The Clinical Significance of PIWIL3 and PIWIL4 Expression in Pancreatic Cancer

Abstract

P-element-induced wimpy testis (PIWI) proteins have been described in several cancers. PIWIL1 and PIWIL2 have been recently evaluated in pancreatic cancer, and elevated expression of PIWIL2 conferred longer survival to patients. However, PIWIL3's and PIWIL4's role in carcinogenesis is rather controversial, and their clinical implication in pancreatic cancer has not yet been investigated. In the present study, we evaluated PIWIL1, PIWIL2, PIWIL3 and PIWIL4 expression in pancreatic cancer-derived cell lines and in one non-tumor cell line as healthy control. Here, we show a differential expression in tumor and non-tumor cell lines of PIWIL3 and PIWIL4. Subsequently, functional experiments with PIWIL3 and/or PIWIL4 knockdown revealed a decrease in the motility ratio of tumor and non-tumor cell lines through downregulation of mesenchymal factors in pro of epithelial factors. We also observed that PIWIL3 and/or PIWIL4 silencing impaired undifferentiated phenotype and enhanced drug toxicity in both tumor- and non-tumor-derived cell lines. Finally, PIWIL3 and PIWIL4 evaluation in human pancreatic cancer samples showed that patients with low levels of PIWIL4 protein expression presented poor prognosis. Therefore, PIWIL3 and PIWIL4 proteins may play crucial roles to keep pancreatic cell homeostasis not only in tumors but also in healthy tissues.

Keywords: EMT; HNF4A; PIWI proteins; PIWIL3; PIWIL4; chemoresistance; motility; pancreatic cancer; survival.

Figure 1

P-element-induced wimpy testis (PIWI) proteins present differential expression in pancreatic cancer (PC), and a late downregulation of PIWIL3 or PIWIL4 in tumor cell lines was found compared to non-tumor cell line. (A) Western blot analysis, and (B) representative micrographs of immunohistochemical staining of a panel of five human PC-derived cell lines and one non-tumor pancreatic cell line (hTERT-HPNE). A human testis tissue was used as positive control. Two independent downregulations of PIWIL3 (top) and PIWIL4 (bottom) were performed to carry out functional experiments with PL45 (C), RWP1 (D) and hTERT-HPNE (E). Crtl: control. kDa: kilodalton. Scr: Scramble. D1–6: Days 1–6. PIWIL3/Actin or PIWIL4/Actin ratio is represented under each protein band. Scale bar: 50 µm.

Figure 2

Downregulation of PIWIL3 and/or PIWIL4 decreased motility of PC and non-tumor cell lines through regulation of epithelial-to-mesenchymal transition (EMT). (A) Micrographs of wound healing assay showed reduced cell motility after PIWIL3 and/or PIWIL4 silencing in both PC-derived cell lines and in the non-tumor pancreatic-derived cell line. Representative images have been taken at 0 and 24 h after scratching. Broken lines indicate migration heads. (B) Statistical analyses of the motility ratio for each cell line according to PIWIL3 and/or PIWIL4 silencing. (C) Representative images from Boyden chamber assay of different cell lines taken at 24 h after seeding. (D) Statistical analyses of the number of migrated cells for each cell line according to PIWIL3 and/or PIWIL4 silencing. (E) Western blot for the expression of PIWIL3 (left) or PIWIL4 (right), Fibronectin, Vimentin, Slug, E-Cadherin and Occludin in PL45 and RWP1 after PIWIL3 or PIWIL4 silencing. The ratio of each protein/Actin ratio is represented under each protein band. Color-coding for each protein downregulation is indicated in the legend box. kDa: kilodalton. Scale bar: 50 µm. *p < 0.05; ***p < 0.001.

Figure 3

PIWIL3 and PIWIL4 impair undifferentiated phenotype. (A) Representative micrographs of undifferentiated pancreatic spheres derived from RWP1 and hTERT-HPNE transfected with siRNA for PIWIL3 (siPIWIL3) or PIWIL4 (siPIWIL4) downregulation individually or in combination. (B) Statistical analyses of number and diameter of spheres according to PIWIL3 and/or PIWIL4 downregulation of RWP1 cell line. (C) Statistical analyses of number and diameter of spheres according to PIWIL3 and/or PIWIL4 downregulation of the non-tumor hTERT-HPNE cell line. Color-coding for each protein downregulation is indicated in the legend box. Scr: scramble. Scale bar: 50 µm. * p < 0.05; *** p < 0.001.

Figure 4

PIWIL3 and PIWIL4 downregulation potentiates the cytotoxic effect of chemotherapy. (A) Cell viability analyses after PIWIL3 and/or PIWIL4 silencing according to Gemcitabine (left) or Nab-Paclitaxel (center) individual treatments or in combination (right) of RWP1 cell line, PL45 (B) and hTERT-HPNE (C) cell lines. (D) Scatterplot and statistical analysis of HNF4A mRNA expression (y axis) and PIWIL4 mRNA expression (x axis) of 178 patient cohort from The Cancer Genome Atlas (TCGA). (E) Representative micrographs of HNF4A low expression (top-left) and high expression top-right). Statistical association between HNF4A and PIWIL4 protein expression of 182 PC samples (bottom). Color-coding for each protein downregulation is indicated in the legend box. Scale bars: 50 µm. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Prognostic impact of PIWIL3 or PIWIL4 in PC patients from the training set. (A) Representative micrographs of tumors with low (left), intermediate (middle) and high PIWIL3 expression (right). (B) Representative micrographs of tumors with low (left), intermediate (middle) and high PIWIL4 expression levels (right). (C) Kaplan–Meier curves according to PIWIL3 protein expression for both progression-free (top) and overall survival (bottom). (D) Kaplan–Meier curves according to PIWIL4 protein expression for both progression-free (top) and overall survival (bottom). p-values were obtained by log-rank test. Scale bars: 50 µm.

Figure 6

Prognostic impact of PIWIL3 or PIWIL4 in PC patients from the validation set. (A) Kaplan–Meier curves according to PIWIL3 protein expression for progression-free survival. (B) Kaplan–Meier curves according to PIWIL3 protein expression for overall survival. (C) Kaplan–Meier curves according to PIWIL4 protein expression for progression-free survival. (D) Kaplan–Meier curves according to PIWIL4 protein expression for overall survival. p-values were obtained by log-rank test.