Deanna J. Campbell

Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2.

Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra‐ and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal‐regulated kinase (ERK)1/2‐ and phosphatidylinositol‐3‐kinase/protein kinase B (AKT)‐signaling pathways through sphingosine 1‐phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2‐ and AKT‐signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)‐mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE‐013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/“duct‐like” structures, which was blocked by treatment with JTE‐013. Conclusion: Our collective data support the hypothesis that CBAs promote CCA cell‐invasive growth through S1PR2. (Hepatology 2014;60:908–918)

Intrahepatic Cholangiocarcinoma Progression: Prognostic Factors and Basic Mechanisms

In this review, we will examine various molecular biomarkers for their potential to serve as independent prognostic factors for predicting survival outcome in postoperative patients with progressive intrahepatic cholangiocarcinoma. Specific rodent models of intrahepatic cholangiocarcinoma that mimic relevant cellular, molecular, and clinical features of the human disease are also described, not only in terms of their usefulness in identifying molecular pathways and mechanisms linked to cholangiocarcinoma development and progression, but also for their potential value as preclinical platforms for suggesting and testing novel molecular strategies for cholangiocarcinoma therapy. Last, recent studies aimed at addressing the role of desmoplastic stroma in promoting intrahepatic cholangiocarcinoma progression are highlighted in an effort to underline the potential value of targeting tumor stromal components together with that of cholangiocarcinoma cells as a novel therapeutic option for this devastating cancer.

Cancer-associated fibroblasts in intrahepatic cholangiocarcinoma.

Purpose of review The aim of this brief review is to provide an up-to-date view of the role played by α-smooth muscle actin-positive cancer-associated fibroblastic cells in promoting intrahepatic cholangiocarcinoma progression. Recent findings An increase in α-smooth muscle actin-positive cancer-associated fibroblastic cells in the stroma of intrahepatic cholangiocarcinoma has recently been demonstrated to accelerate cholangiocarcinoma progression. However, our understanding of the evolving cellular and molecular interactions between these stromal cells and cholangiocarcinoma cells in relation to promoting intrahepatic cholangiocarcinoma progression is only just beginning to be elucidated. Imbalances in multifactorial growth factor/cytokine signaling, activation of Hedgehog-GLI signaling and of proteases involved in extracellular matrix remodeling, and matricellular protein–protein and protein–cholangiocarcinoma cell interactions, as well as hypoxia, all appear to factor into the complex and dynamic interactive mechanisms through which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote intrahepatic cholangiocarcinoma progression. Novel three-dimensional organotypic co-culture models are being developed to facilitate relevant studies of cancer-associated fibroblastic cell/cholangiocarcinoma cell interactions that may more accurately mimic physiologically pertinent features of the tumor. Summary Increasing our understanding of critical interactive pathways by which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote tumor progression can lead to the development of novel multitargeting strategies for intrahepatic cholangiocarcinoma therapy.

Novel organotypic culture model of cholangiocarcinoma progression

Aim:  Recent studies have suggested that increased α‐smooth muscle‐actin positive myofibroblastic cells (α‐SMA positive CAF) in the desmoplastic stroma may relate to a more aggressive cancer and worse survival outcomes for intrahepatic cholangiocarcinoma (ICC) patients. To facilitate investigating cellular and molecular interactions between α‐SMA positive CAF and cholangiocarcinoma cells related to ICC progression, we developed a novel 3‐D organotypic culture model of cholangiocarcinoma that more accurately mimics the stromal microenvironment, gene expression profile and select pathophysiological characteristics of desmoplastic ICC in vivo.

Preclinical assessment of simultaneous targeting of epidermal growth factor receptor (ErbB1) and ErbB2 as a strategy for cholangiocarcinoma therapy

Overexpression of epidermal growth factor receptor (ErbB1) and/or ErbB2 has been implicated in the pathogenesis of cholangiocarcinoma, suggesting that combined ErbB1/ErbB2 targeting might serve as a target‐based therapeutic strategy for this highly lethal cancer. To test this strategy, we investigated targeting with the ErbB1 inhibitor tryphostin AG1517 and the ErbB2 inhibitor tryphostin AG879, in combination and alone, as well as with the dual ErbB1/ErbB2 inhibitor lapatinib, to assess the effectiveness of simultaneous targeting of ErbB1 and ErbB2 signaling over single inhibitor treatments in suppressing cholangiocarcinoma cell growth in vitro and the therapeutic efficacy of lapatinib in vivo. Our in vitro studies were carried out using rat (BDEneu and C611B) and human (HuCCT1 and TFK1) cholangiocarcinoma cell lines. The efficacy of lapatinib to significantly suppress liver tumor growth was tested in an orthotopic, syngeneic rat model of intrahepatic cholangiocarcinoma progression. Our results demonstrated that simultaneous targeting of ErbB1 and ErbB2 signaling was significantly more effective in suppressing the in vitro growth of both rat and human cholangiocarcinoma cells than individual receptor targeting. Lapatinib was an even more potent inhibitor of cholangiocarcinoma cell growth and inducer of apoptosis than either tryphostin when tested in vitro against these respective cholangiocarcinoma cell lines, regardless of differences in their levels of ErbB1 or ErbB2 protein expression and/or mechanism of activation. Lapatinib treatment also produced a significant suppression of intrahepatic cholangiocarcinoma growth when administered early to rats, but was without effect in inhibiting liver tumor growth in rats with more advanced tumors. Conclusion: Our findings suggest that simultaneous targeting of ErbB1 and ErbB2 could be a potentially selective strategy for cholangiocarcinoma therapy, but is likely to be ineffective by itself against advanced cancer. (HEPATOLOGY 2010)

Differential gene expression profiling of cultured neu-transformed versus spontaneously-transformed rat cholangiocytes and of corresponding cholangiocarcinomas☆

Previously, we described an orthotopic cholangiocarcinoma model based on bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEsp cells) compared to high grade malignant erbB-2/neu- transformed BDE1 cholangiocytes (BDEneu cells) into the livers of syngeneic rats, which closely mimics clinical features of early versus advanced stages of the human cancer. We now used gene expression microarray together with quantitative real-time RT-PCR to profile genes differentially expressed in highly tumorigenic BDEneu cells and corresponding tumors compared to less aggressive tumorigenic BDEsp cells and tumors. Genes identified as being commonly overexpressed in parent BDEneu cells, tumors, and in a BDEneu tumor-derived cholangiocarcinoma cell line included Sox17, Krt20, Erbb2, and Sphk1 when respectively compared to BDEsp cells, tumors, and tumor-derived BDEsp cholangiocarcinoma cells. Muc1 was also prominently overexpressed in BDEneu cells and tumor-derived cholangiocarcinoma cells over that expressed in corresponding BDEsp cell lines. Periostin and tenascin-C, which were produced exclusively by cholangiocarcinoma-associated fibroblastic cells, were each significantly overexpressed in BDEneu tumors compared to BDEsp tumors. Interestingly, amphiregulin was representative of a gene found to be significantly underexpressed in vitro in BDEneu cells compared to BDEsp cells, but significantly overexpressed in BDEneu tumors compared to BDEsp tumors, and correlated with BDEneu cholangiocarcinoma progression in vivo. Our data support a unique animal model that recapitulates important molecular features of human cholangiocarcinoma progression, and may serve as a potentially powerful preclinical platform for identifying and rapidly testing novel molecular targeting strategies for cholangiocarcinoma therapy and/or prevention.

Overexpression of periostin and distinct mesothelin forms predict malignant progression in a rat cholangiocarcinoma model

Periostin and mesothelin have each been suggested to be predictors of poor survival for patients with intrahepatic cholangiocarcinoma, although the clinical prognostic value of both of these biomarkers remains uncertain. The aim of the current study was to investigate these biomarkers for their potential to act as tumor progression factors when assessed in orthotopic tumor and three‐dimensional culture models of rat cholangiocarcinoma progression. Using our orthotopic model, we demonstrated a strong positive correlation between tumor and serum periostin and mesothelin and increasing liver tumor mass and associated peritoneal metastases that also reflected differences in cholangiocarcinoma cell aggressiveness and malignant grade. Periostin immunostaining was most prominent in the desmoplastic stroma of larger sized more aggressive liver tumors and peritoneal metastases. In comparison, mesothelin was more highly expressed in the cholangiocarcinoma cells; the slower growing more highly differentiated liver tumors exhibited a luminal cancer cell surface immunostaining for this biomarker, and the rapidly growing less differentiated liver and metastatic tumor masses largely showed cytoplasmic mesothelin immunoreactivity. Two molecular weight forms of mesothelin were identified, one at ∼40 kDa and the other, a more heavily glycosylated form, at ∼50 kDa. Increased expression of the 40‐kDa mesothelin over that of the 50 kDa form predicted increased malignant progression in both the orthotopic liver tumors and in cholangiocarcinoma cells of different malignant potential in three‐dimensional culture. Moreover, coculturing of cancer‐associated myofibroblasts with cholangiocarcinoma cells promoted overexpression of the 40‐kDa mesothelin, which correlated with enhanced malignant progression in vitro. Conclusion: Periostin and mesothelin are useful predictors of tumor progression in our rat desmoplastic cholangiocarcinoma models. This supports their relevance to human intrahepatic cholangiocarcinoma. (Hepatology Communications 2018;2:155–172)

Sa1687 Modeling Cholangiocarcinoma Desmoplasia for Rapidly Identifying Stromal Targeting Agents

A S L D A b st ra ct s sham or bile duct ligation (BDL) and were then treated with chloroquine (an inhibitor of autophagy; 60 mg/kg/day), rapamycin (an activator of autophagy; 30 mg/kg/day) or recombinant PGRN (5 nmol/kg/day) via intraperitoneal implanted minipump for 3 days. Cholangiocyte proliferation and intrahepatic biliary mass were assessed in vivo by immunohistochemistry using proliferating cellular nuclear antigen (PCNA) or cytokeratin 19 (CK19) antibodies respectively. In vitro, a parental mouse cholangiocyte cell line and cells overexpressing the pro-autophagic effector Sirt1 were treated with chloroquin and rapamycin and PGRN for up to 48 hr and proliferation assessed using MTS assays. Expression of the autophagy markers, Beclin, ATG5, ATG7 and LAMP1, and Sirt1 were assessed by qPCR, immunoblotting and immunohistochemistry in livers and cholangiocytes after the above-mentioned treatments. Autophagy was also assessed in vitro in mouse cholangiocyte cell lines using a commercially available lysotracker staining kit. Results: After BDL, markers of autophagy were increased, specifically in cholangiocytes. Strategies to inhibit autophagy (chloroquine) increased intrahepatic biliary mass and induced cholangiocyte proliferation in vivo and in vitro, whereas strategies to stimulate autophagy (rapamycin) reduced intrahepatic biliary mass after BDL and reduced cholangiocyte proliferation in vivo and in vitro. Treatment with recombinant PGRN reduced the expression of autophagy markers in cholangiocytes and increased cholangiocyte proliferation. Associated with these effects was a PGRN-induced suppression of the pro-autophagic molecule, Sirt1. Indeed, the inhibitory effect of PGRN on autophagy was alleviated in cholangiocytes overexpressing Sirt1. Conclusion: Our data suggest that autophagy is upregulated during hyperplastic cholangiocyte proliferation, presumably as a compensatory mechanism to prevent overt proliferation. Furthermore, PGRN may induce cholangiocyte proliferation by inhibiting autophagy via the suppression of Sirt1 expression.

Abstract 2174: Differential gene overexpression in highly malignant erbB2/neu transformed rat BDE1 cholangiocytes versus less aggressive spontaneously transformed BDE1 cholangiocytes

Intrahepatic cholangiocarcinoma is a highly malignant primary hepatobiliary cancer often presenting as advanced disease with a dismal prognosis. Despite advances, the molecular pathogenesis of cholangiocarcinoma progression is still poorly understood. Previously, we described a unique “patient-like” animal model of intrahepatic cholangiocarcinoma based on bile duct inoculation of highly malignant erbB2/neu transformed rat BDE1 cholangiocytes (BDEneu cells) compared to less malignant spontaneously transformed BDE1 cholangiocytes (BDEsp cells) into the livers of syngeneic rats (Sirica, A.E., et al. 2008; 47:1178-1190). The aim of this study was to use gene expression microarray analysis, validated with quantitative real-time RT-PCR, to provide a profile of significantly overexpressed gene targets in highly tumorigenic BDEneu cells and resultant tumors compared to less malignant BDEsp cells and tumors. Our microarray analysis identified 4,711 probe sets that were significantly (q 600-fold and >100-fold, respectively, in the BDE liver tumors when compared to the cultured BDE cell lines, supporting their tumor stromal origin. Quantitative real-time RT-PCR analysis of BDEneu tumors further demonstrated a strong positive correlation between increased levels of mRNA transcripts for selected genes (i.e., Sox 17, keratin 20, sphingosine kinase 1, and amphiregulin) and progressively increased liver tumor size. Interestingly, amphiregulin mRNA, which was determined to be underexpressed in cultured BDEneu cells compared to BDEsp cells, was significantly increased in the BDEneu tumors over that expressed in the BDEsp tumors, and appeared to correlate with progression in BDEneu cholangiocarcinoma. (Supported by R01 CA 39225 and R01 CA 83650 to A.E.S). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2174.