Abstract 2493: Inhibition of phosphoinositide 3-kinase suppressed growth of cancer cells with PIK3CA mutations or loss of PTEN expression via G0/G1 arrest of cell cycle but not apoptosis

Abstract

Abstract Background: The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in cancer by gain-of-function mutation of PIK3CA and loss of PTEN expression, and mediates growth- and anti-apoptotic signals. Therefore, PI3K is considered as a promising therapeutic target to inhibit growth and to induce apoptosis in such cancers. We previously developed a novel PI3K-specific inhibitor ZSTK474 with antitumor efficacy (Yaguchi et al, J. Natl. Cancer Inst. 98: 545-56, 2006; Kong and Yamori, Curr. Med. Chem. 16: 2839-54, 2009). In this study, we have examined the cellular mechanism by which ZSTK474 exerts its antitumor efficacy in human cancer cells with or without PIK3CA/PTEN aberrations. Methods: We examined the antitumor effect of ZSTK474 on various human cancer cell lines with or without PIK3CA/PTEN aberration in vitro and in vivo. Protein expression and apoptosis in ZSTK474-treated and untreated tumor sections were analyzed by immunohistochemistry and TUNEL assay, respectively. Analysis of cell cycle in vitro was determined by flow cytometry and by Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) system introduced to cancer cells. Induction of apoptosis in vitro was estimated by caspase activation using a substrate cleavage assay and by detection of subdiploid cells using flow cytometry. Results: In vivo, ZSTK474 effectively inhibited the growth of human tumor xenograft derived from a PTEN-null prostate cancer cell line PC3. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. Similar result was obtained in another PTEN-negative cell line KM-12 and three PIK3CA-mutated cell lines HCT-15 (E545K), MKN1 (E545K) and SK-OV3 (H1047R). In vitro, ZSTK474 induced marked G0/G1 arrest accompanied by a decrease of nuclear cyclin D1, induction of p27kip1 and p15ink4b, and hypophosphorylation of pRB in PC-3 cells. However, ZSTK474 did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. Similar result was obtained other cell lines with or without PIK3CA /PTEN aberrations. Conclusion: These results clearly indicated that ZSTK474 did not induce apoptosis but rather induced strong G0/G1 arrest in cancer cells with or without PIK3CA/PTEN aberrations, which might cause its therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2493.