Bile acids activate YAP to promote liver carcinogenesis

Abstract

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 the loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, and Yes-associated protein (YAP) 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 pathway dependent on the 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.

Figure 1. FXR/SHP double knockout (DKO) develop spontaneous hepato cellular carcinoma and phenocopy Mst1/2 double knockout (Mst1/2^−/−) mice

A. Gross liver examination shows presence of multiple tumor nodes with 100% penetrance compared to tumor free WT mice (n=20). B. Progressive tumor growth is evident from increasing liver to bodyweight ratio of DKO mice with age. C-D Hepatic oval cell marker, OC21D11 (red) shows increased staining in 8-10 week old DKO (right) compared to WT (left) mice and its quantification is shown in E, DAPI in blue stains the nuclei (n=4-6). F-H H&E staining of a year old normal liver WT (F) and DKO (G&H). DKO mice develop adenomas, whose growth rate exceeds the adjacent host liver (G), and Hepato Cellular Carcinoma, with marked nuclear atypia and mitotic activity (H); the inset shows cholangiofibrosis, versus the normal liver. Arrowheads show fat accumulation, arrows point to focal inflammation and dotted line demarcates the adenoma boundary from the injured liver tissue. Magnification X120 and insets are X250. *P<0.05, **P<0.001 compared to WT.

Figure 2. Bile acid overload is sufficient to induce YAP activation

A. Gene expression analysis in DKO mice indicates induction of YAP targets. B. Immunoblot confirms reduced levels of inactive phospho YAP and activation of its targets in 5-8 weeks old DKO animals compared to WT. C. WT primary hepatocytes upon treatment with DKO serum display YAP activation, which can be mimicked with bile acid, chenodeoxycholic acid (CDCA). CDCA induces YAP target CTGF (D). WT mice fed 1% CA diet (E) or 0.1% DDC diet (F) for 2 weeks display YAP activation compared to chow fed animals. Densitometry was performed on immunoblots for n=3-5 animals per group and fold differences in comparison to control WT are written in parenthesis.

Figure 3. BA overload does not cause overt cell death but alters canonical hippo signaling

Primary hepatocytes were cultured and treated with DMSO (A) and varying doses of CDCA (B-D) overnight. Cells were fixed and TUNEL positive green staining nuclei (marked with white arrows) were counted at least in ten different fields for each of the treatment groups to quantify cell death (E). We performed this experiment in triplicates. **P<0.001 when compared to WT, CDCA 75 and *P<0.01 when compared to CDCA 150. Magnification X120. Immunoblots for hippo pathway kinases were performed in DKO (F), WT animals treated with either 1% CA (G) or 0.1% DDC (H) for 2 weeks. Hippo kinases were altered only in DKO and in CA fed mice with no changes in DDC fed WT animals. However, YAP targets CTGF and Cyclin D1 were induced upon CA and DDC treatment clearly indicating YAP activation in these cholestatic mouse models.

Figure 4. Increased Iqgap1 expression correlates strongly with YAP activation

Iqgap1 is robustly induced in DKO (A), WT (B) and in Iqgap2^−/− animals treated with 1%CA diet (C) compared to their respective controls. (D-F) 1% cholic acid treatment leads to elevated liver enzymes in serum of Iqgap2^−/− (G) Immunoblot shows YAP activation in naïve Iqgap2^−/− mice. (H) Immunohistochemistry reveals abundant YAP expression, with increased nuclear YAP in Iqgap2KO compared to WT mouse livers after 1%CA diet (Insets). Magnification X120. *P< 0.05; compared to WT mice on normal chow.

Figure 5. Iqgap1 is crucial for bile acid mediated hepatocyte proliferation

WT (A-B), Iqgap1^−/−(C-D) and Iqgap2^−/− (E-F) mice were fed 1% CA diet for 2 weeks. Ki-67 staining was performed in these samples to assess cellular proliferation. N= 3-4 mice per group were stained with Ki-67 and the positively stained brown nuclei were quantified (G). *P< 0.001; compared to WT (a) and compared to Iqgap2^−/− (b). Magnification used is X120.

Figure 6. IQGAP1 overexpression is sufficient to increase YAP levels and to increase hepatocyte proliferation

(A). Hepatic Iqgap1 over-expression results in YAP activation, this is quantitated using immunoblots (B). Iqgap1 over-expression for a period of 2 weeks results in slightly elevated serum ALT but not AST levels (C-D) and increased liver size (E-F) with no change in serum bile acid concentration (G). Robust YAP expression is observed in all hepatocytes after Iqgap1 overexpression, which correlates well with the increased proliferation observed with Ki-67 staining (inset). Densitometry on immunoblots for n=3-5 animals per group. Fold differences in comparison to control WT are in parenthesis. Magnification X120 and insets are X250. *P<0.05, compared to WT.

Figure 7. Human biliary disorders exhibit strong YAP and IQGAP1 expression

A. Normal liver: bile duct epithelium has uniform cytoplasmic expression of both YAP and IQGAP1 proteins. Arterial smooth muscle is also positive. IQGAP1 is also expressed in sinusoidal Kupffer cells. B. 2 year old with congenital hepatic fibrosis: all ducts express IQGAP1 and YAP in cytoplasm and YAP in some nuclei. C. Extrahepatic biliary atresia in 2 week old: In addition to duct and ductular expression of both proteins in the cytoplasm, many ductal, ductular and some hepatocyte nuclei are positive for YAP. D. PFIC3 (Progressive Familial Intrahepatic Cholestasis 3)- The expression pattern is identical to that of extra hepatic biliary atresia. (Original magnifications X125, with purple staining for YAP (top) and red staining for IQGAP1 (bottom). E. Human adenoma and hepatocellular carcinoma samples stained with YAP and IQGAP1. F. Model illustrating how elevated BAs cause YAP activation.