ABSTRACT E2f (E2f1-8) transcription factors are critical regulators of cell cycle and their activity is physically regulated by Rb family proteins (Rb, p107 and p130). Disruption of the Rb/E2f interaction is a hallmark of cancer, as defined by Hanahan&Weinberg. The acquired resistance to antigrowth signals resulting from this disruption is thought to be necessary for tumor initiation. However, how does unrestricted E2f activity initiates tumorigenesis in vivo is poorly understood and remains a fundamental gap in our understanding of cancer biology. In particular, whether the differentiation status affect the capacity of a cell to transform upon disruption of Rb/E2f interaction is unknown. In addition, whether unrestricted E2f activates other oncogenic features besides aberrant proliferation is still obscure. Hepatocellular carcinoma (HCC) is the second cancer in terms of death worldwide. Limited understanding of the functional consequences for frequent genetic events hampers the development of efficient therapeutics. The Rb/E2f interaction is disrupted in the vast majority of HCC, as a consequence of several events that target upstream components of the Rb/E2f pathway. Therefore, HCC is a relevant model to investigate the consequences of unrestricted E2f activity for cancer initiation. Accordingly, pan-liver inactivation of Rb family genes (Triple Knock Out, TKO) is sufficient to initiate HCC (TKO HCC) that recapitulate multiple features of the human disease. Studies of carcinogen-induced models of HCC have led to the conclusion that hepatocytes are the sole source of HCC. Specific inactivation of Rb family in hepatocytes triggers a short proliferative burst but fails to initiate HCC. This result challenges the current hepato-centric model in the field and suggests that other lineages can also serve as a cell of origin for HCC. Accordingly, inactivation of Rb family in multiple liver lineages reveals that TKO HCC arises from a periductal progenitor. In particular, our preliminary data indicates that unrestricted E2f activity couples aberrant proliferation with cell fate alteration in this population to initiate HCC. Based on these data, we propose to:1) determine the molecular mechanisms that alter the cell fate of a periductal progenitor to serve as a cell of origin for TKO HCC. 2) determine the individual and compound role of E2f factors in TKO HCC initiation and development. 3) determine therapeutic vulnerabilities in TKO HCC that could serve as novel treatment for patients. We believe that our proposal will address fundamental questions regarding the role of E2f in cancer initiation, as identified above. In addition, we expect that our results will establish that different cell lineages can serve as a cell of origin for HCC, which will have important clinical implications, in particular regarding the classification of patients and the development of therapies tailored for different classes of HCC.