Abstract B62: Protein inhibitor of activated STAT1 (PIAS1) is expressed in prostate cancer and stimulates cellular proliferation through inhibition of p21

Abstract

Abstract Introduction: PIAS (Protein Inhibitors of Activated STAT) proteins have originally been identified as inhibitors of the JAK/STAT pathway but they have a wide role in the regulation of transcription. Besides their DNA binding ability they also act as SUMO-E3 ligases, thus interacting with and modulating the activity of various proteins. Whilst PIAS proteins have previously been reported to regulate androgen receptor activity in prostate cancer, studies that systematically address PIAS protein expression patterns and their role in the regulation of cell growth and cell death in cancer have not been performed yet. The aim of this study is to investigate both in vivo and in vitro expression patterns of PIAS1 in prostate specimens and cell lines and to elucidate the functional role of PIAS1 in the regulation of proliferation, cell cycle progression, and apoptosis. Experimental procedures: In vivo expression of PIAS1 was determined by immunohistochemical analysis of a tissue microarray of benign and malignant human prostate cancer samples (n=90). For in vitro studies, the expression levels of mRNA and protein were determined by Western Blot and Real Time PCR. Localization studies were performed by immunofluorescence and cell fractionation. For down-regulation of PIAS1, cells were transfected with 25nM of siRNA. For overexpression of PIAS1, cells were transfected with 3μg of pEGFP-PIAS1. Proliferation was measured using [3H] thymidine incorporation assay. Apoptosis and cell cycle analysis were determined by FACS after propidium iodide staining. Clonogenicity was determined by a 2-D colony formation assay. Results: Tissue microarray data reveal that PIAS1 expression is significantly higher in malignant areas in comparison to benign tissue in clinical prostate cancer specimens. Furthermore, the staining pattern of PIAS1 correlates with the staining patterns of the proliferation markers PCNA and Ki67, thus suggesting a pro-proliferative role for PIAS1. Most benign and malignant prostate cell lines express PIAS1 mRNA and protein. PIAS1 predominantly shows nuclear localisation. Downregulation of PIAS1 leads to a ∼50% decrease in cell proliferation and clonogenicity after 96 h. At the same time, PIAS1 depletion does not significantly influence apoptosis. Decreased proliferation correlates with an increased nuclear expression of the cell-cycle regulator p21 at mRNA and protein level (up to 6-fold). Furthermore, PIAS1 down-regulation leads to a significant increase of the percentage of cells in G1-phase and a decrease of cells in S-phase of the cell cycle. Overexpression of PIAS1 decreases expression of p21, leading to a significant increase of cells in S-phase, and subsequently to an accelerated proliferation rate. Conclusion: Our data indicate that PIAS1 has pro-proliferative effects in prostate cancer cell lines and tissue. We demonstrate that PIAS1 influences cell cycle progression via the regulation of p21 expression and thereby affects cell proliferation. Our results clearly show that PIAS1 may be a new target for improved prostate cancer therapy. Citation Format: Julia Hoefer, Martin Puhr, Georg Schaefer, Helmut Klocker, Holger Erb, Ian G. Mills, Ludger Hengst, Zoran Culig. Protein inhibitor of activated STAT1 (PIAS1) is expressed in prostate cancer and stimulates cellular proliferation through inhibition of p21 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B62.