The impact of the RASSF1C and PIWIL1 on DNA methylation: the identification of GMIP as a tumor suppressor.
ABSTRACT: INTRODUCTION:Recently we have identified a novel RASSF1C-PIWIL1-piRNA pathway that promotes lung cancer cell progression and migration. PIWI-like proteins interact with piRNAs to form complexes that regulate gene expression at the transcriptional and translational levels. We have illustrated in previous work that RASSF1C modulates the expression of the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway could potentially contribute to lung cancer stem cell development and progression, in part, through modulation of gene methylation of both oncogenic and tumor suppressor genes. Therefore, we tested this hypothesis using a non-small cell lung cancer (NSCLC) cell model to identify Candidate Differentially Methylated Regions (DMRs) modulated by the RASSF1C-PIWIL1-piRNA pathway. MATERIALS AND METHODS:We studied the impact of over-expressing RASSF1C and knocking down RASSF1C and PIWIL1 expression on global gene DNA methylation in the NSCLC cell line H1299 using the Reduced Representation Bisulfite Sequencing (RRBS) method. RESULTS:DMRs were identified by comparing DNA methylation profiles of experimental and control cells. Over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions; and statistically significant candidate genes residing DMR regions in lung cancer cells were identified, including oncogenes and tumor suppressors. One of the hypermethylated genes, Gem Interacting Protein (GMIP), displays tumor suppressor properties. GMIP expression attenuates lung cancer cell migration, and its over-expression is associated with longer survival of lung cancer patients. CONCLUSIONS:The RASSF1C-PIWI-piRNA pathway modulates key oncogenes and tumor suppressor genes. GMIP is hypermethylated by this pathway and has tumor suppressor properties.
Project description:RASSF1C up-regulates important genes involved in lung cancer cell growth, including a stem cell self-renewal gene, piwil1. In this article, we report the identification of small noncoding PIWI-interacting RNAs (piRNAs) in lung cancer cells over-expressing RASSF1C. A piRNA microarray screen was performed using RNA isolated from the lung cancer cell line H1299 stably over-expressing RASSF1C and corresponding control. The piRNA microarray screen identified several piRNAs that are regulated by RASSF1C and we have validated the expression of two up-regulated piRNAs (piR-34871 and piR-52200) and two down-regulated piRNAs (piR-35127 and piR-46545) in lung cancer cells with silenced and over-expressed RASSF1C using RT-PCR. We also assessed the expression of these four piRNAs in lung tumor and matched normal tissues (n = 12). We found that piR-34871 and piR-52200 were up-regulated in 58% and 50%, respectively; while piR-35127 and piR-46545 were down-regulated in 50% in lung tumor tissues tested. The expression of piR-35127 was inversely correlated with RASSF1C expression in 10/12 tumor tissues. Over-expression of piR-35127 and piR-46545 and knock-down of piR-34871 and piR-52200 significantly reduced normal lung and breast epithelial cell proliferation and cell colony formation as well as proliferation of lung cancer cell lines (A549 and H1299) and breast cancer cell lines (Hs578T and MDA-MB-231). This suggests that these novel piRNAs may potentially be involved in regulating lung cell transformation and tumorigenesis. RASSF1C may potentially modulate the expression of its piRNA target genes through attenuation of the AMPK pathway, as over-expression of RASSF1C resulted in reduction of p-AMPK, p21, and p27 protein levels.
Project description:The importance of the Piwi-interacting RNA (piRNA) pathway for germ cell maintenance, genome integrity, DNA methylation and retrotransposon control raises possible roles of this pathway in cancer. Indeed aberrant expression of human PIWI orthologs and Maelstrom has been observed in various cancers. In this study we explored the expression and function of piRNA pathway genes in human ovarian cancer, based on our recent work, which showed widespread expression of piRNA pathway genes in the mammalian. Our work shows that PIWIL1 and MAEL expression is significantly increased in malignant EOC (n = 25) compared to benign tumor tissues (n = 19) and normal ovarian tissue (n = 8). The expression of PIWIL3 is lower in malignant and benign tissues when compared to normal ovary. Sequencing of PIWIL1 transcript revealed that in many tumors deletion of exon 17 leads to the introduction of a premature stop codon in the PIWI domain, likely due to a splicing error. In situ hybridization on tumor sections revealed that L1, PIWIL1, 2 and MAEL are specifically expressed in epithelial cells (cancerous cells) of EOC. Furthermore, PIWIL2 and MAEL are co-expressed in the stromal cells adjacent to tumor cells. Since PIWIL1 and MAEL are up regulated in malignant EOC and expressed in the epithelial cells, we investigated if these two genes affect invasiveness of ovarian cancer cell lines that do not normally express these genes. PIWIL1 and MAEL were transiently over expressed in the ovarian cancer cell line SKOV3, followed by real-time measurements of cell invasiveness. Surprisingly both PIWIL1 and MAEL over expression decreased the invasiveness of SKOV3 cells. Our findings support a growing body of evidence that shows that genes in this pathway are upregulated in cancer. In ovarian cancer we show for the first time that Piwil1 transcript may often be abnormal result in non functional product. In contrast to what has been observed in other cell types, we found that PIWIL1 and MAEL have a repressive effect on cell invasiveness.
Project description:The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.
Project description:Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, PIWI proteins, a subfamily of the PAZ-PIWI domain (PPD) protein family, have emerged as promising candidates for targeted cancer therapy. PPD proteins are essential for small noncoding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the PIWI subfamily partners with PIWI-interacting RNA (piRNA). Both PIWI proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, PIWI proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of PIWI proteins in cancer. Here we report that one of the four PIWI proteins in humans, PIWIL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out the PIWIL1 gene (<i>PIWIL1-</i>KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, few bona fide piRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PIWIL1 does not affect its oncogenic function. Thus, PIWIL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a piRNA-independent function of PIWIL1 in promoting gastric cancer.
Project description:Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis. In addition to the well-recognized microRNAs, recent studies have also shown that epigenetic silencing by CpG island hypermethylation of other classes of non-coding RNAs, such as transcribed ultraconserved regions (T-UCRs) or small nucleolar RNAs (snoRNAs), also occur in human neoplasia. Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development. We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines. Most importantly, these epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation of the LINE-1 repetitive sequences, one of the target genomic loci where the PIWI/piRNA machinery acts as a caretaker in non-transformed cells.
Project description:The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.
Project description:The blood-tumor barrier (BTB) restricts the efficient delivery of anti-glioma drugs to cranial glioma tissues. Increased BTB permeability may allow greater delivery of the therapeutic agents. Increasing evidence has revealed that PIWI proteins and PIWI-interacting RNAs (piRNAs) play an important role in tumor progression. However, whether PIWI proteins and piRNAs regulate BTB permeability remains unclear. In the present study, we demonstrated that the PIWIL1/piRNA-DQ593109 (piR-DQ593109) complex was the predominant regulator of BTB permeability. Briefly, PIWIL1 was upregulated in glioma endothelial cells (GECs). Furthermore, piR-DQ593109 was also overexpressed in GECs, as revealed via a piRNA microarray. Downregulation of PIWIL1 or piR-DQ593109 increased the permeability of the BTB. Moreover, PIWIL1 and piR-DQ593109, which formed a piRNA-induced silencing complex, degraded the long non-coding RNA maternally expressed 3 (MEG3) in a sequenced-dependent manner. Furthermore, restoring MEG3 released post-transcriptional inhibition of Runt related transcription factor 3 (RUNX3) by sponging miR-330-5p. In addition, RUNX3 bounded to the promoter regions and reduced the promoter activities of ZO-1, occludin, and claudin-5, which significantly impaired the expression levels of ZO-1, occludin, and claudin-5. In conclusion, downregulating PIWIL1 and piR-DQ593109 increased BTB permeability through the MEG3/miR-330-5p/RUNX3 axis. These data may provide insight into glioma treatment.
Project description:PIWI-interacting RNA (piRNA) silences the transposons in germlines or induces epigenetic modifications in the invertebrates. However, its function in the mammalian somatic cells remains unknown. Here we demonstrate that a piRNA derived from Growth Arrest Specific 5, a tumor-suppressive long non-coding RNA, potently upregulates the transcription of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a proapoptotic protein, by inducing H3K4 methylation/H3K27 demethylation. Interestingly, the PIWIL1/4 proteins, which bind with this piRNA, directly interact with WDR5, resulting in a site-specific recruitment of the hCOMPASS-like complexes containing at least MLL3 and UTX (KDM6A). We have indicated a novel pathway for piRNAs to specially activate gene expression. Given that MLL3 or UTX are frequently mutated in various tumors, the piRNA/MLL3/UTX complex mediates the induction of TRAIL, and consequently leads to the inhibition of tumor growth.
Project description:Young neurons in the developing brain establish a polarized morphology for proper migration. The PIWI family of piRNA processing proteins are considered to be restrictively expressed in germline tissues and several types of cancer cells. They play important roles in spermatogenesis, stem cell maintenance, piRNA biogenesis, and transposon silencing. Interestingly a recent study showed that de novo mutations of PIWI family members are strongly associated with autism.Here, we report that PIWI-like 1 (PIWIL1), a PIWI family member known to be essential for the transition of round spermatid into elongated spermatid, plays a role in the polarization and radial migration of newborn neurons in the developing cerebral cortex. Knocking down PIWIL1 in newborn cortical neurons by in utero electroporation of specific siRNAs resulted in retardation of the transition of neurons from the multipolar stage to the bipolar stage followed by a defect in their radial migration to the proper destination. Domain analysis showed that both the RNA binding PAZ domain and the RNA processing PIWI domain in PIWIL1 were indispensable for its function in neuronal migration. Furthermore, we found that PIWIL1 unexpectedly regulates the expression of microtubule-associated proteins in cortical neurons.PIWIL1 regulates neuronal polarization and radial migration partly via modulating the expression of microtubule-associated proteins (MAPs). Our finding of PIWIL1's function in neuronal development implies conserved functions of molecules participating in morphogenesis of brain and germline tissue and provides a mechanism as to how mutations of PIWI may be associated with autism.
Project description:OBJECTIVE:Smoking remains a primary etiological factor in head and neck squamous cell carcinoma (HNSCC). Given that non-coding RNAs (ncRNAs), including PIWI-interacting RNAs (piRNAs), have emerged as mediators of initiation and progression in head and neck malignancies, we undertook a global study of piRNA expression patterns in smoking-associated HNSCC. MATERIALS AND METHODS:Using RNA-sequencing data from 256 current smoker and lifelong nonsmoker samples in The Cancer Genome Atlas (TCGA), we analyzed the differential expression patterns of 27,127 piRNAs across patient cohorts stratified by tobacco use, with HPV16 status and tumor status taken into account. We correlated their expression to clinical characteristics and to smoking-induced alterations of PIWI proteins, the functional counterparts of piRNAs. Finally, we correlated our identified piRNAs and PIWI proteins to known chromosomal aberrations in HNSCC to understand their wider-ranging genomic effects. RESULTS AND CONCLUSION:Our analyses implicated a 13-member piRNA panel in smoking-related HNSCC, among which NONHSAT123636 and NONHSAT113708 associated with tumor stage, NONHSAT067200 with patient survival, and NONHSAT081250 with smoking-altered PIWIL1 protein expression. 6 piRNAs as well as PIWIL1 correlated with genomic alterations common to HNSCC, including TP53 mutation, TP53-3p co-occurrence, and 3q26, 8q24, and 11q13 amplification. Collectively, our findings provide novel insights into the etiology-specific piRNA landscape of smoking-induced HNSCC.