2B,C) and YAP expression (Fig. 2D) was observed between 24 or 36 hours after mitogen administration. Accordingly, coactivating activity of YAP evaluated by measuring the expression levels of Birc-5/survivin, a target gene of YAP,17 revealed a 20-fold increase in the expression of this gene in the livers of mice sacrificed 24 hours after the first dose, but only a two-fold increase after the second dose of TCPOBOP (Fig. 2E). These findings suggest that YAP transitions from an active to an inactive state and

is involved in the refractoriness of enlarged livers to a second mitogenic challenge. In this context, it is noteworthy that almost check details no residual hepatocyte proliferation occurs in mice exposed to the second dose of TCPOBOP after the first 36 hours, as also indicated in Fig. 1C. To further investigate the role of the Hippo pathway in mitogen-induced liver enlargement, we cloned

in a lentiviral vector an active form of YAP (Ser127-381Ala, EPZ-6438 mw mYAP)25, 26 lacking the phosphorylation sites needed for its sequestration in the cytoplasm. Infection of mouse livers with mYAP 4 days before the second dose of TCPOBOP (Fig. 3A,B) did override the physiological termination of liver growth. Indeed, whereas no proliferation was observed after the second dose of TCPOBOP in mice transduced with control vector, a significant increase of cell proliferation was observed when TCPOBOP was given to mice infected with the active YAP (6.54% of BrdU-positive IMP dehydrogenase nuclei versus 16.31%; P < 0.01) (Fig. 3,C,D). To investigate whether injection of mYAP could per se lead to hepatocyte proliferation regardless of TCPOBOP administration, we injected untreated

mice with YAP lentiviruses and compared the proliferative response of hepatocytes with that of mice receiving two administrations of TCPOBOP, in the presence or absence of exogenous YAP (Fig. 3E). A further experimental group was treated with control lentiviruses. As shown in Fig. 3F, transduction of hepatocytes with YAP alone induced only a slight and not statistically significant increase of BrdU incorporation over control values. On the other hand, liver transduction with YAP of mice treated with TCPOBOP led to a highly significant increase in proliferation compared with TCPOBOP-treated mice transduced with control virus. These experiments strongly suggest that the Hippo pathway is involved in the refractoriness of enlarged liver to further mitogenic stimuli. Overexpression of YAP has been observed in many human tumors, enough that it is considered a candidate oncogene.22, 27 Despite the fact that TCPOBOP-induced enlarged livers do not grow further once they reach a certain size, they nevertheless develop HCC upon an initiating dose of a chemical carcinogen followed by repeated treatments with the mitogen.