ABSTRACT
The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Humans , Mice , Carcinoma, Hepatocellular/metabolism , Disease Models, Animal , ELAV-Like Protein 1/metabolism , Liver Neoplasms/pathology , RNA/metabolism , SumoylationABSTRACT
BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.
Subject(s)
Bile Duct Neoplasms , Cancer-Associated Fibroblasts , Cholangiocarcinoma , Hepatic Stellate Cells , Protein-Lysine 6-Oxidase , Tumor Microenvironment , Humans , Bile Duct Neoplasms/pathology , Bile Duct Neoplasms/metabolism , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/enzymology , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Cancer-Associated Fibroblasts/enzymology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cholangiocarcinoma/pathology , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/genetics , Cholangiocarcinoma/enzymology , Gene Expression Regulation, Neoplastic , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Hepatic Stellate Cells/enzymology , Neoplastic Stem Cells/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/enzymology , Oxidative Phosphorylation , Protein-Lysine 6-Oxidase/metabolism , Protein-Lysine 6-Oxidase/genetics , Signal TransductionABSTRACT
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.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Membrane Transport Proteins , Sorafenib/pharmacology , Sorafenib/therapeutic useABSTRACT
Introduction: Proteasome inhibition is first line therapy in multiple myeloma (MM). The immunological potential of cell death triggered by defects of the ubiquitin-proteasome system (UPS) and subsequent perturbations of protein homeostasis is, however, less well defined. Methods: In this paper, we applied the protein homeostasis disruptors bortezomib (BTZ), ONX0914, RA190 and PR619 to various MM cell lines and primary patient samples to investigate their ability to induce immunogenic cell death (ICD). Results: Our data show that while BTZ treatment triggers sterile type I interferon (IFN) responses, exposure of the cells to ONX0914 or RA190 was mostly immunologically silent. Interestingly, inhibition of protein de-ubiquitination by PR619 was associated with the acquisition of a strong type I IFN gene signature which relied on key components of the unfolded protein and integrated stress responses including inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR) and general control nonderepressible 2 (GCN2). The immunological relevance of blocking de-ubiquitination in MM was further reflected by the ability of PR619-induced apoptotic cells to facilitate dendritic cell (DC) maturation via type I IFN-dependent mechanisms. Conclusion: Altogether, our findings identify de-ubiquitination inhibition as a promising strategy for inducing ICD of MM to expand current available treatments.
Subject(s)
Interferon Type I , Multiple Myeloma , Humans , Multiple Myeloma/metabolism , Proteasome Inhibitors/pharmacology , Proteasome Endopeptidase Complex/metabolism , Immunogenic Cell Death , Bortezomib/pharmacologyABSTRACT
APN/CD13 is expressed in a variety of cells/tissues and is therefore associated with diverse physiological functions, including proliferation, differentiation, migration, angiogenesis, invasion, metastasis, vasoconstriction, and the regulation of normal and impaired immune function. Increased expression or activity of APN/CD13 has been described for various tumors, such that APN/CD13 is in most cases associated with reduced disease-free and overall survival. The mechanisms that mediate these cellular effects of APN/CD13 have been largely determined and are described here. APN/CD13-regulated signaling pathways include integrin recycling, the regulation of small GTPase activities, cell-ECM interactions, and Erk1/2, PI3K, and Wnt signaling. APN/CD13 is a neo-angiogenesis marker that is not found on normal endothelia, but it is found on neo-angiogenetically active endothelia. Therefore, APN/CD13 represents a specific receptor for so-called "tumor-homing peptides" (NRG peptides). Peptides containing the NRG motif show high-affinity binding to APN/CD13. APN/CD13 thus represents a versatile target for the inhibition of tumor-induced angiogenesis through the tumor-selective administration of, e.g., cytotoxic substances. Furthermore, it enables the molecular imaging of tumor masses and the assessment of (neo)angiogenesis in animal models and in patients. Pharmacological inhibitors of APN/CD13 have been proven to reduce tumor growth and tumor progression in various APN/CD13-positive tumors.
ABSTRACT
Humans are constantly exposed to many environmental pollutants, some of which have been largely acknowledged as key factors in the development of metabolic disorders such as diabetes and obesity. These chemicals have been classified as endocrine-disrupting chemicals (EDCs) and, more recently, since they can interfere with metabolic functions, they have been renamed as metabolism-disrupting chemicals (MDCs). MDCs are present in many consumer products, including food packaging, personal care products, plastic bottles and containers, and detergents. The scientific literature has ever-increasingly focused on insulin-releasing pancreatic ß-cells as one of the main targets for MDCs. Evidence highlights that these substances may disrupt glucose homeostasis by altering pancreatic ß-cell physiology. However, their potential impact on glucagon-secreting pancreatic α-cells remains poorly known despite the essential role that this cellular type plays in controlling glucose metabolism. In the present study, we have selected seven paradigmatic MDCs representing major toxic classes, including bisphenols, phthalates, perfluorinated compounds, metals, and pesticides. By using an in vitro cell-based model, the pancreatic α-cell line αTC1-9, we have explored the effects of these compounds on pancreatic α-cell viability, gene expression, and secretion. We found that cell viability was moderately affected after bisphenol-A (BPA), bisphenol-F (BPF), and perfluorooctanesulfonic acid (PFOS) exposure, although cytotoxicity was relatively low. In addition, all bisphenols, as well as di(2-ethylhexyl) phthalate (DEHP) and cadmium chloride (CdCl2), promoted a marked decreased on glucagon secretion, together with changes in the expression of glucagon and/or transcription factors involved in cell function and identity, such as Foxo1 and Arx. Overall, our results indicated that most of the selected chemicals studied caused functional alterations in pancreatic α-cells. Moreover, we revealed, for the first time, their direct effects on key molecular aspects of pancreatic α-cell biology.
Subject(s)
Endocrine Disruptors , Environmental Pollutants , Humans , Glucagon , Cell Survival , Environmental Pollutants/toxicity , Insulin , Benzhydryl Compounds/toxicity , Endocrine Disruptors/toxicity , Gene ExpressionABSTRACT
Endocrine-disrupting chemicals (EDCs) are chemical substances that can interfere with the normal function of the endocrine system. EDCs are ubiquitous and can be found in a variety of consumer products such as food packaging materials, personal care and household products, plastic additives, and flame retardants. Over the last decade, the impact of EDCs on human health has been widely acknowledged as they have been associated with different endocrine diseases. Among them, a subset called metabolism-disrupting chemicals (MDCs) is able to promote metabolic changes that can lead to the development of metabolic disorders such as diabetes, obesity, hepatic steatosis, and metabolic syndrome, among others. Despite this, today, there are still no definitive and standardized in vitro tools to support the metabolic risk assessment of existing and emerging MDCs for regulatory purposes. Here, we evaluated the following two different pancreatic cell-based in vitro systems: the murine pancreatic ß-cell line MIN6 as well as the human pancreatic ß-cell line EndoC-ßH1. Both were challenged with the following range of relevant concentrations of seven well-known EDCs: (bisphenol-A (BPA), bisphenol-S (BPS), bisphenol-F (BPF), perfluorooctanesulfonic acid (PFOS), di(2-ethylhexyl) phthalate (DEHP), cadmium chloride (CdCl2), and dichlorodiphenyldichloroethylene (DDE)). The screening revealed that most of the tested chemicals have detectable, deleterious effects on glucose-stimulated insulin release, insulin content, electrical activity, gene expression, and/or viability. Our data provide new molecular information on the direct effects of the selected chemicals on key aspects of pancreatic ß-cell function, such as the stimulus-secretion coupling and ion channel activity. In addition, we found that, in general, the sensitivity and responses were comparable to those from other in vivo studies reported in the literature. Overall, our results suggest that both systems can serve as effective tools for the rapid screening of potential MDC effects on pancreatic ß-cell physiology as well as for deciphering and better understanding the molecular mechanisms that underlie their action.
Subject(s)
Benzhydryl Compounds , Endocrine Disruptors , Animals , Dichlorodiphenyl Dichloroethylene , Endocrine Disruptors/toxicity , Humans , Insulin , Mice , PlasticsABSTRACT
Bisphenol-A (BPA) is a widespread endocrine disrupting chemical that constitutes a risk factor for type 2 diabetes mellitus (T2DM). Data from animal and human studies have demonstrated that early exposure to BPA results in adverse metabolic outcomes in adult life. In the present work, we exposed pregnant heterozygous estrogen receptor ß (ERß) knock out (BERKO) mice to 10 µg/kg/day BPA, during days 9-16 of pregnancy, and measured ß-cell mass and proliferation in wildtype (WT) and BERKO male offspring at postnatal day 30. We observed increased pancreatic ß-cell proliferation and mass in WT, yet no effect was produced in BERKO mice. Dispersed islet cells in primary culture treated with 1 nM BPA showed an enhanced pancreatic ß-cell replication rate, which was blunted in pancreatic ß-cells from BERKO mice and mimicked by the selective ERß agonist WAY200070. This increased ß-cell proliferation was found in male adult as well as in neonate pancreatic ß-cells, suggesting that BPA directly impacts ß-cell division at earliest stages of life. These findings strongly indicate that BPA during pregnancy upregulates pancreatic ß-cell division and mass in an ERß-dependent manner. Thus, other natural or artificial chemicals may use this ERß-mediated pathway to promote similar effects.
Subject(s)
Benzhydryl Compounds/toxicity , Estrogen Receptor beta/metabolism , Insulin-Secreting Cells/drug effects , Maternal Exposure/adverse effects , Phenols/toxicity , Prenatal Exposure Delayed Effects/etiology , Prenatal Exposure Delayed Effects/metabolism , Animals , Cell Division/drug effects , Cell Proliferation/drug effects , Estrogen Receptor beta/genetics , Female , Humans , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Knockout , Pregnancy , Prenatal Exposure Delayed Effects/geneticsABSTRACT
Changes in the phenotype that characterizes cancer cells are partly due to altered processing of pre-mRNA by the spliceosome. We have previously reported that aberrant splicing plays an essential role in the impaired response of hepatocellular carcinoma (HCC) to sorafenib by reducing the expression of functional organic cation transporter type 1 (OCT1, gene SLC22A1) that constitutes the primary way for HCC cells to take up this and other drugs. The present study includes an in silico analysis of publicly available databases to investigate the relationship between alternative splicing of SLC22A1 pre-mRNA and the expression of genes involved in the exon-recognition machinery in HCC and adjacent non-tumor tissue. Using Taqman Low-Density Arrays, the findings were validated in 25 tumors that were resected without neoadjuvant chemotherapy. The results supported previous reports showing that there was a considerable degree of alternative splicing of SLC22A1 in adjacent non-tumor tissue, which was further increased in the tumor in a stage-unrelated manner. Splicing perturbation was associated with changes in the profile of proteins determining exon recognition. The results revealed the importance of using paired samples for splicing analysis in HCC and confirmed that aberrant splicing plays an essential role in the expression of functional OCT1. Changes in the exon recognition machinery may also affect the expression of other proteins in HCC. Moreover, these results pave the way to further investigations on the mechanistic bases of the relationship between the expression of spliceosome-associated genes and its repercussion on the appearance of alternative and aberrant splicing in HCC.
Subject(s)
Alternative Splicing , Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Organic Cation Transporter 1/genetics , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Computer Simulation , Datasets as Topic , Exons/genetics , Female , Gene Expression Profiling , Humans , Kaplan-Meier Estimate , Liver/pathology , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Male , Middle Aged , RNA Precursors/genetics , RNA Precursors/metabolism , Spliceosomes/metabolismABSTRACT
Owing to intrinsic and acquired chemoresistance, the response of gastric adenocarcinoma (GAC) to chemotherapy is very poor. Here we have investigated the role of transportome in reducing the intracellular content of anticancer drugs and conferring multidrug resistance (MDR) phenotype. Tumors specimens and paired adjacent tissue were analyzed to determine the MDR signature by TaqMan Low-Density Arrays and single-gene qPCR. Strategies of sensitization were evaluated in vitro using the GAC-derived cell line AGS and in vivo using a subcutaneous xenograft model in immunodeficient nude mice. Several transporters involved in drug uptake and export, which are present in healthy stomach, were highly expressed in GAC. In contrast, the cancer-type OATP1B3 was almost exclusively expressed in tumor tissue. The transportome profile varied depending on tumor anatomical location, differentiation, and stage. Immunofluorescence analysis revealed high MRP1 and MRP4 expression at the plasma membrane of tumor cells as well as AGS cells in culture, in which MRP inhibition resulted in selective sensitization to cytotoxic MRP substrates, such as sorafenib, docetaxel, etoposide, and doxorubicin. In mice with subcutaneous tumors formed by AGS cells, sorafenib alone failed to prevent tumor growth. In contrast, this drug induced a marked inhibitory effect when it was co-administered with diclofenac. In conclusion, MRP1 and MRP4 play an important role in the lack of response of GAC to drugs that are transported by these export pumps. Moreover, agents, such as sorafenib, considered at present useless to treat GAC, may become active antitumor drugs when co-administered with non-toxic MRP inhibitors, such as diclofenac.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Stomach Neoplasms/drug therapy , ATP-Binding Cassette Transporters/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , Diclofenac/administration & dosage , Docetaxel/administration & dosage , Doxorubicin/administration & dosage , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/genetics , Female , Humans , Male , Mice, Nude , Middle Aged , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Sorafenib/administration & dosage , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
The presence of genome-wide DNA hypermethylation is a hallmark of lower grade gliomas (LGG) with isocitrate dehydrogenase (IDH) mutations. Further molecular classification of IDH mutant gliomas is defined by the presence (IDHmut-codel) or absence (IDHmut-noncodel) of hemizygous codeletion of chromosome arms 1p and 19q. Despite the DNA hypermethylation seen in bulk tumors, intra-tumoral heterogeneity at the epigenetic level has not been thoroughly analyzed. To address this question, we performed the first epigenetic profiling of single cells in a cohort of 5 gliomas with IDH1 mutation using single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq). Using the Fluidigm HT IFC microfluidics platform, we generated chromatin accessibility maps from 336 individual nuclei, and identified variable promoter accessibility of non-coding RNAs in LGGs. Interestingly, local chromatin structures of several non-coding RNAs are significant factors that contribute to heterogeneity, and show increased promoter accessibility in IDHmut-noncodel samples. As an example for clinical significance of this result, we identify CYTOR as a poor prognosis factor in gliomas with IDH mutation. Open chromatin assay points to differential accessibility of non-coding RNAs as an important source of epigenetic heterogeneity within individual tumors and between molecular subgroups. Rare populations of nuclei that resemble either IDH mutant molecular group co-exist within IDHmut-noncodel and IDHmut-codel groups, and along with non-coding RNAs may be an important issue to consider for future studies, as they may help guide predict treatment response and relapse.A web-based explorer for the data is available at shiny.turcanlab.org.
Subject(s)
Brain Neoplasms/genetics , Cell Nucleus/genetics , Chromatin/genetics , Epigenesis, Genetic/genetics , Glioma/genetics , Isocitrate Dehydrogenase/genetics , Brain Neoplasms/pathology , Cell Nucleus/pathology , Chromatin/pathology , Cohort Studies , Glioma/pathology , Humans , Mutation/genetics , Sequence Analysis, RNA/methodsABSTRACT
Introduction: Chemotherapy remains the only option for advanced cancer patients when other alternatives are not feasible. Nevertheless, the success rate of this type of therapy is often low due to intrinsic or acquired mechanisms of chemoresistance. Among them, drug extrusion from cancer cells through ATP-binding cassette (ABC) proteins plays an important role. ABC pumps are primary active transporters involved in the barrier and secretory functions of many healthy cells. Areas covered: In this review, we have used The Cancer Genome Atlas (TCGA) database to explore the relationship between the expression of the major ABC proteins involved in cancer chemoresistance in the most common types of cancer, and the drugs used in the treatment of these tumors that are substrates of these pumps. Expert opinion: From unicellular organisms to humans, several ABC proteins play a major role in detoxification processes. Cancer cells exploit this ability to protect themselves from cytostatic drugs. Among the ABC pumps, MDR1, MRPs and BCRP are able to export many antitumor drugs and are expressed in several types of cancer, and further up-regulated during treatment. This event results in the enhanced ability of tumor cells to reduce intracellular drug concentrations and hence the pharmacological effect of chemotherapy.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , ATP-Binding Cassette Transporters/metabolism , Databases, Genetic , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/genetics , Up-RegulationABSTRACT
Although the multi-tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)-CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA-CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up-regulation in tumor tissue was found for miR-141 and miR-330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral-mediated transduction of eCCA (EGI-1 and TFK-1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up-regulated in CCA. Conclusion: The reason for impaired hOCT1-mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib.
Subject(s)
Bile Duct Neoplasms/drug therapy , Cholangiocarcinoma/drug therapy , Down-Regulation/genetics , Octamer Transcription Factor-1/genetics , Protein Kinase Inhibitors/pharmacology , Sorafenib/pharmacology , Animals , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathology , Cell Line, Tumor/drug effects , Cholangiocarcinoma/genetics , Cholangiocarcinoma/pathology , DNA Methylation/genetics , Disease Models, Animal , Drug Resistance/genetics , Genetic Therapy/methods , Humans , Immunoblotting , Male , RNA, Messenger/genetics , Random Allocation , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction/methods , Statistics, NonparametricABSTRACT
INTRODUCTION: Anticancer chemotherapy often faces the problem of intrinsic or acquired drug refractoriness due in part to efficient mechanisms of defense present or developed, respectively, in cancer cells. Owing to their polarity and/or high molecular weight, many cytostatic agents cannot freely cross the plasma membrane by simple diffusion and hence depend on SLC proteins to enter cancer cells. The downregulation of these transporters and the appearance of either inactivating mutations or aberrant splicing, hamper the possibility of anticancer drugs to interact with their intracellular targets. Areas covered: In addition to specific literature, we have revised Gene database of the NCBI PubMed resources and information publicly available at NIH 'The Cancer Genome Atlas' (TCGA) (update November 2018) to evaluate the relationship between the profile of expression of SLC transporters playing a major role in the transportome and accounting for drug uptake, in healthy and tumor tissue, and their ability to recognize as substrate several antitumor drugs frequently used in the treatment of different types of cancer, which could affect the overall response to chemotherapy based on regimens including these drugs. Expert commentary: Changes in the transportome may affect the overall response to chemotherapy based on drugs taken up by SLC transporters.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Neoplasms/drug therapy , Solute Carrier Proteins/genetics , Antineoplastic Agents/administration & dosage , Biological Transport , Genome, Human , Humans , Neoplasms/genetics , Solute Carrier Proteins/metabolism , Tissue Distribution , Treatment OutcomeABSTRACT
BACKGROUND AND PURPOSE: The expression of the human organic cation transporter-1 (hOCT1, gene SLC22A1) is reduced in hepatocellular carcinoma (HCC). The molecular bases of this reduction and its relationship with the poor response of HCC to sorafenib were investigated. EXPERIMENTAL APPROACH: HCC transcriptomes from 366 samples available at TCGA were analysed. Alternative splicing was determined by RT-PCR. The role of miRNAs in SLC22A1 downregulation was investigated. Expression of Oct1 was measured in rodent HCC models (spontaneously generated in Fxr-/- mice and chemically-induced in rats). hOCT1 was overexpressed in human hepatoma cells (HuH7 and HepG2). Sorafenib and regorafenib uptake was determined by HPLC-MS/MS. KEY RESULTS: hOCT1 overexpression enhanced sorafenib, but not regorafenib, quinine-inhibitable uptake by hepatoma cells. In rodent HCC, Oct1 was downregulated, which was accompanied by impaired sorafenib uptake. In mice with s.c.-implanted HCC, sorafenib inhibited the growth of hOCT1 overexpressing tumours. In human HCC, hOCT1 expression was inversely correlated with SLC22A1 promoter methylation, whereas demethylation with decitabine enhanced hOCT1 expression in hepatoma cells. Increased proportion of aberrant hOCT1 mRNA variants was found in HCC samples. In silico analysis identified six miRNAs as candidates to target hOCT1 mRNA. When overexpressed in HepG2 cells a significant hOCT1 mRNA decay was induced by hsa-miR-330 and hsa-miR-1468. Analysis of 39 paired tumour/adjacent samples from TCGA revealed that hsa-mir-330 was consistently upregulated in HCC. CONCLUSION AND IMPLICATIONS: Impaired hOCT1 expression/function in HCC, in part due to epigenetic modifications, plays an important role in the poor pharmacological response of this cancer to sorafenib.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Epigenesis, Genetic/drug effects , Liver Neoplasms/drug therapy , Octamer Transcription Factor-1/antagonists & inhibitors , Sorafenib/pharmacology , Animals , CHO Cells , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Cricetulus , Drug Screening Assays, Antitumor , Epigenesis, Genetic/genetics , Gene Expression Profiling , Gene Silencing/drug effects , HEK293 Cells , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Male , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
BACKGROUND: Sorafenib is the drug of choice in the treatment of advanced hepatocellular carcinoma (HCC). Beneficial effects are limited by mechanisms of chemoresistance, which include downregulation and/or impaired function of plasma membrane transporters accounting for drug uptake. The organic cation transporter 1 (OCT1) plays a major role in sorafenib uptake and decreased expression in HCC has been associated with poorer response. METHODS: The multicenter retrospective TRANSFER study involved tumor biopsies from 39 patients with advanced HCC and sorafenib therapy for ≥4 wk. Endpoint was the relationship between clinicopathological features and immunohistological result. Immunostaining was performed using specific primary anti-OCT1-head and anti-OCT1-tail antibodies. Tumors were classified according to a simplified staining score as absent, weak, moderate or strong, taking into account the localization of the staining at the plasma membrane as positive or negative. RESULTS: Results confirmed OCT1 downregulation in half of the cases investigated (10% absent, 38% weak). However, only one third of tumors expressing OCT1 displayed plasma membrane location (15% vs. 36% cytosolic expression). When comparing HCC with and without OCT1 expression, no different sorafenib response was found. When tumors expressing OCT1 at the plasma membrane were considered separately, a marked longer survival was found (Log Rank p<0.001). No association between OCT1 expression at the plasma membrane with tumor stage, previous treatment with TACE or radiological response was seen.In conclusion, these results indicate that the presence of OCT1 at the plasma membrane, rather than its expression levels, is related to better outcome of HCC patients treated with sorafenib.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Niacinamide/analogs & derivatives , Octamer Transcription Factor-1/metabolism , Phenylurea Compounds/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Membrane/metabolism , Female , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Middle Aged , Niacinamide/pharmacokinetics , Niacinamide/therapeutic use , Octamer Transcription Factor-1/deficiency , Phenylurea Compounds/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , Retrospective Studies , SorafenibABSTRACT
BACKGROUND AIMS: Primary hematopoietic stem and progenitor cells (HSPCs) are key components of cell-based therapies for blood disorders and are thus the authentic substrate for related research. We propose that ubiquitous small-volume diagnostic samples represent a readily available and as yet untapped resource of primary patient-derived cells for cell- and gene-therapy studies. METHODS: In the present study we compare isolation and storage methods for HSPCs from normal and thalassemic small-volume blood samples, considering genotype, density-gradient versus lysis-based cell isolation and cryostorage media with different serum contents. Downstream analyses include viability, recovery, differentiation in semi-solid media and performance in liquid cultures and viral transductions. RESULTS: We demonstrate that HSPCs isolated either by ammonium-chloride potassium (ACK)-based lysis or by gradient isolation are suitable for functional analyses in clonogenic assays, high-level HSPC expansion and efficient lentiviral transduction. For cryostorage of cells, gradient isolation is superior to ACK lysis, and cryostorage in freezing media containing 50% fetal bovine serum demonstrated good results across all tested criteria. For assays on freshly isolated cells, ACK lysis performed similar to, and for thalassemic samples better than, gradient isolation, at a fraction of the cost and hands-on time. All isolation and storage methods show considerable variation within sample groups, but this is particularly acute for density gradient isolation of thalassemic samples. DISCUSSION: This study demonstrates the suitability of small-volume blood samples for storage and preclinical studies, opening up the research field of HSPC and gene therapy to any blood diagnostic laboratory with corresponding bioethics approval for experimental use of surplus material.
Subject(s)
Blood Specimen Collection/methods , Blood Specimen Collection/standards , Cell Separation/methods , Cell Separation/standards , Cell- and Tissue-Based Therapy/methods , Leukocytes/pathology , Thalassemia/blood , Blood Preservation/methods , Blood Preservation/standards , Cell Proliferation , Cell Survival , Cells, Cultured , Cryopreservation , Feasibility Studies , Freezing , Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/physiology , Humans , Leukocyte Count , Leukocytes/physiology , Serologic Tests , Thalassemia/pathologyABSTRACT
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).