Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway

Abstract

Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.

Keywords: Hippo; Piwi; Ras; Warts; piRNA; transposon.

Figure 1.

Wts-RNAi and activated Ras^V12 induce ectopic expression of Piwi in wing discs. (A) hsFLP,Act-Gal4>UAS-RFP control clones (red) mark the nucleus. Wts-RNAi;Ras^V12 (B), Wts-RNAi (C), and Ras^V12 (D) wing disc clones express Piwi. (E) Cells with nuclear endogenous Piwi (left; Nu) and cytoplasmic PIWI (right; Cy) foci coexist in the developing female Wts-RNAi;Ras^V12 larval gonad, stained in the same well as discs. (Blue) DAPI; (red) UAS-RFP clones; (green) α-Piwi. (Left panels) DAPI/RFP merge. (Right panels) RFP/α-Piwi merge.

Figure 2.

piRNA machinery and Piwi-bound piRNAs in WRR-1 cells. (A) Piwi localizes to the nucleus of ovarian somatic sheet (OSS) and WRR-1 cells and is undetectable in two Ras^V12 lines (R3 and R7). (Blue) DAPI; (red) α-Piwi. (B) Transcript levels of piRNA pathway components in Ras^V12 (R3 and R7), WRR-1, ovary, and OSS cells. (C) Piwi mRNA levels are shown in OSS cells compared with WRR-1 cells by quantitative PCR (qPCR). Dspt4 served as a control. (D) Piwi-bound piRNAs originating from clusters in WRR-1 cells show a 5′ U bias, similar to OSS cells. (E) Genomic clusters generate primarily 23- to 29-nt piRNAs associated with Piwi (Piwi immunoprecipitation [IP] #1 and #2). (F) Genomic distribution of small RNAs cloned from whole (total) and Piwi-bound (immunoprecipitation) fractions in OSS and WRR-1 cells and in control ovaries. (G) piRNA abundance at clusters in WRR-1, OSS cells, and ovaries. Clusters are ranked by piRNA abundance in WRR-1 cells. piRNAs from genomic clusters associate with Piwi in immunoprecipitations from OSS and WRR-1, shown as enrichment [log₂(RPM IP/total)].

Figure 3.

The effects of depleting piRNA pathway components. (A) Piwi staining in the indicated knockdown cells. Immunofluorescence was performed 5 d after transfection of dsRNAs against each indicated gene. Piwi protein is undetectable in most Piwi-depleted cells and aggregates in cytoplasmic foci in Zuc and Armi knockdowns (insets, arrowheads). (Blue) DAPI; (red) α-Piwi. (B) Piwi, Armi, and Zuc knockdowns impair piRNA production; Aub or Ago3 depletion has no effect. (C, top panel) Piwi, Zuc, and Armi knockdowns decrease piRNA levels at most clusters, including flamenco. (Bottom panel) Depletion of Aub or Ago3 shows little to no effect on piRNA production at clusters. Depletion of GFP (control) has no effect.

Figure 4.

The primary pathway generates piRNAs matching a broad spectrum of transposons. (A) The most abundant piRNAs matching to transposable elements (TEs) in WRR-1 cells correspond primarily to somatic and intermediate transposons (gypsy elements, idefix, or ZAM), consistent with their production from the flamenco locus. These piRNAs are associated with Piwi. (Bottom) Fewer piRNAs are derived from germline-specific transposons. (B) piRNA levels decrease in Piwi, Zuc, or Armi knockdowns but remain largely unaltered upon depletion of Aub or Ago3. (C,D) piRNAs matching consensus TEs present a 5′ U bias (C) and are selectively bound to Piwi (D) in WRR-1 cells, in contrast to 21-nt endo-siRNAs originating from TEs. (E) Piwi, Zuc, or Armi depletion selectively abolishes the production of 23- to 29-nt TE-matching piRNAs.

Figure 5.

Transposons are regulated by the primary piRNA machinery. Transposon transcripts are up-regulated in Piwi, Zuc, and Armi knockdowns and remain unchanged in Aub and Ago3 knockdowns. Colored dots represent the expression of a transposon as log₁₀(RPM) and its somatic or germline classification.

Figure 6.

Piwi promotes the growth of WRR-1 cells and affects gene expression programs. (A) Proliferation of WRR-1 cells decreases upon Piwi knockdown, as compared with control (nontransfected) and GFP knockdown cells. A representative EdU pulse-labeling assay is presented as the percentage of EdU-labeled cells in the population detected following a 5-h pulse. (B) One-hundred-fifty-six genes producing piRNAs in WRR-1 cells show susceptibility to depletion of primary piRNA components at the steady-state RNA level (RNA-seq). For 91 piRNA-producing genes, mRNA levels increase more than twofold upon knockdown of Piwi, Zuc, or Armi, including 22 common up-regulated genes. Transcript levels of 64 genes decrease upon depletion of primary components, 12 of which are down-regulated in knockdowns of Piwi, Zuc, and Armi.