Increased expression of the RNA-binding protein Sam68 protects prostate cancer cell from apoptosis

Prostate cancer develops as an androgen-dependent hyper-proliferation of prostatic gland epithelial cells and it is initially treated with an anti-androgen therapy. However, after initial remission, it often evolves into a highly aggressive and metastatic androgen-independent cancer, for which a successful therapy has not yet been established. Our laboratory has previously described the activation of the tyrosine kinase Src in a subset of advanced prostate carcinomas. In these patients, activation of src correlated with tyrosine phosphorylation of Sam68, a mitotic substrate of Src-like kinases. Sam68 is an RNA binding protein belonging to the STAR family (signal transduction and RNA metabolism) involved in pre-mRNA splicing, processing and stability. Herein we have investigated the role of Sam68 in prostate cancer cells. Sam68 is highly expressed in patients with advanced prostate carcinomas (Gleason sum 6-9) as we determined by immunohistochemistry and Western blot analysis. These results were also confirmed at the mRNA level by real-time PCR analysis from the same patients. In addition, immunohistochemical analysis of carcinomas of different tissues (thyroid, liver and testicular tumors) also showed that up-regulation of Sam68 is a frequent event. Hence, our results suggest that an elevated expression of Sam68 represent an advantage for the physiology of the cell. To define the role of Sam68 in the cell and to understand the effects of altering its cellular concentration, we performed transfection experiments to deplete the endogenous Sam68 from LNCaP prostate cancer cells by RNAi . Cell proliferation assays with MTS revealed that Sam68-depleted cells grow at a slower rate than cells transfected with scrambled siRNA used as control. Moreover, silencing of Sam68 reduced the percentage of BrdU-positive cells, indicating that the proliferation rate was reduced after down-regulation of Sam68. To determine whether sam68 was required for the response of cacner cells to cytotoxic drugs, we exposed scrambled or Sam68 depleted cells to various concentration of etoposide or ciplatin. Cell staining with Annexin V and propidium iodide showed that Sam68 depletion sensitises LNCaP to cisplatin and etoposide treatment. We are currently determining on a large scale the expression profile of LNCap cells in which Sam68 has been down regulated by hybridising the mRNAs of these cells to a microarray chip containing most of the human genes (Affymetrix U133 2.0 plus) and to a chip containing biomarkers for prostate tumors (Super-array Prostate Cancer Biomarker Array).