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Background: Multiple lines of evidence support that the Hedgehog (Hh) signalling has a role in the maintenance and progression of different human cancers. Therefore, inhibition of the Hh pathway represents a valid anticancer therapeutic approach for renal cell carcinoma (RCC) patients. NVP-LDE225 is a Smoothened (Smo) antagonist that induces dose-related inhibition of Hh and Smo-dependent tumour growth. Methods: We assayed the effects of NVP-LDE225 alone or in combination with everolimus or sunitinib on the growth and invasion of human RCC models both in vitro and in vivo. To this aim, we used a panel of human RCC models, comprising cells with acquired resistance to sunitinib - a multiple tyrosine kinase inhibitor approved as a first-line treatment for RCC. Results: NVP-LDE225 cooperated with either everolimus or sunitinib to inhibit proliferation, migration, and invasion of RCC cells even in sunitinib-resistant (SuR) cells. Some major transducers involved in tumour cell motility, including paxillin, were also efficiently inhibited by the combination therapy, as demonstrated by western blot and confocal microscopy assays. Moreover, these combined treatments inhibited tumour growth and increased animal survival in nude mice xenografted with SuR RCC cells. Finally, lung micrometastasis formation was reduced when mice were treated with NVP-LDE225 plus everolimus or sunitinib, as evidenced by artificial metastatic assays. Conclusions: Hedgehog inhibition by NVP-LDE225 plus sunitinib or everolimus bolsters antitumour activity by interfering with tumour growth and metastatic spread, even in SuR cells. Thus, this new evidence puts forward a new promising therapeutic approach for RCC patients.

Protein kinase A type I (PKAI) and its regulatory subunit RI alpha are overexpressed in cancer cells and are induced by mitogenic hormones and growth factors in nontransformed cells. RI alpha/PKAI are directly involved in the G1>S transition and cell proliferation of non-transformed human breast MCF-10A cells. Retroviral vector-mediated overexpression of RI alpha in these cells (MCF-10A RI alpha) confers the ability to grow in serum-free medium. p53 controls a G1 check point before transition to the S phase, playing a key role in the regulation of cell proliferation and in the preservation of DNA integrity. In this study we evaluated the interaction of p53 and RI alpha on cell cycle progression and cell proliferation of MCF-10A cells. Retroviral vector-mediated overexpression of wild-type p53 in the MCF-10A neo and MCF-10A RI alpha cells determined a marked inhibition of RI alpha protein expression in MCF-10A-p53 cells and induced G0/G1 accumulation, cell gowth arrest and changes in cell morphology not due to apoptosis in both MCF-10A-p53 and MCF-10A RI alpha-p53 cells. On the other hand, in the MCF-10A RI alpha cells we observed an increased expression of the endogenous p53, although these cells were still able to proliferate. These results suggest that overexpression of wildtype p53 acts in a dominant fashion to abrogate the RI alpha induction of G1>S transition and cell proliferation. Moreover, overexpression of RI alpha leads to increased synthesis of endogenous p53 which, however, is unable to interfere with the RI alpha-dependent mitogenic signalling.