for PIWI research in cancer [49].
A few years after PIWI's discovery, a report of piRNA was found for The biogenesis of piRNAs involves two phases. The first half of the
the first time in 2002 in Tetrahymena (Fig. 2), where Tetrahymena PAZ- piRNA biogenesis is the initiation phase, and the last half is the effector
Piwi domain protein (TWI1P), a PIWI homolog, was found associated phase or piRNA-mediated silencing phase [67]. Before the initiation
with small RNA of ~26-31 nucleotides (nts), which was required to form phase, the RNA pol II transcribes the piRNA gene into a long single-
transcriptionally active macronucleus by eliminating DNA [34]. Later, stranded [68] unspliced precursor transcript, termed ‘pre-piRNA’ in a
several groups solidified this concept in 2006 and 2007, establishing promoter-independent manner from the cluster harbouring highly re­
previously uncharacterized 30 nts long small RNAs associated with a petitive regions of the genome, including diverse TEs [69]. Among fruit
PIWI homolog of Drosophila, zebrafish, mice, rats, and even humans. flies, the germ cell's piRNAs are marked epigenetically by RHINO, which
Such RNAs were piRNAs found to be single-stranded, roughly 26-31 nts is the heterochromatin-bound factor [70,71] and forms the RDC com­
long, with a strong U bias at the 1st position. These RNAs were later plex (RHINO, DEADLOCK, and CUFF). This complex attaches to the
discovered to have a unique 2'O methylation at their 3' site, which chromatin of the piRNA cluster (dual strand), which encodes pre-piRNA
provided stability. Several studies have reported their role in gene from both genomic strands, followed by its transcription through RNA
regulation in germline and somatic cells, including cancers [50–61]. The pol II [72]. Furthermore, CUFF interferes with the association of the
discovery of piRNA and its RNAi-mediated regulatory components as a cleavage and polyadenylation specificity factor (CPSF) complex, thereby
part of the maintenance of GSCs has now expanded beyond and has preventing premature stoppage of RNA pol II activity and protecting the
become a promising agent in modulating cellular networks in cancer and processed transcripts from degradation by the RAT1, an exonuclease
other somatic events. Moreover, the maintenance of GSCs could indicate [73]. Although the pre-piRNAs are transcribed by RNA pol II, there is the
its possible role in fetal development. These enlightened and encouraged absence of splicing signatures and poly (A) tails. Hence, their export to
scientists to learn more about piRNA biogenesis in the hopes of the cytoplasm requires BOOTLEGGER and NXF3. Moreover, the
unlocking the key to its functionality, which could be used to treat BOOTLEGGER is contracted to the piRNA cluster, which then brings
human diseases. Hence, a thorough understanding of their biogenesis is NXF3, which specifically binds the precursors, allowing them to be
essential. exported to the biogenesis site [72] (Fig. 3B).

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Fig. 3. Biogenesis of pre-piRNA and the proteins involved in the process. A) PIWI protein showing its domains and bound piRNA being processed. B) Generation of
pre-piRNAs from chromatin. C) Nuage and its components associated with the biogenetic process.

3.2. Nuage - the house of piRNA biogenesis works in the 3'-5' direction generating the 3' ends of the pre-piRNAs,
which is recognized by the PAZ domain [85]. The PAZ domain recog­
piRNA biogenesis differs from that of siRNA and miRNA in their nizes the 2-nts overhang at the 3' ends, and the Piwi domain cleaves the
independency on RNase III endonuclease, i.e., DICER, and is similar to target piRNA by forming a PIWI-piRNA complex [86]. The piRNA-
siRNA concerning the presence of 2'-O-methylation [74]. The produc­ binding pocket of the PIWI protein determines the final length of a
tion and loading of piRNAs occur in the cytoplasm localized in a piRNA; for instance, PIWIL1-associated piRNAs are about 30 nts in size,
membrane-less region called the Nuage [54,75–77], which contains and PIWIL2-associated piRNAs are 26 nts long [86]. This defined the
many conserved components such as Vasa (VAS) [78], Spindle E (SPN-E) length of a piRNA, which is found within a range of 24 to 32 nts [62]. A
[79] and Maelstrom (MAEL) [67,80] (Fig. 3C). Nuage, otherwise known study on mice showed that MILI endonuclease generates two fragments,
as Yb bodies, chromatoid bodies, pi-bodies, or piP-bodies, is based on one of which is the pre-piRNA intermediate that generates phased
the type of species and cells. They recognize and process pre-piRNAs piRNA, and the other is released from the complex and eventually de­
exported to the cytoplasm from the nucleus [67]. In addition to being grades [82]. Finally, methyltransferase HEN1 adds a characteristic 2'
the center for piRNA biogenesis and piRISC formation [80], VAS and methyl modification to the 3' ends of piRNAs, resulting in their stabili­
SPN-E are required for the nuclear localization of ZFRP8, which is zation [87].
involved with the biogenesis of primary piRNAs, playing a role similar to Besides the nucleus, mitochondria are also involved in piRNA
that of PIWI and MAEL [81]. Moreover, Nuage not only serves as the site biogenesis. In Drosophila, after primary piRNA precursors are shuttled to
to produce primary piRNAs (explained in the next section), it may also the nuage, an unknown nuclease processes them into piRNA in­
be indirectly responsible for initiating secondary piRNA biogenesis. termediates, which then bind piwi to form a piwi-piRNA intermediate
complex [88]. This is followed by translocation of the complex to the
3.3. Primary biogenesis of piRNAs mitochondrial surface by ARMI, which helps unwind the piRNA in­
termediates [89]. Finally, ZUC, also known as MitoPLD, a highly
The initiation phase of biogenesis begins by loading the 5' mono­ conserved mitochondria-anchored endonuclease found on the outer
phosphate pre-piRNA transcript to the PIWI protein. The presence of the membrane of mitochondria [90], cuts the intermediate precursor com­
monophosphate at the 5' is required to load almost all argonaute pro­ plex at each 25th or 40th nt, generating short 5' and 3' ends of piRNAs
teins [81]. The loading also requires additional proteins, such as [91] (Fig. 4). The final ZUC processing is mediated by two mitochondrial
Armitage (ARMI), co-chaperone Shutdown (SHU), Yb - an RNA helicase proteins, GASZ and DAEDALUS [92], which act as scaffolds [88].
with Tudor domain, specific for the somatic cells [63,82], and Zucchini
(ZUC) [83], where ZUC generates most of the Aubergine (AUB)-bound 3.4. Secondary biogenesis of piRNAs
piRNAs [82,84]. At first, the pre-piRNAs get loaded on the PIWI, having
a steric preference for 5' terminal 1U. Then, they are trimmed at their 3′ The effector phase of biogenesis begins by loading AUB, a PIWI
ends by an exonuclease named TRIMMER and its cofactor PAPI that protein, to primary piRNA sequences (antisense to TE). This will guide

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Fig. 4. Primary biogenesis of piRNA and role of PIWI in the process.

Fig. 5. Secondary biogenesis of piRNA and role of PIWI in the process. Light purple represents antisense, and orange represents sense strand.

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the AUB to search for complementary TE mRNA transcripts within the Further, how similar the biogenesis process would be in humans? Apart
cytoplasm. When an AUB-bound piRNA recognizes a transcript with from PIWILs, what else might be playing the role? Where could this
sequence complementarity, AUB cuts the target transcript 10 nts from process be taking place in the case of somatic cells? Remain unexplored.
the piRNA's 5' end, producing the 5' ends of the recently formed pre-
piRNA, which gets incorporated into Argonaute3 (AGO3) that con­ 4. Types of gene regulation mediated by piRNA and PIWI
tains adenosine in position 10 (10A) for further processing and forms
secondary piRNA [65]. As the AGO3-bound piRNAs are sense (5'-3') to The piRNAs and PIWI regulate gene expression transcriptionally,
the TEs, these further enhance the production of the piRNAs derived post-transcriptionally, and epigenetically to maintain genome stability
from the cluster, recognizing the transcripts complementary to the and regulate other biological processes. These mechanisms are discussed
cluster, producing piRNAs antisense (3'-5') in nature. This interplay in the next section.
linking the AUB-bound piRNAs and those of AGO3-bound is widely
termed the ‘Ping-pong cycle’ [82] (Fig. 5). This cycle occurs at different
4.1. Transcriptional regulation
sub-compartments within nuage, to which AGO3 gets recruited by
directly interacting with Krimper (KRIMP). KRIMP, the stable Nuage
There are reports of PIWI unable to show any slicer activity but could
component, coordinates the assembly of another complex, known as the
silence TEs co-transcriptionally within the nucleus [57,95–97]. Thus,
ping-pong piRNA processing (4P) complex through AGO3 and AUB
the silencing process mediated by the PIWI in the nucleus is independent
(methylated). The binding of KRIMP with AGO3 is stable, unlike binding
of the Piwi’s slicer activity. This silencing is mediated by Piwi/MAEL
with AUB, which is transient [93]. Another regulator, EXD1 [94], sup­
predominantly at the transcriptional level by blocking the recruitment of
presses TEs in mammalian male germlines by enhancing the secondary
Pol II (Fig. 6A). Thus, transcriptional gene silencing (TGS) appears to be
piRNA biogenesis pathway in collaboration with its partner, TDRD12
the primary mechanism by which PIWI suppresses TEs. However, an
[89].
additional layer of PTGS cannot be ruled out [95]. In addition, the
It is evident from the biogenesis that the specificity of piRNA-
cluster-derived piRNAs can act as antisense oligonucleotides for TE
mediated gene modulation is primarily determined by the bound PIWI
transcripts. Despite having no enzymatic function other than binding to
proteins and sequence complementarity with the target mRNA. More
their targets, piRNAs assemble with PIWI proteins to form piRISCs and
research is needed to fully comprehend the downstream processing of
repress TEs co-transcriptionally by causing heterochromatinization of
piRNA maturation and regulation of cellular function in humans.
the target loci (Fig. 6A) [98]. piRNA guides PIWI proteins like MIWI, and

Fig. 6. Different modes of gene regulations are mediated by piRNA and PIWI. A) TGS mediated by PIWI/piRNA by blocking RNA pol II or heterochromatin the target.
B) PTGS mediated by PIWI-piRNA via cleavage and blockage of PolyA tail, inhibiting translation. C) Gene silencing by epigenetic regulation through histone
methylation and methylation of the promoter regions containing CpG islands and inducing heterochromatinization.

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AGO3, which act as an RNase, cleaving and repressing their cognate TE 5. Aberrant expression of piRNA and PIWIL in cancer
transcripts [99,100]. Moreover, the piRNAs and PIWI are found to
interfere with gene regulation at the post-transcriptional level, as Several studies have illustrated that piRNAs and PIWILs show a
described here. remarkably varied expression level among different cell types, ranging
from pluripotent stem cells to highly differentiated somatic and cancer
cells [115].
4.2. Post-transcriptional regulation

PIWI-bound piRNAs cleave the TE transcripts post-transcriptionally 5.1. Cancer-specific expressional aberration of PIWILs
by slicer activity [99]. The cleaved products are either degraded or
serve as the ping-pong mechanism's precursor for another set of piRNAs Abnormal expression of PIWILs is reported in several cancers
[52,54], as described previously. Besides the endonuclease activity of [12–14,116–120] listed in Table 1. Interestingly, PIWI is found to act as
PIWI, the copy number of piRNAs also seems to be involved in regulating biomarkers and has also been linked with the hallmarks of cancer, [119]
target transcripts [101]. PTGS also involves the degradation of target including cancer proliferation, apoptosis, and their ability to invade and
mRNA transcript (Fig. 6B). For instance, piR-30840 in human T-lym­ metastasize [15].
phocytes interacts with TRAMP complex and nuclear exosomes, pro­
moting the decay of the target mRNAs [102,103]. Because mRNA 5.2. Cancer-specific dysregulated expression of piRNAs
polyadenylation is essential for maintaining its stability and translation,
any factor that interferes with Poly-A can skew mRNA expression and Several piRNAs and their ectopic expression have been well docu­
protein synthesis (Fig. 6B). piRISC containing MIWI and CAF1 is found mented in cancerous tissues from various anatomic positions, such as
to induce mRNA deadenylation in mouse elongating spermatids, breast, bladder, kidney, colon, head, neck, ovaries, lung, prostate, thy­
impacting PTGS [104]. AUB and AGO3 in Drosophila, with their asso­ roid, stomach, uterus and gastric mucosa, etc. [121–126].
ciated piRNAs, interact with SMAUG to assemble the CCR4 complex
onto specific mRNA, resulting in their degradation through dead­ Table 1
enylation of the mRNA [105,106]. Aberrant expression of PIWIs and their role in cancer.
Sl. PIWIL Cancer type Action on cancer Reference
No type
4.3. Epigenetic regulation
1. PIWIL1 CRC Increases lymph node [222]
This is also the key mechanism through which PIWI family proteins metastasis and invasion
eOC Represses invasion [223]
control gene expression. They modulate the activity of DNA methyl­ Renal cell carcinoma Enhances tumor growth [224]
transferases (DNMTs), which trigger modifications at the chromatin or (RCC)
the histone proteins. The PIWI-piRNA complexes are known to meth­ GC Associated with cell [140]
ylate CpG islands of the target genes and the TEs by directing DNMTs proliferation
Myeloid leukemia Inhibits cell growth and [223]
[107] (Fig. 6C). Further, it has been seen that PIWILs and piRNA can
(LAML) causes programmed cell
regulate the DNMTs in type I endometrial cancer, where PIWIL1 en­ death
hances the expression of DNMT1 [108], and piR-823 does the same for BLCA Increases apoptosis by [179]
DNMT1, DNMT3A, and DNMT3B in BC [109]. The PIWI-piRNA com­ binding with with piRABC
plexes also recruit histone methyltransferases (HMTs), Suv39H1, and and upregulating Tnfsf4
Sarcomas (SARC) Drives tumor growth and [225]
SETDB1 that control the degree of methylation of H3K and H4K, colony formation by
affecting gene regulation (Fig. 6C) [110]. Disruption of nuclear locali­ modulating tumor
zation of PIWI results in activation of the TE, which is indicated by an suppressor genes
enhancement of histone marks responsible for activation (H3K79me2 LC Promotes proliferation by [116]
inhibiting apoptosis
and H3K4me2) and a decline in histone marks responsible for repression
Endometrial Enhances lymph node [226]
(H3K9me2/3) of several TEs [111,112]. Piwi knockdown also reduces adenocarcinoma metastasis through PSCA
H3K9me3 on actively transcribed TE, which could have recruited HP1 (EAC) and TBX2
and mediated the establishment of heterochromatin, repressing the GBM Promotes cell [227]
transcriptional process [95]. Experiments on Drosophila also showed proliferation, invasion,
and migration
that PIWI and HP1a could regulate the silencing of TEs and various 2. PIWIL2 BC Enhances metastasis by [164]
genes specific to a particular tissue [113]. Association of piRNA with methylating latexin.
DNMTs was also found, where it modulates the activity of cell cycle NSCLC Promotes cell proliferation [228]
regulator (Cdkn2b) by changing the DNA methylation and consecutively by regulating G2/M stage
through CDK2 and cyclin
histone (H3) activity along the promoter region [106]. Furthermore, an
A
intriguing study discovered that piRNA-guided epigenetic changes play BLCA Act as a tumor suppressor [229]
a role in cardiomyocyte apoptosis, where HAAPIR, a heart-apoptosis- CRC Function as an oncogene [161]
associated piRNA, binds to NAT10, an RNA acetyltransferase, and im­ by promoting metastasis
proves the stability and translation efficacy of Tfec (transcription factor 3. PIWIL3 GBM Inhibits tumor growth, [118]
migration and invasion
EC) mRNA transcript by promoting ac4C acetylation. Tfec then activates Malignant Enhances progression and [230]
downstream proapoptotic gene BCL2-interacting killer (Bik), which Melanoma metastasis
triggers the apoptotic process [114]. GC Promotes migration, [170]
As the key players of the RNAi pathway, PIWILs and piRNAs are proliferation and invasion
4. PIWIL4 BC Promotes the survivability [231]
known for maintaining genome stability besides regulating genes. They
of cancer cells, and their
do this either by binding to specific TEs and genes or modulating them migration
epigenetically. However, unlike miRNAs, their mode of action, such as RB Promotes progression of [141]
their targeting pattern, is not conserved. Moreover, as each step of gene RB by controlling the cell
regulation can be modulated by PIWI and piRNA, they embody a robust cycle through OTX2, p16,
PCNA
and effective tool that can be utilized to develop disease therapeutics.

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A study on non-small cell lung carcinoma (NSCLC) stated that piRNA pathogenesis. This evidence (refer to Table 2.) speculated the possible
in the intron could silence lamc2 in a concentration dependant way bridge connecting the dysregulated expression of our small RNA of in­
[127]. Among the several reasons behind the dysregulation of any terest, its effector protein, and the potential modulation that might be
piRNAs expression in any disease is single nucleotide polymorphism taking place in cancers because of their aberrant expressions. Further­
(SNP), which can sometimes affect the genomic origin [128] or function more, scientists are working hard to learn extensively regarding the role
of the piRNA. One such example is reported in CRC, where the SNP of these piRNAs and PIWILs in the cancer machinery, as well as how
located within the piRNA revealed a change in its functionality. An SNP, their involvement could be used to benefit humanity.
rs11776042, located within piR-015551 (thymine to cytosine; T > C)
could modify the secondary structure of the piRNA, thus influencing its 6. Mechanistic aspects of piRNA and PIWILs in oncogenetic
effect on the development of CRC [129]. events
Dysfunction of piRNAs and PIWILs and other factors such as genetic
polymorphisms like SNPs in piRNAs can disturb the expression of It is now known that PIWILs and piRNAs are linked with most of the
oncogenic or tumor suppressor target genes, leading to cancer cancer features, like the proliferation of the cancerous cells [130],

Table 2
Aberrant expression of piRNAs and their roles in cancer.
Sl. Cancer type Differentially expressed Expression Action on cancer References
No piRNA Level

1. BC piR-4987 ↑ Associated with lymph node positivity and serve as tumor marker [174]
piR-20365, piR-20485, ↑ [174]
piR-20582
piR-021285 ↓ Suppresses proliferation and invasion by epigenetic modification of ARHGAP11A [232]
piR-36712 ↓ Suppresses cell proliferation, invasiveness, and ability to migrate [233]
Shows negative correlation with axillary lymph node metastasis [233]
piR-36743 ↓ Inhibits growth post-transcriptionally [129]
piR-932 ↑ Promotes epithelial-mesenchymal transition [164]
piR-008114 ↓ [234] [235]
pi-sno75 ↓ Induces apoptosis by activating trail epigenetically [236]
piR-36984 ↑ [235]
2. GC piR-823 ↓ Inhibits cell growth [237]
piR-651 ↑ Promotes cancer development [144]
piR-55891 ↑ [238]
3. RCC piR-32051, piR-39894, ↑ Enhances metastasis [239]
piR-43607
4. ccRCC piR-57125 ↓ Suppresses metastasis [127]
piR-30924, piR-38756 ↑ Promotes metastasis [127]
piR-30924 ↑ Enhances metastasis and cell proliferation [173]
5. HCC piR-Hep1 ↑ Promotes cancer cell survival, ability to migrate and invasiveness [127]
Liver Cancer piR_LLi_24894 ↑ [129]
6. PDAC piR-017061 ↓ [240]
7. MM piR-823 ↑ Promotes tumorigenicity through cell cycle regulators and apoptosis-related [143]
proteins; Facilitates tumorigenicity by enhancing angiogenesis and contributes in
poor prognosis
8. LC piR-55490 ↓ Causes gain in the proliferation rate by suppressing the activation Akt/mTOR [241]
pathway
piR-34871, piR-52200 ↑ Promotes EMT and enhances cell proliferation by blocking cell cycle arrest [129]
piR-35127, piR-46545 ↓
piR-L-163 ↓ Accelerates DNA synthesis and accumulation of cells in G2-M phase [233,234]
piR-L-138 ↑ Promotes chemoresistivity [242]
9. NSCLC piR-L-163 ↓ [241]
piR-651 ↑ Promotes tumor formation through upregulation of cyclin d1 and ckd4 [117]
10. BLCA piR-60152 ↓ Overexpression inhibits cell proliferation, and colony formation, and promoted [241]
cell death
piR-60152 ↓ Inhibits cell proliferation and enhances apoptosis [173] [179]
11. GBM piR-30188 ↓ Suppresses proliferation, invasion, and induces apoptosis [118,129]
piR-8041 ↓ Reduces cell proliferation through apoptosis and cell cycle arrest. [121]
piR-33221 ↓ Increases blood-tumor barrier (BTB) permeability for glioma cells [129]
piR-57139 ↓ Promotes the translocation of chemotherapeutics into glioma tissues [129]
12. Glioma piR-36041, piR-49820 ↑ Negatively correlated with disease prognosis [243]
piR-598 Promotes cell proliferation [241]
13. CRC piR‑1245 ↑ Tumor progression, advanced T stage, distant metastasis and poor survival [176]
piR-823 ↑ Enhance cell proliferation, invasion and inhibit apoptosis [138] [244]
piR-54265 ↑ Enhance proliferation, metastasis, inhibits apoptosis and indicate poor survival; [129,245,246]
Acts as biomarker
piR-18849, piR-19521 ↑ Associated with poorer degree of differentiation and lymph node metastasis [175]
piR-020619, piR-020450 ↑ Contributes to cancer initiation [247]
14. Fibrosarcoma piR-39980 ↓ Enhance apoptosis and suppress proliferation [13]
15. Colon cancer piR-28876, piR-23210, ↓ [125]
piR-32159, piR-5937,
16. PCa piR-23209 ↓ [125]
piR-19166 ↓ Suppress migration and metastasis [177]
piR-001773, piR-017184 ↑ Promotes growth of tumor [248]
piR-31470 ↑ Promote proliferation, oxidative stress and DNA damage [249]
17. Papillary Thyroid piR-35413 ↓ Involved in the development and progression [234]
Cancer (PTC) piR-13643, piR-21238 ↑ Distinguishes malignant thyroid nodules from benign, as a biomarker [250]

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T. Chattopadhyay et al. BBA - Reviews on Cancer 1877 (2022) 188772

Fig. 7. Oncologic events reported being regulated by different piRNAs and PIWILs.

escape from cell death [130], the metastatic ability of tumors [131], its genes could be a source of genic piRNAs, indicating their contribution to
ability to invade [131], and its ability to show cellular senescence, etc. the turnover of histone mRNA during the cell cycle [147]. A study in LC
[130–133]. We have discussed these aspects (Fig. 7) in the next section. also revealed that the axis of RASSF1C-PIWIL1-piRNA could promote
epithelial-mesenchymal transition (EMT) by increasing epidermal
6.1. Cell cycle growth factor receptor (EGFR) signalling and inhibiting the cell cycle
arrest, thereby increasing cell proliferation [129,148].
The piRNA can maintain the equilibrium between self-renewal and
cell division, and any disruption in this equilibrium could significantly 6.2. Programmed cell death (PCD)
impact cellular physiology leading to tumorigenesis [134]. This was
evident in Drosophila, where the PIWI-piRNA pathway was reported to Also known as apoptosis, PCD is the terminal pathway of cellular
be involved in regeneration, ageing and allowing cells to proliferate physiology and is one of the significant events targeted by cancer cells.
indefinitely [135]. In humans, PIWIL1 marked the first report showing Although a large portion of its relevance to cancer is now known, the
its role in self-renewal of stem cells. Its overexpression caused malig­ hidden role of piRNAs and PIWILS remains sparsely known and is being
nancies among the germ cells [136], and its inhibition arrested cells in investigated. There is evidence that PIWIL2 inhibits apoptosis by over­
the G2/M stage in BC by regulating CDK4, CDK6, CDK8, TβRI, and TβRII expressing and inducing up-regulated expression of antiapoptotic genes
positively [137]. Similarly, PIWIL2 contributed to tumorigenesis of such as Bcl-XL via the STAT3/BCL- XL and STAT3/CYCLIN D1 signalling
testicular seminomas through the STAT3/BCL-XL/CYCLIN signalling pathways [49]. Furthermore, silencing of piwil2 induced apoptosis in
pathway [138] and of HeLa cells by regulating G1 phase progression transformed testicular germ cell lines by suppressing stat3 [49]. PIWIL2
through cyclin-dependent kinase inhibitor (CDKI) expression as a result can also repress the transcription of p53 by preventing the p38-induced
of p53 suppression [139]. In addition to this, silencing of piwil4 in phosphorylation of p53 [149] through the assembly of the PIWIL2/
Retinoblastoma (RB) and piwil1 in GC led to cellular senescence by STAT3/c-Src triple protein-protein complex [139]. Further, along with
arresting the cells at the G2-M phase [140] [141]. Further, in glioma, the pathway mentioned above, PIWIL2 inhibits apoptosis by regulating
piwil1 silencing caused G0-G1 phase arrest and dysregulated expression the TGFβ pathway in HEK293 cells and has a role in Fas-mediated
of cell cycle-related proteins like Cyclin D1[142]. The above studies apoptosis and p53 phosphorylation [149]. In addition, piwil2 over­
revealed the role of PIWIL in regulating the cell cycle of tumorigenic expression inhibits cell apoptosis by acting on STAT3 in BC and colon
cells. cancer [150]. In chronic myeloid leukaemia (CML), the ectopic
piRNAs have also been shown to regulate the cell cycle process. An expression of piwil1 was found to suppress growth and induce apoptosis
antagomir of piRNA-823 was found to regulate the cell cycle by inhib­ [151].
iting cyclin D1, CDK4 and decreasing the level of phosphorylated Rb Furthermore, there have been studies where a piRNA has been
(pRb) in CRC and multiple myeloma (MM) [138,143]. Also, piR-651 discovered to be oncogenic or tumor suppressive. A survey in CRC
inhibition could block G2/M phase cells of GC [144], and its upregula­ showed piR-823 to reduce apoptosis [138]. piR-39980 induces apoptosis
tion in NSCLC decreased G0/G1 phase cells and elevated those from the in fibrosarcoma by inducing DNA fragmentation and condensed nuclei
G2/M phase regulating cell proliferation by cyclin D1 and CDK4 [117]. formation by regulating RRM2 [13]. The same piRNA inhibited
Another piRNA, piR-8041, has been shown to arrest cells at the G1/S apoptosis in osteosarcoma (OS), accumulating γ-H2AX in the nuclei
checkpoint and induce apoptosis, possibly by downregulating [14]. Additionally, piR-L-138 was found to protect the LSCC cells from
extracellular-signal-regulated kinase1/2 (ERK1/2) mitogen-activated CDDP-induced apoptosis, resulting from the interaction between piR-L-
protein kinase (MAPK) signalling [145], MAP3K7 [146], which en­ 138 and p60-MDM2 [152].
codes for transforming growth factor (TGF)—activated kinase in Glio­
blastoma (GBM) [121]. An exclusive study on core histone proteins
(H2A, H2B, H3, H4) revealed that all replication dependant histone

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T. Chattopadhyay et al. BBA - Reviews on Cancer 1877 (2022) 188772

6.3. Invasion and metastasis 6.4. Angiogenesis

The lethality of cancer is represented by its ability to relocate its This is another physiological phenomenon engrossed with carcino­
position by either invading or metastasizing all over the body genesis, which recreates a survival-friendly environment for cancer
[142,153,154]. Several studies have also implicated PIWILs and piRNA cells. While evaluating the role of piRNA-823, its antagomir modulates
in cancer invasion and metastasis. For instance, piwil1 was found to have the angiogenic activity in MM by reducing VEGF secretion, which is one
a positive correlation with lymph node metastasis in epithelial ovarian of the major cytokines with pro-angiogenic activity [143,181]. More­
cancer (eOC) [153]. Additionally, its decreased expression led to inhi­ over, in OC, piwil1 expression was positively correlated with
bition of GBM by regulating cellular invasiveness and their migratory angiopoietin-2 (Ang-2) and tyrosine kinase receptor with immunoglob­
ability through mmp-2 and mmp-9 [142]. Another discovery about ulin and EGF homology domain 2 (Tie-2), which are part of vascular
metastasis was its linkage with changes in methylation of DNA. In BC growth factors. [182]. These findings indicate that angiogenesis may
[155], protease urokinase-type plasminogen activator (UPA) was modulate tumorigenesis through the PIWIL -piRNA pathway.
involved in invasion and metastasis, and its promoter hypomethylation In recent years, there has been an explosion of publications on piR­
was linked to poor patient outcomes in PCa [156]. Another study also NAs and PIWILs. Many of these have emphasized their importance in
proposed that MILI, which is engaged in LINE1 methylation [157], could cancer biologies, such as their role in tumor growth, cell cycle pro­
be involved here and used as a marker for predicting tumor node gression, cell death, invasion or metastatic ability, and angiogenesis.
metastasis stage (TNM) and disease prognosis in these cancers [158]. piRNAs can also be used as a biomarker for cancer diagnosis because
PIWIL1 is also found to be negatively related to tumor stage and invasion specific piRNA expressions are linked to various clinical variables of
in cervical squamous cell carcinoma (CESC) [159], while it was posi­ cancer. Further technological advancements have enabled the decryp­
tively associated with TNM stage in GC [154], lymph node metastasis in tion of a wide range of cancer piRNomes. So far, we've discussed the role
hepatocellular cancer (HCC) [160], and metastasis in pancreatic ductal of piRNAs and their critical biogenesis factor PIWIL in cancer; now, we'll
adenocarcinoma (PDAC) by interacting with APC/C [159]. talk about HNSCC and discuss our current understanding of the disease
Piwil2, known to act in the pathophysiology of cancer [161], was and the potential contribution of piRNAs and PIWILs in this cancer.
found to be used as the prognostic biomarker for metastasis and invasion
of colon cancer [162] and PCa [163], metastasis in BC [164], lymph 7. ncRNA crosstalk in head and neck cancer
node metastasis in eOC [153] and muscle invasion in BLCA [165],
migration in GBM cells [166] (through MMP-9, N-cadherin, vimentin, Intriguingly, ncRNAs, including miRNA, circRNA, and lncRNA
and E-cadherin), and invasion in cervical cancer [167]. Another study [183], have been shown to participate in the progression of HNSCC and
stated that PIWIL2 facilitates NME/NM23 nucleoside diphosphate ki­ modulate sensitivity or resistance to various chemotherapeutic drugs.
nase 2 (NME2) (a transcription factor) binding to the G-quadruplex These ncRNAs have been extensively reviewed individually by Weg M.
within the promoter of c-Myc, a proto-oncogene and induces its tran­ et al., Shenglan et al., Carlo et al., Jing et al., and others [184–188].
scription, which further enhances RhoA expression and modulates F- Hence, we shall be discussing here their crosstalks implicated in HNSCC.
actin filaments, affecting tumor cell invasion and migration in HCC These ncRNAs and the regulatory networks of their targets have been
[168]. The binding of piR-Hep1 with PIWIL2 promotes growth and in­ involved in the dysregulated profiles of protein-coding genes associated
vasion in HCC by increasing the level of akt in its phosphorylated form with oncogenic events of various cancer types, including HNSCC. Such
through the PI3K/AKT signalling [169]. Further, PIWIL3 has been found RNA-RNA interactions modulate the post-transcriptional regulation of
to regulate the level of mta1, mmp-9, mmp-2, and RhoC, which promotes genes by communicating or co-regulating each other. This network is
invasion in GC [170]. Also, reports show PIWIL4 to promote invasion in named competing for endogenous RNA (ceRNA), and it has been linked
HeLa and C33A cells [171]. to various biological events. This machinery is influenced by multiple
These studies prompted the involvement of piRNAs in cancer factors, such as the abundance and subcellular localization of the
biology. In clear cell renal cell carcinoma (ccRCC), a decrease in interacting ceRNA components, binding affinity of ncRNAs to their
expression of piR-57125 promoted the degree of metastasis [172]. In sponges, etc. Its imbalance must have been playing an essential role in
contrast, two other piRNAs, piR-30924 and piR-38756, showed an the initiation and progression of cancers such as HNSCC [189].
elevated expression in ccRCC samples in primary and distant metastasis Among ceRNAs, circRNAs are a novel subtype of ncRNAs, which has
compared to the nonmetastatic ones. This revealed different piRNAs a crucial role in cancer progression by sponging miRNAs. Several studies
behave differently in the same cancer [173]. In BC, the enhanced piR- on the circRNA have shown that they are differentially expressed in
4987 positively correlates with metastasis to the lymph nodes, and a various types of HNSCC. Their crosstalk as ceRNA (circRNA/miRNA/
similar observation was noted for another piRNA [174], piR-18849, mRNA) regulates the expression of cancer-specific genes and cancer-
among the CRC patients [175]. In addition, piR-823, piR-1245 [176] specific phenomena, namely proliferation of the cancer cells, cell
and piR-54265 [177,178] showed a positive correlation with their death, migration, and their ability to invade [190–195]. Similarly,
expression and CRC staging, indicating that they promote metastasis and lncRNA/miRNA/mRNA and pseudogene/miRNA/mRNA are also re­
invasion [178]. Similarly, through an in vivo study, piR-19166 showed a ported as ceRNA involved in the oncogenic transformation of HNSCC
negative correlation with metastasis in PCa through suppression of cttn, [196–200] [27]. For instance, a lncRNA zfas1 modulates cancer pro­
mt1-mmp, mmp2, and mmp9 [177]. Furthermore, piR-hep1 [179] is gression and invasion through the downregulation of miR-150-5p,
positively associated with invasiveness and the metastasizing ability of which mainly works reversely by upregulating eif4e target mRNA
HCC [169]. piR-004918 and piR-019308 were also associated with [201]. Similarly, hsa_circ_0042666/tgfbr3/miR-2238 and Cdr1as/miR-
metastasis in GC [180], and piR-823, in addition to its role in CRC, was 7/ccne1/pik3cd are two examples of circRNA-mediated regulatory
identified to regulate CSCs in luminal BC [109]. Also, piR-Hep could mechanisms found involved in laryngeal squamous cell cancer devel­
induce the growth and invasiveness of HCC by binding with PIWIL2 and opment [202–204]. Pseudogenes, also called “genomic fossils”, are
affecting PI3K/AKT signalling [169]. Similarly, another piRNA, piRABC, another subtype of ncRNA contributing to cancer development through
influenced cancer development in BLCA through binding with PIWIL1 the ceRNA mechanism [205,206]. Other ceRNAs reported in HNSCC and
[179]. Thus, several studies emphasized that PIWI and piRNAs their functions are listed in Table S1 and shown in Fig. 8. This tempted
contribute to metastasis and disease progression [180]. us to think if a similar kind of crosstalk exists between piRNA, target,
and other sponging genes modulating the oncogenesis of HNSCC
(Fig. 8). This might unveil a new layer of gene regulations in HNSCC,
which might shed light on finding new paths for discovering novel

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T. Chattopadhyay et al. BBA - Reviews on Cancer 1877 (2022) 188772

Fig. 8. Involvement of ceRNAs in HNSCC tumorigenesis. A) miRNA sponged ceRNA-mediated regulations implicated in tumorigenic events of HNSCC. B) piRNAs
might regulate target genes implicated in HNSCC through ceRNA gene regulatory mechanisms.

treatment modalities for this malignancy. likely interplay of molecular mechanisms. Silencing piwil2 caused an
increase in p53, which resulted in apoptosis. Moreover, the migration
8. Role of piRNA and PIWIL in head and neck cancer ability of one of the OSCC cells (Tca8113) was inhibited by 50% after
piwil2 silencing. This reduction in migration of Tca8113 cells was
With a survival rate of 35%, HNSCC is a well-known lethal cancer confirmed by a decrease in the protein level of N-CADHERIN, VIMEN­
worldwide. As their pathology is unclear and not much explored, an TIN, MMP9, and an elevation in E-CADHERIN. The above findings
improvement in understanding the mechanisms at its molecular level, suggested that piwil2 acts as an oncogene in OSCC as it increases pro­
which includes genetic and epigenetic changes, is necessary to advance liferation, inhibits apoptosis, and promotes metastasis. Thus, combining
toward novel diagnostic and therapeutic strategies [207]. A thorough piwil2 inhibition with any chemotherapeutic drug may significantly
examination of a wide range of ncRNAs, particularly piRNAs, which improve the treatment outcome of OSCC (Fig. 9) [208].
have received little attention in HNSCC, is needed to help open prom­ Because smoking is a known primary etiological factor of HNSCC
ising avenues that could lead to novel diagnostic and therapeutic [209,210], another group led by Weg M Ongkeko decided to see if there
strategies. was a link with PIWILs [211]. Firstly, they evaluated the presence of any
aberration among the PIWILs (PIWIL1, PIWIL2, PIWIL3, and PIWIL4) by
8.1. Role of piwil in HNSCC analyzing their expression profiles at mRNA levels of patients falling
under the category of smokers as well as non-smokers from TCGA. From
As previously stated, PIWIL2 has been shown to regulate metastasis this analysis, only PIWIL genes were retained among the significantly
and invasion in cancers such as eOC and PC [153,163]. These studies dysregulated genes in all the cohorts considered in this study. Among the
encouraged a group of researchers led by Hong Chang to explore if any PIWILs, piwil1 was the only one upregulated among the different groups
such phenomenon with PIWIL2 exists in OSCC [208]. This study was studied. As piRNAs and PIWILs go hand in hand, they investigated the
initiated by collecting cancer and para-cancer tissues from about 60 correlation of the piRNAs with PIWIL1 among the HNSCC smoking pa­
OSCC patients, followed by gene expression analysis of these tissues. tients. It was found that the piRNA, NONHSAT081250, had a lower
During this analysis, Piwil2 was found upregulated, and its expression expression among smoking-induced HNSCC patients. Further, the
was consistent in different OSCC cell lines at their mRNA level. Further, downregulated expression of NONHSAT081250 was found among pa­
expression profiles from OSCC patients were classified into cohorts tients where piwil1 was comparatively overexpressed. To further inves­
based on piwil2 expression and TNM stage. From this, it was observed tigate the role of the duo (piRNA and PIWIL) in smoking-induced
that those with higher piwil2 expression revealed a poor prognosis. HNSCC, they examined the association between these and the copy
Furthermore, higher piwil2 expression was associated with poor differ­ number variations (CNV) recorded in HNSCC from the Broad Institute
entiation status compared to lower piwil2 expression, and such patients Firehose database. This analysis found that the HNSCC-associated CNVs
were more likely to develop lymph node metastasis. These cohort were linked with lower expression of NONHSAT108298 and NON­
studies suggest that the poor prognosis of patients with elevated piwil2 HSAT081250 and higher expression of piwil1 (Fig. 9) [211]. This pro­
expression may be due to a higher TNM stage, correlating PIWIL2 vided new insight into the possible relationship between piRNA, their
expression with OSCC progression. biogenesis factor PIWI, and smoking-induced HNSCC.
After that, various in-vitro experiments were carried out to reveal the

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T. Chattopadhyay et al. BBA - Reviews on Cancer 1877 (2022) 188772

Fig. 9. piRNA and PIWILs in HNSCC tumorigenesis and their possible roles in HNSCC therapeutics.

8.2. Role of piRNAs in HNSCC among HPV16 or 18 HNSCC patients.

Interestingly, 5 of the 11 piRNAs were significantly linked with
The first link between the dysregulation of piRNAs and their asso­ overall survival outcome among HPV positive cohort. Furthermore, the
ciation with patient survival in HNSCC was formulated using a combi­ most aberrantly expressed piRNA, FR140858, showed 100% sequence
natorial approach of in-silico and experimental study, along with other complementarity with a region of chromosome 7 corresponding to a
categories of ncRNAs such as lncRNAs and miRNAs. This study was also gene, minichromosome maintenance complex component 7 (mcm7),
led by Weg M Ongkeko [184]. They initiated this study by importing the whose expression showed correlation with that of HPV viral oncogene
RNA-sequencing data of HNSCC and tumor-paired adjacent normal from E7 (Fig. 9). Thus, silencing of this piRNA could potentially increase
the TCGA, which consisted of tumor-normal small RNA-seq. Datasets for mcm7 expression, and with further validation, it could emerge as a po­
piRNA profiling. The expression analysis showed 61 piRNAs showing tential prognostic biomarker [213].
significant (FDR < 0.05) change in their expression compared to the Weg M Ongkeko also conducted a similar study in which, in addition
paired normal tissue. As the patients' age and their HPV status were to identifying HPV-associated piRNAs in HNSCCs, a correlation between
found to produce diverse survival outcomes in HNSCC patients [212], expression of HPV-related piRNAs and retrotransposons as well as
they limited their study cohort to only HPV-negative patients under the retrotransposon-like genes was determined to identify potential targets
age of 85. To further evaluate the ncRNAs with prognostic potential, the for these piRNAs [214]. From this study, a total of 58 piRNAs were found
previously obtained differentially expressed piRNAs were studied, from aberrantly expressed among HNSCC smokers with HPV16 compared to
which 6 piRNAs were found as a predictor for the overall outcome of the the smokers without HPV. In addition, 39 piRNAs showed aberrant
patients. This study also found a strong correlation between one of the expression between HNSCC non-smokers with HPV16 and the non-
six piRNAs and TP53 mutation and the survival of HPV-negative and smokers without HPV. Among these, 30 piRNAs were dysregulated in
HPV-positive HNSCC patients. The key piRNA discovered was piR- both cohorts. This study also reported that only the HPV-upregulated
34736, whose dysregulation was confirmed using qRT-PCR in multiple piRNAs positively correlate with PIWIL4’s expression in HNSCC. They
normal oral epithelial and HNSCC cell lines. The increase in piRNA discovered that the HPV-upregulated piRNAs were negatively correlated
expression in HNSCC was associated with a significant decrease in sur­ with the expression of the retrotransposon-derived family (RTL) mem­
vivability and progression in the tumor stage, indicating that piRNA has bers. Furthermore, a positive correlation was found between the HPV-
a distinct oncogenic function in HNSCC [184]. induced upregulation of piRNAs and DNMT3L and several RTL genes,
Another in silico study was conducted by Wan L Lam and colleagues which is dysregulated in various cancers like pancreatic cancer (PAAD),
to elucidate the possible connection between the profile of the piRNA GC, and HCC, suggesting their oncogenic and tumor-suppressive role.
transcriptome and the clinicopathologic features of HNSCC [213]. This Furthermore, they discovered that overexpression of piRNAs, such as
study identified 247 piRNAs significantly differentially expressed in the NONHSAT059231, NONHSAT077463, and NONHSAT144936, was
tumor. Additionally, 25 piRNAs were found to express exclusively in associated with a mutation in PTEN, and that of NONHSAT102574 and
non-malignant tissues, while 87 piRNAs were expressed in tumor tis­ NONHSAT144936 were associated with a mutation in NOTCH. Finally,
sues. They also assessed the expression of these piRNAs in HPV16 and they also showed the clinical relevance of the upregulated piRNAs
HPV18, as these are known to have the highest oncogenic potential. NONHSAT077364 and NONHSAT144936 by investigating their corre­
They also discovered that 11 of them were showing aberrant expression lation with higher pathologic stages using the Kruskal-Wallis test.

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T. Chattopadhyay et al. BBA - Reviews on Cancer 1877 (2022) 188772

(Fig. 9) [214]. A handful of studies involving Piwils and piRNAs have been done till
Besides smoking, consumption of alcohol is a well-known etiological today concerning HNSCC malignancy. Among PIWILs, PIWIL1, PIWIL2,
factor in HNSCC [215,216], and its effects on piRNAs expression were and PIWIL4 are reported to be associated with oncogenic events of
studied by Saad, Maarouf A et al. using a combination of in silico and HNSCC and its risk factors. A few transcriptome profiling studies have
experimental approaches. Here, the RNA-seq. Data of drinkers and non- revealed dysregulated expression of piRNAs and their link to the clini­
drinkers groups were imported from TCGA and analysed, which yielded copathological progression of HNSCC. Moreover, some experimental
3,223 piRNAs differentially expressed with a fold change of more than 2. approaches showed piRNA-related gene silencing is involved in the
Among these, only five (piR-58510, piR-34946, piR-43219, piR-38034, oncogenic transformation like metastasis and chemosensitivity of
and piR-70732) had a fold change of >2, uniformly in all patients. chemotherapeutic drug CDDP. These studies have sparked curiosity
One of the piRNAs, piR-34946, was further found to be significantly among the researchers to unveil the possible role of piRNA and PIWILs in
deregulated in both the cohorts with alcohol and tobacco usage, which the tumorigenesis of HNSCC. However, there are many aspects of this
suggested their possible synergistic effect on piRNAs expression in study that remain unexplored.
HNSCC. Furthermore, it was found that piR-58510 and piR-35373 were
under-expressed, which was correlated with the longer survival rate 9. Conclusion and future aspects
among HNSCC patients [122].
Additionally, they checked for the presence of dysregulated piRNA With the advancement of sequencing technologies, researchers have
and their association with piwil4 concerning the pathogenesis of HNSCC better understood the non-coding portion of the genome. This has
due to alcohol consumption [122]. Interestingly, they found an upre­ prompted the development of new therapeutics, such as RNAi therapy,
gulated expression of piR-35373, piR-266308, piR-58510, and piR- which has improved the prognosis of diseases, particularly cancer [220].
38034, which were highly elevated in the presence of 0.1% of ethanol While there is ample evidence of piRNA and PIWIL regulating cellular
treatment. Further, they found a significant upregulation of piR-35373, function in many cancers [220,221], their mechanism of action remains
piR-266308, and piR-38034 with acetaldehyde treatment in both OKF4 to be elucidated. To better understand the mechanism underlying piRNA
and OKF6 cells. These piRNAs were the same as those found in response and PIWIL functionality, we must first get more insights into their dis­
to alcohol treatment, indicating that they may play an oncogenic role in covery, origin, biogenesis, and the mechanisms adopted to regulate gene
HNSCC carcinogenesis (Fig. 9). Moreover, piR-35373 was also found to expression, which we have elaborated on in this review.
overlap with 3-phosphoglycerate dehydrogenase (phgdh), speculating its Despite advances in the treatment of HNSCC, the alarming incidence
ability to regulate phgdh in HNSCC. Surprisingly, they also discovered rate, especially among the tobacco and alcohol consuming population,
that expression of only piwil4 showed aberration (increase in expression) encouraged us to review the status of HNSCC concerning ncRNA
when exposed to both ethanol and acetaldehyde (Fig. 9) [122]. research. As ncRNAs play a crucial role in tumorigenesis, we wonder if
Another study involving piRNA profiling done by Wu, Lihong et al. in piRNA and PIWIL will contribute to the same. Currently, HNSCC studies
a 4-NOQ induced OSCC mouse model identified that known and novel are mostly limited to piRNAs and PIWILs profiling. Due to a lack of
piRNAs were found significantly dysregulated in the OSCC compared to experimental studies, their ability to play a key role in translational
the control [217]. The RNA-seq identified 462 dysregulated genes research and therapy remains a long way off. These voids prompted us to
within the 4NQO-induced mouse models compared to the healthy mouse raise a few critical questions: What possible roles do these piRNAs and
models with logFC ≥1, p-value <0.05. Additionally, they analyzed the PIWILs play in the pathophysiology of HNSCC? Can piRNA cause
GSE31056 dataset to uncover the commonly dysregulated genes of apoptosis in HNSCC, and if so, how? Can any piRNA prevent HNSCC
OSCC between humans and mice. This revealed that 502 genes were tumor metastasis? Is it possible for piRNA to induce cellular senescence
significantly dysregulated, with 260 under-expressed and 189 overex­ and inhibit tumor cell proliferation in HNSCC? What are the potential
pressed in humans with logFC ≥1, p<0.05. Finally, when mouse and genes regulated by piRNAs in HNSCC, and does this regulation act as a
human genes were compared, 22 differentially expressed genes (DEGs) boon or a bane in HNSCC therapy? Can piRNA and PIWIL be used as
were common, among which 11 were discovered to be involved in a predictive or therapeutic markers in HNSCC? Answering them might
regulatory network with the 11 piRNAs through target binding. Litera­ enlighten the role of piRNA and PIWIL in HNSCC and may also serve as a
ture studies on these targets revealed mmp13 to be involved in metas­ springboard for developing a new therapeutic realm for HNSCC in the
tasis of squamous cell carcinoma cells and lamc2 expression to promote future.
the malignant progression of OSCC. Further, mamdc2 exerts an anti-
tumorigenic effect in OSCC, and epigenetic silencing of mal causes Author contributions
tumorigenesis of OSCC. These target genes, with their corresponding
piRNA regulating them, enlighten the role of these piRNAs in OSCC All the authors equally participated in the preparation of the
[217]. Several significant genes, piRNAs, and their pathways have been manuscript.
essayed here. Further research could reveal the underlying molecular
mechanism, which could then be used to develop better therapeutics. Declaration of Competing Interest
So far, we have reviewed the ability of piRNA and PIWILs in OSCC
and HNSCC, but their ability to act as any therapeutic reagent has not The authors declare that they have no known competing financial
been discussed. A group of researchers investigated the potential role of interests or personal relationships that could have appeared to influence
piRNA in the chemosensitivity and chemo resistivity of well-known the work reported in this paper.
chemotherapeutic agents, such as platinum, in OSCC. CDDP-based
chemotherapy, the first platinum-based drug [218], was investigated Data availability
in this study. The expression of piR-1037 was upregulated post-CDD
chemotherapy (Fig. 9). Suppression of this piRNA was seen to promote No data was used for the research described in the article.
chemosensitivity as CDDP could positively influence its expression in
the OSCC cells. In addition, piR-1037 could elevate XIAP (X-linked in­ Appendix A. Supplementary data
hibitor of apoptosis) at its protein level, regulating it post-
transcriptionally by protecting its mRNA or protein from decay. Supplementary data to this article can be found online at https://doi.
Furthermore, inhibition of piR-1037 inhibited the properties of cancer org/10.1016/j.bbcan.2022.188772.
progression in OSCC cells. Thus, piR-1037 proved to be a critical
mechanistic factor in OSCC chemotherapy [219].

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