Abstract

It is generally accepted that PIWI proteins are predominately expressed in the germline but absent in somatic tissues. Their best-characterized role is to suppress transposon expression, which ensures genomic stability in the germline. However, increasing evidence has suggested that PIWI proteins are linked to the hallmarks of cancer defined by Weinberg and Hanahan, such as cell proliferation, anti-apoptosis, genomic instability, invasion and metastasis. This provides new possibilities for anticancer therapies through the targeting of PIWI proteins, which may have fewer side effects due to their potential classification as a CTA (cancer/testis antigen). Furthermore, PIWI has been proposed to act as a diagnostic and prognostic marker for many types of cancer, and even to differentiate early- and late-stage cancers. We herein summarize the latest progress in this exciting field, hoping to encourage new investigations of PIWIs in cancer biology that will help to develop new therapeutics for clinical application.

Introduction

The argonaute family consists of well-conserved proteins of ∼95 kDa [1]. They regulate gene expression through the complementary recognition of their RNA or DNA targets with tiny regulatory RNAs, such as microRNAs (miRNAs), short-interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs) [2,3]. According to the phylogenetic analysis, argonaute family proteins can be classified into two subclasses: AGO and PIWI [4,5]. In contrast to the ubiquitous expression of AGO proteins, PIWI expression is mostly restricted to germ cells [6]. The PIWI family was first identified in a genetic screen for mutants that affect the asymmetric division of stem cells in the Drosophila germline [7,8]. PIWI proteins have been extensively investigated [9,10]. The PIWI family is highly conserved in structure and function in a wide variety of organisms. PIWI has several different members depending on the species. There are four members in human: PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2 [11]; three in mice: MIWI, MILI, and MIWI2 [12–14]; three in Drosophila: PIWI, AUB (Aubergine), and AGO3 [7,8,15–17]; two in zebrafish: ZILI and ZIWI [18,19]; and two in Caenorhabditis elegans: PRG-1 and PRG-2 [20]. All of these proteins share three characteristic domains: PAZ, MID, and PIWI [21]. PIWI proteins bind piRNAs that are generally of 26–31 nucleotides in length [10]. Mutations of PIWI proteins in mice, Drosophila, zebrafish, and some other species commonly cause defects in gametogenesis [8,10,13,14,16–20,22–24], indicating the evolutionarily conserved essential roles for PIWI proteins in germline development. Accumulating evidence has shown that PIWIs function in piRNA biogenesis and associate with mature piRNAs to form the piRNA-induced silencing complex (piRISC) in the germline, which protects the integrity of the genome by silencing transposable elements [9]. Transposable elements in eukaryotic genomes are mobile genetic fragments that have the ability to self-propagate, which may lead to genome unstability. PIWI protein bound to a transposon-derived piRNA will translocate to the nucleus and silence transposons epigenetically. So, deletion of PIWI will cause infertility.

Forty-four years have passed since the declaration of war against cancer in 1971 when US President Richard Nixon signed the National Cancer Act [25]. Considerable researches from science and medicine have revealed the molecular mechanisms underlying cancer development and progression [26], but cancer still remains a big health problem, and hundreds of thousands of people still face the threat of death from cancer every day. Undoubtedly, cancer development is an extremely complex process [26]. Thus, new investigations in tumorigenesis and cancer treatments are urgently needed.

Recent studies have linked PIWI expression to tumorigenesis. PIWI proteins have been preliminarily associated with some phenotypic hallmarks of cancer, such as sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, mediating genome instability and mutation, and promoting cell growth [26,27]. The molecular details underlying the oncogenic activities of PIWI remain to be fully addressed, but PIWIs have been found to regulate the occurrence of those cancer hallmarks [27,28]. Because PIWI proteins are predominantly expressed in germ cells and are ectopically expressed in tumors, they are candidate cancer/testis antigens (CTAs) [29,30]. CTAs are expressed predominantly in the testis and in various types of cancers. Owing to their restricted expression in testis and tumors, they have been used as the targets of immunotherapy with no or little negative side effects. Therefore, PIWI proteins could serve as good targets for better chemotherapeutic drugs to treat tumors in humans. This review will summarize the latest research progress on the roles of PIWIs in tumorigenesis and cancer progression in order to encourage new investigations of PIWIs in cancers [27,28,31,32].

PIWIs Are Involved in Tumorigenesis and Progression

PIWIs were first discovered to be aberrantly expressed in cancer [33]. A large body of work, especially clinical investigations, has linked all four human PIWI proteins to tumorigenesis (Table 1). While other factors in cancer have extensively been characterized, PIWIs are relatively new players in cancers. Most studies of PIWIs in human cancers were published only very recently. The expressions of PIWI proteins were increased in various cancers, such as prostate cancer, cervical cancer, and colon cancer (Table 1). Many kinds of cancer cell lines were also found to over-express PIWI proteins. Based on results from these clinical samples and cancer cell lines, PIWIs have been linked to some of the hallmarks of cancer [26], prominently suggesting an oncogenic role of PIWI (Fig. 1 and Table 2).

Table 1.

Expression of PIWI members in tumor tissues and cells

PIWI piRNAs Human diseases Examined tumor samples
 
Detection levels
 
Reference 
Tissues Cell lines RNA PR 
PIWIL1 (HIWI)  Cervical cancer Yes   Yes [34
 Colon cancer Yes   Yes [35
 Colorectal cancer Yes MGC803, SGC7901, BGC823, AGS, N87, MKN45, LoVo, CL187, HT29, RKO, SW480, HCT116, HepG2, BEL7402, SMMC7721, E30, E70, E140, E180, E410, E450, E510, YES2, T12, PG, GLC82, H446, H460, H1299, A549  Yes [36
 Endometrial cancer Yes   Yes [37
 Esophageal cancer Yes KYSE70, KYSE140, KYSE450  Yes [38
 Gastric cancer Yes   Yes [39
 Gastric cancer Yes NCI-SNU-1, NCI-SNU-5, NCI-SNU-16, AGS, NCI-N87, RF-48, MGC803 Yes Yes [40
 Glioma Yes U251, U87, LN229 Yes Yes [41
 Liver cancer Yes HCCLM3, MHCC97H, MHCC97L, SMMC7721, HepG2 Yes Yes [42
 Pancreatic cancer Yes  Yes Yes [43
 Soft-tissue sarcoma Yes  Yes  [44
 Sarcoma Yes MFH  Yes [45
 Seminoma Yes  Yes  [33
 Breast cancer Yes MDA-MB-231, MCF-7 Yes Yes [46
 Lung cancer  SSCloAldebr cells Yes Yes [45
 Cervical cancer Yes HeLa, SiHa, C33A Yes Yes [47
 Hepatocellular carcinoma Yes MHCC97L, MHCC97H Yes Yes [48
 Epithelial ovarian cancer Yes  Yes Yes [49
 Ovarian cancer Yes   Yes [50
PIWIL2 (HILI)a  Breast cancer Yes MDA-MB-231 Yes Yes [51
 Breast cancer Yes   Yes [52
 Breast, cervical, and other cancers Yes THP-1, CCRF, Jurkat, H9, Raji, Daudi, HEL, Dami, HL-60, K562, PBL985, HCT-8, CaCo-2, HT-29, SW480, CT26CL25, HeLa, CaoV3, HEY1B, MDA-MB-231, MDA-MB-468, MCF-7, H1299, LL2, HepG2, Huh7, N2a, C8161, INS-1, SW872, 3B11(EL) Yes Yes [53
 Cervical cancer Yes   Yes [54
 Colorectal cancer Yes   Yes [55
 Ovarian cancer  A2780, CP70, CDDP, MCP2, MCP3, MCP8, 2008, 2008C13  Yes [56
 b Yes MDA-MB-231, NIH3T3, GC-1, HeLa Yes Yes [57
 Gastric cancer Yes   Yes [39
 Colon cancer Yes SW620, SW480 Yes Yes [58
 Ovarian cancer Yes   Yes [50
 Testicular seminoma Yes TERA1 Yes Yes [59
 Breast cancer Yes MCF-7, ZR-75.1, SKBR3 Yes Yes [60
piR-932   Breast cancer stem cells Yes  [61
HILI+ 78 up+73 down Cervical cancer  HeLa Yes  [62
HILI- 79 up+61 down Cervical cancer  HeLa Yes  [62
PIWIL3  Gastric cancer Yes   Yes [39
 Ovarian cancer Yes   Yes [50
PIWIL4 (HIWI2)  Gastric cancer Yes   Yes [39
 Cervical cancer Yes HeLa, C33A Yes Yes [63
 Renal cell carcinoma Yes  Yes  [64
 Breast cancer Yes MCF-7, ZR-75.1, SKBR3 Yes Yes [60
 Ovarian cancer Yes   Yes [50
 piR-823 Gastric cancer Yes MGC-803, SGC-7901 Yes  [65
 piR-823 Multiple myeloma Yes RPMI8226, U266, ARH-77, KM3 Yes Yes [66
 piR-651 Gastric, colon, lung, and breast cancer Yes HepG2, HeLa, Bcap-37 MSTO-211H, NCI-H446, MGC-803, SGC-7901 Yes  [67
 piR-4987, piR-20365, piR-20485, piR-20582, piR-19825(No), piR-17458(No)  Yes  Yes  [68
 piR-Hep1 Hepatocellular carcinoma Yes  Yes  [69
 ∼70 piRNAs Breast cancer  Exponentially growing and growth-arrested MCF-7 cells Yes  [70
 >100 BC piRNAs Breast cancer Yes  Yes  [60
 197 piRNAs differentially expressed Bladder cancer Yes  Yes  [71
PIWI piRNAs Human diseases Examined tumor samples
 
Detection levels
 
Reference 
Tissues Cell lines RNA PR 
PIWIL1 (HIWI)  Cervical cancer Yes   Yes [34
 Colon cancer Yes   Yes [35
 Colorectal cancer Yes MGC803, SGC7901, BGC823, AGS, N87, MKN45, LoVo, CL187, HT29, RKO, SW480, HCT116, HepG2, BEL7402, SMMC7721, E30, E70, E140, E180, E410, E450, E510, YES2, T12, PG, GLC82, H446, H460, H1299, A549  Yes [36
 Endometrial cancer Yes   Yes [37
 Esophageal cancer Yes KYSE70, KYSE140, KYSE450  Yes [38
 Gastric cancer Yes   Yes [39
 Gastric cancer Yes NCI-SNU-1, NCI-SNU-5, NCI-SNU-16, AGS, NCI-N87, RF-48, MGC803 Yes Yes [40
 Glioma Yes U251, U87, LN229 Yes Yes [41
 Liver cancer Yes HCCLM3, MHCC97H, MHCC97L, SMMC7721, HepG2 Yes Yes [42
 Pancreatic cancer Yes  Yes Yes [43
 Soft-tissue sarcoma Yes  Yes  [44
 Sarcoma Yes MFH  Yes [45
 Seminoma Yes  Yes  [33
 Breast cancer Yes MDA-MB-231, MCF-7 Yes Yes [46
 Lung cancer  SSCloAldebr cells Yes Yes [45
 Cervical cancer Yes HeLa, SiHa, C33A Yes Yes [47
 Hepatocellular carcinoma Yes MHCC97L, MHCC97H Yes Yes [48
 Epithelial ovarian cancer Yes  Yes Yes [49
 Ovarian cancer Yes   Yes [50
PIWIL2 (HILI)a  Breast cancer Yes MDA-MB-231 Yes Yes [51
 Breast cancer Yes   Yes [52
 Breast, cervical, and other cancers Yes THP-1, CCRF, Jurkat, H9, Raji, Daudi, HEL, Dami, HL-60, K562, PBL985, HCT-8, CaCo-2, HT-29, SW480, CT26CL25, HeLa, CaoV3, HEY1B, MDA-MB-231, MDA-MB-468, MCF-7, H1299, LL2, HepG2, Huh7, N2a, C8161, INS-1, SW872, 3B11(EL) Yes Yes [53
 Cervical cancer Yes   Yes [54
 Colorectal cancer Yes   Yes [55
 Ovarian cancer  A2780, CP70, CDDP, MCP2, MCP3, MCP8, 2008, 2008C13  Yes [56
 b Yes MDA-MB-231, NIH3T3, GC-1, HeLa Yes Yes [57
 Gastric cancer Yes   Yes [39
 Colon cancer Yes SW620, SW480 Yes Yes [58
 Ovarian cancer Yes   Yes [50
 Testicular seminoma Yes TERA1 Yes Yes [59
 Breast cancer Yes MCF-7, ZR-75.1, SKBR3 Yes Yes [60
piR-932   Breast cancer stem cells Yes  [61
HILI+ 78 up+73 down Cervical cancer  HeLa Yes  [62
HILI- 79 up+61 down Cervical cancer  HeLa Yes  [62
PIWIL3  Gastric cancer Yes   Yes [39
 Ovarian cancer Yes   Yes [50
PIWIL4 (HIWI2)  Gastric cancer Yes   Yes [39
 Cervical cancer Yes HeLa, C33A Yes Yes [63
 Renal cell carcinoma Yes  Yes  [64
 Breast cancer Yes MCF-7, ZR-75.1, SKBR3 Yes Yes [60
 Ovarian cancer Yes   Yes [50
 piR-823 Gastric cancer Yes MGC-803, SGC-7901 Yes  [65
 piR-823 Multiple myeloma Yes RPMI8226, U266, ARH-77, KM3 Yes Yes [66
 piR-651 Gastric, colon, lung, and breast cancer Yes HepG2, HeLa, Bcap-37 MSTO-211H, NCI-H446, MGC-803, SGC-7901 Yes  [67
 piR-4987, piR-20365, piR-20485, piR-20582, piR-19825(No), piR-17458(No)  Yes  Yes  [68
 piR-Hep1 Hepatocellular carcinoma Yes  Yes  [69
 ∼70 piRNAs Breast cancer  Exponentially growing and growth-arrested MCF-7 cells Yes  [70
 >100 BC piRNAs Breast cancer Yes  Yes  [60
 197 piRNAs differentially expressed Bladder cancer Yes  Yes  [71

PR, protein; No, not confirmed by PCR.

aPIWIL2-like proteins examined here.

bHuman testicular seminoma, prostate cancer, breast cancer, gastrointestinal cancer, ovarian cancer, and endometrial cancer, and mouse breast tumor, rhabdomyosarcoma and medulloblastoma.

Table 2.

Involvement of PIWI members in tumorigenesis and progression

PIWI piRNA Human disease Hallmarks of tumor Mechanical points Reference 
PIWIL1 (HIWI)  Seminoma Promoting cell proliferation  [33
 Gastric cancer Promoting cell proliferation Inducing cell cycle arrest in G2/M phase, possibly has some relationship to proliferation marker Ki67 [40
 Hepatocellular carcinoma Promoting cell proliferation Possibly has some relationship to PCNA (proliferating cell nuclear antigen) [72
 Sarcoma Promoting cell proliferation Decreasing cellular differentiation state, thus keeping indefinite proliferation. Inversely correlating with tumor suppressor genes (TSGs), such as p15, p21 and p27 [73
 Cervical squamous cell carcinoma Promoting cell migration and invasion  [34
 Hepatocellular carcinoma Promoting cell migration and invasion  [72,48
 Sarcoma Genomic instability Controlling two common transposons, IAP and Line1, directly and correlating with global DNA hypermethylation [73
 Breast cancer Promoting cell growth  [46
 Glioma cancer Promoting cell growth, invasion and migration Suppressing cell growth related to apoptosis and the cell cycle by altering p21, cyclin D1, Bcl-2, and Bax. Inhibiting cell migration and invasion by reducing the expression of MMP2 and MMP9 [74
 Epithelial ovarian cancer Having a repressive effect on cell invasiveness  [49
 Lung cancer Cancer stem cell self-renewal Maintenance of lung cancer stem cell populations [45
PIWIL2 (HILI)a  Colon cancer Promoting cell proliferation  [58
 Colon cancer Promoting migration and invasion Activating MMP9 transcriptional activities [58
 Breast cancer Promoting cell proliferation Predominantly expressed in breast cancer stem cell population, possibly participating in maintaining the infinite proliferation potential of CSCs. Activating STAT3/cyclin D1 pathway, promoting cell proliferation [51
 b Inhibition of apoptosis and promotion of proliferation Activating STAT3/Bcl-xL signaling pathway to inhibit apoptosis and promote proliferation [51,57
 Cervical cancer and liver cancer Anti-apoptosis Directly associating with STAT3 via its PAZ domain and forming a PIWIL2/STAT3/c-Src triple protein complex. Furthermore, STAT3 is phosphorylated by c-Src and translocated to the nucleus, where it then binds to the p53 promoter and represses its transcription [75
 Breast cancer Cell migration and invasion  [52
 Colorectal carcinoma Cell migration and invasion  [55
 Ovarian cancer Genomic instability Involved in chromatin decondensation, especially in response to exposure to the DNA damaging agent cisplatin, by regulating histone modifications such as acetylation and methylation, thus enhancing cancer cell survival [56
  Genomic instability Mediating DNA repair through an axis of Piwil2, histone acetylation, chromatin relaxation, and upstream DNA damage response (DDR) pathways [77
 Breast, cervical, and other cancers Promoting tumor cell survival and proliferation Up-regulating the nuclear expression of NF-kB, further increasing STAT3 and Bcl2 gene expression. Participating in cell cycle progress by promoting the G0/1 transition into S phase [53
  Cell proliferation, migration, and invasion Ectopically expressed piwil2 stable cell line of mouse embryonic fibroblasts [78
piRNAs   HILI, along with piRNAs, plays a role in LINE1 suppression in HeLa cancer cells [62
piR-932 Breast cancer  The combination of piR-932 and PIWIL2 may be a positive regulator in the process of breast cancer stem cells through promoting the methylation of Latexin [61
PIWIL3      
PIWIL4 (HIWI2)  Colon cancer Cell migration and invasion  [76
 Cervical cancer Cell migration and invasion  [63
 Cervical cancer Promoting cell growth and proliferation by inhibiting apoptosis Impairing apoptosis through the p14ARF/p53 pathway, without affecting the cell cycle, thus playing an oncogenic role in cervical cancer [63
piR-823 Gastric cancer Suppressing tumor cell growth  [65
piR-823 Multiple myeloma Regulating de novo DNA methylation and angiogenesis piR-823 decreased the expression of DNMT3A and 3B, which in turn led to decrease in global DNA methylation and reexpression of methylation-silenced tumor suppressor, p16INK4A. piR-823 also induced reduction of vascular endothelial growth factor secretion, with consequent decreased proangiogenic activity [66
piR-651 Gastric, colon, lung, and breast cancer Promoting cell growth Gastric cancer cells arrested at the G2/M phase by piR-651 inhibitor [67
piR-Hep1 Hepatocellular carcinoma Promoting cell viability, motility, and invasiveness  [69
piRABC Bladder cancer Inhibiting bladder cancer cell proliferation, colony formation, and promote cell apoptosis Regulating TNFSF4 [69
PIWI piRNA Human disease Hallmarks of tumor Mechanical points Reference 
PIWIL1 (HIWI)  Seminoma Promoting cell proliferation  [33
 Gastric cancer Promoting cell proliferation Inducing cell cycle arrest in G2/M phase, possibly has some relationship to proliferation marker Ki67 [40
 Hepatocellular carcinoma Promoting cell proliferation Possibly has some relationship to PCNA (proliferating cell nuclear antigen) [72
 Sarcoma Promoting cell proliferation Decreasing cellular differentiation state, thus keeping indefinite proliferation. Inversely correlating with tumor suppressor genes (TSGs), such as p15, p21 and p27 [73
 Cervical squamous cell carcinoma Promoting cell migration and invasion  [34
 Hepatocellular carcinoma Promoting cell migration and invasion  [72,48
 Sarcoma Genomic instability Controlling two common transposons, IAP and Line1, directly and correlating with global DNA hypermethylation [73
 Breast cancer Promoting cell growth  [46
 Glioma cancer Promoting cell growth, invasion and migration Suppressing cell growth related to apoptosis and the cell cycle by altering p21, cyclin D1, Bcl-2, and Bax. Inhibiting cell migration and invasion by reducing the expression of MMP2 and MMP9 [74
 Epithelial ovarian cancer Having a repressive effect on cell invasiveness  [49
 Lung cancer Cancer stem cell self-renewal Maintenance of lung cancer stem cell populations [45
PIWIL2 (HILI)a  Colon cancer Promoting cell proliferation  [58
 Colon cancer Promoting migration and invasion Activating MMP9 transcriptional activities [58
 Breast cancer Promoting cell proliferation Predominantly expressed in breast cancer stem cell population, possibly participating in maintaining the infinite proliferation potential of CSCs. Activating STAT3/cyclin D1 pathway, promoting cell proliferation [51
 b Inhibition of apoptosis and promotion of proliferation Activating STAT3/Bcl-xL signaling pathway to inhibit apoptosis and promote proliferation [51,57
 Cervical cancer and liver cancer Anti-apoptosis Directly associating with STAT3 via its PAZ domain and forming a PIWIL2/STAT3/c-Src triple protein complex. Furthermore, STAT3 is phosphorylated by c-Src and translocated to the nucleus, where it then binds to the p53 promoter and represses its transcription [75
 Breast cancer Cell migration and invasion  [52
 Colorectal carcinoma Cell migration and invasion  [55
 Ovarian cancer Genomic instability Involved in chromatin decondensation, especially in response to exposure to the DNA damaging agent cisplatin, by regulating histone modifications such as acetylation and methylation, thus enhancing cancer cell survival [56
  Genomic instability Mediating DNA repair through an axis of Piwil2, histone acetylation, chromatin relaxation, and upstream DNA damage response (DDR) pathways [77
 Breast, cervical, and other cancers Promoting tumor cell survival and proliferation Up-regulating the nuclear expression of NF-kB, further increasing STAT3 and Bcl2 gene expression. Participating in cell cycle progress by promoting the G0/1 transition into S phase [53
  Cell proliferation, migration, and invasion Ectopically expressed piwil2 stable cell line of mouse embryonic fibroblasts [78
piRNAs   HILI, along with piRNAs, plays a role in LINE1 suppression in HeLa cancer cells [62
piR-932 Breast cancer  The combination of piR-932 and PIWIL2 may be a positive regulator in the process of breast cancer stem cells through promoting the methylation of Latexin [61
PIWIL3      
PIWIL4 (HIWI2)  Colon cancer Cell migration and invasion  [76
 Cervical cancer Cell migration and invasion  [63
 Cervical cancer Promoting cell growth and proliferation by inhibiting apoptosis Impairing apoptosis through the p14ARF/p53 pathway, without affecting the cell cycle, thus playing an oncogenic role in cervical cancer [63
piR-823 Gastric cancer Suppressing tumor cell growth  [65
piR-823 Multiple myeloma Regulating de novo DNA methylation and angiogenesis piR-823 decreased the expression of DNMT3A and 3B, which in turn led to decrease in global DNA methylation and reexpression of methylation-silenced tumor suppressor, p16INK4A. piR-823 also induced reduction of vascular endothelial growth factor secretion, with consequent decreased proangiogenic activity [66
piR-651 Gastric, colon, lung, and breast cancer Promoting cell growth Gastric cancer cells arrested at the G2/M phase by piR-651 inhibitor [67
piR-Hep1 Hepatocellular carcinoma Promoting cell viability, motility, and invasiveness  [69
piRABC Bladder cancer Inhibiting bladder cancer cell proliferation, colony formation, and promote cell apoptosis Regulating TNFSF4 [69

aPIWIL2-like proteins examined here.

bHuman testicular seminoma, prostate cancer, breast cancer, gastrointestinal cancer, ovarian cancer and endometrial cancer, and mouse breast tumor, rhabdomyosarcoma and medulloblastoma.

Figure 1.

Regulation of some hallmarks of cancer by the PIWI family Cancer hallmarks are involved in the pathogenesis of some or all cancers, which is proposed by Weinberg. PIWIs may contribute to the formation of many hallmarks of cancer, such as the promotion of cell division, resistance to cell death, evasion of growth suppression, maintenance of genomic integrity, and facilitation of cell migration and invasion.

Figure 1.

Regulation of some hallmarks of cancer by the PIWI family Cancer hallmarks are involved in the pathogenesis of some or all cancers, which is proposed by Weinberg. PIWIs may contribute to the formation of many hallmarks of cancer, such as the promotion of cell division, resistance to cell death, evasion of growth suppression, maintenance of genomic integrity, and facilitation of cell migration and invasion.

PIWIs are involved in promoting cell proliferation

One of the most fundamental characteristics of cancer cells is their capacity to sustain unlimited proliferation [26]. Investigations in the relationship of PIWIs with this hallmark of cancer have been carried out. Current investigations only focused on PIWIL1 and PIWIL2. The involvement of PIWI proteins in cancer was first reported in seminomas in which PIWILI might participate in enhancing the proliferation of germ cells but not somatic cells [33]. Very recently, the hiwi gene was also reported to promote the growth of human breast tumors [46], gastric tumors [40], and hepatocellular carcinoma cells [72]. The piwil2 gene was suggested to modulate the proliferation of colon cancer [58] and breast cancer [51,53]. In Drosophila, PIWI and AUB were demonstrated to contribute to the growth of malignant brain tumors [79].

But how do PIWIs promote tumor cell growth? Cell-cycle progression enhances cancer cell growth, which illustrates why cell-cycle promoters serve as good candidates for tumor oncogenes. Inhibition of hiwi suppressed the growth of human gastric cancer cells and induced cell-cycle arrest at the G2/M phase of the cell cycle [40]. The PIWIL2-like (PL2L) protein PL2L60, a splice isoform of PIWIL2, but not the other isoforms (PL2L80, PL2L50, and PL2L40), is predominantly expressed in various types of human and mouse tumor cells. PL2L60 was found to participate in cell-cycle progression by promoting the G0/1 to S phase transition [53]. The identification of the isoforms of PL2L proteins will provide a novel insight into the molecular mechanisms of tumorigenesis and a new strategy for cancer diagnostics and anticancer drug development.

Normal tissues maintain cell number homeostasis to ensure normal tissue architecture and function by strictly controlling the production and release of growth-promoting signals. Cancer cells attain uncontrollable cell proliferation by deregulating these signals. In breast cancer MDA-MB-231 cells, PIWIL2 was demonstrated to activate the STAT3/cyclin D1 pathway, thus promoting cell proliferation [51]. PL2L60 was found to promote tumor cell survival and proliferation in vitro by up-regulating the nuclear expression of NF-κB, further increasing STAT3 and Bcl-2 expression [53].

The theory of cancer stem cells (CSCs) in tumorigenesis has recently been proposed, which accounts for the heterogeneity of tumor tissue by a hierarchy-based model [80]. In this model, CSCs possess the ability to self-renew and to generate differentiated tumor cells, thus being responsible for the overall organization of a tumor. Indeed, PIWIs have been found to correlate with CSCs and with the undifferentiated state that they uniquely possess. In Drosophila, female piwi mutants exhibit germline stem cell tumors that are sustained by elevated Dpp signaling [81]. In human, the hiwi gene was shown to be involved in the maintenance of lung cancer stem cell populations [45], and piwil2 is involved in maintaining the indefinite proliferation potential of CSCs to promote breast tumor growth [51]. If cancer cells are to proliferate indefinitely, they must be maintained in an undifferentiated state. Increased HIWI in sarcoma precursors inhibits cellular differentiation in vitro and generated sarcomas in vivo, and the inducible down-regulation of HIWI in human sarcomas inhibits growth and re-establishes differentiation [73]. These data indicate that the up-regulation of PIWI proteins can maintain the status of cancer stem cells and/or decrease the cellular differentiation state, thus allowing indefinite proliferation to contribute to tumorigenesis.

PIWIs are involved in inhibiting cell apoptosis

The notion that programmed cell death by apoptosis serves as a natural barrier to cancer development has been established over the last two decades [82–84]. The loss of tumor suppressor activity and/or the promotion of oncogenic factors in cancer cells will result in an inability to react to cell death signaling. Tumors may avoid this cell death by increasing the expression of antiapoptotic regulators (Bcl-2 and Bcl-xL) or by down-regulating proapoptotic factors (Bax, Bim, and Puma) [26]. Current data are very limited; however, antiapoptotic effects of PIWI proteins have indeed been found in cancer cells [51,57,63,74,75].

Silencing hiwi in glioma cells was found to increase cell-cycle arrest and promote cell apoptosis. The expressions of p21, cyclin D1, Bcl-2, and Bax were also significantly altered [74]. In MDA-MB-231 and NIH-3T3 cells, PIWIL2 was demonstrated to act as an oncogene by activating the STAT3/Bcl-xL signaling pathway to inhibit apoptosis and to promote proliferation [51,57]. In HeLa cells, Lu et al. [75] verified that PIWIL2 is directly associated with STAT3 protein via its PAZ domain and forms a PIWIL2/STAT3/c-Src triple protein complex. Furthermore, STAT3 is phosphorylated by c-Src and translocates to the nucleus where it binds to the p53 promoter and represses its transcription. This demonstrates that PIWIL2 plays an anti-apoptotic role in tumor cells as a positive regulator of the STAT3 signaling pathway and provides novel insights into the role of PIWIL2 in tumorigenesis. In HeLa cells, PIWIL4 was also found to impair apoptosis through the p14ARF/p53 pathway without affecting the cell cycle, thus playing an oncogenic role in cervical cancer [63]. It is possible that PIWIL4 can directly regulate p14ARF/p53 pathway by inducing H3K9 methylation at the p14ARF locus [85]. However, evidence is needed to verify this hypothesis. These data indicate that the high expression of PIWI proteins in cancer cells can promote apoptosis resistance of cancer cells by regulating apoptosis-related factors.

PIWIs are involved in facilitating cell migration and invasion

Carcinomas from epithelial tissues were found to progress to higher pathological grades of malignancy, reflecting local invasion and distant metastasis [26]. PIWI proteins have been reported to be related to cancer cell migration and invasion. In clinical cases, the elevated expression of PIWIL1 was found to be associated with the invasion of cervical squamous cell carcinoma (CSCC) and, interestingly, had a statistically significant positive correlation with human papillomavirus 16 (HPV16) but had no correlation with patient's age or histological grade [34]. In hepatocellular carcinoma (HCC) cell lines, PIWIL1 expression was increased in parallel with the metastatic potential of HCC cell lines, i.e. the expression levels of PIWIL1 in MHCC97L, MHCC97H, and HCCLM3 cells were significantly higher than that in SMMC7721 and HepG2 cells, and depletion of PIWIL1 caused decreased invasion and metastasis [72,48]. In contrast, a very recent report indicated that PIWIL1 has a repressive effect on cell invasiveness in epithelial ovarian cancer [49]. piwil2 was expressed in the cytoplasm (Cyt), nucleus (N), or both the cytoplasm and nucleus (C-N) of invasive and metastatic breast cancers, showing a potential relationship with cancer cell migration and invasion [52]. In colorectal carcinomas, a high level of PIWIL2 expression was significantly correlated with a low degree of differentiation and with deep invasion and perineural invasion [55]. The expression levels of PIWIL4 and EIF2C2–4 in colon cancer samples were significantly increased in advanced tumors with distant metastases, suggesting that these proteins may promote tumor invasion [76]. PIWIL4 can also promote cervical cancer cell invasion, based on transwell invasion assays employing HeLa and C33A cells [63].

At present, the mechanisms underlying the invasion and metastasis of carcinoma cells are largely unknown, but they have been schematized as a sequence of discrete steps often termed the invasion-metastasis cascade [26,86]. During these steps, epithelial-mesenchymal transition (EMT) is a prominent regulatory program by which transformed epithelial cells can acquire the abilities to invade, to resist apoptosis, and to disseminate [26]. Some transcription factors, including Slug, Snail, Goose-coid, Twist, and ZEB1, are highly expressed by metastatic cells and have been suggested to play a role in inducing EMT [86]. However, whether PIWIs influence or are influenced by these transcription factors and/or EMT remains to be investigated. Clearly, cell invasion and metastasis are strongly influenced by the extracellular matrix (ECM). One molecule of the ECM, matrix metallopeptidase 9 (MMP9), has previously been shown to play a critical role in mediating the tumor microenvironment and to be involved in cancer cell invasion and metastasis [87]. Knockdown of hiwi was found to inhibit the migration and invasion of glioma cells by reducing the expression of MMP2 and MMP9 [74]. PIWIL2 can promote cell migration and invasion through the activation of MMP9 in SW620 and SW480 colon cancer cell lines [58].

PIWIs are involved in mediating genomic integrity

Genomic instability is a recently emerging hallmark of cancer. The defects in genome maintenance and repair are selectively advantageous and are therefore responsible for tumor progression [26]. Epigenetic mechanisms, such as DNA methylation and histone modifications [88–90], and the caretakers of the genome, such as genes associated with DNA damage, influence genomic stability [91]. Although available data are few, PIWI proteins have been revealed to mediate genome integrity in cancer cells and this regulation is related to epigenetic mechanisms.

In hiwi-transformed sarcoma precursors, i.e. mesenchymal stem cells (MSCs), hiwi levels are inversely correlated with the levels of known TSGs (tumor suppressor genes), such as p15, p21, and p27; and hiwi-mediated tumorigenesis is associated with global DNA hypermethylation at non-promoter CpG regions and is reversible by DNA-methyltransferase inhibitors [73]. In addition, piwil2 is involved in chromatin decondensation in mouse embryonic fibroblasts (MEFs), especially in response to exposure to the DNA damaging agent cisplatin (a genotoxic agent used for cancer chemotherapy) [56] because it regulates the levels of histone modifications such as acetylation and methylation, thus enhancing cancer cell survival [56]. Furthermore, Yin et al. [77] reported that piwil2 can mediate DNA repair through an axis of histone acetylation, chromatin relaxation, and upstream DDR (DNA damage response) pathways. These observations may explain why PIWIL2 is over-expressed in some cancers.

Normally, in each cell generation, genome maintenance systems detect and resolve defects in the DNA to ensure that spontaneous mutation rates are extremely low. However, in tumorigenesis, cancer cells often have enhanced rates of mutations [92,93], and hiwi orthologs have been implicated in transposon silencing to maintain genomic integrity in normal germline cells [17,94], implying that hiwi is a caretaker of the genome and not an oncogene. That is to say, cancer cells could have decreased expression of HIWIs and increased transposon activities. Strangely, current clinical cases showed up-regulated expression of HIWI proteins in cancers. Siddiqi et al. [73] demonstrated that in PIWIL1-expressing sarcoma precursors, the expression levels of two common transposons (IAP and Line1) were severely reduced. Whether these reductions of transposon activities are the main causes of tumorigenesis or are accompanying unimportant negative effects of tumorigenesis remains to be further clarified. As we know, genomes of cancer cells are unstable. How to address the different effects of PIWIs on the germline (maintenance of genomic integrity) and cancer cells is a very interesting topic.

PIWIs May Be Biomarkers for Cancer Diagnosis and Prognosis

Most of the current investigations about PIWIs' association with tumorigenesis and cancer progression are from the reports of clinicopathologic data. These data suggested that PIWIs could be used as biomarkers for clinical diagnosis and prognosis related to poor outcome. All four human PIWI homologs have been examined, but most of these investigations focused on PIWIL1 and PIWIL2. However, clinical diagnosis measures and therapeutic medicines targeting PIWIs have not been developed thus far.

It has been shown that hiwi expression is increased gradually in normal gastric tissues, atrophic gastritis, intestinal metaplasia and gastric cancers, suggesting that hiwi may be involved in the development of gastric cancer [40]. In cervical cancer, hiwi was shown to facilitate chemoresistance and was suggested to be a cancer stem cell marker [47]. In pancreatic ductal adenocarcinoma, the elevated levels of hiwi mRNA and protein have no general impact on patients' survival. However, patients with the abnormal expression of hiwi mRNA showed a significantly increased risk for tumor-related death [43]. In patients with soft-tissue sarcomas, high level of hiwi mRNA was correlated with high risk of tumor-related death [44]. In colorectal cancer, high expression of HIWI was a significant prognostic factor for the overall survival of patients, especially for those patients at early stages or without lymph node metastasis [36]. In glioma, hiwi expression was greatly increased with the ascending of tumor grades, showing the correlation of greater positive hiwi with poorer outcome [41]. In HCC, HIWI was associated with larger tumor size and intra-hepatic metastasis, and it was also an independent risk factor for the overall survival and recurrence-free survival, particularly in patients with low serum α-fetoprotein and low Edmondson-Steiner grade [42]. In esophageal cancer, cytoplasmic HIWI, not nuclear HIWI, is significantly related to higher histological grade, clinical stage, and poorer clinical outcome [38]. Besides its use as a single marker, HIWI could increase the detection accuracy (∼80%) in colon adenocarcinoma when used in combination with synuclein-γ (SNCG), phosphatase of regenerating liver-3 (PRL-3), and arrest-defective protein 1 (ARD1) homolog A [35]. In contrast, in endometrioid adenocarcinoma, PIWIL1 was not associated with its clinicopathological features [37].

Enhanced expressions of piwil2 were found in human testicular seminomas, prostate cancer, breast cancer, gastrointestinal cancer, ovarian cancer, and endometrial cancer, and also in mouse breast cancer, rhabdomyosarcoma and medulloblastoma [51,52,57]. Interestingly, the enhanced expression of piwil2 was not seen in human testicular non-seminomas tumors, and the corresponding PIWIL2 isoform is PL2L60A, not PL2L80A [59]. This increased expression of piwil2 in various cancers suggests that PIWIL2 can be a useful prognostic factor with potential diagnostic and therapeutic applications. In patients with colorectal carcinomas, higher levels of PIWIL2 expression were found to be correlated with decreased survival rate [55]. In breast cancer, piwil2 was predominantly expressed in cancer stem cells, 81% of carcinomas in situ and 90% of invasive carcinomas [51]. He et al. [54] also reported that piwil2 was expressed in various stages of cervical cancers. Interestingly, piwil2 showed the potential to be used as a complementary biomarker for p16, a surrogate indicator of high-risk human papillomavirus (HR-HPV) infection, to improve the sensitivity and specificity of current screening methods for cervical cancers [54]. In colon cancer, piwil2 expression was significantly correlated with more aggressive clinical and pathological parameters with poorer 5-year, metastasis-free survival and overall survival [58]. Another study showed that the increased expressions of EIF2C1 and PIWIL2 were significantly associated with colon cancer, suggesting that EIF2C1 and PIWIL2 may represent novel colon cancer biomarkers with early diagnostic significance [76]. Interestingly, over-expression of piwil2 was found to contribute to the cisplatin resistance in human ovarian cancer cell lines, suggesting that PIWIL2 was a marker for cisplatin resistance in cancer chemotherapy [56]. This study demonstrated the important clinical significance of PIWIL2 for addressing the recurrence of cancer after chemotherapy.

PIWIL4 is also up-regulated in human cervical cancer tissues compared with the adjacent normal tissues, suggesting that it could be used as a new therapeutic target in the future [63]. Expression of piwil4, but not piwil1, piwil2, and piwil3, was significantly higher in the renal cell carcinomas than that in the corresponding normal renal tissues [64], suggesting that only piwil4 has clinical significance in renal cell carcinomas.

In addition to single member of the PIWI family, combined multiple members are also identified to have clinical significance. In breast cancer, PIWIL2 and PIWIL4, not PIWIL1 and PIWIL3, had high expression levels in clinical patients [60]. In gastric cancer, the expression of PIWIL1-4 was significantly correlated with the T-stage, lymph node metastasis and clinical TNM (cTNM), and elevated PIWIL1 and PIWIL2 expressions were associated with poorer overall survival, and PIWIL1 expression was suggested to be an independent prognostic factor [39]. In ovarian cancer, the expression of PIWIL1-4 in primary and metastatic tumors from patients with stage III epithelial ovarian cancer was significantly enhanced [50]. However, which member of the PIWI family is comparatively more important in tumorigenesis still remains to be investigated.

PIWI-interacting RNAs Are Related to Tumorigenesis

Although PIWI proteins are important for tumorigenesis, PIWI-interacting RNAs (piRNAs) also play important roles in this process. piRNAs are highly expressed in reproductive tissues, but are also expressed in the brain [95], and a normal human plasma-derived exosome had 1.31% piRNAs [96]. Some piRNAs can be associated with HIWI2 in somatic cells including cancer cells [97]. Many piRNAs in exponentially growing or growth-arrested human breast cancer MCF-7 cells have also been identified [70]. So far, some piRNAs, such as human piR-Hep1, piR-823, piR-651, piR-4987, piR-20365, piR-20485, piR-20582, and piRABC (Tables 1 and 2), have been found to be related to the hallmarks of cancers [60,62,65,67–69,71,98,99]. However, few literatures are available. piR-823 was found to contribute to tumorigenesis by regulating de novo DNA methylation and angiogenesis in multiple myeloma [66]. piR-932 was suggested to bind PIWIL2 to promote the methylation of Latexin in breast cancer stem cells [61]. Nevertheless, how piRNAs in complex with PIWI proteins participate in tumorigenesis, invasion and metastasis remains to be further elucidated.

Future Directions in PIWI Research

Since the discovery of PIWI as an essential regulator of gametogenesis, the unexpected roles of the PIWI family in cancer have expanded, beyond the promotion of cell proliferation, to the inhibition of cell death, the escape from growth suppression, the enhancement of cell migration and invasion, and the maintenance of genomic instability [27,28]. Studies reviewed here suggested that PIWIs might play an important role in modulating the occurrence of some cancer hallmarks in cancer cells. These data demonstrated the prognostic and therapeutic potential of PIWIs [26]. However, current investigations are preliminary, and the potential roles of PIWI proteins in regulating each cancer hallmark need to be further examined. Of note, low expression of HIWIs in human testicular tumorigenesis (both in primary seminoma and non-seminoma testicular tumors) has been reported [100], which is opposite to the previous findings in somatic cell cancers. Further studies should be carried out to clarify this inconstancy. In addition, current clinical data are still limited, and whether PIWIs can act as key markers for clinical diagnosis and prognosis remains to be further investigated.

Undoubtedly, current evidence on the molecular details of PIWIs involved in tumorigenesis and development is still lacking. The detailed mechanisms that underlie how PIWIs, PIWI-associated piRNAs or other RNA species contribute to each of these hallmarks or even emerging new hallmarks remain to be explored. This is partly due to the limited clinical samples in the current investigations. More easily manipulated materials, such as cell lines and animal models should be employed in future studies. Unfortunately, no data from animal models are currently available to elucidate the molecular mechanism. It should be noted that the question whether over-expression of PIWI members is merely a cause or a result of tumorigenesis still remains to be answered. So, in vivo over-expression animal model would be a good starting point in the future [101]. Very interesting finding of the relationship between PIWIs and cancer stem cells may bring up an important direction in future investigations, since the piwi genes have a strong conserved association with stem cells, and can even be used as a stem cell marker in many invertebrate animals [10]. Therefore, ectopic expression of the piwi genes in cancers may promote stemness. But whether and how the piwi genes promote stemness in cancers remain to be investigated. Furthermore, in most cases, evidence is still lacking to clarify whether the PIWI proteins are directly or indirectly leading to the described outcomes. So, more detailed studies are needed in the future. Such investigations will significantly broaden our knowledge of tumorigenesis and may lead to novel therapeutic applications targeting PIWIs or their related pathways.

Although our current understanding of how PIWI is involved in tumorigenesis is still very limited, some features of PIWIs' expression would make them ideal candidates for therapeutic intervention. PIWIs can act as CTAs, and targeting them may have no or little side effects on somatic tissue for anticancer therapy. However, chemotherapeutic drugs still remain to be developed. Unlike ordinary oncogenic proteins, PIWI functions have been implicated in almost all cancer-related processes such as initiation, development, migration, and invasion [78]. As such, suppressing its expression in the relevant tumors or combining with other therapeutics might provide new strategies to treat cancer. Indeed, in an xenograft mouse model, silencing of hiwi in lung cancer significantly inhibited tumor growth [102]. This shRNA-mediated delivery strategy appeared to be an effective therapeutic approach for lung cancer, and therefore provides some useful clues for RNAi gene therapy in solid cancers. The modification of the epitopes of tumor antigens could increase the binding affinity and stability and subsequently overcome the immune tolerance existing in the patients. Modifying an epitope of PL2L60 was found to enhance the activity of the native epitope to induce cytotoxic T lymphocytes [103]. This result suggested that the novel epitopes identified for PL2L60 could be used as novel candidates for the immunotherapy of patients with PL2L60-expressing tumors. Enhanced immunotherapy may be a good way to treat cancers; however, more studies are needed. Taken together, there is much hope for cancer therapy by targeting PIWIs for future drug development. However, this is just a beginning and much more work remains to be done.

In summary, well-conserved PIWI family members have been suggested to be involved in tumorigenesis and cancer progression despite their classical role in spermatogenesis. Because PIWI cancer biology is a new emerging research field, most of the current investigations are scattered and non-systematic. More profound and comprehensive investigations and detailed mechanistic studies are required before it can be clinically used.

Funding

This work was supported by the grants from Key Disciplines Group Construction Project of Pudong Health Bureau of Shanghai (No. PDWxq2014-9), Shanghai Health and Family Planning Commission (No. 201440050), Science and Technology Commission of Shanghai Municipality (No. 15ZR1437100), the National Natural Science Foundation of China (No. 81200468), and the Starting-Up Fund for Chinese-American Research Institute for Diabetic Complications from Wenzhou Medical University (No. QTJ13007).

Acknowledgement

The authors would like to thank Dr Wuzi Dong (Northwest A & F University, Yangling, China) for his help in preparing the manuscript.

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