2). However, no lung click here metastases were observed in the tumor-bearing Trp53KO;Tgfbr2KO mice. Assessment of the gene status of the normal liver and tumors of the various genotypes confirmed the tissue specific recombination and deletion status as expected (Supporting Fig. 1). Based on our analysis of the Trp53KO versus Trp53KO;Tgfbr2KO mice, it was clear that mice lacking p53 and with intact Tgfbr2 developed tumors at a younger age, had increased liver to body weight ratios, and displayed overall worse survival rates compared with the mice lacking both p53 and Tgfbr2. Subsequently, we conducted a series of studies assessing candidate mechanisms that may be responsible for the protumorigenic effects

of TGF-β in the setting of loss of p53 in the liver. We initially focused on AFP, a gene frequently overexpressed in human liver cancer that may promote HCC formation.25 AFP has been shown to be regulated by both p53 and TGF-β19, 20 and is thought to play a pathogenic role in liver cancer by acting as a growth factor and immunosuppressor.26, 27 Afp mRNA levels were analyzed in tumor and nontumor tissue isolated from mice of various genotypes (Fig. 3A). Afp Messenger RNA (mRNA) was expressed at very low levels in normal liver

tissue harvested from Control mice, consistent with previous reports.28, 29 There was no significant difference in the median level of Afp mRNA detected in the normal livers of Tgfbr2KO mice compared with Control mice (P = 0.7104). A significant increase in Afp mRNA levels was observed in normal Trichostatin A concentration tissue from Trp53KO mice and normal tissue from Trp53KO;Tgfbr2KO mice (Trp53KO and Trp53KO;Tgfbr2KO versus Control, P = 0.0003 and 0.0047, respectively). This moderate increase over basal levels in normal liver is consistent with the role of p53 in Afp repression. Analysis of the levels of Afp mRNA

in Trp53KO tumors revealed two distinct subsets of tumors: a high Afp-expressing group and a moderate/low Afp-expressing group (Fig. 3A). This was in contrast to tumors from Trp53KO;Tgfbr2KO mice which all had moderate/low Afp expression. The ratio of Afp mRNA expression (tumor:normal liver, Janus kinase (JAK) T/N) was also calculated for Trp53KO and Trp53KO;Tgfbr2KO mice (Fig. 3B). In the Trp53KO mice the ratio of Afp mRNA expression in tumors versus normal liver in a subset of tumors was higher than in tumors arising in the Trp53KO;Tgfbr2KO mice (P = 0.0426). Although we observed increased Afp in a subset of Trp53KO tumors, it is clear that elevated Afp levels are not the sole mechanism responsible for increased liver tumor formation in the Trp53KO mice. Therefore, we determined if there were other concurrent mechanisms that may help explain how Tgfbr2 cooperates with loss of p53 to promote liver tumor formation. One possible mechanism could be through regulation of the TGF-β signaling pathway itself. TGF-β1 has been shown to be upregulated in a number of tumors, including HCC.