IGFBP-3 therapy inhibits prostate cancer growth by enhancing apoptosis and suppressing angiogensis: role of phosphorylation

C46 Epidemiological studies have implicated high circulating IGF-I and low circulating IGFBP-3 levels with increased risk of developing prostate cancer. IGFBP-3 promotes apoptosis by both IGF-dependent and -independent mechanisms in many cancer models, including prostate. In cultured prostate cancer cells, IGFBP-3 is rapidly internalized and localized to the nucleus, where its interactions with the nuclear receptor RXR alpha are important in apoptosis induction. However, little is known about the intrinsic mechanisms regulating the apoptotic actions of IGFBP-3. Proteomic and bioinformatic analysis of IGFBP-3 reveals multiple consensus phosphorylation sites for kinases including CK2, PKA, PKC and cdc2. We have previously reported that phosphorylation of IGFBP-3 at Ser-156 by DNA-PK enhances its nuclear accumulation, and is essential for its ability to interact with RXR and induce apoptosis in 22RV1 and LAPC4 prostate cancer cells. Indeed, IGFBP-3-S156A is completely unable to induce apoptosis in our cell system. Using specific chemical inhibitors and siRNA, we now investigated the contribution of other protein kinases to the regulation of IGFBP-3-induced apoptosis. Importantly, preventing the activation of CK2 enhanced the apoptotic potential of IGFBP-3. We mapped two potential CK2 phosphorylation sites to the central region of IGFBP-3: S167 and S175. These sites were mutated to Ala, and the resulting constructs were transfected in to 22RV1 and LAPC4 cells. Wt-IGFBP-3 and IGFBP-3-S175A induced apoptosis to a comparable extent, however, overexpression of IGFBP-3-S167A was significantly more apoptotic. These effects were specific to apoptosis-induction, however, since wtIGFBP-3 and IGFBP-3/S167A had comparable effects on cell growth and proliferation, assessed by MTT and BrdU incorporation assays. Interestingly, IGFBP-3-S167A was able to induce apoptosis even in the absence of active DNA-PK, while the DNA-PK inactive IGFBP-3-S156A mutant regained the ability to induce apoptosis when CK2 activity was inhibited chemically or by using siRNA. Together, these data reveal two key regulatory phosphorylation sites in the central region of IGFBP-3. Phosphorylation of S156 by DNA-PK promotes apoptosis, whilst phosphorylation of S167 by CK2 limits the ability of IGFBP-3 to induce apoptosis. Interestingly, our data suggest that the anti-apoptotic phosphorylation event induced by CK2 is dominant. Pre-treatment of 22RV1 cells with IGFBP-3-siRNA also limits the ability of high doses of CK2 inhibitor to induce apoptosis. These effects can be reversed by the addition of exogenous IGFBP-3 protein, suggesting that inhibition of IGFBP-3 action by CK2 may be a key mechanism through which CK2 induces cell survival. IGFBP-3-S167A, but not wild type IGFBP-3, can partially antagonize the survival effects of overexpressing CK2, suggesting a potentially important relationship between IGFBP-3 and CK2 in regulating cell survival and apoptosis in prostate cancer.
 IGFBP-3 infusion into SCID mice with 22RV1 xenograts reduced tumor size by 40%, dramatically enhanced apoptosis, and suppressed angiogenesis.
 IMPACT: IGFBP-3 is currently being explored as a potential therapy for many cancers, including prostate. Regulation of IGFBP-3 by kinases and phosphatases may modulate the development and effectiveness of IGFBP-3 as an anti-cancer therapy.