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1.
Gastroenterology ; 153(3): 812-826, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28624577

RESUMO

BACKGROUND & AIMS: Agents that induce an immune response against tumors by altering T-cell regulation have increased survival times of patients with advanced-stage tumors, such as melanoma or lung cancer. We aimed to characterize molecular features of immune cells that infiltrate hepatocellular carcinomas (HCCs) to determine whether these types of agents might be effective against liver tumors. METHODS: We analyzed HCC samples from 956 patients. We separated gene expression profiles from tumor, stromal, and immune cells using a non-negative matrix factorization algorithm. We then analyzed the gene expression pattern of inflammatory cells in HCC tumor samples. We correlated expression patterns with the presence of immune cell infiltrates and immune regulatory molecules, determined by pathology and immunohistochemical analyses, in a training set of 228 HCC samples. We validated the correlation in a validation set of 728 tumor samples. Using data from 190 tumors in the Cancer Genome Atlas, we correlated immune cell gene expression profiles with numbers of chromosomal aberrations (based on single-nucleotide polymorphism array) and mutations (exome sequence data). RESULTS: We found approximately 25% of HCCs to have markers of an inflammatory response, with high expression levels of the CD274 molecule (programmed death-ligand 1) and programmed cell death 1, markers of cytolytic activity, and fewer chromosomal aberrations. We called this group of tumors the Immune class. It contained 2 subtypes, characterized by markers of an adaptive T-cell response or exhausted immune response. The exhausted immune response subclass expressed many genes regulated by transforming growth factor beta 1 that mediate immunosuppression. We did not observe any differences in numbers of mutations or expression of tumor antigens between the immune-specific class and other HCCs. CONCLUSIONS: In an analysis of HCC samples from 956 patients, we found almost 25% to express markers of an inflammatory response. We identified 2 subclasses, characterized by adaptive or exhausted immune responses. These findings indicate that some HCCs might be susceptible to therapeutic agents designed to block the regulatory pathways in T cells, such as programmed death-ligand 1, programmed cell death 1, or transforming growth factor beta 1 inhibitors.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Fator de Crescimento Transformador beta1/genética , Imunidade Adaptativa/genética , Idoso , Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patologia , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Tolerância Imunológica/genética , Imuno-Histoquímica , Imunofenotipagem , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais , Transcriptoma , Microambiente Tumoral/imunologia , beta Catenina/genética
2.
Gut ; 66(3): 530-540, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-26658144

RESUMO

OBJECTIVE: Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance. DESIGN: HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts. RESULTS: Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC). CONCLUSIONS: Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Somatomedinas/metabolismo , Idoso , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/uso terapêutico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Somatomedinas/antagonistas & inibidores , Somatomedinas/genética , Sorafenibe , Esferoides Celulares , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Hepatol ; 66(5): 952-961, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28126467

RESUMO

BACKGROUND & AIMS: Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy. METHODS: Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n=164) and intrahepatic cholangiocarcinoma (iCCA) (n=149). RESULTS: Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliary-derived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p<0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p=0.008), showed significant upregulation of transforming growth factor (TGF)-ß signaling and enrichment of inflammation-related and immune response signatures (p<0.001). Stem-cell tumors were characterized by spalt-like transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p<0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs. CONCLUSIONS: Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-ß signaling. LAY SUMMARY: Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided.


Assuntos
Neoplasias dos Ductos Biliares/genética , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Neoplasias Hepáticas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/classificação , Neoplasias dos Ductos Biliares/patologia , Carcinoma Hepatocelular/classificação , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/classificação , Colangiocarcinoma/patologia , Instabilidade Cromossômica , Feminino , Humanos , Neoplasias Hepáticas/classificação , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Adulto Jovem
4.
Gastroenterology ; 151(6): 1192-1205, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27614046

RESUMO

BACKGROUND & AIMS: Effective treatments are urgently needed for hepatocellular carcinoma (HCC), which is usually diagnosed at advanced stages. Signaling via the insulin-like growth factor (IGF) pathway is aberrantly activated in HCC by IGF2 overexpression. We aimed to elucidate the mechanism of IGF2 overexpression and its oncogenic activities and evaluate the anti-tumor effects of reducing IGF2 signaling. METHODS: We obtained 228 HCC samples from patients who underwent liver resection, 168 paired non-tumor adjacent cirrhotic liver samples, and 10 non-tumor liver tissues from patients undergoing resection for hepatic hemangioma. We analyzed gene expression, microRNA, and DNA methylation profiles for all samples, focusing on genes in the IGF signaling pathway. IGF2 was expressed in SNU449 and PLC5 HCC cells and knocked down with small hairpin RNAs in Hep3B and Huh7 cell lines. We analyzed these cells for proliferation, apoptosis, migration, and colony formation. We performed studies in mice engineered to express Myc and Akt1 in liver, which develop liver tumors, with or without hepatic expression of Igf2. Mice with xenograft tumors grown from HCC cells were given a monoclonal antibody against IGF1 and IGF2 (xentuzumab), along with sorafenib; tumor growth was measured and tissues were analyzed by immunohistochemistry and immunoblots. RESULTS: Levels of IGF2 messenger RNA and protein were increased >20-fold in 15% of human HCC tissues compared with non-tumor liver tissues. Methylation at the fetal promoters of IGF2 was reduced in the HCC samples and cell lines that overexpressed IGF2, compared with those that did not overexpress this gene, and non-tumor tissues. Tumors that overexpressed IGF2 had gene expression patterns significantly associated with hepatic progenitor cell features, stellate cell activation, NOTCH signaling, and an aggressive phenotype (P < .0001). In mice engineered to express Myc and Akt1 in liver, co-expression of Igf2 accelerated formation of liver tumors, compared to mice with livers expressing only Myc and Akt1, and shortened survival times (P = .02). The antibody xentuzumab blocked phosphorylation of IGF1 receptor in HCC cell lines and reduced their proliferation and colony formation. In mice with xenograft tumors, injection of xentuzumab, with or without sorafenib, slowed tumor growth and increased survival times compared to vehicle or sorafenib alone. Xentuzumab inhibited phosphorylation of IGF1 receptor and AKT and reduced decreased tumor vascularization compared with vehicle. CONCLUSIONS: A large proportion of HCC samples were found to overexpress IGF2, via demethylation of its fetal promoter. Overexpression of IGF2 accelerates formation of liver tumors in mice with hepatic expression of MYC and AKT1, via activation of IGF1 receptor signaling. An antibody against IGF1 and IGF2 slows growth of xenograft tumors and increases survival of these mice.


Assuntos
Anticorpos Neutralizantes/farmacologia , Carcinoma Hepatocelular/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Hepáticas/genética , RNA Mensageiro/metabolismo , Animais , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Metilação de DNA , Epigênese Genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Patológica/tratamento farmacológico , Niacinamida/análogos & derivados , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Transdução de Sinais/genética , Sorafenibe , Ensaio Tumoral de Célula-Tronco , Regulação para Cima
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