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Survivin is a unique member of the inhibitor of apoptosis (IAP) proteins that is overexpressed in numerous cancers through poorly defined mechanisms. One such mechanism may be through constitutive activation of the insulin-like growth factor-I (IGF-I) signaling pathway, implicated in the development and progression of prostate cancer. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that IGF-I induces Survivin expression, and that silencing Survivin by lentiviral-mediated small hairpin RNA (shRNA) represses IGF-I-stimulated cell growth, implicating Survivin as a mediator of this growth response. Moreover, our data support that the induction of Survivin by IGF-I occurs through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. Use of various Survivin promoter-luciferase constructs revealed that the CDE and CHR response elements in the proximal region of the Survivin promoter are involved in this IGF-I response. Transforming growth factor (TGF-β) signaling antagonists similarly activated the Surivin promoter and rendered cells refractory to further promoter activation by IGF-I. IGF-I suppressed levels of phospho-Smads 2 and 3 with kinetics similar to that of Survivin induction. Suppression of TGF-β signaling, either by TGF-β receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-β receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTOR. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin by IGF-I. Taken together, these results suggest that IGF-I signaling through a PI3K/Akt/mTORC1 mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-β signaling.

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Although hyper-activated mTOR is well recognized as being pivotal to prostate cancer growth and progression, the underlying mechanisms by which it promotes such responses remain incompletely understood. Here we show that rapamycin activates Smads 1 and 5 in human prostate cancer cells and tissues through blocking mTORC1 kinase. ShRNA-based gene silencing and gene overexpression approaches reveal that Smads 1 and 5 mediate while Smad8 represses rapamycin-induced cell death and expression of the BMP transcriptional target Id1 in human prostate cancer cell lines. Moreover, such phospho-Smad1/5-mediated rapamycin responses were blocked by LDN-193189 (a BMPRI kinase inhibitor) or Noggin (a BMP antagonist) in LNCaP prostate cancer cells. Likewise, the mTOR kinase inhibitors Ku-0063794 and WYE-354 each enhanced phosphorylation of Smad1/5. Intriguingly, silencing Raptor alone enhanced while silencing Rictor repressed the phosphorylation of Smad1/5, indicating that mTORC1 represses while mTORC2 activates BMP signaling. Immunohistochemical analysis showed increased levels of phospho-Smad1/5 concomitant with suppression of phospho-S6 and Survivin levels in PC3 human prostate cancer xenografts in athymic mice administered rapamycin (i.p., 5 mg/kg/day, 2 to 6 days). Moreover, we show that compared to prostate tumor tissue from untreated patients, levels of phospho-Smad1/5 were significantly elevated in the prostate tumor tissue of high-risk prostate cancer patients who received 8 weeks of the rapalog everolimus as part of a neoadjuvant clinical trial prior to undergoing local definitive therapy by radical prostatectomy. Taken together, our data implicate Smads 1, 5 and 8 as potential prognostic markers and therapeutic targets for mTOR inhibition therapy of prostate cancer.