Systems biology approach for targeted therapy of liver cancer P13K/AKT/MTOR pathway inhibitors : an ally or rival for sorafenib'e
Hepatocellular carcinoma (HCC) is one of the leading causes of cancerrelated mortality worldwide. It is the second most frequent cause of cancer death in men, and the sixth in women due to its aggressive behavior and resistance to conventional therapies. Sorafenib (Nexavar, BAY43-9006), a multi-kinase inhibitor with anti-angiogenic functions, is the only FDA-approved molecular-targeted agent for the treatment of patients with advanced HCC. Yet, Sorafenib shows limited overall survival benefit associated with resistance and tumor recurrence. Current mono-target- or single pathway-centric drug designs are not sufficient for effective therapy of advanced HCC. Negative results of clinical trials on targeted therapies for advanced HCC are due to clinical heterogeneity, complexity of cirrhotic background and interconnected regulation of cancer hallmarks through compensatory signaling pathways with redundant functions. Secretion of growth factors, pro-inflammatory and immune-suppressive cytokines and chemokines in the tumor microenvironment and consequent activation of tumor-promoting signaling cascades confer resistance to Sorafenib treatment. RAF/MEK/ERK and PI3K/AKT/mTOR are the major tumorpromoting signaling pathways with regulatory functions in all hallmarks of HCC. They have redundant functions and inhibition of one pathway can stimulate compensatory signaling from the other pathway. Since Sorafenib targets angiogenic VEGFR and PDGFR kinases and RAF/MEK/ERK signaling, the primary mechanism of resistance to Sorafenib and tumor recurrence in HCC patients emerges to be the compensatory signaling from the PI3K/AKT pathway. Therefore, we anticipated that combined treatment with Sorafenib and PI3K/AKT inhibitors could reverse drug resistance in HCC. In this study, we analyzed the synergistic effects of Sorafenib and PI3K/AKT inhibitors on HCC cell growth and migration, determined possible mechanisms underlying synergistic mechanism of action by transcriptome analysis, and further showed that combination therapy leads to tumor regression in PTEN-deficient HCC xenografts in vivo. We showed that PTEN-deficient HCC cells with constitutively active PI3K/AKT signaling depend on the alpha isoform of PI3K (p110-α) for survival and co-targeting these cells with the isoform specific PI3K inhibitor (PI3Ki- α, PIK-75) overcomes resistance to Sorafenib. Indeed, while dual-targeting of PTENdeficient HCC cells with Sorafenib and PI3Ki-α results in synergistic growth inhibition, dual-targeting these cells with Sorafenib and a beta isoform specific inhibitor of PI3K (PI3Ki-α, TGX-221) leads to an antagonistic increase in tumor growth compared to single treatment with Sorafenib, since inhibiting p110-! promotes compensatory signaling from p110-α. We also investigated the cytotoxic effects of inhibiting Akt kinase by using an Akt2 isoform-specific inhibitor (Akti-2) and a general Akt inhibitor targeting both isoforms (Akti-1,2). Akti-1,2 and Akti-2 did not induce anti-growth or pro-apoptotic mechanisms, but they were highly effective in reducing migration. Akt2 isoform is specifically overexpressed in HCC, and is correlated with its progression. Moreover, the Akt2 isoform-specific role of Akt kinase on migration has been demonstrated in breast cancer, and depletion of AKT2, but not AKT1, was shown to promote regression of PTEN-deficient prostate cancer xenografts. Based on these findings, we predicted a prominent role of Akt2 in PTEN-deficient HCC cells and examined the therapeutic efficacy of combined treatment of Sorafenib and Akti-2 in vivo in athymic mice bearing Mahlavu tumor xenografts. After 3 weeks of treatment, tumor growth was reduced significantly in all tested groups (Sorafenib, Akti-2 and Combination) compared to the control group. Substantial intra-tumoral necrosis produced a temporary increase in tumor size but resulted in significant reduction in tumor weight (p<0.001) in combination-treated mice compared to Sorafenib-treated mice and further produced tumor regressions. In this study, we showed cytotoxic activity of a new PI3K α soform specific kinase inhibitor (PI3Ki-α / PIK-75) at 0.1 µM that acts synergistically with Sorafenib in vitro, determined the predominant role of PI3K isoform p110α in PTEN-deficient HCC cells, and revealed synergistic anti-tumor effect of combining Sorafenib with a new Akt isoform 2 specific kinase inhibitor (Akti-2) in vitro and in vivo.