BackgroundDespite huge efforts invested in the last years in the field of conventional and targeted cancer therapies, hepatocellularcarcinoma (HCC) remains largely incurable. Currently, less than 40% of HCC patients are eligible for curative approaches whilethe rest must undergo either conventional chemotherapy or targeted pharmacological therapy with multikinase inhibitorsorafenib. However, both approaches often fail to fully eradicate the disease, as cancer cells circumvent the block imposed bythe drug, resulting in cancer relapse. This process, termed evasive resistance, is a poorly understood yet extremely relevantunmet medical need in the field of HCC.HypothesisTo date, the molecular mechanisms governing evasive resistance have remained elusive, thus precluding the design of neweffective therapies against HCC. We hypothesize that when evasive resistance occurs, cancer cells upregulate compensatorymechanisms that allow them to escape the therapy and proliferate. We further hypothesize that thorough transcriptomic andphosphoproteomic profiling applied to in vivo and in vitro models of HCC can identify these alternative pathways.AimsThe aim of this project is to identify the molecular mechanisms governing the emergence of evasive resistance to sorafenibtreatment in HCC. To accomplish this task we will undertake several complementary approaches involving the extensive use ofgenetic animal models of HCC and in vitro techniques. The outcomes of these different approaches will allow us to monitor thetranscriptomic and phosphoproteomic changes occurring in HCC cells while they become competent to evade therapy.Experimental DesignEvasive resistance to sorafenib treatment in HCC will be investigated by exploiting genetic HCC animal models and thenumerous HCC cell lines available at the moment. Once HCC progression has started, mice will be treated either with sorafenibor vehicle, tumor growth will be monitored and transcriptomic/phosphoproteomic analyses will be performed before and afterthe development of resistance to sorafenib. Given the mounting evidence pointing to cancer stem cells as crucial players inestablishing tumorigenicity, metastasis and chemoresistance, particular attention will be paid to isolate and characterize thesetumor-propagating cells at the transcriptomic and phosphoproteomic level. This is not trivial since the most relevant alterationsleading to the emergence of sorafenib resistance are likely to occur in the cancer stem cell compartment. Similarly, HCC celllines (either commercial or obtained from murine HCC models) will be cultured in the presence or absence of sorafenib andused for the fast assessment of the signaling pathways cancer cells rely on under sorafenib treatment. In particular we plan toperform a synthetic lethality siRNA screening on sorafenib-resistant HCC cells in order to uncover key nodes in the signalingcircuitry that drive the emergence of evasive resistance.Expected ResultsGiven the extremely complex nature of evasive resistance development, we expect that only the integration of in vivo and invitro approaches plus the combined expertise of the members of the ERC Synergy MERiC network in which this project iscomprised will be instrumental to achieve the best possible understanding of evasive resistance, with immediate potential for thedesign of effective therapies against HCC.