Razumilava, Nataliya

Cholangiocarcinoma (CCA) is a highly lethal cancer arising from the biliary tract epithelium. The cancer biology of this neoplasm is not well understood. To date, only a few CCA cell lines have been reported, which were mostly developed from Asian patients. In this study, we report and characterize a new intrahepatic CCA cell line, LIV27, derived from a surgically resected tumor in a 67-year-old Caucasian woman with primary sclerosing cholangitis (PSC). LIV27 cells grow well in collagen-coated flasks or plates with a doubling time of 57.8 h at passage 14. LIV27 cells have high tumorigenicity in nude mice and stain positive for CK7 and CK19, markers that differentiate CCA from hepatocellular carcinoma. Karyotype analysis showed that LIV27 is aneuploid. We established a single-locus short tandem repeat profile for the LIV27 cell line. This newly established cell line will be a useful model for studying the molecular pathogenesis of, and developing novel therapies for, cholangiocarcinoma.

Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH ( pCMV‐Shh ), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 ( Gli1lacZ/lacZ ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro . EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6 . The HH ligands Shh and Indian HH ( Ihh ) were expressed in epithelial cells, whereas the transcriptional effectors Gli1 , Gli2 , and Gli3 and the HH receptor Patched1 ( Ptch1 ) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.

Cholangiocarcinoma (CCA) is a lethal hepatobiliary neoplasm originating from the biliary apparatus. In humans, CCA risk factors include hepatobiliary inflammation and fibrosis. The recently identified interleukin (IL)−1 family member, IL‐33, has been shown to be a biliary mitogen which also promotes liver inflammation and fibrosis. Our aim was to generate a mouse model of CCA mimicking the human disease. Ectopic oncogene expression in the biliary tract was accomplished by the Sleeping Beauty transposon transfection system with transduction of constitutively active AKT (myr‐AKT) and Yes‐associated protein. Intrabiliary instillation of the transposon–transposase complex was coupled with lobar bile duct ligation in C57BL/6 mice, followed by administration of IL‐33 for 3 consecutive days. Tumors developed in 72% of the male mice receiving both oncogenes plus IL‐33 by 10 weeks but in only 20% of the male mice transduced with the oncogenes alone. Tumors expressed SOX9 and pancytokeratin (features of CCA) but were negative for HepPar1 (a marker of hepatocellular carcinoma). Substantive overlap with human CCA specimens was revealed by RNA profiling. Not only did IL‐33 induce IL‐6 expression by human cholangiocytes but it likely facilitated tumor development in vivo by an IL‐6–sensitive process as tumor development was significantly attenuated in Il‐6–/– male animals. Furthermore, tumor formation occurred at a similar rate when IL‐6 was substituted for IL‐33 in this model. Conclusion: The transposase‐mediated transduction of constitutively active AKT and Yes‐associated protein in the biliary epithelium coupled with lobar obstruction and IL‐33 administration results in the development of CCA with morphological and biochemical features of the human disease; this model highlights the role of inflammatory cytokines in CCA oncogenesis. (Hepatology 2015;61:1627–1642)

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary neoplasm with limited therapeutic options. Thus, new therapeutic strategies are needed to advance our treatment armamentarium for this devastating disease. MicroRNAs represent a new class of endogenous nucleic acids regulating development, cell death, and carcinogenesis. Recent studies have indicated that the miR-106b∼25 cluster, which is upregulated in cancer, has potential antiapoptotic properties. Meanwhile, we have demonstrated that Hedgehog (Hh) signaling is activated in CCA and also has a strong antiapoptotic effect. Based on this information, we posited that Hh signaling may regulate mir-106b∼25 expression. Due to the dependence of CCA on antiapoptotic signals, we conducted a study to determine the role of miR-106b∼25 in CCA apoptosis and unveil its regulatory mechanisms. Experimental procedures: Immortalized benign and malignant human cholangiocyte cell lines were used for the experiments. MicroRNA expression was analyzed using real-time quantitative RT-PCR (qRT-PCR) assays. The effect of the microRNAs was studied using Lipofectamine-based transfection with precursor microRNAs or locked-nucleic acid antagonists (LNAs) to respectively potentiate or decrease microRNAs expression. Functional apoptosis studies employed DAPI staining with fluorescent microscopic analysis of nuclear morphology, or biochemical measurement of caspase-3/7 activity. Western blotting and luciferase assays were conducted to identify potential messenger RNA targets. Results: Expression of miR-106b∼25 was enhanced in malignant vs. benign cholangiocyte cell lines. Treatment with the known antagonist of Hh signaling, cyclopamine, resulted in downregulation of the cluster. Because CCA cells express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in vivo, we next examined TRAIL-induced apoptosis in CCA cells. TRAIL-mediated apoptosis was attenuated by transfection with miR-25 and enhanced by LNAs. Computational analysis identified the TRAIL death receptor 4 (DR4) as a potential target of this cluster. Indeed, we observed downregulation of DR4 cellular protein levels during overexpression of miR-106b∼25 and upregulation of DR4 upon cluster inhibition with LNAs. The 3’untranslated region (UTR) of the DR4 mRNA was cloned into a luciferase reporter, and the potential direct regulation of DR4 by miR-106b∼25 examined. Indeed, the regulation of DR4 by miR-106b∼25 was confirmed by this luciferase assay demonstrating a direct inhibitory effect of the miR-106b∼25 polycistron on the DR4 3’UTR. Conclusions: The miR-106b∼25 cluster is overexpressed in malignant cholangiocyte cell lines, likely by a Hh signaling pathway. miR-106b∼25 inhibits apoptosis, at least in part, via downregulation of DR4. LNA targeted inhibition of miR-106b∼25 sensitizes CCA cells to apoptosis and may represent a new therapeutic strategy for this cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2011-199

AbstractBackgroundCholangiocarcinomas (CCAs) are highly desmoplastic neoplasms with a tumour microenvironment plentiful in myofibroblasts (MFBs).MFB‐derivedPDGF‐BBsurvival signalling is a mediator ofCCAcell resistance to apoptotic stimuli. This raises the concept that targetingPDGFR‐β, a cognate receptor ofPDGF‐BB, represents a potential strategy for the treatment of humanCCA.AimsHerein, we examine a role for inhibitingPDGFR‐β in restoringCCAcell sensitivity to apoptotic stimuliin vitroandin vivo.MethodsWe employed humanCCAsamples from 41 patients (19 intrahepatic and 22 extrahepaticCCAsamples), the humanCCAcell linesKMCH‐1 andHUCCT‐1 as well as shPDGFR‐β‐KMCH‐1 and human myofibroblasticLX‐2 cells for these studies.In vivo‐experiments were conducted using a syngeneic rat orthotopicCCAmodel.ResultsOf severalMFB‐derived growth factors profiled,PDGF‐BBandCTGFwere most abundantly expressed; however, onlyPDGF‐BBattenuated tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) cytotoxicity. Co‐culturingCCAcells withPDGF‐BB‐secretingMFBs significantly decreasedTRAIL‐inducedCCAcell apoptosis when compared with monoculture conditions; this cytoprotective effect was abrogated in the presence of the tyrosine kinase inhibitors imatinib mesylate or linifanib, which inhibitPDGFR‐β. Consistent with these findings,MFB‐imparted cytoprotection also was abolished whenPDGFR‐β was knocked down as demonstrated in shPDGFR‐β‐KMCH‐1 cells. Finally, administration of imatinib mesylate increasedCCAcell apoptosis and reduced tumour growth in a rodentin vivo‐CCAmodel that mimics the human disease.ConclusionsTargetingPDGFR‐β sensitizesCCAcells to apoptotic stimuli and appears to be therapeuticin vivo.