The importance of androgen signaling in prostate cancer (PCa) has long been recognized and is underscored by findings of intracrine production of androgens in castration-recurrent PCas. Nonetheless, genes that are critical for PCa cell proliferation and invasive potential and that are subject to androgen regulation remain largely elusive. Mounting evidence indicates that secondary transcription factors play a critical role in mediating the effects of androgens on PCa cells. Using the FHL2 gene as a model, we have identified an indirect mechanism of androgen action in which the effects of androgens are mediated by Serum Response Factor (SRF), a transcription factor that regulates expression of genes involved in mitogenic responses and cytoskeletal organization. To explore the generality of this concept, we transfected LNCaP cells with siRNAs targeting SRF or control siRNAs and cultured cells in the presence or absence of androgens. RNA derived from biological triplicates was analyzed using Human Genome U133 Plus 2.0 Arrays (Affymetrix). Applying criteria of at least 2-fold change in expression upon androgen treatment in control-transfected cells and no androgen regulation or effects on basal expression levels upon loss of SRF, we identified 158 androgen- and SRF-responsive genes. Androgen- and SRF-dependency was confirmed by real time RT-PCR. Ingenuity pathway analysis, which assigned roles in cell cycle, cell-cell interactions, cell morphology and cellular organization to these genes, identified cancer as the primary disease associated with this gene profile. The identified genes correspond to less than 6 percent of androgen responsive genes in LNCaP cells. To verify the clinical relevance of this gene signature, we compared it to mRNA expression data sets generated using RNA from laser capture microdissected human prostate samples representing varying degrees of malignancy (normal (17), BPH (10), PIN (4), Gleason pattern (GP) 3 (31), 4 (20) and 5 (10), lymph node metastasis (LN) (7)) on the same platform. We found that the 158 gene profile is sufficient to separate benign and malignant prostate tissues. Closer examination revealed that 28, 69, 71, 56, and 67 probe sets are differentially expressed (p<0.05) in PIN, GP3, GP4, GP5 and LN compared to normal prostate epithelium, respectively. Significant differences were also found between paired cancer and normal tissues (8) and cancer versus metastatic samples (4) from the same patient. Using a linear regression model, we demonstrated that 67 transcripts correlated significantly with tumor aggressiveness. Taken together, our data validate the clinical relevance of the indirect SRF-dependent mechanism of androgen action in PCa and highlight the importance of the androgen/SRF signaling axis as a valid therapeutic target in PCa. Supported by NIH, TJ Martell Foundation, Mayo Clinic prostate SPORE (HVH) and Belgian-American Educational Foundation (HVH).
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-45.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO