Vascular endothelial growth factor (VEGF) is a survival factor in metastatic human prostate cancer cells via induction of myeloid cell leukemia-1 (Mcl-1)

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Introduction and Objectives: Acquisition of apoptosis resistance is a crucial characteristic of metastatic cancer cells. Aberrant VEGF expression and signaling has been correlated to human prostate cancer (PCa) progression. Previously we demonstrated that elevated serum VEGF levels are associated with clinical PCa bone metastasis. However, the role of VEGF signaling in PCa metastasis remains elusive. In this study, we investigated VEGF autocrine signaling in a PCa ARCaP progression model, which has been shown to undergo epithelial-to-mesenchymal transition (EMT or ARCaP_E to ARCaP_M).

Design: Expression of VEGF receptors (VEGFR1/Flt, VEGFR2/Flk, and neuropilin-1/NRP1) was evaluated in PCa cell lines by RT-PCR and western blot. Effects of VEGF, VEGF siRNA, NRP1 siRNA, or VEGFR antagonists on proliferation and apoptosis of ARCaP cells were analyzed by MTS assay or FACS. Effects of VEGF signaling on the expression of Mcl-1 were studied by RT-PCR and western blot using ARCaP EMT model. VEGF-induced activation of tyrosine kinase c-Src and signal transducers and activators of transcription 3 (STAT3), were analyzed by western blot.

Results: VEGF receptors, including Flt, Flk, and NRP1, are differentially expressed by ARCaP cells upon EMT. Expression of NRP1 was significantly increased in bone-metastatic ARCaP_M tumor tissues, as well as in clinical PCa tumor specimens. Treatment with VEGF siRNA effectively induced apoptosis in bone-metastatic ARCaP_M cells, whereas exogenous recombinant VEGF rescued the effects of VEGF siRNA, suggesting VEGF may be a survival factor in PCa cells. Mcl-1, an antiapoptotic factor that has been correlated to higher Gleason grades and metastasis in clinical PCa specimens, was increased in PCa cells with high propensity for bone metastasis (e.g. ARCaP_M and C4-2). VEGF rapidly induced Mcl-1 transcription at a low concentration, and this induction can be abrogated by VEGF siRNAs or pharmacological inhibitors of VEGF signals, suggesting that autocrine activation of VEGF-Flk-NRP1 signaling is responsible for the induction of Mcl-1. c-Src and STAT3 were shown to mediate VEGF induction of Mcl-1, thereby conferring PCa cells apoptotic resistance.

Conclusion: Autocrine VEGF signaling, mediated by c-Src/STAT3/Mcl-1, contributes to apoptotic resistance of PCa cells. This signaling pathway is activated by EMT through interaction with bone microenvironment. A coordinated regulation of VEGF and Mcl-1 may contribute to PCa growth and metastasis and hence an attractive therapeutic target.