Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (beta1wt /TRAMP) mice as well as in mice carrying a conditional ablation of beta1 integrins in the prostatic epithelium (beta1pc-/- /TRAMP). Since JNK is known to be suppressed by beta1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in beta1pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in beta1pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of beta1wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the beta1/IGF-IR crosstalk and report that beta1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that beta1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.

This study was carried out to dissect the mechanism by which beta1 integrins promote resistance to radiation. For this purpose, we conditionally ablated beta1 integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of beta1 to promote resistance to radiation was also analyzed by using an inhibitory antibody to beta1, AIIB2, in a xenograft model. The role of beta1 integrins and of a beta1 downstream target, c-Jun amino-terminal kinase 1 (JNK1), in regulating radiation-induced apoptosis in vivo and in vitro was studied. We show that beta1 integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, beta1 integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of beta1 on resistance to radiation in vitro and in vivo. Finally, given the established cross-talk between beta1 integrins and type1 insulin-like growth factor receptor (IGF-IR), we analyzed the ability of IGF-IR to modulate beta1 integrin levels. We report that IGF-IR regulates the expression of beta1 integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that beta1 integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation.

BACKGROUND: Prostate-specific antigen (PSA) is a pivotal downstream target gene of the androgen receptor (AR), and a serum biomarker to monitor prostate cancer (PrCa) progression. It has been reported that PSA transactivates AR, but the mechanistic requirements of this response have not been investigated. METHODS: We studied the localization of PSA, AR, and Src in intracellular compartments of synthetic androgen (R1881)-stimulated LNCaP and C4-2B PrCa cells, using immunofluorescence and subcellular fractionation approaches. We also investigated the effect of downregulation of PSA on AR expression by immunoblotting and real-time PCR using short hairpin RNA (shRNA) and small interfering RNA (siRNA). Src activity was analyzed by immunoblotting. RESULTS: R1881 stimulation induced nuclear localization of both PSA and AR in LNCaP and C4-2B PrCa cells as well as increased phosphorylation of Src. Stable shRNA or transient siRNA knockdown of PSA resulted in reduced AR protein levels as well as AR mRNA levels in C4-2B cells. Similar to C4-2B cells, ablation of AR levels upon silencing of PSA was also confirmed in VCaP cells, another androgen-independent cell line. Silencing of PSA did not cause significant changes in Src activation; besides, Src regulation by integrins did not appear to affect AR transcriptional activity. CONCLUSIONS: PSA localizes to nuclei of androgen-stimulated PrCa cells, and controls AR mRNA and protein levels. This regulatory loop is specific for PSA, does not involve known AR activators such as Src and AKT, and may contribute to AR signaling under conditions of increasing PSA levels in patients.