SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS: SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS: SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/ß-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS: SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY: Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.

Inhibition of metalloprotease hyperactivity in cystic cholangiocytes halts the development of polycystic liver diseases.

OBJECTIVE: Polycystic liver diseases (PCLDs) are genetic disorders characterised by progressive bile duct dilatation and/or cyst development. Their pathogenesis is a consequence of hyperproliferation, hypersecretion and microRNA alterations in cholangiocytes. Here we evaluate the role of matrix metalloproteases (MMPs) in the hepatic cystogenesis of PCLDs. DESIGN: Metalloprotease activity was measured by microfluorimetric assays in normal and polycystic cholangiocyte cultures from humans and rats, and gene expression by real time quantitative PCR. The role of cytokines, oestrogens and growth factors present in the cystic fluid of PCLD patients was evaluated for MMP activity. The MMP inhibitor marimastat was examined for cystic expansion in vitro and in polycystic kidney (PCK) rats. RESULTS: Polycystic human and rat cholangiocytes displayed increased MMP activity, which was associated with increased mRNA levels of different MMPs. Interleukin (IL)-6 and IL-8, and 17ß-oestradiol, all stimulated MMP activity in human cholangiocytes. The presence of antibodies against IL-6 and/or IL-8 receptor/s inhibited baseline MMP hyperactivity of polycystic human cholangiocytes but had no effect on normal human cholangiocytes. MMP-3 was overexpressed in cystic cholangiocytes from PCLD human and PCK rat livers by immunohistochemistry. Marimastat reduced MMP hyperactivity of polycystic human and rat cholangiocytes and blocked the cystic expansion of PCK cholangiocytes cultured in three-dimensions. Chronic treatment of 8-week-old PCK rats with marimastat inhibited hepatic cystogenesis and fibrosis. CONCLUSIONS: PCLDs are associated with cholangiocyte MMP hyperactivity resulting from autocrine/paracrine stimulation by IL-6 and IL-8. Inhibition of this MMP hyperactivity with marimastat decreased hepatic cystogenesis in vitro and in an animal model of PCLD, offering a potential therapeutic tool.

Molecular Mechanisms of Cholangiocarcinogenesis: New Potential Targets for Therapy.

Cholangiocarcinoma (CCA) is a heterogeneous group of dysplastic disorders affecting the biliary epithelium. It is the second most common primary liver tumor which accounts for around 3% of all gastrointestinal cancers. CCA is very deadly due to its aggressiveness, late diagnosis and high chemoresistance. The incidence is increasing worldwide and the therapeutic options are very limited. Radiotherapy, chemotherapy, surgery and/or liver transplantation may be indicated in patients who meet certain criteria, but chances of success are low. There is therefore increasing interest in understanding the molecular mechanisms involved in the pathogenesis of this cancer type and in identifying new targets for therapy. Current strategies are based on targeting key signaling pathways involved in proliferation, survival, apoptosis and migration. In this review, the most relevant molecular mechanisms involved in the pathogenesis of CCA are discussed and the main preclinical and clinical studies are highlighted. Moreover, future directions in basic and clinical research are indicated.