Farnesoid X receptor (FXR), the primary bile acid-sensing nuclear receptor, also is known for its anticancer properties. It is known that FXR deficiency in mice results in spontaneous hepatocellular carcinoma (HCC), but the mechanisms are not completely understood. We report that sustained activation of the Wnt/␤-catenin pathway is associated with spontaneous HCC in FXR-knockout (KO) mice. HCC development was studied in FXR-KO mice at 3, 8, and 14 months of age. No tumors were observed at either 3 or 8 months, but the presence of HCC was observed in 100% of the FXR-KO mice at the age of 14 months. Further analysis revealed no change in ␤-catenin activation in the livers of 3-month-old FXR-KO mice, but a moderate increase was observed in 8-month-old FXR-KO mice. ␤-Catenin activation further increased significantly in 14-month-old tumor-bearing mice. Further analysis revealed that two independent mechanisms might be involved in ␤-catenin activation in the livers of FXR-KO mice. Activation of canonical Wnt signaling was evident as indicated by increased Wnt4 and dishevelled expression along with glycogen synthase kinase-3␤ inactivation. We also observed decreased expression of E-cadherin, a known regulator of ␤-catenin, in FXR-KO mice. The decrease in E-cadherin expression was accompanied by increased expression of its transcriptional repressor, Snail. Consistent with the increased HCC in FXR-KO mice, we observed a significant decrease in FXR expression and activity in human HCC samples. Taken together, these data indicate that a temporal increase in the activation of Wnt/␤-catenin is observed during spontaneous HCC development in FXR-KO mice and is potentially critical for tumor development.

Background/Aims: Hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and hepatoblastoma (HB) are the main hepatic malignancies with limited treatment options and high mortality. Recent studies have implicated Hippo Kinase pathway in cancer development but detailed analysis of Hippo Kinase signaling in human hepatic malignancies, especially CC and HB, is lacking. Methods: We investigated Hippo Kinase signaling in HCC, CC and HB using cells and patient samples. Results: Increased expression of yes-associated protein (Yap), the downstream effector of the Hippo Kinase pathway, was observed in HCC cells and siRNA-mediated knockdown of Yap resulted in decreased survival of HCC cells. The density-dependent activation of Hippo Kinase pathway characteristic of normal cells was not observed in HCC cells and CCLP cells, a cholangiocarcinoma cell line. Immunohistochemistry of Yap in HCC, CC and HB tissues indicated extensive nuclear localization of Yap in majority of tissues. Western blot analysis performed using total cell extracts from patient samples and normal livers showed extensive activation of Yap. Marked induction of glypican-3, CTGF and Survivin, the three Yap target genes was observed in the tumor samples. Further analysis revealed significant decrease in expression and activity of Lats kinase, the main upstream regulator of Yap. However, no change in activation of Mst-2 kinase, the upstream regulator of Lats kinase was observed. Conclusions: These data show that Yap induction mediated by inactivation of Lats is observed in hepatic malignancies. These studies highlight Hippo Kinase pathway as a novel therapeutic target for hepatic malignancies.

It is known that the liver undergoes size increase and differentiation simultaneously during the postnatal period. Cells in the liver undergo a period of well-controlled proliferation to achieve the adult liver-to-body weight ratio. The postnatal liver growth is also accompanied by simultaneous hepatic differentiation. However, the mechanisms of liver size regulation and differentiation are not completely clear. Herein we report that yes-associated protein (Yap), the downstream effector of the Hippo Kinase signaling pathway, plays a role in liver size regulation and differentiation during the postnatal liver growth period. Postnatal liver growth was studied in C57BL/6 mice over a time course of postnatal days (PND) 0- 30. Analysis of nuclear Yap by Western blot indicated peak Yap activation between PND15–20, which coincided with increased cyclin D1 expression and liver cell proliferation. Analysis of postnatal liver development in Yap+/− mice revealed a significant decrease in the liver-to-body weight ratio compared with Yap+/+ mice at PND15 and -30. Yap+/− mice exhibited a significant decrease in postnatal liver cell proliferation, but no change in apoptosis was observed. Furthermore, global gene expression analysis of Yap+/− livers revealed a role of Yap in regulation of genes involved in bile acid metabolism, retinoic acid metabolism, ion transport, and extracellular matrix proteins. Taken together, these data indicate that Yap plays a role in both cell proliferation and possibly in hepatic differentiation during postnatal liver development.

Therapeutic strategies based on a specific oncogenic target are better justified when elimination of that particular oncogene reduces tumorigenesis in a model organism. One such oncogene, Musashi-1 (Msi-1), regulates translation of target mRNAs and is implicated in promoting tumorigenesis in the colon and other tissues. Msi-1 targets include the tumor suppressor adenomatous polyposis coli (Apc), a Wnt pathway antagonist lost in ∼80% of all colorectal cancers. Cell culture experiments have established that Msi-1 is a Wnt target, thus positioning Msi-1 and Apc as mutual antagonists in a mutually repressive feedback loop. Here, we report that intestines from mice lacking Msi-1 display aberrant Apc and Msi-1 mutually repressive feedback, reduced Wnt and Notch signaling, decreased proliferation, and changes in stem cell populations, features predicted to suppress tumorigenesis. Indeed, mice with germline Apc mutations (Apc Min ) or with the Apc 1322T truncation mutation have a dramatic reduction in intestinal polyp number when Msi-1 is deleted. Taken together, these results provide genetic evidence that Msi-1 contributes to intestinal tumorigenesis driven by Apc loss, and validate the pursuit of Msi-1 inhibitors as chemo-prevention agents to reduce tumor burden.

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Most colorectal cancers are thought to be initiated by mutation of the tumor suppressor Adenomatous polyposis coli gene (APC). APC mutations that result in loss of function lead to deregulation of Wnt signaling and inappropriate proliferation within intestinal crypts. Understanding APC regulation within normal intestinal cells is important for developing methods to restore function in a pathological state. Using cultured human colonocytes, we have previously shown that translation of APC can be blocked by the RNA binding protein, Musashi 1 (MSI1) and that MSI1 is a Wnt target. We propose that a double negative feedback loop between APC and MSI1 functions in maintenance of intestinal cell homeostasis. To explore the MSI1/APC interaction in vivo, we analyzed intestinal tissue from MSI1 knock-out mice (Msi1-/-). We found higher levels of APC and less Wnt signal activation in intestines from the Msi1-/- mice. There were also more enterocytes within the intestinal crypts of Msi1-/-mice, indicating an increase in differentiation. These three observations provide confirmation of the proposed APC/MSI1 double negative feedback loop in vivo. Evidence from cultured human cells and mouse models supports a role for an APC/MSI1 double negative feedback loop in the response of intestinal cells to Wnt signaling. However, the underlying mechanisms remain to be determined. Proteins that bind to a specific motif in the 3’UTR of target mRNA can induce translation inhibition or promotion as well as stabilization or destabilization of the mRNA. We already established that MSI1 blocks APC translation; however, the effect of MSI1 on APC mRNA stability was unknown. Here, we show that MSI1 binding to the 3’ UTR of APC mRNA stabilizes the message. In contrast, Numb, another established MSI1 target, does not appear to be stabilized by MSI1 binding. If MSI1 bound to the 3’UTR of APC mRNA stabilizes the message, but inhibits translation, then something likely leads to MSI1 release when APC translation resumes. One possibility is that MSI1 protein is inherently unstable. In this case, MSI1 binds to APC mRNA and blocks translation but is rapidly degraded and therefore releases APC mRNA for translation unless additional MSI1 is made. Because MSI1 is a Wnt target, a cell stimulated by Wnt ligand should continually replace the MSI1, therefore APC mRNA translation would remain blocked until the Wnt signal is removed. Our data showing that MSI1 has a relatively long half-life does not support this model. Current work is aimed to explore whether MSI1 release from APC mRNA is regulated by post-translational modifications or other protein-protein interactions. In conclusion, by acting as a Wnt antagonist, APC blocks MSI1 transcription. On the other hand, MSI1 binds to and stabilizes APC mRNA while also blocking APC translation. The MSI/APC double negative feedback loop appears important for maintaining homeostasis of intestinal epithelial cells. Citation Format: Andy R. Wolfe, Erick Spears, Amanda Ernlund, Kristi L. Neufeld. Musashi 1 stabilizes and blocks translation of Adenomatous Polyposis Coli mRNA in intestinal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2013-1110

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