Impact of liver diseases and pharmacological interactions on the transportome involved in hepatic drug disposition.

The liver plays a pivotal role in drug disposition owing to the expression of transporters accounting for the uptake at the sinusoidal membrane and the efflux across the basolateral and canalicular membranes of hepatocytes of many different compounds. Moreover, intracellular mechanisms of phases I and II biotransformation generate, in general, inactive compounds that are more polar and easier to eliminate into bile or refluxed back toward the blood for their elimination by the kidneys, which becomes crucial when the biliary route is hampered. The set of transporters expressed at a given time, i.e., the so-called transportome, is encoded by genes belonging to two gene superfamilies named Solute Carriers (SLC) and ATP-Binding Cassette (ABC), which account mainly, but not exclusively, for the uptake and efflux of endogenous substances and xenobiotics, which include many different drugs. Besides the existence of genetic variants, which determines a marked interindividual heterogeneity regarding liver drug disposition among patients, prevalent diseases, such as cirrhosis, non-alcoholic steatohepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, viral hepatitis, hepatocellular carcinoma, cholangiocarcinoma, and several cholestatic liver diseases, can alter the transportome and hence affect the pharmacokinetics of drugs used to treat these patients. Moreover, hepatic drug transporters are involved in many drug-drug interactions (DDI) that challenge the safety of using a combination of agents handled by these proteins. Updated information on these questions has been organized in this article by superfamilies and families of members of the transportome involved in hepatic drug disposition.

Relationship between cholestasis and altered progesterone metabolism in the placenta-maternal liver tandem.

BACKGROUND: In intrahepatic cholestasis of pregnancy (ICP), there are elevated maternal serum levels of total bile acids, progesterone, and some sulfated metabolites, such as allopregnanolone sulfate, which inhibits canalicular function. AIM: To investigate the relationship between cholestasis and the expression of crucial enzymes involved in progesterone metabolism in the liver and placenta. METHODS: Obstructive cholestasis was induced by bile duct ligation (BDL). RT-qPCR (mRNA) and western blot (protein) were used to determine expression levels. Srd5a1 and Akr1c2 enzymatic activities were assayed by substrate disappearance (progesterone and 5α-dihydroprogesterone, respectively), measured by HPLC-MS/MS. RESULTS: BDL induced decreased Srd5a1 and Akr1c2 expression and activity in rat liver, whereas both enzymes were up-regulated in rat placenta. Regarding sulfotransferases, Sult2b1 was also moderately up-regulated in the liver. In placenta from ICP patients, SRD5A1 and AKR1C2 expression was elevated, whereas both genes were down-regulated in liver biopsies collected from patients with several liver diseases accompanied by cholestasis. SRD5A1 and AKR1C2 expression was not affected by incubating human hepatoma HepG2 cells with FXR agonists (chenodeoxycholic acid and GW4064). Knocking-out Fxr in mice did not reduce Srd5a1 and Akr1c14 expression, which was similarly down-regulated by BDL. CONCLUSION: SRD5A1 and AKR1C2 expression was markedly altered by cholestasis. This was enhanced in the placenta but decreased in the liver, which is not mediated by FXR. These results suggest that the excess of progesterone metabolites in the serum of ICP patients can involve both enhanced placental production and decreased hepatic clearance. The latter may also occur in other cholestatic conditions.

Sensitization of cholangiocarcinoma cells to chemotherapy through BCRP inhibition with ß-caryophyllene oxide.

Cholangiocarcinomas (CCAs) are cancers originated in the biliary tree, which are characterized by their high mortality and marked chemoresistance, partly due to the activity of ATP-binding cassette (ABC) export pumps, whose inhibition has been proposed as a strategy for enhancing the response to chemotherapy. We have previously shown that ß-caryophyllene oxide (CRYO) acts as a chemosensitizer in hepatocellular carcinoma by inhibiting ABCB1, MRP1, and MRP2. Here, we have evaluated the usefulness of CRYO in inhibiting BCRP and improving the response of CCA to antitumor drugs. The TCGA-CHOL cohort (n = 36) was used for in silico analysis. BCRP expression (mRNA and protein) was assayed in samples from intrahepatic (iCCA) and extrahepatic (eCCA) tumors (n = 50) and CCA-derived cells (EGI-1 and TFK-1). In these cells, BCRP-dependent mitoxantrone transport was determined by flow cytometry. At non-toxic concentrations, CRYO inhibited BCRP function, which enhanced the cytostatic effect of drugs used in the treatment of CCA. The BCRP ability to confer resistance to a panel of antitumor drugs was determined in Chinese hamster ovary (CHO) cells with stable BCRP expression. At non-toxic concentrations, CRYO markedly reduced BCRP-induced resistance to known substrate drugs (mitoxantrone and SN-38) and cisplatin, gemcitabine, sorafenib, and 5-FU but not oxaliplatin. Neither CRYO nor cisplatin alone significantly affected the growth of BCRP-expressing tumors subcutaneously implanted in immunodeficient mice. In contrast, intratumor drug content was enhanced when administered together, and tumor growth was inhibited. In sum, the combined treatment of drugs exported by BCRP with CRYO can improve the response to chemotherapy in CCA patients.

TGFß-induced circLTBP2 predicts a poor prognosis in intrahepatic cholangiocarcinoma and mediates gemcitabine resistance by sponging miR-338-3p.

Background & Aims: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer worldwide with an increasing incidence and limited therapeutic options. Therefore, there is an urgent need to open the field to new concepts for identifying clinically relevant therapeutic targets and biomarkers. Here, we explored the role and the clinical relevance of circular RNA (circRNA) circLTBP2 in iCCA. Methods: Transforming growth factor ß (TGFß)-regulated circRNAs were identified by dedicated microarrays in human HuCC-T1 iCCA cell line, and their clinical relevance was evaluated in independent cohorts of patients. Gain and loss of function of circLTBP2 combined with functional tests was performed in vitro and in vivo in mice. RNA pulldown, microRNA sequencing, and RNA immunoprecipitation were performed to explore the sponging activity of circLTBP2. Results: CircLTBP2 (has_circ_0032603) was identified as a novel TGFß-induced circRNA in several cholangiocarcinoma cell lines. CircLTBP2 promotes tumour cell proliferation, migration, and resistance to gemcitabine-induced apoptosis in vitro and tumour growth in vivo. Mechanistically, circLTBP2 acts as a competitive RNA regulating notably the activity of the tumour suppressor microRNA miR-338-3p, leading to the overexpression of its pro-metastatic targets. The restoration of miR-338-3p levels in iCCA cells reversed the pro-tumourigenic effects driven by circLTBP2, including the resistance to gemcitabine-induced apoptosis. In addition, circLTBP2 expression predicted a reduced survival, as detected in not only tumour tissues but also serum extracellular vesicles isolated from patients with iCCA. Conclusions: CircLTBP2 is a novel effector of the pro-tumourigenic arm of TGFß and a clinically relevant biomarker easily detected from liquid biopsies in iCCA. Impact and implications: Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer with limited therapeutic options. Opening the field to new concepts is urgently needed to improve the survival of patients. Here, we evaluated the role and the clinical relevance of circular RNA. We report that TGFß-induced circLTBP2 contributes to CCA carcinogenesis and may constitute a clinically relevant prognostic biomarker detected in liquid biopsies.

Evaluation of potential targets to enhance the sensitivity of cholangiocarcinoma cells to anticancer drugs.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly lethal cancer originated in the biliary tree. Available treatments for CCA are scarcely effective, partly due to mechanisms of chemoresistance, such as aberrant activation of Wnt/ß-catenin pathway and dysfunctional p53. AIM: To evaluate the impact of enhancing the expression of negative regulators of the Wnt/ß-catenin pathway (AXIN1, AXIN2, and GSK3B) and the tumor suppressor gene TP53. METHODS: Gene expression in paired samples of CCA and adjacent non-tumor liver tissue was determined by RT-qPCR and immunohistochemistry (IHC). Using lentiviral vectors, CCA cells were transduced with genes of interest to assess their impact on the resistome (TLDA), apoptosis (annexin V/propidium iodide), and decreased cell viability (MTT). RESULTS: IHC revealed marked nuclear localization of ß-catenin, consistent with Wnt/ß-catenin pathway activation. In silico analysis with data from TCGA showed heterogeneous down-regulation of AXIN1, AXIN2, and GSK3B in CCA. Enhancing the expression of AXIN1, AXIN2, and GSK3B in CCA cells was not enough to block the activity of this signaling pathway or significantly modify resistance to 5-FU, gemcitabine, and platinated drugs. Consistent with impaired p53 function, CDKN1A was down-regulated in CCA. Forced TP53 expression induced p21 up-regulation and reduced cell proliferation. Moreover, the resistome was modified (FAS, BAX, TYMP, and CES2 up-regulation along with DHFR, RRM1, and BIRC5 down-regulation), which was accompanied by enhanced sensitivity to some antitumor drugs, mainly platinated drugs. CONCLUSION: Enhancing TP53 expression, but not that of AXIN1, AXIN2, and GSK3B, in CCA cells may be a useful strategy to sensitize CCA to antitumor drugs.

Role of organic cation transporter 3 (OCT3) in the response of hepatocellular carcinoma to tyrosine kinase inhibitors.

Impaired function of organic cation transporter 1 (OCT1) in hepatocellular carcinoma (HCC) has been associated with unsatisfactory response to sorafenib. However, some patients lacking OCT1 at the plasma membrane (PM) of HCC cells still respond to sorafenib, suggesting that another transporter may contribute to take up this drug. The aim of this study was to investigate whether OCT3 could contribute to the uptake of sorafenib and other tyrosine kinase inhibitors (TKIs) and whether OCT3 determination can predict HCC response to sorafenib. Cells overexpressing OCT3 were used to determine the ability of this carrier to transport sorafenib. Immunostaining of OCT3 was performed in HCC samples obtained in the TRANSFER study. Considering the intensity of staining and the number of OCT3-positive cells, tumors were classified as having absent, weak, moderate, or strong OCT3 expression and were also categorized according to the presence or absence of PM staining. Functional in vitro studies revealed that OCT3 is also able to mediate sorafenib uptake. Other TKIs, such as regorafenib, lenvatinib, and cabozantinib can also interact with this transporter. In silico studies suggested that the expression of OCT3 is better preserved in HCC than that of OCT1. In HCC samples, OCT3 was expressed at the PM of cancer cells, and its presence, detected in 26% of tumors, was associated with better outcomes in patients treated with sorafenib. In conclusion, analysis by immunohistochemistry of OCT3 in the PM of tumor cells may help to predict the response of HCC patients to sorafenib and potentially to other TKIs.

Lacking Immunotherapy Biomarkers for Biliary Tract Cancer: A Comprehensive Systematic Literature Review and Meta-Analysis.

BACKGROUND: Immunotherapy has recently been incorporated into the spectrum of biliary tract cancer (BTC) treatment. The identification of predictive response biomarkers is essential in order to identify those patients who may benefit most from this novel treatment option. Here, we propose a systematic literature review and a meta-analysis of PD-1, PD-L1, and other immune-related biomarker expression levels in patients with BTC. METHODS: Prisma guidelines were followed for this systematic review and meta-analysis. Eligible studies were searched on PubMed. Studies published between 2017 and 2022, reporting data on PD-1/PD-L1 expression and other immune-related biomarkers in patients with BTC, were considered eligible. RESULTS: A total of 61 eligible studies were identified. Despite the great heterogeneity between 39 studies reporting data on PD-L1 expression, we found a mean PD-L1 expression percentage (by choosing the lowest cut-off per study) of 25.6% (95% CI 21.0 to 30.3) in BTCs. The mean expression percentages of PD-L1 were 27.3%, 21.3%, and 27.4% in intrahepatic cholangiocarcinomas (iCCAs-15 studies), perihilar-distal CCAs (p/dCCAs-7 studies), and gallbladder cancer (GBC-5 studies), respectively. Furthermore, 4.6% (95% CI 2.38 to 6.97) and 2.5% (95% CI 1.75 to 3.34) of BTCs could be classified as TMB-H and MSI/MMRd tumors, respectively. CONCLUSION: From our analysis, PD-L1 expression was found to occur approximately in 26% of BTC patients, with minimal differences based on anatomical location. TMB-H and MSI molecular phenotypes occurred less frequently. We still lack a reliable biomarker, especially in patients with mismatch-proficient tumors, and we must need to make an effort to conceive new prospective biomarker discovery studies.

Relevance of the organic anion transporting polypeptide 1B3 (OATP1B3) in the personalized pharmacological treatment of hepatocellular carcinoma.

Although pharmacological treatment is the best option for most patients with advanced hepatocellular carcinoma (HCC), its success is very limited, partly due to reduced uptake and enhanced efflux of antitumor drugs. Here we have explored the usefulness of vectorizing drugs towards the organic anion transporting polypeptide 1B3 (OATP1B3) to enhance their efficacy against HCC cells. In silico studies (RNA-Seq data, 11 cohorts) and immunohistochemistry analyses revealed a marked interindividual variability, together with general downregulation but still expression of OATP1B3 in the plasma membrane of HCC cells. The measurement of mRNA variants in 20 HCC samples showed the almost absence of the cancer-type variant (Ct-OATP1B3) together with marked predominance of the liver-type variant (Lt-OATP1B3). In Lt-OATP1B3-expressing cells, the screening of 37 chemotherapeutical drugs and 17 tyrosine kinase receptors inhibitors (TKIs) revealed that 10 classical anticancer drugs and 12 TKIs were able to inhibit Lt-OATP1B3-mediated transport. Lt-OATP1B3-expressing cells were more sensitive than Mock parental cells (transduced with empty lentiviral vectors) to some Lt-OATP1B3 substrates (paclitaxel and the bile acid-cisplatin derivative Bamet-UD2), but not to cisplatin, which is not transported by Lt-OATP1B3. This enhanced response was abolished by competition with taurocholic acid, a known Lt-OATP1B3 substrate. Tumors subcutaneously generated in immunodeficient mice by Lt-OATP1B3-expressing HCC cells were more sensitive to Bamet-UD2 than those derived from Mock cells. In conclusion, Lt-OATP1B3 expression should be screened before deciding the use of anticancer drugs substrates of this carrier in the personalized treatment of HCC. Moreover, Lt-OATP1B3-mediated uptake must be considered when designing novel anti-HCC targeted drugs.

Role of Intracellular Drug Disposition in the Response of Acute Myeloid Leukemia to Cytarabine and Idarubicin Induction Chemotherapy.

Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.

Impact of Aberrant ß-Catenin Pathway on Cholangiocarcinoma Heterogeneity.

The poor prognosis of most cases of advanced cholangiocarcinoma (CCA) constitutes a severe problem in modern oncology, which is aggravated by the fact that the incidence of this liver cancer is increasing worldwide and is often diagnosed late, when surgical removal is not feasible. The difficulty of dealing with this deadly tumor is augmented by the heterogeneity of CCA subtypes and the complexity of mechanisms involved in enhanced proliferation, apoptosis avoidance, chemoresistance, invasiveness, and metastasis that characterize CCA. Among the regulatory processes implicated in developing these malignant traits, the Wnt/ß-catenin pathway plays a pivotal role. Alteration of ß-catenin expression and subcellular localization has been associated with worse outcomes in some CCA subtypes. This heterogeneity, which also affects cellular and in vivo models commonly used to study CCA biology and anticancer drug development, must be taken into account for CCA investigation to more accurately extrapolate basic laboratory research to the clinical situation. A better understanding of the altered Wnt/ß-catenin pathway in relationship with the heterogeneous forms of CCA is mandatory for developing novel diagnostic tools and therapeutic strategies for patients suffering from this lethal disease.

Serum lipidome unravels a diagnostic potential in bile acid diarrhoea.

OBJECTIVE: Bile acid diarrhoea (BAD) is debilitating yet treatable, but it remains underdiagnosed due to challenging diagnostics. We developed a blood test-based method to guide BAD diagnosis. DESIGN: We included serum from 50 treatment-naive patients with BAD diagnosed by gold standard 75selenium homotaurocholic acid test, 56 feature-matched controls and 37 patients with non-alcoholic fatty liver disease (NAFLD). Metabolomes were generated using mass spectrometry covering 1295 metabolites and compared between groups. Machine learning was used to develop a BAD Diagnostic Score (BDS). RESULTS: Metabolomes of patients with BAD significantly differed from controls and NAFLD. We detected 70 metabolites with a discriminatory performance in the discovery set with an area under receiver-operating curve metric above 0.80. Logistic regression modelling using concentrations of decanoylcarnitine, cholesterol ester (22:5), eicosatrienoic acid, L-alpha-lysophosphatidylinositol (18:0) and phosphatidylethanolamine (O-16:0/18:1) distinguished BAD from controls with a sensitivity of 0.78 (95% CI 0.64 to 0.89) and a specificity of 0.93 (95% CI 0.83 to 0.98). The model was independent of covariates (age, sex, body mass index) and distinguished BAD from NAFLD irrespective of fibrosis stage. BDS outperformed other blood test-based tests (7-alpha-hydroxy-4-cholesten-3-one and fibroblast growth factor 19) currently under development. CONCLUSIONS: BDS derived from serum metabolites in a single-blood sample showed robust identification of patients with BAD with superior specificity and sensitivity compared with current blood test-based diagnostics.

Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis, and prognostication of cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). METHODS: EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 44), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC aetiology (n = 56), and hepatocellular carcinoma (n = 34) and healthy individuals (n = 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC = 0.947; odds ratio [OR] =36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC = 0.992; OR = 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC = 0.941; OR = 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. CONCLUSIONS: Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Mitochondrial RNA methyltransferase TRMT61B is a new, potential biomarker and therapeutic target for highly aneuploid cancers.

Despite being frequently observed in cancer cells, chromosomal instability (CIN) and its immediate consequence, aneuploidy, trigger adverse effects on cellular homeostasis that need to be overcome by anti-stress mechanisms. As such, these safeguard responses represent a tumor-specific Achilles heel, since CIN and aneuploidy are rarely observed in normal cells. Recent data have revealed that epitranscriptomic marks catalyzed by RNA-modifying enzymes change under various stress insults. However, whether aneuploidy is associated with such RNA modifying pathways remains to be determined. Through an in silico search for aneuploidy biomarkers in cancer cells, we found TRMT61B, a mitochondrial RNA methyltransferase enzyme, to be associated with high levels of aneuploidy. Accordingly, TRMT61B protein levels are increased in tumor cell lines with an imbalanced karyotype as well as in different tumor types when compared to control tissues. Interestingly, while TRMT61B depletion induces senescence in melanoma cell lines with low levels of aneuploidy, it leads to apoptosis in cells with high levels. The therapeutic potential of these results was further validated by targeting TRMT61B in transwell and xenografts assays. We show that TRM61B depletion reduces the expression of several mitochondrial encoded proteins and limits mitochondrial function. Taken together, these results identify a new biomarker of aneuploidy in cancer cells that could potentially be used to selectively target highly aneuploid tumors.

Expression of Chemoresistance-Associated ABC Proteins in Hepatobiliary, Pancreatic and Gastrointestinal Cancers.

Hepatobiliary, pancreatic, and gastrointestinal cancers account for 36% of the ten million deaths caused by cancer worldwide every year. The two main reasons for this high mortality are their late diagnosis and their high refractoriness to pharmacological treatments, regardless of whether these are based on classical chemotherapeutic agents, targeted drugs, or newer immunomodulators. Mechanisms of chemoresistance (MOC) defining the multidrug resistance (MDR) phenotype of each tumor depend on the synergic function of proteins encoded by more than one hundred genes classified into seven groups (MOC1-7). Among them, the efflux of active agents from cancer cells across the plasma membrane caused by members of the superfamily of ATP-binding cassette (ABC) proteins (MOC-1b) plays a crucial role in determining tumor MDR. Although seven families of human ABC proteins are known, only a few pumps (mainly MDR1, MRP1-6, and BCRP) have been associated with reducing drug content and hence inducing chemoresistance in hepatobiliary, pancreatic, and gastrointestinal cancer cells. The present descriptive review, which compiles the updated information on the expression of these ABC proteins, will be helpful because there is still some confusion on the actual relevance of these pumps in response to pharmacological regimens currently used in treating these cancers. Moreover, we aim to define the MOC pattern on a tumor-by-tumor basis, even in a dynamic way, because it can vary during tumor progression and in response to chemotherapy. This information is indispensable for developing novel strategies for sensitization.

Clinical relevance of biomarkers in cholangiocarcinoma: critical revision and future directions.

Cholangiocarcinoma (CCA) is a malignant tumour arising from the biliary system. In Europe, this tumour frequently presents as a sporadic cancer in patients without defined risk factors and is usually diagnosed at advanced stages with a consequent poor prognosis. Therefore, the identification of biomarkers represents an utmost need for patients with CCA. Numerous studies proposed a wide spectrum of biomarkers at tissue and molecular levels. With the present paper, a multidisciplinary group of experts within the European Network for the Study of Cholangiocarcinoma discusses the clinical role of tissue biomarkers and provides a selection based on their current relevance and potential applications in the framework of CCA. Recent advances are proposed by dividing biomarkers based on their potential role in diagnosis, prognosis and therapy response. Limitations of current biomarkers are also identified, together with specific promising areas (ie, artificial intelligence, patient-derived organoids, targeted therapy) where research should be focused to develop future biomarkers.

Metabolic-associated fatty liver disease: From simple steatosis toward liver cirrhosis and potential complications. Proceedings of the Third Translational Hepatology Meeting, organized by the Spanish Association for the Study of the Liver (AEEH).

This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC).

Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. METHODS: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. RESULTS: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. CONCLUSION: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. LAY SUMMARY: Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.

The altered serum lipidome and its diagnostic potential for Non-Alcoholic Fatty Liver (NAFL)-associated hepatocellular carcinoma.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is affecting more people globally. Indeed, NAFLD is a spectrum of metabolic dysfunctions that can progress to hepatocellular carcinoma (NAFLD-HCC). This development can occur in a non-cirrhotic liver and thus, often lack clinical surveillance. The aim of this study was to develop non-invasive surveillance method for NAFLD-HCC. METHODS: Using comprehensive ultra-high-performance liquid chromatography mass-spectrometry, we investigated 1,295 metabolites in serum from 249 patients. Area under the receiver operating characteristic curve was calculated for all detected metabolites and used to establish their diagnostic potential. Logistic regression analysis was used to establish the diagnostic score. FINDINGS: We show that NAFLD-HCC is characterised by a complete rearrangement of the serum lipidome, which distinguishes NAFLD-HCC from non-cancerous individuals and other HCC patients. We used machine learning to build a diagnostic model for NAFLD-HCC. We quantified predictive metabolites and developed the NAFLD-HCC Diagnostic Score (NHDS), presenting superior diagnostic potential compared to alpha-fetoprotein (AFP). Patients' metabolic landscapes show a progressive depletion in unsaturated fatty acids and acylcarnitines during transformation. Upregulation of fatty acid transporters in NAFLD-HCC tumours contribute to fatty acid depletion in the serum. INTERPRETATION: NAFLD-HCC patients can be efficiently distinguished by serum metabolic alterations from the healthy population and from HCC patients related to other aetiologies (alcohol and viral hepatitis). Our model can be used for non-invasive surveillance of individuals with metabolic syndrome(s), allowing for early detection of NAFLD-HCC. Therefore, serum metabolomics may provide valuable insight to monitor patients at risk, including morbidly obese, diabetics, and NAFLD patients. FUNDING: The funding sources for this study had no role in study design, data collection, data analyses, interpretation or writing of the report as it is presented herein.