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1.
Gut ; 2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907830

RESUMO

OBJECTIVE: Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer usually arising on a background of chronic liver injury involving inflammatory and hepatic regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM-2) is predominantly expressed in hepatic non-parenchymal cells and inhibits Toll-like receptor signalling, protecting the liver from various hepatotoxic injuries, yet its role in liver cancer is poorly defined. Here, we investigated the impact of TREM-2 on liver regeneration and hepatocarcinogenesis. DESIGN: TREM-2 expression was analysed in liver tissues of two independent cohorts of patients with HCC and compared with control liver samples. Experimental HCC and liver regeneration models in wild type and Trem-2-/- mice, and in vitro studies with hepatic stellate cells (HSCs) and HCC spheroids were conducted. RESULTS: TREM-2 expression was upregulated in human HCC tissue, in mouse models of liver regeneration and HCC. Trem-2-/- mice developed more liver tumours irrespective of size after diethylnitrosamine (DEN) administration, displayed exacerbated liver damage, inflammation, oxidative stress and hepatocyte proliferation. Administering an antioxidant diet blocked DEN-induced hepatocarcinogenesis in both genotypes. Similarly, Trem-2-/- animals developed more and larger tumours in fibrosis-associated HCC models. Trem-2-/- livers showed increased hepatocyte proliferation and inflammation after partial hepatectomy. Conditioned media from human HSCs overexpressing TREM-2 inhibited human HCC spheroid growth in vitro through attenuated Wnt ligand secretion. CONCLUSION: TREM-2 plays a protective role in hepatocarcinogenesis via different pleiotropic effects, suggesting that TREM-2 agonism should be investigated as it might beneficially impact HCC pathogenesis in a multifactorial manner.

2.
Clin Cancer Res ; 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32933994

RESUMO

PURPOSE: Biliary tract cancer (BTC) is a heterogeneous group of rare gastrointestinal malignancies with dismal prognosis often associated with inflammation. We assessed the prognostic value of IL6 and YKL-40 compared with CA19-9 before and during palliative chemotherapy. We also investigated in mice whether IL6R inhibition in combination with gemcitabine could prolong chemosensitivity. EXPERIMENTAL DESIGN: A total of 452 Danish participants with advanced (locally advanced and metastatic) BTC were included from six clinical trials (February 2004 to March 2017). Serum CA19-9, IL6, and YKL-40 were measured before and during palliative treatment. Associations between candidate biomarkers and progression-free survival (PFS) and overall survival (OS) were analyzed by univariate and multivariate Cox regression. Effects of inhibiting IL6R and YKL-40 were assessed in vitro, and of IL6R inhibition in vivo. RESULTS: High pretreatment levels of CA19-9, IL6, and YKL-40, and increasing levels during treatment, were associated with short PFS and OS in patients with advanced BTC. IL6 provided independent prognostic information, independent of tumor location and in patients with normal serum CA19-9. ROC analyses showed that IL6 and YKL-40 were predictive of very short OS (OS < 6 months), whereas CA19-9 was best to predict OS > 1.5 years. Treatment with anti-IL6R and gemcitabine significantly diminished tumor growth when compared with gemcitabine monotherapy in an in vivo transplant model of BTC. CONCLUSIONS: Serum IL6 and YKL-40 are potential new prognostic biomarkers in BTC. IL6 provides independent prognostic information and may be superior to CA19-9 in certain contexts. Moreover, anti-IL6R should be considered as a new treatment option to sustain gemcitabine response in patients with BTC.

4.
Nat Rev Gastroenterol Hepatol ; 17(9): 557-588, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32606456

RESUMO

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.

5.
RNA ; 26(11): 1726-1730, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32669295

RESUMO

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in diseases such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin, primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA that requires relatively few RNA sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.

6.
Hepatology ; 2020 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32304327

RESUMO

Cholangiocarcinoma (CCA) encompasses a heterogeneous collection of malignancies for which diagnostic biomarkers are lacking and population screening is otherwise infeasible due its status as a rare disease. Coupled with high post-surgical recurrence rates among the minority of patients diagnosed at resectable stages, systemic clinical management will inevitably be required for the majority of CCA patients with recurrent and advanced disease. In this review, we discuss the therapeutic potential of different classes of molecular targets at various stages of development in CCA, including those targeted at the tumor epithelia (oncogenic, developmental, metabolic, epigenomic) and the tumor microenvironment (angiogenesis, checkpoint regulation). Further, we discuss the successes and failures of CCA targeted therapies, emphasizing key lessons learned that should pave the way for future molecular target evaluation in this uncommon yet bona fide target-rich disease.

7.
iScience ; 23(4): 101008, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32268280

RESUMO

HOTAIR was proposed to regulate either HoxD cluster genes in trans or HoxC cluster genes in cis, a mechanism that remains unclear. We have identified a 32-nucleotide conserved noncoding element (CNE) as HOTAIR ancient sequence that likely originated at the root of vertebrate. The second round of whole-genome duplication resulted in one copy of the CNE within HOTAIR and another copy embedded in noncoding transcript of HOXD11. Paralogous CNEs underwent compensatory mutations, exhibit sequence complementarity with respect to transcripts directionality, and have high affinity in vitro. The HOTAIR CNE resembled a poised enhancer in stem cells and an active enhancer in HOTAIR-expressing cells. HOTAIR expression is positively correlated with HOXC11 in cis and negatively correlated with HOXD11 in trans. We propose a dual modality of HOTAIR regulation where transcription of HOTAIR and its embedded enhancer regulates HOXC11 in cis and sequence complementarity between paralogous CNEs suggests HOXD11 regulation in trans.

8.
Cells ; 9(3)2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32183400

RESUMO

: Cholangiocarcinoma (CCA) comprises a group of heterogeneous biliary cancers with dismal prognosis. The etiologies of most CCAs are unknown, but primary sclerosing cholangitis (PSC) is a risk factor. Non-invasive diagnosis of CCA is challenging and accurate biomarkers are lacking. We aimed to characterize the transcriptomic profile of serum and urine extracellular vesicles (EVs) from patients with CCA, PSC, ulcerative colitis (UC), and healthy individuals. Serum and urine EVs were isolated by serial ultracentrifugations and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. EVs transcriptome was determined by Illumina gene expression array [messenger RNAs (mRNA) and non-coding RNAs (ncRNAs)]. Differential RNA profiles were found in serum and urine EVs from patients with CCA compared to control groups (disease and healthy), showing high diagnostic capacity. The comparison of the mRNA profiles of serum or urine EVs from patients with CCA with the transcriptome of tumor tissues from two cohorts of patients, CCA cells in vitro, and CCA cells-derived EVs, identified 105 and 39 commonly-altered transcripts, respectively. Gene ontology analysis indicated that most commonly-altered mRNAs participate in carcinogenic steps. Overall, patients with CCA present specific RNA profiles in EVs mirroring the tumor, and constituting novel promising liquid biopsy biomarkers.

9.
Nat Commun ; 11(1): 168, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924754

RESUMO

Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5'-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5'-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sequência de Bases , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Humanos , RNA/genética , RNA/fisiologia , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , RNA não Traduzido/genética , RNA não Traduzido/fisiologia , Elementos Reguladores de Transcrição , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Zigoto
10.
Hepatology ; 71(1): 196-213, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31211856

RESUMO

Cholangiocarcinoma (CCA) mortality rates are increasing as a result of rising incidence and limited curative treatment(s) for patients with advanced disease. NOTCH pathway reactivation has been reported in biliary malignancies to conflicting degrees, hindering prioritization of key therapeutic targets within the network and identification of candidate responder patients for NOTCH-directed therapies. We analyzed genomic data from 341 patients with CCA and identified NOTCH1 significantly increased in a subgroup characterized by distinct stromal infiltration. Network-wide imbalance of the NOTCH pathway was seen in CCA, including correlation of NOTCH1 with NOTCH3 and NOTCH ligands. Given the diversity of observed NOTCH receptor engagement, γ-secretase modulation was rationalized as a therapeutic option. Indeed, subcutaneous transplantation of sensitive and resistant CCA cell lines pretreated with a γ-secretase inhibitor (GSi) cocktail demonstrated the antineoplastic effects of GSi in a subset of CCA and led to the development of a 225-gene responder signature. This signature was validated in an independent cohort of 119 patients. Further, this signature was enriched in liver tumors initiated by hydrodynamic injections of activated-NOTCH as compared with the AKT-RAS-driven tumors. Candidate GSi-responder patients were characterized by distinct transcriptomes overlapping with previous hepatobiliary metastasis and stemness, unique stromal properties, and dysfunctional intratumoral immune infiltration. Pan-cancer analysis identified 41.9% of cancer types to harbor prospective GSi-responder patients, which was adapted into a 20-gene GSi-sensitivity score metric capable of discriminating nanomolar versus micromolar sensitivity to a cell-permeable GSi (Z-LLNle-CHO) across 60 diverse tumor lines (area under the curve = 1). Conclusion: We have established a GSi-responder signature with evidence across several patient cohorts, as well as in vitro and in vivo models, to enable precision medicine application of NOTCH-directed therapy in CCA as well as prospectively across diverse malignancies.

11.
Nat Commun ; 10(1): 5220, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31745086

RESUMO

The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.


Assuntos
Ductos Biliares/metabolismo , Efrina-B1/metabolismo , Hepatopâncreas/metabolismo , Receptores da Família Eph/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Ductos Biliares/embriologia , Diferenciação Celular/genética , Efrina-B1/genética , Efrina-B3/genética , Efrina-B3/metabolismo , Perfilação da Expressão Gênica , Hepatopâncreas/embriologia , Ligantes , Morfogênese/genética , Mutação , Ligação Proteica , Receptores da Família Eph/genética , Transdução de Sinais/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
12.
Nat Rev Gastroenterol Hepatol ; 16(12): 748-766, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666728

RESUMO

Primary liver cancer (PLC) is the fourth most frequent cause of cancer-related death. The high mortality rates arise from late diagnosis and the limited accuracy of diagnostic and prognostic biomarkers. The liver is a major regulator, orchestrating the clearance of toxins, balancing glucose, lipid and amino acid uptake, managing whole-body metabolism and maintaining metabolic homeostasis. Tumour onset and progression is frequently accompanied by rearrangements of metabolic pathways, leading to dysregulation of metabolism. The limitation of current therapies targeting PLCs, such as hepatocellular carcinoma and cholangiocarcinoma, points towards the importance of deciphering this metabolic complexity. In this Review, we discuss the role of metabolic liver disruptions and the implications of these processes in PLCs, emphasizing their clinical relevance and value in early diagnosis and prognosis and as putative therapeutic targets. We also describe system biology approaches able to reconstruct the metabolic complexity of liver diseases. We also discuss whether metabolic rearrangements are a cause or consequence of PLCs, emphasizing the opportunity to clinically exploit the rewired metabolism. In line with this idea, we discuss circulating metabolites as promising biomarkers for PLCs.


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Neoplasias Hepáticas/metabolismo , Biomarcadores/metabolismo , Doença Crônica , Progressão da Doença , Humanos , Hepatopatias/complicações , Hepatopatias/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/fisiopatologia , Redes e Vias Metabólicas , Metabolômica , Prognóstico , Biologia de Sistemas
13.
Oncogenesis ; 8(11): 61, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641101

RESUMO

Cholangiocarcinoma (CCA) is a particularly aggressive hepatobiliary malignancy, for which the molecular mechanisms underlying the malignant phenotype are still poorly understood, and novel and effective therapeutic strategies are limited. The pro-survival protein kinase CK2 is frequently overexpressed in cancer and is receiving increasing interest as an anti-tumor drug target. Its precise role in CCA biology is still largely unknown. Here we show that expression of the CK2α and α' catalytic subunits and of the ß regulatory subunit is increased in human CCA samples. Increased expression of CK2 subunits was shown in CCA cell lines compared to non-transformed cholangiocytes. We used chemical inhibition of CK2 and genetic modification by CRISPR/Cas9 to explore the contribution of CK2 to the malignant phenotype of CCA cells. Disruption of CK2 activity results in cell death through apoptosis, reduced invasion and migration potential, and G0/G1 cell cycle arrest. Importantly, CCA cells with a reduced CK2 activity are more sensitive to chemotherapy. Altogether, our results demonstrate that CK2 significantly contributes to increased proliferative potential and augmented growth of CCA cells and indicate the rationale for its targeting as a promising pharmacologic strategy for cholangiocarcinoma.

14.
Trends Cancer ; 5(6): 335-350, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31208696

RESUMO

Cholangiocarcinoma (CCA) comprises a heterogeneous collection of malignancies arising within the biliary tract, characterized by late diagnosis, innate chemoresistance, and abysmal prognosis. Sequencing data have uncovered recurrent mutations in diverse epigenetic regulators, implicating epigenetic destabilization at the root of these tumors. However, few studies have characterized biliary tumor epigenomes. In this Opinion article, we argue that an epigenome-oriented approach to CCA could establish diverse interconnections between many key aspects of research on this disease, including molecular heterogeneity, diverse cells of origin, and prominent tumor microenvironments. Moreover, we discuss plausible causes of epigenome dysregulation in biliary tumors, including genetic, epigenetic, metabolic, microenvironmental, and physiological factors. Lastly, we assess the translational potential of epigenomics in CCA to uncover robust biomarkers and therapeutic opportunities for this growing group of patients with limited treatment options.

16.
Clin Epidemiol ; 11: 323-331, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118820

RESUMO

Introduction: Spontaneous (non-traumatic) subarachnoid hemorrhage (ntSAH) is frequently suspected in the emergency department, but the incidence rate is low. Diagnosis registers can provide valuable data for research in uncommon conditions like ntSAH. Unfortunately, validity vary in the registers. We aimed to assess the validity of the ntSAH diagnosis in the Danish National Patient Register (DNPR) and secondly to describe patients misclassified as ntSAH and to calculate the incidence rate of ntSAH. Methods: From the DNPR we extracted information on patients at least 18 years of age on admission registered with a diagnosis of ntSAH and admitted to a hospital in the Capital Region of Denmark between January 1, 2008 and December 31, 2014. Two independent investigators reviewed the medical records to assess if the diagnosis could be confirmed. Those not confirmed were categorized according to a predeveloped case report form. Results: We identified 1101 patients with a diagnosis of ntSAH; medical records were accessible for 1069 (97.7%) and 842 (78.8%) met the inclusion criteria. The diagnosis was confirmed in 537 patients (63.8% (95% confidence interval: 60.5-67.0%)). Among patients where ntSAH was not confirmed, 122 (40.0%) had a traumatic subarachnoid hemorrhage (tSAH), ntSAH had been suspected in 57 (18.9%) patients but was not substantiated during admission, while parenchymal hemorrhages were identified in 45 (14.5%) patients. The incidence rate was 5.5 (95% CI: 4.9-6.1) per 100,000 persons per year. Conclusion: We found a positive predictive value (PPV) of 63.8% for the diagnosis of non-traumatic subarachnoid hemorrhage in the Danish National Patient Register. The low PPV suggests that care should be taken if unvalidated data are used for research and that results of previous studies should be interpreted with caution.

17.
Hepatology ; 70(4): 1246-1261, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30972782

RESUMO

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.

18.
Liver Int ; 39 Suppl 1: 32-42, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30829432

RESUMO

The complexity of cholangiocarcinoma (CCA) cellularity and the molecular perturbation mechanisms that underlie the diversity of growth patterns of this malignancy remain a clinical concern. Tumours of the biliary system display significant intrinsic chemoresistance, caused by significant stromal involvement and genome-wide tumour heterogeneity, hampering disease remission and palliation as well as promoting the metastatic behaviour. It is crucial to advance our present understanding of the risk and molecular pathogenesis of CCA. This will facilitate the delineation of patient subsets based on molecular perturbations and adjust for precision therapies.


Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Terapia de Alvo Molecular , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/patologia , Humanos , Medicina de Precisão
19.
Br J Pharmacol ; 176(6): 787-800, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30592786

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Epigênese Genética/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Fator 1 de Transcrição de Octâmero/antagonistas & inibidores , Sorafenibe/farmacologia , Animais , Células CHO , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Epigênese Genética/genética , Perfilação da Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar
20.
Int J Cancer ; 144(11): 2782-2794, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30485423

RESUMO

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were performed using next-generation sequencing (NGS). Key oncogenic alterations were identified by targeted NGS and cell lines carrying potentially actionable mutations were treated with corresponding specific inhibitors. PDCL fully resembled morphological features of the primary cancers in vitro and in vivo over extended period in culture. Genomic alterations as well as transcriptome profiles showed high similarity with primary tumors and remained stable during long-term culturing. Targeted-NGS confirmed that key oncogenic mutations such as TP53, KRAS, CTNNB1 as well as actionable mutations (e.g. MET, cKIT, KDR) were highly conserved in PDCL and amenable for individualized therapeutic approaches. Integrative genomic and transcriptomic approaches further demonstrated that PDCL more closely resemble molecular and prognostic features of PLC than established cell lines and are valuable tool for direct target evaluation. Our integrative analysis demonstrates that PDCL represents refined model for discovery of relevant molecular subgroups and exploration of precision medicine approaches for the treatment of this deadly disease.


Assuntos
Linhagem Celular Tumoral/patologia , Neoplasias Hepáticas/patologia , Medicina de Precisão/métodos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Análise Mutacional de DNA , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Camundongos , Mutação , Cultura Primária de Células/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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