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SUMMARY Elevated bile acid levels increase hepatocellular carcinoma by unknown mechanisms. Here we show that mice with a severe defect in bile acid homeostasis due to loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, YAP (Yes Associated Protein) activation, and develop spontaneous liver tumorigenesis. This phenotype mirrors mice with loss of hippo kinases or overexpression of their downstream target YAP. Bile acids act as upstream regulators of YAP via a novel pathway dependent on induction of the scaffold protein Iqgap1. Patients with diverse biliary dysfunctions exhibit enhanced Iqgap1 and nuclear YAP expression. Our findings reveal an unexpected mechanism for bile acid regulation of liver growth and tumorigenesis via the Hippo pathway.

IQGAPs are multidomain scaffolding proteins that integrate Rho GTPase and Ca2؉ /calmodulin signals with cell adhesive and cytoskeletal reorganizational events. Targeted disruption of the murine Iqgap2 gene resulted in the age-dependent development of apoptosis and hepatocellular carcinoma (HCC), characterized by the overexpression of IQGAP1, the loss of membrane E-cadherin expression, the cytoplasmic translocation (and activation) of ␤-catenin, and the overexpression of a nuclear target of ␤-catenin, cyclin D1. In normal hepatocytes, IQGAP2 was found to exist as one component of a multifunctional scaffolding complex comprising IQGAP1, ␤-catenin, and E-cadherin, with no evidence for direct IQGAP1-IQGAP2 interactions. Interbreeding of Iqgap2 ؊/؊ mice into the Iqgap1 ؊/؊ background resulted in the phenotypic correction of the preexisting hepatopathy, decreases in the incidence and sizes of HCC tumors, and the normalization of overall survival rates compared to those of Iqgap2 ؊/؊ mice, suggesting that maximal penetrance of the Iqgap2 ؊/؊ HCC phenotype requires the coordinate expression of IQGAP1. These results identify Iqgap2 as a novel tumor suppressor gene specifically linked to the development of HCC and the activation of the Wnt/␤-catenin signaling pathway, while also suggesting that IQGAP1 and IQGAP2 retain functionally divergent roles in hepatocellular carcinogenesis.

BackgroundIQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC.MethodsIQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA.ResultsIQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6).ConclusionsWe conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.