ABSTRACT
cJun NH2-terminal kinase (JNK) inhibition has been suggested as a potential treatment for insulin resistance and steatosis through activation of the transcription factor PPARα. However, the long-term consequences have not been evaluated. We found that hepatic JNK deficiency alters bile acid and cholesterol metabolism, resulting in hepatic expression of FGF15 and activation of ERK in cholangiocytes, which ultimately promotes their proliferation. Genetic inactivation of PPARα identifies PPARα hyperactivation as the molecular mechanism for these deleterious effects. Our analysis indicates that hepatic PPARα activation is oncogenic: PPARα deficiency protects mice against carcinogen-induced hepatocellular carcinoma under high fat diet (HFD) condition. These surprising results urge the re-consideration of using JNK inhibitors or PPAR agonists for the treatment of metabolic syndrome.
INTRODUCTION
Liver cancer is the fifth most common cancer and the second leading cause of cancer deaths worldwide (El-Serag, Davila et al., 2003, Parkin, Bray et al., 2005). Metabolic syndrome is a newly recognized, but important risk factor thought to contribute to the increased incidence of hepatocellular carcinoma (HCC) (Klein, Dawson et al., 2014). Steatosis contributes to HCC development due to its association with oxidative stress and inflammation (Smedile & Bugianesi, 2005). Metabolic disorders are common among obese and diabetic patients, and hepatocellular injuries can occur due to increased fat accumulation in the liver. Non-alcoholic fatty liver disease (NAFLD) is extremely frequent in these patients (Caldwell, Crespo et al., 2004), and body weight excess is commonly associated with advanced disease (Neuschwander-Tetri, Brunt et al., 2003). Recent studies have led to the identification of hepatic cJun NH2-terminal kinase (JNK) as a signal transduction pathway that is critically required for obesity-induced insulin resistance and steatosis (Manieri & Sabio, 2015). The cJun NH2-terminal kinase (JNK) signaling pathway contributes to the development of obesity and insulin resistance (Sabio & Davis, 2010). Indeed, mice deficient for Jnk in hepatocytes are resistant to high fat diet (HFD)-induced insulin resistance and steatosis (Vernia, Cavanagh-Kyros et al., 2014); therefore, this signaling pathway represents a potential target for therapeutic intervention. Biochemical studies demonstrate that JNK suppresses PPARα activation in hepatocytes, affecting lipid metabolism and steatosis through the hepatokine FGF21 (encoded by a PPARα target gene) (Vernia, Cavanagh-Kyros et al., 2016, Vernia et al., 2014).
The transcription factor PPARα plays a pivotal role in intracellular free fatty acid (FFA) and triglyceride metabolism by regulating genes involved in fatty acid transport and degradation in mitochondria and peroxisomes (Evans, Barish et al., 2004, Gulick, Cresci et al., 1994, Unger & Zhou, 2001). PPARα is expressed primarily in liver, heart, and muscle and is a major regulator of fatty acid transport, catabolism and energy homeostasis (Memon, Tecott et al., 2000). PPARα activation in the liver is increased in metabolic diseases and obesity (Memon et al., 2000), and PPARα agonists appear to be therapeutically beneficial in diabetes. In fact, PPARα protects against steatosis in the mouse (Ip, Farrell et al., 2003) and suppresses hepatic inflammation (Teoh, Williams et al., 2010). However, PPARα deficiency in mice increases susceptibility to diethylnitrosamine (DEN)-induced HCC (Zhang, Chu et al., 2014), but long-term studies in rodents showed an association of PPARα agonists with hepatic carcinogenesis (Holden & Tugwood, 1999). These findings conflict with the growth inhibitory effects reported for PPARα agonists in cancer cell lines, including HCC cell lines (Maggiora, Oraldi et al., 2010, Panigrahy, Kaipainen et al., 2008, Yamasaki, Kawabe et al., 2011). PPARα may therefore cause context-specific actions on liver cancer development.
The activation of PPARα modifies bile acid (BA) synthesis, conjugation and transport (Li & Chiang, 2009). Altered regulation of BA may protect against steatosis but could increase liver cancer development due to changes in FGF protein levels. Although FGF19 improves the glycemic response and reduces liver steatosis, it also induces liver cancer (Shapiro, Kolodziejczyk et al., 2018). These contrasting potential functions of BAs prompted us to examine whether lack of JNK in hepatocytes, and the resulting hyperactivation of PPARα, could alter BA homeostasis with subsequent deleterious effects. Elucidation of the contribution of JNK and PPARα to liver carcinogenesis may help the development of effective treatments against this malignancy.
RESULTS
Hepatic JNK-deficiency alters bile acid homeostasis
We have previously shown that hepatic JNK deficiency results in the activation of the nuclear transcription factor PPARα and protection against diet-induced insulin resistance and steatosis (Vernia et al., 2014). The activation of PPARα caused altered BA metabolism (Li & Chiang, 2009). We therefore examined BA in hepatic JNK deficient mice (LDKO) and control mice (LWT) at 6 months of age. We found that total BA concentration in the blood of LDKO mice were significantly increased compared with LWT mice (Fig 1A). The increase in circulating BA concentration is consistent with the possibility that LDKO mice may suffer from cholestasis.
The analysis of bile collected from the gallbladder of LDKO and LWT mice revealed significantly increased amounts of BA relative to the amount of cholesterol and phosphatidylcholine (PC) (Fig 1B). Hepatic expression of genes related to hepatic PC synthesis (Scd2, Chpt1, and Chkb) or hepatocyte-mediated transport of PC (Abcb4 and Atp8b1) and BA (Abc11 and Slc10a1) was markedly increased in LDKO mice (Supplementary Fig EV1A, B). Similarly, increased expression of genes related to cholesterol synthesis (Hmgcs1, Hmgcr) and BA synthesis (Baat, Cyp8b1 and Cyp27a) was detected in LDKO mice (Fig 1C). These data are consistent with altered biosynthesis and secretion of both cholesterol and BA through PPARα activation.
Hepatic JNK-deficiency causes cholestasis and liver damage
It is established that cholangitis is a major risk factor for the development of cholangiocarcinoma (de Groen, Gores et al., 1999). We therefore examined the liver of mature adult LDKO and LWT mice. No evidence of hepatic disease was found in LWT mice. Similarly, analysis of hepatic sections prepared from young adult LDKO mice (age 4 months) did not indicate the presence of liver pathology (Figure EV1C). However, at age 10 months, 82% of LDKO mice displayed multifocal bile duct hyperplasia together with fibrosis and inflammatory cell infiltrates (Fig 1D). Cholangiocytes stained positively with PCNA, a marker for proliferation (Fig 1D). These changes were associated with increased expression of myeloid genes (Cd68 and Lyz) and inflammatory cytokines (Ifng, Tnf, Il10, and Il12a) in the liver (Fig 1E), and increased liver damage, as suggested by the high levels of liver enzymes (ALT, AST, and γ-GT) in the blood of LDKO mice (Fig 1F). Theremaining LDKO mice exhibited cholangiocarcinoma (6%) or appeared to be healthy (12%). At age 14 months, 95% of LDKO mice displayed cholangiocarcinoma (Fig 2A) associated with fibrosis (Fig 2B) and a large increase in liver mass together with a significant increase in ALT and AST (Fig 2C). The remaining LDKO mice (6%) exhibited cystic livers with bile duct hyperplasia. Histological analysis indicated increased staining of glutamine synthetase (GS) in liver tumor lesions, together with neoplastic nodules with positive staining of the ductular markers CK19 and Sox9 (Fig 2D). Together, these data confirm that the majority of mature mice with compound deficiency of JNK1 and JNK2 progressively develop cholangiocarcinoma.
The development of bile duct hyperplasia and cholangiocarcinoma in LDKO mice was associated with increased hepatic expression of Cytokeratin 19 (Krt19), a cholangiocyte-specific epithelial marker, the G protein-coupled BA receptor 1 (Gpbar1) and the apical sodium-dependent BA transporter (Asbt, gene symbol Slc10a2) both expressed in cholangiocytes (Keitel, Reinehr et al., 2007) (Dawson, Lan et al., 2009) (Figure 2E). The Notch receptor ligand Jagged-1 promotes the formation of intrahepatic bile ducts (Piccoli & Spinner, 2001), and was overexpressed in the liver of LDKO mice (Fig 2E). Moreover, Bone morphogenetic protein 4 (Bmp4) mediates cholestasis-induced fibrosis (Fan, Shen et al., 2006) and cooperates with FGF to promote the development of cholangiocytes from hepatoblasts (Yanai, Tatsumi et al., 2008); expression of hepatic Bmp4 was increased in LDKO mice compared with LWT mice (Fig 2E). Hepatoblasts maturate to cholangiocytes through activation of ERK pathway (Yang, Wang et al., 2017) and BA can increase proliferation by ERK activation through FXR/FGF15/FGR4 pathway (Li & Chiang, 2015). We evaluated this pathway in 6 months-old mice, before cancer has developed. In concordance with elevated BA production, we found high FXR activation as suggested by the high levels of its target genes (Shp and Fgr4) observed in LDKO mice (Fig 2F). Moreover, while control mice did not expressed Fgf15, we could detect Fgf15 in LDKO livers (Fig 2F). In agreement with these results, histological analysis indicated increased staining of ERK phosphorylation in cholangiocytes from LDKO mice compared with LWT mice (Fig 2G). Together, these changes in FXR/FGF15/FGR4/ERK pathway may contribute to cholangiocyte maturation and proliferation from hepatoblast resulting in bile duct hyperplasia and development of cholangiocarcinoma detected in LDKO mice.
PPARα deficiency reduces liver cancer in JNK1/2 deficient liver
To confirm that hyperactivation of PPARα is involved in the development of cholangiocarcinoma in LDKO mice, we ablated the Ppara gene in LDKO mice. In the resulting JNK1/2 plus PPARα liver-deficient-mice (LPPARαDKO), both tumor burden and incidence were clearly reduced (Fig 3A) compared with LDKO mice. The major changes in BA were also reversed (Fig 3B). This is consistent with reduced hepatic expression in LPPARαDKO mice of genes involved in cholesterol and BA synthesis (Hmgcr, Baat, Cyp8b1 and Cyp27a) and hepatocyte-mediated BA transport (Abc11, Abc4, Abcg5, Abcg8) (Fig 3C). Histological analyses indicated that PPARα deficiency increased liver steatosis, but reduced hallmarks of carcinogenesis (anisokaryosis, apoptosis, ductogenesis, dysplasia and mitosis) in LPPARαDKO compared with LDKO mice (Fig 3D). Moreover, CK19 and SOX9 staining were increased in LDKO mice compared with LPPARαDKO in agreement with cholangiocyte proliferation. Furthermore, although liver tumor lesions in LDKO mice became prominently stained for glutamine synthetase (GS), reduced staining for glutamine synthetase was observed in the liver of LPPARαDKO mice (Fig 3D). In addition, RT-qPCR analysis indicated that both inflammation and cholangiocarcinoma markers were reduced in LPPARαDKO mice compared with LDKO mice (Fig 3E, F). This evidence suggests that PPARα deficiency protected against the promotion of cholangiocyte proliferation in mice lacking hepatocyte JNK1/2. To evaluate whether PPARα deficiency and subsequent normalization of BA production blunted the FXR/FGF15/FGR4/ERK pathway, we evaluated FXR target gene expression. Hepatic RT-qPCR analysis indicated that Fgf15, Shp and Fgr4 expression were reduced in LPPARαDKO mice compared with LDKO mice (Fig 3G). This is consistent with the observation of lower levels of ERK activation, detected by immunohistochemistry, in LPPARαDKO cholangiocytes (Fig 3H).
PPARα deficiency protects against DEN-induced HCC development in HFD-fed mice
Our analysis suggests that activation of PPARα may promote liver tumor development by altering BA physiology. Increased BA induces the synthesis and secretion of inflammatory cytokines in liver, which consequently results in liver injury (Miyake, Wang et al., 2000). Recently, it has been shown that increased hepatic BA controls HCC development in HFD-fed mice (Xie, Wang et al., 2016). To evaluate the role of PPARα in this context, we administered DEN to WT and PPARαKO mice on postnatal day 14, and 6 weeks later placed the animals on either a normal chow diet or a high-fat diet (HFD) in which 60% of calories are fat-derived (Park, Lee et al., 2010). Livers were examined for signs of HCC 8 months after DEN injection (Fig 4A). On normal chow diet, the two genotypes showed no significant differences in tumor number or size (Figu EV2). In contrast, on HFD, the mean number of tumors per animal was lower in PPARαKO mice compared with WT counterparts (Fig 4B). Moreover, tumors were smaller in PPARαKO mice than in WT mice (Fig 4B). This protection against HCC development in HFD-fed PPARαKO mice explained the better survival (Fig 4C). RT-qPCR analysis of tumor and non-tumor tissues from WT and PPARαKO mice revealed reduced expression of the cell-cycle genes Cdk2, Ccna1, Foxm1, and Cdc25c in non-tumor samples from PPARαKO mice and enhanced expression of the cell-cycle regulatory genes p21, Trp53, p19 and p57 in PPARαKO tumor tissue (Fig 4D). Collectively, these data indicate that the absence of PPARα protects against DEN-induced HCC in HFD-fed mice.
PPARα deficiency protects from liver damage on HFD
DEN induces hepatocyte death associated with enhanced compensatory proliferation and augmented HCC development (Das, Garlick et al., 2011, Hui, Bakiri et al., 2007, Maeda, Kamata et al., 2005). DEN-induced apoptosis in liver tissue, measured by caspase 3 cleavage, was reduced in HFD-fed PPARαKO mice (Fig 4E). Moreover, analysis of ALT and AST levels induced by acute DEN treatment in HFD-fed mice revealed less liver damage in PPARαKO mice than in WT counterparts (Fig 4F). Additionally, ERK and STAT3, targets of FGF15/19 that modulate HCC development (Uriarte, Fernandez-Barrena et al., 2013, Uriarte, Latasa et al., 2015, Zhou, Luo et al., 2017), were less activated in livers of PPARαKO mice (Fig 4G).
However, there was no significant reduction in blood levels of the cytokines IL6, TNFα, IL1β, CCL2, and IFNγ in HFD-fed PPARαKO mice, suggesting that the lower acute DEN-induced liver damage was not associated with reduced inflammation (Fig EV3). After acute DEN injection, HFD-fed PPARαKO mice showed higher levels of the chemokines CXCL2 and CCL3 (Fig EV3), correlating with higher levels of markers of infiltration by immune cells (Fig EV4). These results suggest that PPARα in the liver, and not in hematopoietic cells, promotes HCC development in HFD-fed WT animals.
To confirm the role of PPARα in hepatocytes, we tested HCC development in chimeras created by transplanting WT or PPARαKO bone marrow (BM) into lethally irradiated WT or PPARαKO recipients. Chronic-DEN-induced HCC development was strongly suppressed in reconstituted PPARαKO mice compared with reconstituted WT mice, irrespective of donor BM genotype. On HFD, reconstituted PPARαKO mice also developed significantly smaller tumors than their WT counterparts, again irrespective of donor BM genotype (Fig 4H). Despite the established role of PPARα as an immune-modulator (Daynes & Jones, 2002), our bone marrow transplantation experiments show that loss of PPARα in immune cells does not contribute to the protection observed in PPARαKO mice. Our analysis indicates that that the protection against HCC in HFD-fed PPARαKO mice is not primarily mediated by bone-marrow-derived cells.
DISCUSSION
The growing occurrence of liver cancer is due, in part, to an increasing prevalence of established risk factors, such as obesity and physical inactivity (Torre, Bray et al., 2015). HCC, in particular, is strongly associated with obesity and often appears after years of liver steatosis (Caldwell et al., 2004). In addition, long-term elevated BA levels are a risk factor for liver cancer development (Zhang, Zhou et al., 2015) and patients having elevated BA concentration and diabetes have a higher risk of developing HCC (Cui, Martin et al., 2018, Wu, Ge et al., 2010). Indeed, increased BA can lead to inflammation, apoptosis and necrosis of hepatocytes (Allen, Jaeschke et al., 2011, Jansen, Ghallab et al., 2017).
PPARα is an important modulator of liver metabolism controlling lipid and BA homeostasis, and its activation has been shown to decrease fatty liver disease (Abdelmegeed, Yoo et al., 2011, Li & Chiang, 2009, Yeon, Choi et al., 2004). We report that JNK mediated repression of PPARα causes changes in BA homeostasis which suppress cholangiocyte proliferation. Consequently, JNK-deficiency stimulates cholangiocyte proliferation and promotes the development of cholangiocarcinoma. This increased proliferation is mediated by the altered BA metabolism and the elevated hepatic expression of FXR/FGF15/FGR4 that triggers ERK activation in cholangiocytes. Our results have strong translational implications for obesity treatment. Activation of FXR by BA triggers the secretion of FGF15/FGF19 in humans (Inagaki, Choi et al., 2005), and the beneficial effects of FGF family on the obese metabolic profile has been well characterized (Nies, Sancar et al., 2015). However, their clinical use has been debated due to their implication in promoting liver cancer formation by stimulating proliferation (Cui et al., 2018, Zhou et al., 2017). In fact, it has been recently suggested that this deleterious effect of FGF15/FGF19 is more evident under HFD conditions (Cui et al., 2018). Our results support this observation as we describe a tumorigenic effect of PPARα under HFD condition and predict a deleterious effect of using FGF analogs for the treatment of obese patients.
Hepatic PPARα is an important mediator of this regulatory cascade. In fact, PPARα deficiency dramatically suppresses the phenotypes induced by JNK deficiency. The role of PPARα in liver cancer is still unclear. While some studies have demonstrated that PPARα activation might promote liver cancer (Hays, Rusyn et al., 2005, Nishimura, Dewa et al., 2007, Peters, Cattley et al., 1997), others indicate that PPARα activation may be neutral or suppress liver cancer development (Cheung, Akiyama et al., 2004, Heindryckx, Colle et al., 2009, Morimura, Cheung et al., 2006, Takashima, Ito et al., 2008). This could be due to different experimental conditions used in these studies. It is established that the consumption of a HFD causes PPARα activation (Soltis, Kennedy et al., 2017). The HFD also causes enhanced BA production and secretion which leads to cell damage and apoptosis, and the compensatory proliferation of surrounding cells (Sun, Beggs et al., 2016, Yoshitsugu, Kikuchi et al., 2019). Here we demonstrate that in WT mice PPARα promotes tumor development in a HFD DEN-induced model of liver cancer. These results contrast with the lack of effects of PPARα over tumor progression in WT mice fed with ND. The pro-tumorigenic effect of PPARα activation in WT mice is due, in part, to an alteration in BA metabolism that drives ERK activation. These conclusions are consistent with a recent report demonstrating that another nuclear receptor, PPARδ, is activated by HFD and promotes intestinal stem cells hyperproliferation driving to colon cancer (Beyaz, Mana et al., 2016). The data presented here identify obesity-induced PPARα activation as a critical factor in HCC development and progression. Moreover, our study provides evidence that in obesity the effect of PPARα is bone-marrow independent, and that a major inductor of liver cancer is the inhibition of the hepatic JNK.
Because of the role of JNK/PPARα/FGF signaling in lipid metabolism and carcinogenesis and their possible use for steatosis and obesity treatment, it is fundamental to understand the mechanism and conditions in which these signaling pathways might contribute to carcinogenic progression. JNK inhibition and PPARα activation are both potential therapeutic targets in obesity but our results urge to consider the risk of HCC development and other secondary effects during long-term treatments.
MATERIALS & METHODS
Animals
PPARα knock out mice (PPARαKO) (B6;129S4-Pparatm1Gonz/J; RRID:IMSR_JAX:008154) and Albumin cre mice (B6.Cg-Speer6-ps1Tg(Alb-cre)21Mng/J; RRID:IMSR_JAX:003574) were purchased from the Jackson Laboratory and backcrossed for 10 generations to the C57BL/6J background (Jackson Laboratory; RRID:IMSR_JAX:000664). Mice with compound JNK1/2 deficiency in hepatocytes (LDKO) have been described (Das et al., 2011, Das, Sabio et al., 2009). Genotypes were identified by PCR analysis of genomic DNA isolated from mouse tails. All experiments were performed in male mice. For tumor studies, PPARαKO mice at postnatal day 14 received a single i.p. injection of 50 mg/kg body weight diethylnitrosamine (DEN, Sigma-Aldrich, N0258) dissolved in saline. Six weeks after DEN treatment, mice were put on a high-fat diet (HFD, Research Diet Inc.) or standard chow diet ad libitum until sacrifice 8 months after DEN injection. One group of HFD-fed mice was used for Kaplan-Meier analysis. For acute response studies, 6-week-old PPARαKO mice and WT mice were fed the HFD for 13 weeks, given a single 100 mg/kg body weight i.p. injection of DEN, and sacrificed after 48 hours. Radiation chimeras were generated by exposing 2-month-old DEN-injected recipient mice to 2 x 650 Gy ionizing radiation and reconstituting with 2×107 cells from donor bone marrow by tail vein injection. Two weeks after bone marrow transplant, mice were fed the HFD ad libitum until sacrifice 8 months after DEN injection. Before sacrifice, blood samples were taken for analysis of ALT/AST and cytokines. In all cases, mice were euthanized after overnight starvation. Mice were housed in a pathogen-free animal facility and kept on a 12-hour light/dark cycle at constant temperature and humidity. All animal experiments conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC, enforced in Spanish law under Real Decreto 53/2013 and the Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts Medical School.
Serum analysis
Plasma activities of ALT and AST were assessed with the ALT and AST Reagent Kit (Biosystems Reagents) using a Benchmark Plus microplate spectrophotometer (Bio-Rad). Plasma concentration of non-sulfated bile acids were measured with the Bile Acid Assay Kit (Sigma-Aldrich) using a Fluoroskan Ascent fluorescence multiwell plate reader (Thermo Labsystems). Serum cytokine concentrations were measured by multiplexed ELISA (Millipore) with a Luminex 200 analyzer.
Biochemical analysis
Total liver proteins were extracted in lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA pH 8.0, 50 mM NaF, 1 mM sodium glycerophosphate, 5 mM pyrophosphate, 0.27 M sucrose, 1% Triton X-100, 0.1 mM PMSF, 0.1% 2-mercaptoethanol, 1 mM sodium ortovanadate, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Extracts were separated by SDS– PAGE and transferred to 0.2 µm pore size nitrocellulose membranes (Bio-Rad). Blots were probed with primary antibodies to caspase-3 (#9662), cleaved caspase-3 (#9661), phospho ERK (#9101; RRID:AB_330744), ERK (#9102), phospho STAT3 (#9145; RRID:AB_2491009), and Vinculin (V4505, Sigma; RRID:AB_477617). All antibodies were used at 1:1000 dilution. After washes, membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare), and signal was detected using an enhanced chemiluminescent substrate for the detection of horseradish peroxidase (Clarity Western ECL substrate; Bio-Rad).
Histochemistry
Histology was performed using tissue fixed in 10% formalin for 24h, dehydrated and embedded in paraffin. Sections (7 μm) were cut and stained using hematoxylin and eosin (American Master Tech Scientific). Sections were also incubated with Bouińs fluid overnight, counter-stain with hematoxylin (Sigma), and then stained with Masson-Trichrome stain (American Master Tech Scientific). Immunohistochemistry was performed by staining tissue sections with antibodies against PCNA (biotinylated from thermofisher MS-106-B; RRID:AB_64272), SOX9 (Abcam ab3697; RRID:AB_304012), glutamine synthetase (Abcam ab73593; RRID:AB_2247588), cytokeratin 19 (Abcam ab15463; RRID:AB_2281021) or phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology #9101). Streptavidin-conjugated horseradish peroxidase (Biogenex), and the substrate 3,3’-diaminobenzidene (Vector Laboratories) were used followed by brief counter-staining with Mayer’s hematoxylin (Sigma).
Analysis of biliary lipids
Bile was collected from the gall bladder following cholecystectomy. We determined cholesterol and phospholipids using an enzymatic assay (Wako). Total bile acids were measured using Hall’s Bile Stain Kit (American MasterTech). Bile acid species were examined by a modification of the method described by (Ye, Liu et al., 2007) using an HPLC-MS/MS (6410 Triple Quad LC/MS, Agilent Technologies). Chromatographic separation was achieved with gradient elution using a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm, 5 µm) kept at 35°C and a flow rate of 500 µl/min. Initial mobile phase was 80:20 methanol/water, both containing 5 mM ammonium acetate and 0.01% formic acid, pH 4.6, and it was changed to 97:3 methanol/water over 9 min and then returned to 80:20 in 1 min. Electrospray ionization (ESI) in negative mode was used, with the following conditions: gas temperature 350°C, gas flow 8 l/min, nebulizer 10 psi, capillary voltage 2500 V. MS/MS acquisition was performed in multiple reaction monitoring (MRM) mode using the specific m/z transitions: [M-H]- ion to 80,2 for taurine-conjugated bile acids and [M-H]- ion to 74 for glycine-conjugated bile acids. Free bile acids did not generate characteristic ion fragments, as reported by others (Ye et al., 2007), and transition from un-fragmented precursor molecular ions 407.1 to 407.1, 391.3 to 391.3 and 375.3 to 375.3 were selected for trihydroxylated, dihydroxylated and monohydroxylated free bile acids, respectively.
Real time q-PCR
Total RNA was isolated from liver and tumor tissue using the RNeasy Mini Kit (Qiagen) with on-column DNase I-digestion. Complementary DNA was synthesized with the High-Capacity Complementary DNA Reverse Transcription Kit (Applied Biosystems). Taqman© assays were performed using the probes listed in Table 1 (Applied Biosystems). Sequences of primers used for quantitative real-time-polymerase chain reaction (qRT-PCR) are provided in Table 2. Expression levels were normalized to Gapdh and Actb mRNA. qRT-PCR was performed using the Fast SYBR Green system (Applied Biosystems) in a 7900HT Fast Real-time PCR thermal cycler (Applied Biosystems). A dissociation curve program was employed after each reaction to verify purity of the PCR products. The expression of mRNA was examined by quantitative PCR analysis using a 7500 Fast Real Time PCR machine.
QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analysis
Differences between groups were examined for statistical significance using 2-tailed unpaired Student’s t test (with Welch’s correction when variances were different) or ANOVA coupled to Bonferroni’s post-test. Kaplan-Meier analysis was performed using the log-rank test. Statistical details and experimental n are specified in figure legends. Statistical analysis were performed with the GraphPad Prism 7 software (RRID:SCR_002798).
Author Contributions
G.S. and A.M. conceived and supervised this project, E.M. and A.M. performed the experiments and prepared figures, G.S, A.M., R.J.D. and E.M. designed, developed the hypothesis, G.S, R.J. D, E.M, A.M., M J. M, J J.G. M analyzed and interpreted the data. L. E-L., T-L, M. E. R, D. S, C.C., F. J. C., L. L-V, T.B, J. C-K participated in the experiments. E.M., A.M and G.S. wrote the manuscript with input from all authors.
Conflict of Interest
The authors declare no potential conflict of interest.
ACKNOWLEDGMENTS
We thank S. Bartlett for English editing and David Garlick (University of Massachusetts Medical School) for pathological examination of tissue sections. CNIC Advanced Imaging and Animal facility for technical support.
G.S. (RYC-2009-04972), F.J.C (RYC-2014-15242) are investigators of the Ramón y Cajal Program. E.M was awarded La Caixa fellowship. This work was funded by grants supported in part by funds from European Regional Development Fund (ERDF): to G.S. European Union’s Seventh Framework Programme (FP7/2007-2013) ERC 260464, EFSD/Lilly European Diabetes Research Programme Dr Sabio, 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (Investigadores-BBVA-2017) IN[17]_BBM_BAS_0066, MINECO-FEDER SAF2016-79126-R, and Comunidad de Madrid IMMUNOTHERCAN-CM S2010/BMD-2326 and B2017/BMD-3733; F.J.C. EXOHEP-CM S2017/BMD-3727 and the COST Action CA17112.; F.J.C. MINECO Retos SAF2016-78711, the AMMF Cholangiocarcinoma Charity 2018/117, NanoLiver-CM Y2018/NMT-4949, UCM-25-2019, ERAB EA/18-14,. F.J.C. is a Gilead Liver Research Scholar. R.J.D: Grant DK R01 DK107220 from the National Institutes of Health; and to JJGM: PI16/00598 from Carlos III Institute of Health, Spain. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).