PIWIL2 downregulation in colon cancer promotes transposon activity and pro-tumorigenic phenotypes

Reactivation of transposable elements (TEs) in somatic tissues, particularly of LINE-1, is associated with disease by causing gene mutations and DNA damage. Previous work has shown that the PIWI pathway is crucial for TE suppression in the germline. However, the status and function of this pathway is not well characterized in differentiated somatic cells and there is lack of consensus on the role of the pathway in somatic tumorigenesis. To shed light on this conundrum, we examined the PIWI pathway in colon cancer through combining bioinformatic analyses and cell-based assays. Shifted Weighted Annotation Network (SWAN) analysis revealed that the pathway experiences significant allelic losses in colon cancer and that PIWIL2, the main catalytic component of the pathway responsible for TE silencing, experiences the highest percent deletions. PIWIL2 is downregulated in colon tumors of advanced stage, nodal metastasis, and in certain subtypes, correlating with poor survival, while it is also downregulated in ulcerative colitis, an inflammatory bowel disease that predisposes to colon cancer. PIWIL2 depletion in colon epithelial Caco2 cells leads to increased anchorage-independent growth, loss of transposon-targeting - non-canonical - piRNAs, increased LINE-1 levels and activity, and in DNA damage, altogether highlighting a tumor-suppressing role of PIWIL2 in the colon. Overall design: Caco2 wild type cells and Caco2 cells with a CRISPR/Cas9 - mediated PIWIL2 knckout were grown to confluency and RNA was isolated by adding 1ml of Trizol (Invitrogen # 15596018), incubating the sample at RT for 5 minutes then using the PureLink RNA mini kit (Invitrogen #12183018A) for total RNA, using the whole transcriptome protocol of the kit. Final RNA concentrations and purity were determined by using a NanoDrop spectrophotometer. short RNA sequencing was performed using the NovaSeq SE50 strategy. Sequenced reads were first processed using Cutadapt (v1.15) to trim the 3' adapter sequence (5'-AGATCGGAAGAGCACACGTCT-3'). Adapter-trimmed reads were analyzed using the piPipes small RNA pipeline to quantify transposon-mapped reads and normalize sense and antisense counts as counts per million (CPM) uniquely mapped to the hg19 genome(Han et al., 2015b). In parallel, reads were aligned to Repbase and known piRNA clusters using Bowtie2(Langmead and Salzberg, 2012, Langmead et al., 2019) and only successfully aligned reads were retained. These reads were then assessed for size distribution and 5' nucleotide signature using the TBr2_basicanalyses.pl script (Rosenkranz et al., 2015). smallRNA-Seq