Elisa Lozano

Expression of SLC22A1 variants may affect the response of hepatocellular carcinoma and cholangiocarcinoma to sorafenib

Reduced drug uptake is an important mechanism of chemoresistance. Down‐regulation of SLC22A1 encoding the organic cation transporter‐1 (OCT1) may affect the response of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CGC) to sorafenib, a cationic drug. Here we investigated whether SLC22A1 variants may contribute to sorafenib chemoresistance. Complete sequencing and selective variant identification were carried out to detect single nucleotide polymorphisms (SNPs) in SLC22A1 complementary DNA (cDNA). In HCC and CGC biopsies, in addition to previously described variants, two novel alternative spliced variants and three SNPs were identified. To study their functional consequences, these variants were mimicked by directed mutagenesis and expressed in HCC (Alexander and SK‐Hep‐1) and CGC (TFK1) cells. The two novel described variants, R61S fs*10 and C88A fs*16, encoded truncated proteins unable to reach the plasma membrane. Both variants abolished OCT1‐mediated uptake of tetraethylammonium, a typical OCT1 substrate, and were not able to induce sorafenib sensitivity. In cells expressing functional OCT1 variants, OCT1 inhibition with quinine prevented sorafenib‐induced toxicity. Expression of OCT1 variants in Xenopus laevis oocytes and determination of quinine‐sensitive sorafenib uptake by high‐performance liquid chromatography‐dual mass spectrometry confirmed that OCT1 is able to transport sorafenib and that R61S fs*10 and C88A fs*16 abolish this ability. Screening of these SNPs in 23 HCC and 15 CGC biopsies revealed that R61S fs*10 was present in both HCC (17%) and CGC (13%), whereas C88A fs*16 was only found in HCC (17%). Considering all SLC22A1 variants, at least one inactivating SNP was found in 48% HCC and 40% CGC. Conclusion: Development of HCC and CGC is accompanied by the appearance of aberrant OCT1 variants that, together with decreased OCT1 expression, may dramatically affect the ability of sorafenib to reach active intracellular concentrations in these tumors. (Hepatology 2013;53:1065–1073)

Cocarcinogenic Effects of Intrahepatic Bile Acid Accumulation in Cholangiocarcinoma Development

Bile acid accumulation in liver with cholangiolar neoplastic lesions may occur before cholestasis is clinically detected. Whether this favors intrahepatic cholangiocarcinoma development has been investigated in this study. The E. coli RecA gene promoter was cloned upstream from Luc2 to detect in vitro direct genotoxic ability by activation of SOS genes. This assay demonstrated that bile acids were not able to induce DNA damage. The genotoxic effect of the DNA-damaging agent cisplatin was neither enhanced nor hindered by the hepatotoxic and hepatoprotective glycochenodeoxycholic and glycoursodeoxycholic acids, respectively. In contrast, thioacetamide metabolites, but not thioacetamide itself, induced DNA damage. Thus, thioacetamide was used to induce liver cancer in rats, which resulted in visible tumors after 30 weeks. The effect of bile acid accumulation on initial carcinogenesis phase (8 weeks) was investigated in bile duct ligated (BDL) animals. Serum bile acid measurement and determination of liver-specific healthy and tumor markers revealed that early thioacetamide treatment induced hypercholanemia together with upregulation of the tumor marker Neu in bile ducts, which were enhanced by BDL. Bile acid accumulation was associated with increased expression of interleukin (IL)-6 and downregulation of farnesoid X receptor (FXR). Bile duct proliferation and apoptosis activation, with inverse pattern (BDL > thioacetamide + BDL >> thioacetamide vs. thioacetamide > thioacetamide + BDL > BDL), were observed. In conclusion, intrahepatic accumulation of bile acids does not induce carcinogenesis directly but facilitates a cocarcinogenic effect due to stimulation of bile duct proliferation, enhanced inflammation, and reduction in FXR-dependent chemoprotection. Implications: This study reveals that bile acids foster cocarcinogenic events that impact cholangiocarcinoma. Mol Cancer Res; 12(1); 91–100. ©2013 AACR.

Role of the Plasma Membrane Transporter of Organic Cations OCT1 and Its Genetic Variants in Modern Liver Pharmacology

Changes in the uptake of many drugs by the target cells may dramatically affect the pharmacological response. Thus, downregulation of SLC22A1, which encodes the organic cation transporter type 1 (OCT1), may affect the response of healthy hepatocytes and liver cancer cells to cationic drugs, such as metformin and sorafenib, respectively. Moreover, the overall picture may be modified to a considerable extent by the preexistence or the appearance during the pathogenic process of genetic variants. Some rare OCT1 variants enhance transport activity, whereas other more frequent variants impair protein maturation, plasma membrane targeting or the function of this carrier, hence reducing intracellular active drug concentrations. Here, we review current knowledge of the role of OCT1 in modern liver pharmacology, which includes the use of cationic drugs to treat several diseases, some of them of great clinical relevance such as diabetes and primary liver cancer (cholangiocarcinoma and hepatocellular carcinoma). We conclude that modern pharmacology must consider the individual evaluation of OCT1 expression/function in the healthy liver and in the target tissue, particularly if this is a tumor, in order to predict the lack of response to cationic drugs and to be able to design individualized pharmacological treatments with the highest chances of success.

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 17β-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.

Lack of mitochondrial DNA impairs chemical hypoxia-induced autophagy in liver tumor cells through ROS-AMPK-ULK1 signaling dysregulation independently of HIF-1α.

Alterations in mitochondrial DNA (mtDNA) and autophagy activation are common events in tumors. Here we have investigated the effect of mitochondrial genome depletion on chemical hypoxia-induced autophagy in liver tumor cells. Human SK-Hep-1 wild-type and mtDNA-depleted (Rho) cells were exposed to the hypoxia mimetic agents CoCl2 and deferoxamine (DFO). Up-regulation of HIF-1α, but not HIF-2α was observed. The expression of several HIF-1α target genes was also found. In human SK-Hep-1 and mouse Hepa 1-6 liver tumor cells, but not in the counterpart Rho derived lines, chemical hypoxia increased the abundance of autophagosomes and autolysosomes. In wild-type and Rho cells, chemical hypoxia induced down-regulation of HIF-1α-dependent autophagy inhibitors Bcl-2 and mTOR, whereas activation of AMPK/ULK1-mediated pro-autophagy pathway occurred only in wild-type cells. Chemical (compound C) and genetic (shRNA) inhibition of AMPK activation resulted in reduced autophagy. ATP levels were similar in both cell types, whereas constitutive and chemical hypoxia-induced reactive oxygen species (ROS) generation was lower in Rho cells. In wild-type cells, the antioxidant N-acetylcysteine blocked CoCl2- and DFO-induced AMPK and autophagy activation, but not endoplasmic reticulum stress induced by CoCl2. Enhanced Bax-α/Bcl-2 ratio and cell death was induced by hypoxia mimetic agents more markedly in wild-type than in Rho cells. Upon blocking autophagy activation with 3-methyladenine, DFO-induced cell death was partially prevented whereas that induced by CoCl2 was increased, but only in wild-type cells. These results suggest that mitochondrial dysfunction associated with the lack of mtDNA impairs the signaling pathways mediated by ROS, controlling autophagy activation in liver tumor cells, which may contributes to cancer development.

Role of macrophages in bile acid-induced inflammatory response of fetal lung during maternal cholestasis

Infant respiratory distress syndrome (iRDS) in babies born from women with intrahepatic cholestasis of pregnancy (ICP) has been associated with intrauterine exposure to high bile acid levels. Here, we have investigated the role of macrophages in hypercholanemia-induced changes in maternal and fetal lung. Obstructive cholestasis in pregnant rats (OCP) was maintained from day 14 of gestation to term. Gene expression was determined by RT-QPCR, Western blot, and immunofluorescence. The maternal-fetal bile acid pool was radiolabelled using [3H]-taurocholate. OCP resulted in increased bile acids in maternal and fetal organs, including lungs. This was accompanied by structural changes in lung tissue, more marked in fetuses (peribronchial edema, collapse of alveolar spaces and deposits of hyaline material in the alveolar lumen), and infiltration of lung tissue by inflammatory cells. The abundance of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF) was also increased in OCP group. Phospholipase A2-IIA (PLA2), the key enzyme in surfactant degradation, was mainly immunodetected in macrophages, which also expressed the bile acid receptor TGR5. The overall expression of PLA2 was markedly enhanced in maternal and fetal lungs of OCP group and in control maternal BALF cells incubated with bile acids. In neonates born from OCP mothers, the enhanced expression of erythropoietin suggested the presence of hypoxia due to iRDS. In conclusion, these results indicate that the accumulation of bile acids due to maternal cholestasis triggers an inflammatory response in the maternal and fetal lungs together with enhanced macrophage-associated PLA2 expression, which may play an important role in iRDS development.Key MessagesMaternal cholestasis causes respiratory distress syndrome in rat neonates.Cholestasis in pregnant rats causes bile acid accumulation in the fetal lung.This induces lung macrophages infiltration and inflammatory response.Alveolar macrophages co-express phospholipase A2-IIA and TGR5, but not FXR.Bile acid accumulation stimulates phospholipase A2-IIA, but not TGR5, expression.

Molecular Bases Of Chemoresistance In Cholangiocarcinoma.

The multidrug resistance (MDR) phenotype accounts for the poor response of cholangiocarcinoma to available antitumor drugs. This is an important limitation to the use of pharmacological approaches, both as adjuvant therapies and for treating advanced CCA when surgical removal is not possible. MDR is the result of a complex combination of defense mechanisms against toxic compounds already present in cholangiocytes, which play a role in the physiology of these cells by protecting the biliary epithelium from the toxins reaching the biliary tree with the blood that perfuses this tissue, or that are secreted by hepatocytes into bile, to which cholangiocytes are exposed. These mechanisms of chemoresistance (MOC) are also present, usually with enhanced efficacy, in tumors derived from cholangiolar cells. The present review article is an update of the state-of-the-art regarding the MOC involved in the poor response of CCA to antitumor drugs. These MOC have been classified as: changes in the amount of drug in the cells due to decreased uptake (MOC-1a) or enhanced efflux (MOC-1b); altered proportions between prodrug, active drug and inactive metabolites (MOC-2); changes in the molecular targets of antitumor drugs (MOC-3); an enhanced ability of tumor cells to repair drug-induced DNA damage (MOC-4), and an impaired apoptosis/survival balance (MOC-5).

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/β-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.

Protective role of biliverdin against bile acid-induced oxidative stress in liver cells.

The accumulation of bile acids affects mitochondria causing oxidative stress. Antioxidant defense is accepted to include biotransformation of biliverdin (BV) into bilirubin (BR) through BV reductase α (BVRα). The mutation (c.214C>A) in BLVRA results in a non-functional enzyme (mutBVRα). Consequently, homozygous carriers suffering from cholestasis develop green jaundice. Whether BVRα deficiency reduces BV-dependent protection against bile acids is a relevant question because a screening of the mut-BLVRA allele (a) in 311 individuals in Greenland revealed that this SNP was relatively frequent in the Inuit population studied (1% a/a and 4.5% A/a). In three human liver cell lines an inverse correlation between BVRα expression (HepG2>Alexander>HuH-7) and basal reactive oxygen species (ROS) levels was found, however the ability of BV to reduce oxidative stress and cell death induced by deoxycholic acid (DCA) or potassium dichromate (PDC) was similar in these cells. The transduction of BVRα or mutBVRα in human placenta JAr cells with negligible BVRα expression or the silencing of endogenous BVRα expression in liver cells had no effect on DCA-induced oxidative stress and cell death or BV-mediated cytoprotection. DCA stimulated both superoxide anion and hydrogen peroxide production, whereas BV only inhibited the latter. DCA and other dihydroxy-bile acids, but not PDC, induced up-regulation of both BVRα and heme oxygenase-1 (HO-1) in liver cells through a FXR independent and BV insensitive mechanism. In conclusion, BV exerts direct and BVRα-independent antioxidant and cytoprotective effects, whereas bile acid accumulation in cholestasis stimulates the expression of enzymes favoring the heme biotransformation into BV and BR.

Chemoresistance and chemosensitization in cholangiocarcinoma

One of the main difficulties in the management of patients with advanced cholangiocarcinoma (CCA) is their poor response to available chemotherapy. This is the result of powerful mechanisms of chemoresistance (MOC) of quite diverse nature that usually act synergistically. The problem is often worsened by altered MOC gene expression in response to pharmacological treatment. Since CCA includes a heterogeneous group of cancers their genetic signature coding for MOC genes is also diverse; however, several shared traits have been defined. Some of these characteristics are shared with other types of liver cancer, namely hepatocellular carcinoma and hepatoblastoma. An important goal in modern oncologic pharmacology is to develop novel strategies to overcome CCA chemoresistance either by increasing drug specificity, such as in targeted therapies aimed to inhibit receptors with tyrosine kinase activity, or to increase the amounts of active agents inside CCA cells by enhancing drug uptake or reducing efflux through export pumps. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.