Loss of LKB1/STK11 expression is an early event in prostate cancer development and predicts therapeutic response to p38α inhibitor

Abstract

INTRODUCTION & OBJECTIVES: Prostate cancer is the most prevalent male cancer and the second leading cause of cancer-related death in men. Several genetic changes have been reported to occur in prostate cancer. Of note, it has been recently shown that conditional Lkb1 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). Recent evidence indicates that overexpression of p38 and overactivation of p38 signaling occur in benign prostate hyperplasia and more markedly in prostate cancer patients, enhancing cell proliferation and cell survival. In this study we evaluated the role of LKB1 in prostate carcinogenesis and the association existing between the activity of the LKB/AMPK pathway and the p38 signaling cascade, with the aim to identify novel therapeutic targets. MATERIAL & METHODS: This study was performed on tissue specimens derived from 22 radical retropubic prostatectomies from patients with prostate cancer. Moreover tissue microarrays containing 150 prostate cancer samples were performed. Prostate cancer cell lines, PC3,DU145 and VCaP, and primary culture of prostate tissue samples were used. Immunoblotting analyses, Quantitative real-time PCR and semiquantitative multiplex PCR were performed. The transient transfection experiments were performed using TransIT-Prostate Transfection Kit. Cell proliferation was determined using the Cell Proliferation Reagent WST-1. RESULTS: LKB1 expression was significantly decreased in prostate cancer samples compared to surrounding normal tissue. Moreover, LKB1 protein levels decreased throughout prostate carcinogenesis, with a significant reduction already evident in high-grade PIN lesions and a complete loss in adenocarcinomas. These findings were confirmed by tissue microarrays containing 150 prostate cancer smaples. To get insight into the role of LKB1/p38 activity balance in prostate cancer, we tested 2 cell lines, one carrying LKB1 wt alleles (PC3) and the other carrying mutant LKB1 (DU145), which both express all p38 isoforms with predominant expression of p38α. During proliferation, DU145 cells showed high p38 and low AMPK activity, while PC3 cells showed low p38 and high AMPK activity. Inhibition of p38α caused a marked increase in P-AMPK in PC3, but not in DU145 cells, consistent with the role of LKB1 as a main upstream activator of AMPK. Importantly, reconstitution of wt LKB1 function in DU145 cells restored P-AMPK activation in response to p38α blockade. Inhibition of p38 differentially affects DU145 and PC3 cell growth, being cytotoxic in DU145 and cytostatic in PC3 cells. Molecular characterization showed activation of apoptosis in DU145 and triggering of the autophagic pathway in PC3 cells upon treatment with p38 inhibitors (p38i). Reconstitution of wt LKB1 function promoted resistance to p38 blockade by reducing apoptosis in DU145 cells. Conversely, inhibition of autophagy or of AMPK triggered apoptosis in PC3 cells treated with p38i. Notably, p38i treatment of tumour biopsies showed an inverse correlation between LKB1 levels and p38i-dependent cell death.