406 HIGH-THROUGHPUT SCREEN FOR GENES THAT SELECTIVELY PROMOTE GROWTH OF ANDROGEN INDEPENDENT PROSTATE CANCER CELLS

CaP cells under normoxic, acute hypoxic (1% oxygen for forty-eight hours), and chronic hypoxic conditions (cultured over six months). Comprehensive mRNA profiling was performed using an Agilent 44K array platform, and we investigated the association between elevated genes and tumor behavior by the applications of Ingenuity Pathways Analysis. From the results, we identified the Vav3 gene and closely analyzed the details of the gene in vitro. We validated the expression by quantitative RT-PCR and Western blot analyses. To analyze the function of Vav3, knockdown by siRNA was performed. Next, we transfected LNCaP with the Vav3 cDNA expression vector (control cells were transfected with neo expression vector) and isolated stable clones with high or low expression. The growth of cancer cells was evaluated in vitro. Also, we examined cell invasiveness and migration using the Matrigel invasion assay. For cell cycle analysis, flow cytometric analysis of propidium iodide-stained nuclei was carried out. RESULTS: The results were as follows: (1) Vav3 mRNA and protein expression levels were increased in LNCaP cells under chronic hypoxia, respectively; (2) chronic hypoxia increased both the invasiveness and migration of LNCaP cells; (3) LNCaP cells under chronic hypoxia showed less invasiveness and cell motility as well as suppressed their growth rates by interrupting Vav3 expression using Vav3 siRNA; (4) Vav3 expression positively correlated with the growth, invasiveness, and motility of LNCaP cells; and (5) interrupting the Vav3 signaling axis with siRNA led to the apoptosis of LNCaP cells via the mitochondrial pathway, with increased activation of caspases-3, and -9 and cleaved PARP. CONCLUSIONS: Our results showed that chronic hypoxia promoted the malignant potential of prostate cancer cells via the overexpression of Vav3. Therefore, targeting the Vav3 signaling pathway could prove to be a promising approach for the treatment of progressive or castration resistant prostate cancer.