**2. PIWIs and piRNAs as potential biomarkers and therapeutic tools**

### **2.1. PIWI proteins as biomarkers**

PIWIL1 overexpression was correlated with poor 5-year survival in malignant glioma, hepatocellular, gastric cancer, colon cancer, pancreatic ductal adenocarcinoma, and ovarian cancer [1]. In hepatocarcinoma, PIWIL1 high levels of expression were correlated with larger tumor size, intrahepatic metastasis, overall survival, and recurrence-free survival [41]. PIWIL2 upregulation was observed in breast cancer, colon cancer, gastrointestinal stromal tumors, renal cell carcinoma, and endometrial carcinoma. In colorectal cancer, PIWIL2 overexpression was associated with aggressive clinico-pathological characteristics and poorer prognostic [30]. In esophageal cancer, PIWIL2 was significantly correlated to high histological grade, advanced clinical stage, and poorer clinical outcomes. Furthermore, PIWIL2 upregulation was identified in a cancer cell subpopulation expressing OCT4 and NANOG stemness factors, suggesting a role in stem-cell maintenance and self-renewal. In breast cancer, PIWIL2 was mainly located in cancer stem cells and showed different expression patterns, with slight nuclear localization in preneoplastic lesions and cytoplasmic/nucleus topography in invasive and metastatic cancers. Its upregulation was correlated with poorer survival [33]. PIWIL2 can promote invasion and metastasis among various cancers by increasing c-Myc expression through binding of NM/ NM23 nucleoside diphosphate kinase 2 (NME2) to G4-motif region within c-Myc. In hepatocellular carcinoma, nuclear co-expression of PIWIL2 and PIWIL4 had a worse prognostic phenotype [71]. In ovarian cancer, PIWIL2 overexpression was associated with cisplatin resistance and poorer prognostic [28]. PIWIL3 and PIWIL4 overexpression were associated with worse prognosis in breast cancer. In gastric cancer, PIWIL1-4 upregulation was significantly associated with TNM stage histological grade and lymph node metastasis [15, 40, 62].

### **2.2. PiRNAS as biomarkers**

integration [56]. A widespread hypomethylation of the genome during progression could induce both aberrant reactivation and propagation of TEs with reactive ectopic overexpression of the PIWI-piRNA pathway. High levels of piRNAs and PIWI proteins observed in somatic cancers could promote abnormal extensive DNA methylation, resulting in silencing of TSGs and acquisition of an aberrant stem-like state [57, 58]. The hypothesis that piRNAs guide PIWIL1 and PIWIL2 to transcriptionally silence TSGs is consolidated by analysis of their orthologs in mice, in which MILI and MIWI2 mutants fail to establish DNA methylation of TEs [59]. Furthermore, by suppressing expression of particular TEs, aberrant reactivation of the PIWI-piRNA axis in cancer may induce genomic and chromosomal instability. Thus, this pathway could be implicated in cancer not only in repression of TEs but also in inactivation of various mRNAs not exposed to germline cells. The second hypothesis is the participation of this pathway in a putative biological system that can differentiate self from nonself RNA [60]. The third hypothesis is the ectopic and coordinated reactivation and overexpression in cancer of cancer/testis antigens (CTA) normally restricted to the germline and including molecules of the PIWI-piRNA axis. As observed in normal gametogenesis, CTAs promote immortalization, implantation, and migration that could correspond in carcinogenesis to transformation, invasion, and metastasis, respectively (**Figure 1**). In this ectopic context, reactivation of the PIWI-piRNA pathway could

confer properties of proliferative germline stem cells to cancer cells [61–68].

**2.1. PIWI proteins as biomarkers**

272 Chromatin and Epigenetics

**2. PIWIs and piRNAs as potential biomarkers and therapeutic tools**

PIWIL1 overexpression was correlated with poor 5-year survival in malignant glioma, hepatocellular, gastric cancer, colon cancer, pancreatic ductal adenocarcinoma, and ovarian cancer [1]. In hepatocarcinoma, PIWIL1 high levels of expression were correlated with larger tumor size, intrahepatic metastasis, overall survival, and recurrence-free survival [41]. PIWIL2 upregulation was observed in breast cancer, colon cancer, gastrointestinal stromal tumors, renal cell carcinoma, and endometrial carcinoma. In colorectal cancer, PIWIL2 overexpression was associated with aggressive clinico-pathological characteristics and poorer prognostic [30]. In esophageal cancer, PIWIL2 was significantly correlated to high histological grade, advanced clinical stage, and poorer clinical outcomes. Furthermore, PIWIL2 upregulation was identified in a cancer cell subpopulation expressing OCT4 and NANOG stemness factors, suggesting a role in stem-cell maintenance and self-renewal. In breast cancer, PIWIL2 was mainly located in cancer stem cells and showed different expression patterns, with slight nuclear localization in preneoplastic lesions and cytoplasmic/nucleus topography in invasive and metastatic cancers. Its upregulation was correlated with poorer survival [33]. PIWIL2 can promote invasion and metastasis among various cancers by increasing c-Myc expression through binding of NM/ NM23 nucleoside diphosphate kinase 2 (NME2) to G4-motif region within c-Myc. In hepatocellular carcinoma, nuclear co-expression of PIWIL2 and PIWIL4 had a worse prognostic phenotype [71]. In ovarian cancer, PIWIL2 overexpression was associated with cisplatin resistance and poorer prognostic [28]. PIWIL3 and PIWIL4 overexpression were associated with PiRNAs are increasingly investigated as diagnostic and prognostic biomarkers [69]. PiRNAs are small RNAs of only 24–32 nt in length which can pass through cell membrane. Like miR-NAs, piRNAs are not degraded in circulation and possess the capability to resist incubation and storage conditions used in laboratories. They are thus detectable in patient samples like blood plasma and serum, saliva, sputum, and urine. Furthermore, piRNAs possess higher sensitivity and specificity compared to an existing lncRNA and miRNA-based biomarkers. They are highly expressed in germinal tissues, but also although at lower levels in various somatic normal tissues, saliva, and plasma-derived exosomes. Furthermore, piRNAs present higher specificity and sensitivity, when compared to miRNAs. In breast cancer, 8 piRNAs were identified as independent prognostic markers and associated with overall survival [63]. Moreover, piR-4987 upregulation in peripheral blood was correlated with lymph node metastasis [48]. In gastric cancer, piR-651 high level of expression in peripheral blood was associated with poor differentiation, advanced TNM stage, and metastasis. A three-piRNA signature, including piR-59056, piR-54878, and piR-62701, could separate patients by risk of recurrence [5]. PiR-651 was also upregulated in other cancers, including lung, colon, and breast tumors. In clear cell renal cell carcinoma, piR-30924 and piR-57125 were shown to be independent prognostic predictors in nonmetastatic patients. In colon cancer, piR59056, piR-54878, and piR-62701 were associated with recurrence-free survival. Few studies have investigated on interactions between piRNAs and PIWI proteins in cancer [70, 72–75]. Actually, it remains unclear whether PIWI proteins independently possess cytoplasmic pro-oncogenic properties that promote cancer cell proliferation, invasion, and metastasis or PIWI proteins located in the nucleus epigenetically modulate numerous biological processes by edifying ribonucleoproteic complexes with piRNAs.

### **2.3. PIWI proteins and piRNAs as therapeutic tools**

Recent studies have revealed the potential for piRNAs as therapeutic tools that target PIWI proteins and oncoproteins [71]. Mounting evidence has revealed that upregulation of PIWI proteins was negatively correlated with patient survival, and that downregulation of PIWI proteins could reduce the number of G2/M phase cells and enhance expression of p53 protein, thus inhibiting proliferation and promoting apoptosis. In addition, PIWI proteins could increase resistance to chemotherapy drugs such as cisplatin, and their downregulation could increase sensitivity of cancer cells to cisplatin. In this way, synthetic piRNAs targeting PIWI genes are potential pertinent tools in transcriptional silencing during cancer progression, and PIWI antibodies targeting PIWI proteins constitute another approach to antagonize cancer cells proliferation at post-transcriptional level. Synthetic piRNAs could also block synthesis of cancer-related proteins by binding to mRNAs. Compared to miRNAs, piRNAs do not require enzymes processing and have better specificity to targets. In a mouse model, artificial piRNAs could be obtained through expression of sense and antisense transcripts, resulting in epigenetic silencing of target genes [1, 72]. Furthermore, piRNAs could be considered as tools to regulate expression levels of TSGs. A lncRNA (*GAS5*)/SnoRNA-derived piRNA enhanced activation of *TRAIL* gene by site-specifically recruiting MLL/COMPASS-like complexes with induction of H3K4 methylation and H3K27 demethylation, resulting in transcriptional activation of *TRAIL* and inhibition of tumor growth [64].

**Author details**

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