Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo

Journal of Hepatology, 2009;51(4): 725-733. Published: 2009.06.11

Pippa Newell, Sara Toffanin, Augusto Villanueva, Derek Y. Chiang, Beatriz Minguez, Laia Cabellos, Radoslav Savic, Yujin Hoshida, Kiat Hon Lim, Pedro Melgar-Lesmes, Steven Yea, Judit Peix, Kemal Deniz, M. Isabel Fiel, Swan Thung, Clara Alsinet, Victoria Tovar, Vincenzo Mazzaferro, Jordi Bruix, Sasan Roayaie, Myron Schwartz, Scott L. Friedman, Josep M. Llovet

Abstract

Background/Aims The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. Methods Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. Results Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. Conclusions Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

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