RESUMO
BACKGROUND: NAFLD caused by abnormalities in hepatic lipid metabolism is associated with an increased risk of developing HCC. The molecular mechanisms underlying the progression of NAFLD-related HCC are not fully understood. We investigated the molecular mechanism and role of KDM6B downregulation in NAFLD-related HCC after the KDM6B gene was identified using microarray analysis as commonly downregulated in mouse NAFLD-related HCC and human nonhepatitis B and nonhepatitis C viral-HCC. METHODS: The 5-hydroxymethylcytosine levels of KDM6B in HCC cells were determined using glycosylated hydroxymethyl-sensitive PCR. Microarray and chromatin immunoprecipitation analyses using KDM6B-knockout (KO) cells were used to identify KDM6B target genes. Lipotoxicity was assessed using a palmitate-treated cell proliferation assay. Immunohistochemistry was used to evaluate KDM6B expression in human HCC tissues. RESULTS: KDM6B expression levels in HCC cells correlated with the 5-hydroxymethylcytosine levels in the KDM6B gene body region. Gene set enrichment analysis revealed that the lipid metabolism pathway was suppressed in KDM6B-KO cells. KDM6B-KO cells acquired resistance to lipotoxicity (p < 0.01) and downregulated the expression of G0S2, an adipose triglyceride lipase/patatin like phospholipase domain containing 2 (ATGL/PNPLA2) inhibitor, through increased histone H3 lysine-27 trimethylation levels. G0S2 knockdown in KDM6B-expressed HCC cells conferred lipotoxicity resistance, whereas ATGL/PNPLA2 inhibition in the KDM6B-KO cells reduced these effects. Immunohistochemistry revealed that KDM6B expression was decreased in human NAFLD-related HCC tissues (p < 0.001), which was significantly associated with decreased G0S2 expression (p = 0.032). CONCLUSIONS: KDM6B-disrupted HCC acquires resistance to lipotoxicity via ATGL/PNPLA2 activation caused by epigenetic downregulation of G0S2 expression. Reduced KDM6B and G0S2 expression levels are common in NAFLD-related HCC. Targeting the KDM6B-G0S2-ATGL/PNPLA2 pathway may be a useful therapeutic strategy for NAFLD-related HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Camundongos , Humanos , Animais , Hepatopatia Gordurosa não Alcoólica/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Lipase/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genéticaRESUMO
Hepatocellular carcinoma (HCC) imposes a huge global burden, arising from various etiological factors such as hepatitis virus infection and metabolic syndrome. While prophylactic vaccination and antiviral treatment have decreased the incidence of viral HCC, the growing prevalence of metabolic syndrome has led to an increase in non-viral HCC. To identify genes downregulated and specifically associated with unfavorable outcome in non-viral HCC cases, screening analysis was conducted using publically available transcriptome data. Among top 500 genes meeting the criteria, which were involved in lipid metabolism and mitochondrial function, a serine transporter located on inner mitochondrial membrane SFXN1 was highlighted. SFXN1 protein expression was significantly reduced in 33 of 105 HCC tissue samples, and correlated to recurrence-free and overall survival only in non-viral HCC. Human HCC cells with SFXN1 knockout (KO) displayed higher cell viability, lower fat intake and diminished reactive oxygen species (ROS) production in response to palmitate administration. In a subcutaneous transplantation mouse model, high-fat diet feeding attenuated tumorigenic potential in the control cells, but not in the SFXN1-KO cells. In summary, loss of SFXN1 expression suppresses lipid accumulation and ROS generation, preventing toxic effects from fat overload in non-viral HCC, and predicts clinical outcome of non-viral HCC patients.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Síndrome Metabólica , Camundongos , Animais , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Síndrome Metabólica/complicações , Espécies Reativas de Oxigênio , Antivirais/uso terapêuticoRESUMO
BACKGROUND: Recent advances in immune checkpoint blockade (ICB) have improved patient prognosis in mismatch repair-deficient and microsatellite instability-high colorectal cancer (dMMR/MSI-H CRC); however, PD-1 blockade has faced a challenge in early progressive disease. We aimed to understand the early event in ICB resistance using an in vivo model. METHODS: We subcutaneously transplanted the MC38 colon cancer cells into C57BL/6 mice, intraperitoneally injected anti-PD-1 antibody and then isolated ICB-resistant subclones from the recurrent tumors. RESULTS: Comparative gene expression analysis discovered seven genes significantly downregulated in the ICB-resistant cells. Tumorigenicity assay of the MC38 cells knocked out each of the seven candidate genes into C57BL/6 mice treated with anti-PD-1 antibody and bioinformatics analysis of the relationship between the expression of the seven candidate genes and the outcome of cancer patients receiving immunotherapy identified Rtp4, an interferon-stimulated gene and a chaperon protein of G protein-coupled receptors, as a gene involved in ICB resistance. Immunohistochemical analysis of transplanted tumor tissues demonstrated that anti-PD-1 antibody failed to recruit T lymphocytes in the Rtp4-KO MC38 cells. Mouse and human RTP4 expression could be silenced via histone H3 lysine 9 (H3K9) trimethylation, and public transcriptome data indicated the high expression level of RTP4 in most but not all of dMMR/MSI-H CRC. CONCLUSIONS: We clarified that RTP4 could be silenced by histone H3K9 methylation as the early event of ICB resistance. RTP4 expression could be a promising biomarker for predicting ICB response, and the combination of epigenetic drugs and immune checkpoint inhibitors might exhibit synergistic effects on dMMR/MSI-H CRC.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Instabilidade de Microssatélites , Chaperonas Moleculares/genética , Chaperonas Moleculares/uso terapêuticoRESUMO
The invasiveness of pancreatic cancer and its resistance to anticancer drugs define its malignant potential, and are considered to affect the peritumoral microenvironment. Cancer cells with resistance to gemcitabine exposed to external signals induced by anticancer drugs may enhance their malignant transformation. Ribonucleotide reductase large subunit M1 (RRM1), an enzyme in the DNA synthesis pathway, is upregulated during gemcitabine resistance, and its expression is associated with worse prognosis for pancreatic cancer. However, the biological function of RRM1 is unclear. In the present study, it was demonstrated that histone acetylation is involved in the regulatory mechanism related to the acquisition of gemcitabine resistance and subsequent RRM1 upregulation. The current in vitro study indicated that RRM1 expression is critical for the migratory and invasive potential of pancreatic cancer cells. Furthermore, a comprehensive RNA sequencing analysis showed that activated RRM1 induced marked changes in the expression levels of extracellular matrixrelated genes, including Ncadherin, tenascinC and COL11A. RRM1 activation also promoted extracellular matrix remodeling and mesenchymal features, which enhanced the migratory invasiveness and malignant potential of pancreatic cancer cells. The present results demonstrated that RRM1 has a critical role in the biological gene program that regulates the extracellular matrix, which promotes the aggressive malignant phenotype of pancreatic cancer.
Assuntos
Antineoplásicos , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular , Neoplasias Pancreáticas , Ribonucleosídeo Difosfato Redutase , Humanos , Acetilação , Gencitabina , Histonas , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Ribonucleosídeo Difosfato Redutase/genética , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Although histone H3K4 methyltransferase SETD1A is overexpressed in various cancer types, the molecular mechanism underlying its overexpression and its target genes in pancreatic ductal adenocarcinoma (PDAC) remain unclarified. We conducted immunohistochemical staining for SETD1A in 105 human PDAC specimens to assess the relationship between SETD1A overexpression and clinicopathological features. The function and target genes of SETD1A were investigated using human pancreatic cancer cell lines. SETD1A expression was upregulated in 51.4% of patients with PDAC and was an independent prognostic factor associated with shorter disease-free survival after resection (p < 0.05). Knockdown and overexpression of SETD1A showed that SETD1A plays a crucial role in increasing the proliferation and motility of PDAC cells. SETD1A overexpression increased tumorigenicity. RNA sequencing of SETD1A-knockdown cells revealed downregulation of RUVBL1, an oncogenic protein ATP-dependent DNA helicase gene. ChIP analysis revealed that SETD1A binds to the RUVBL1 promoter region, resulting in increased H3K4me3 levels. Knockdown of RUVBL1 showed inhibition of cell proliferation, migration, and invasion of PDAC cells, which are similar biological effects to SETD1A knockdown. High expression of both SETD1A and RUVBL1 was an independent prognostic factor not only for disease-free survival but also for overall survival (p < 0.05). In conclusion, we identified RUVBL1 as a novel downstream target gene of the SETD1A-H3K4me3 pathway. Co-expression of SETD1A and RUVBL1 is an important factor for predicting the prognosis of patients with PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Relevância Clínica , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Prognóstico , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias PancreáticasRESUMO
Immune checkpoint blockade (ICB) treatment improves the prognosis of several types of solid tumors, however, responsiveness to ICB therapy remains low in pancreatic ductal adenocarcinoma (PDACs), which has a rich tumor microenvironment (TME). The TME is composed of various stromal cells, including cancer-associated fibroblasts (CAFs), which contribute to the establishment of an immunosuppressive microenvironment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an innate immune pathway that results in the upregulation of immune cell recruiting-cytokines and anti-tumor efficacy. In this study, we aimed to investigate the impact of cGAS-STING expression and the presence of CAFs upon immune cell infiltration in PDACs. cGAS and STING co-expressing PDAC cases showed favorable survival, with many cytotoxic CD8 + T cell infiltrations from the stromal component adjacent to the cancer cells toward cancer cells, but not in cGAS-STING signaling defected PDAC cases. The signatures of tumor-restrain CAFs were expressed in tumors with cGAS-STING signaling. Finally, transwell co-culture experiments demonstrated that immune cell infiltration was impeded by the presence of CAFs, but not by activation of cGAS-STING signaling. In conclusion, pro-infiltration signals, such as cGAS-STING, and characterization of CAFs are crucial in defeating CAF barricades and encouraging immune cell infiltration in PDACs.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Proteínas de Membrana , Nucleotidiltransferases , Neoplasias Pancreáticas , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Humanos , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Microambiente TumoralRESUMO
Comprehensive analysis of clinical samples has recently identified molecular and immunological classification of hepatocellular carcinoma (HCC), and the CTNNB1 (ß-catenin)-mutated subtype exhibits distinctive characteristics of immunosuppressive tumor microenvironment. For clarifying the molecular mechanisms, we first established human and mouse HCC cells with exon 3 skipping of ß-catenin, which promoted nuclear translocation and activated the Wnt/ß-catenin signaling pathway, by using newly developed multiplex CRISPR/Cas9-based genome engineering system. Gene set enrichment analysis indicated downregulation of immune-associated gene sets in the HCC cells with activated ß-catenin signaling. Comparative analysis of gene expression profiles between HCC cells harboring wild-type and exon 3 skipping ß-catenin elucidated that the expression levels of four cytokines were commonly decreased in human and mouse ß-catenin-mutated HCC cells. Public exome and transcriptome data of 373 human HCC samples showed significant downregulation of two candidate cytokine genes, CCL20 and CXCL2, in HCC tumors with ß-catenin hotspot mutations. T cell killing assays and immunohistochemical analysis of grafted tumor tissues demonstrated that the mouse Ctnnb1Δex3 HCC cells evaded immunosurveillance. Taken together, this study discovered that cytokine controlled by ß-catenin signaling activation could contribute to immune evasion, and provided novel insights into cancer immunotherapy for the ß-catenin-mutated HCC subtype.
Assuntos
Carcinoma Hepatocelular/metabolismo , Éxons , Evasão da Resposta Imune , Neoplasias Hepáticas/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Mutação , beta Catenina/genéticaRESUMO
The activity of histone acetyltransferases (HATs) plays a central role in an epigenetic modification in cooperation with HDACs (histone deacetyl transferases). It is likely that malfunction of this enzymatic machinery controlling epigenetic modification is relevant to carcinogenesis and tumor progression. However, in pancreatic cancer, the clinical relevance of HAT activity and histone acetylation has remained unclear. We identified that H3 acetylation was expressed in all pancreatic cancer patients, indicating that H3 acetylation may be essential in pancreatic cancer cells. We also found that the HAT inhibitor C646 augmented anti-tumor effects in vitro by inhibiting cell proliferation and cell cycle progression concomitantly with suppression of acetylated H3K9 and H3K27 expression. C646 or p300 and CBP (CREB-binding protein)-specific siRNA treatment inhibited the transcription of the G2/M cell cycle regulatory proteins cyclin B1 and CDK1 (cyclin-dependent kinase 1). C646 treatment also inhibited tumor growth in vivo in a xenograft mouse model. C646 could be an effective therapeutic agent for pancreatic cancer. The epigenetic status of pancreatic cancers based on their level of histone H3 acetylation may influence patient survival. Epigenetic stratification according to H3K27 acetylation could be useful for predicting disease prognosis as well as the therapeutic efficacy of C646 in pancreatic cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Pontos de Checagem da Fase M do Ciclo Celular , Proteína Cofatora de Membrana/metabolismo , Idoso , Animais , Apoptose , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of hepatocellular carcinoma (HCC) is a multistep process with a complex interaction of various genetic backgrounds and the tumor microenvironment. In addition to the development of rational approaches to epidemiologic research, early detection, and diagnosis, considerable progress has been made in systemic treatment with molecular-targeted agents for patients with advanced HCC. Moreover, encouraging reports of recent clinical trials of combination therapy with immune-checkpoint inhibitors (ICIs) has raised high hopes. Each HCC is the result of a unique combination of somatic alterations, including genetic, epigenetic, transcriptomic, and metabolic events, leading to conclusive tumoral heterogeneity. Recent advances in comprehensive genetic analysis have accelerated molecular classification and defined subtypes with specific characteristics, including immune-associated molecular profiles reflecting the immune reactivity in the tumor. In considering the development of therapeutic strategies in combination with immunotherapy, proper interpretation of molecular pathological characterization could lead to effective therapeutic deployment and enable individualization of the management of HCC. Here, we review distinctive molecular alterations in the subtype classification of HCC, current therapies, and representative clinical trials with alternative immune-combination approaches from a molecular pathological point.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Terapia Combinada , Humanos , Imunoterapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Microambiente TumoralRESUMO
Stemness factors control microRNA expression in cancer stem cells. Downregulation of miR-100 and miR-125b is associated with tumor progression and prognosis of various cancers. Comparing miRNA profiling of patient-derived tumorsphere (TS) and adherent (2D) hepatocellular carcinoma cells, miR-100 and miR-125b are identified to have association with stemness. In TS cells, miR-100 and miR-125b were downregulated comparing to 2D cells. The finding was reproduced in Hep3B cells. Overexpression of stemness factors NANOG, OCT4 and SOX2 by introduction of gene constructs in Hep3B cells suppressed these two miRNA expression levels. Treatment of chromeceptin, an IGF signaling pathway inhibitor, decreased numbers of TS and inhibited the AKT/mTOR pathway. Stable cell line of miR-100 and miR-125b overexpression decreased IGF2 expression and inhibited tumor growth in the xenograft model. In conclusion, miR-100 and miR-125b have tumor suppressor role in hepatocellular carcinoma through inhibiting IGF2 expression and activation of the AKT/mTOR pathway.
Assuntos
Carcinoma Hepatocelular/genética , Fator de Crescimento Insulin-Like II/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Regulação para Cima , Benzopiranos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , PrognósticoRESUMO
Genomic analyses have recently discovered the malignant subtype of human intrahepatic cholangiocarcinoma (ICC) characterized by frequent mutations of chromatin remodeling gene ARID1A; however, the biological and molecular functions still remain obscure. We here examined the clinical and biological significances of ARID1A deficiency in human ICC. Immunohistochemical analysis demonstrated that the loss of ARID1A was an independent prognostic factor for overall survival of ICC patients (P = 0.023). We established ARID1A-knockout (KO) cells by using the CRISPR/Cas9 system from two human cholangiocarcinoma cell lines. ARID1A-KO cells exhibited significantly enhanced migration, invasion, and sphere formation activity. Microarray analysis revealed that ALDH1A1, a stemness gene, was the most significantly elevated genes in ARID1A-KO cells. In addition, ALDH enzymatic activity as a hallmark of cancer stem cells was markedly high in the KO cells. ARID1A and histone deacetylase 1 were directly recruited to the ALDH1A1 promoter region in cholangiocarcinoma cells with undetectable ALDH1A1 expression by chromatin immunoprecipitation assay. The histone H3K27 acetylation level at the ALDH1A1 promoter region was increased in cells when ARID1A was disrupted (P < 0.01). Clinically, inverse correlation between ARID1A and ALDH1A1 expression was also identified in primary ICC (P = 0.018), and ARID1A-negative and ALDH1A1-positve ICCs showed worse prognosis than only ARID1A-negative cases (P = 0.002). In conclusion, ARID1A may function as a tumor suppressor in ICC through transcriptional downregulation of ALDH1A1 expression with decreasing histone H3K27 acetylation. Our studies provide the basis for the development of new epigenetic approaches to ARID1A-negative ICC. Immunohistochemical loss of ARID1A is an independent prognostic factor in intrahepatic cholangiocarcinoma patients. ARID1A recruits HDAC1 to the promoter region of ALDH1A1, a stemness gene, and epigenetically suppresses ALDH1A1 expression with decreasing histone H3K27 acetylation in cholangiocarcinoma cells.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/patologia , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Células-Tronco Neoplásicas/patologia , Retinal Desidrogenase/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Família Aldeído Desidrogenase 1/genética , Apoptose , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histonas/genética , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Retinal Desidrogenase/genética , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and fibrosis, is predicted to be the leading cause of cirrhosis and hepatocellular carcinoma (HCC) in the next decade. Although recent evidence suggests the importance of fibrosis as the strongest determinant of HCC development, the molecular mechanisms underlying NASH-induced carcinogenesis still remain unclear. Here we performed RNA sequencing analysis to compare gene expression profiles of activated fibroblasts prepared from two distinct liver fibrosis models: carbon tetrachloride-induced fibrosis as a model without obesity and HCC and genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet, which develop steatosis, NASH, and eventually HCC. Our data showed that activated fibroblasts exhibited distinct gene expression patterns in each etiology, and that the 'pathways in cancer' were selectively upregulated in the activated fibroblasts from MC4R-KO mice. The most upregulated gene in these pathways was fibroblast growth factor 9 (FGF9), which was induced by metabolic stress such as palmitate. FGF9 exerted anti-apoptotic and pro-migratory effects in fibroblasts and hepatoma cells in vitro and accelerated tumor growth in a subcutaneous xenograft model. This study reveals upregulation of cancer-associated gene expression in activated fibroblasts in NASH, which would contribute to the progression from NASH to HCC.
Assuntos
Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Regulação para Cima , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Fator 9 de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de NeoplasiasRESUMO
BACKGROUND: Mechanistic target of rapamycin (mTOR) pathway is essential for the growth of gastric cancer (GC), but mTOR inhibitor everolimus was not effective for the treatment of GCs. The Cancer Genome Atlas (TCGA) researchers reported that most diffuse-type GCs were genomically stable (GS). Pathological analysis suggested that some diffuse-type GCs developed from intestinal-type GCs. METHODS: We established patient-derived xenograft (PDX) lines from diffuse-type GCs, and searched for drugs that suppressed their growth. Diffuse-type GCs were classified into subtypes by their gene expression profiles. RESULTS: mTOR inhibitor temsirolimus strongly suppressed the growth of PDX-derived diffuse-type GC-initiating cells, which was regulated via Wnt-mTOR axis. These cells were microsatellite unstable (MSI) or chromosomally unstable (CIN), inconsistent with TCGA report. Diffuse-type GCs in TCGA cohort could be classified into two clusters, and GS subtype was major in cluster I while CIN and MSI subtypes were predominant in cluster II where PDX-derived diffuse-type GC cells were included. We estimated that about 9 and 55% of the diffuse-type GCs in cluster II were responders to mTOR inhibitors and checkpoint inhibitors, respectively, by identifying PIK3CA mutations and MSI condition in TCGA cohort. These ratios were far greater than those of diffuse-type GCs in cluster I or intestinal-type GCs. Further analysis suggested that diffuse-type GCs in cluster II developed from intestinal-type GCs while those in cluster I from normal gastric epithelial cells. CONCLUSION: mTOR inhibitors and checkpoint inhibitors might be useful for the treatment of a subset of diffuse-type GCs which may develop from intestinal-type GCs.
Assuntos
Neoplasias Gástricas/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Humanos , Camundongos , Instabilidade de Microssatélites , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a heterogeneous disease with various etiological factors, and ranks as the second leading cause of cancer-related mortality worldwide due to multi-focal recurrence. We herein identified three major subtypes of HCC by performing integrative analysis of two omics data sets, and clarified that this classification was closely correlated with clinicopathological factors, immune profiles and recurrence patterns. METHODS: In the test study, 183 tumor specimens surgically resected from HCC patients were collected for unsupervised clustering analysis of gene expression signatures and comparative analysis of gene mutations. These results were validated by using genome, methylome and transcriptome data of 373 HCC patients provided from the Cancer Genome Atlas Network. In addition, omics data were obtained from pairs of primary and recurrent HCC. FINDINGS: Comprehensive molecular evaluation of HCC by multi-platform analysis defined three major subtypes: (1) mitogenic and stem cell-like tumors with chromosomal instability; (2) CTNNB1-mutated tumors displaying immune suppression; and (3) metabolic disease-associated tumors, which included an immunogenic subgroup characterized by macrophage infiltration and favorable prognosis. Although genomic and epigenomic analysis explicitly distinguished between HCC with intrahepatic metastasis (IM) and multi-centric HCC (MC), the phenotypic similarity between the primary and recurrent tumors was not correlated to the IM/MC origin, but to the classification. INTERPRETATION: Identification of these HCC subtypes provides further insights into patient stratification as well as presents opportunities for therapeutic development. FUND: Ministry of Education, Culture, Sports, Science and Technology of Japan (16H02670 and 18K19575), Japan Agency for Medical Research and Development (JP15cm0106064, JP17cm0106518, JP18cm0106540 and JP18fk0210040).
Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Imunomodulação , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunomodulação/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Prognóstico , TranscriptomaRESUMO
Although genomic analysis have recently discovered the malignant subtype of human pancreatic ductal adenocarcinoma (PDAC) characterized by frequent mutations of histone demethylase KDM6A, the biological and molecular roles still remain obscure. We herein elucidated the clinical and biological impacts of KDM6A deficiency on human PDAC and identified the therapeutic potential by pathological and molecular evaluation. Immunohistochemical analysis suggested that loss of KDM6A in cancerous tissues was an independent prognostic factor for both recurrence-free and overall survival in the 103 tumor specimens surgically resected from patients with PDAC. We established KDM6A knocked out cells by using the CRISPR/Cas9 system and KDM6A-expressed cells by doxycycline-inducible system from each two human PDAC cell lines, respectively. KDM6A knockout enhanced aggressive traits of human PDAC cell lines, whereas KDM6A overexpression suppressed them. Microarray analysis revealed reduced expression of 22 genes including five well-known tumor suppressors, such as CDKN1A, and ChIP-PCR analysis displayed depleted enrichment of histone H3 lysine 27 acetylation (H3K27ac) at the promoter regions of the five candidates. The epigenetic alterations were induced by the impaired recruitment of histone acetyltransferase p300, which cooperatively interacted with KDM6A. Consistent with these results, the KDM6A knockout cells demonstrated higher vulnerability to histone deacetylase (HDAC) inhibitors through the reactivation of CDKN1A in vitro and in vivo than the KDM6A wild-type. In conclusion, KDM6A exhibited essential roles in human PDAC as a tumor suppressor and KDM6A deficiency could be a promising biomarker for unfavorable outcome in PDAC patients and a potential surrogate marker for response to HDAC inhibitors.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Histona Desmetilases/deficiência , Proteínas Nucleares/deficiência , Neoplasias Pancreáticas/tratamento farmacológico , Acetilação , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Epigênese Genética , Técnicas de Inativação de Genes , Xenoenxertos , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , PrognósticoRESUMO
Histone modification plays important molecular roles in development and progression of cancers. Dysregulation of histone H3 arginine (R) methylation is still unknown in primary cancer, including gastric cancer (GC). Although PRMT6 contributes to asymmetric dimethylation at H3R2 (H3R2me2as) in cancer cells, its molecular functions are poorly understood in GC. In this study, we assessed H3R2me2as and PRMT6 expression levels in 133 primary GC tissues by immunohistochemistry. Increased H3R2me2as was found in 68 GC (51.1%) cases and independently related to poor prognosis. PRMT6 was overexpressed in 70 GC (52.6%) and strongly correlated with the global H3R2me2as levels (P < 0.001). By analyzing biological functions of PRMT6 in GC cell lines by lentivirus-based systems, PRMT6 overexpression enhanced global H3R2me2as and invasiveness in vitro, while PRMT6 knockout (PRMT6-KO) suppressed these effects and tumorigenicity in vivo. ChIP and microarray assays demonstrated that PRMT6-KO GC cells decreased the enrichments of H3R2me2as at the promoter regions of PCDH7, SCD and IGFBP5, resulting in upregulation of their gene expression. PRMT6 was recruited to the promoter regions of PCDH7 and SCD in the PRMT6-overexpressed cells. Knockdown of tumor suppressor PCDH7 in the PRMT6-KO GC cells elevated cell migration and invasion. PRMT6 expression inversely correlated with PCDH7 expression in primary GC (P = 0.021). Collectively, our findings strongly indicate that H3R2me2as is a strong prognostic indicator of GC patients, and PRMT6-overexpressing GC cells may acquire invasiveness through direct transcriptional inhibition of PCDH7 by increasing H3R2me2as level. Thus, inhibition of the PRMT6-H3R2me2as pathway could be a promising new therapeutic strategy in GC.
Assuntos
Histonas/metabolismo , Proteínas Nucleares/fisiologia , Proteína-Arginina N-Metiltransferases/fisiologia , Neoplasias Gástricas/metabolismo , Animais , Arginina/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/fisiologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Masculino , Metilação , Camundongos , Protocaderinas , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and poor patient prognosis. We have previously established an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. In this study, we explored low-molecular-weight drugs selectively eliminating mouse and human DGC cells. METHODS: We derived mouse gastric cancer (GC) cell lines from DGC of the DCKO mice demonstrating enhanced tumourigenic activity in immunodeficient mice and acquired tolerance to cytotoxic anti-cancer agents. RESULTS: We performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates selectively killing the GC cell lines. The most potent drug mestranol, an oestrogen derivative, and other oestrogen receptor modulators specifically attenuated cell viability of the GC cell lines by inducing apoptosis preceded by DNA damage. Moreover, mestranol could significantly suppress tumour growth of the GC cells subcutaneously transplanted into nude mice, consistent with longer survival time in the female DCKO mice than in the male. Expectedly, human E-cadherin-mutant and -low gastric cancer cells showed higher susceptibility to oestrogen drugs in contrast to E-cadherin-intact ones in vitro and in vivo. CONCLUSIONS: These findings may lead to the development of novel therapeutic strategies targeting DGC.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/classificação , Antineoplásicos/uso terapêutico , Proteínas Cdh1/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Pancreatic neuroendocrine tumors (PanNETs) have considerable malignant potential. Frequent somatic mutations and loss of DAXX protein expression have been found in PanNETs. DAXX is known as a transcriptional repressor; however, molecular functions underlying DAXX loss remain unclear in PanNETs. We evaluated DAXX expression by immunohistochemistry in 44 PanNETs. DAXX-knockdown (KD) and -knockout (KO) PanNET cells were analyzed for in vitro and vivo The target genes were screened by microarray and chromatin immunoprecipitation (ChIP) assays for DAXX, histone H3.3 and H3K9me3 complex. In clinicopathological features, low DAXX expression was significantly correlated with nonfunctional tumors, higher Ki-67 index and WHO grade. Microarray and ChIP assays of DAXX-KD/KO identified 12 genes as the direct targets of DAXX transcriptional repressor. Among them, expression of five genes including STC2 was suppressed by DAXX/H3.3/H3K9me3 pathway. DAXX-KD/KO cells enhanced sphere forming activity, but its effect was suppressed by knockdown of STC2 In xenograft models, tumorigenicity and tumor vessel density were significantly increased in DAXX-KO cells with high expression of STC2. Clinically, higher recurrence rate was recognized in PanNETs with low expression of DAXX and high expression of STC2 than others (P = 0.018). Our data suggest that DAXX plays as a tumor suppressor and DAXX/H3.3 complex suppresses target genes by promoting H3K9me3 in PanNETs. Combination of DAXX loss and its target gene STC2 overexpression might be effective biomarkers and therapeutic candidates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Histonas/metabolismo , Tumores Neuroendócrinos/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Proteínas Correpressoras , Feminino , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Chaperonas Moleculares , Tumores Neuroendócrinos/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genéticaRESUMO
Metabolic syndrome is a newly identified risk factor for hepatocellular carcinoma (HCC); however, tumor-specific biomarkers still remain unclear. We performed cross-species analysis to compare gene signatures of HCC from human patients and melanocortin 4 receptor-knockout mice, which develop HCC with obesity, insulin resistance, and dyslipidemia. Unsupervised hierarchical clustering and principle component analysis of 746 differentially expressed orthologous genes classified HCC of 152 human patients and melanocortin 4 receptor-knockout mice into two distinct subgroups, one of which included mouse HCC and was causatively associated with metabolic risk factors. Nine genes commonly overexpressed in human and mouse metabolic disease-associated HCC were identified; fatty acid binding protein 4 (FABP4) was remarkably enriched in intratumoral activated hepatic stellate cells (HSCs). Subclones constitutively expressing FABP4 were established from a human HSC cell line in which expression levels of inflammatory chemokines, including IL-1A and IL-6, were up-regulated through NF-κB nuclear translocation, resulting in recruitment of macrophages. An immunohistochemical validation study of 106 additional human HCC samples indicated that FABP4-positive HSCs were distributed in tumors of 38 cases, and the FABP4-high group consisted of patients with nonviral and nonalcoholic HCC (P = 0.027) and with multiple metabolic risk factors (P < 0.001) compared with the FABP4-low group. Thus, FABP4 overexpression in HSCs may contribute to hepatocarcinogenesis in patients with metabolic risk factors by modulation of inflammatory pathways.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Células Estreladas do Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a Ácido Graxo/genética , Células Estreladas do Fígado/patologia , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Fatores de RiscoRESUMO
Decrease in blood concentration of branched-chain amino acids, especially leucine, is known to promote liver carcinogenesis in patients with chronic liver disease, but the mechanism is unclear. We herein established hepatocellular carcinoma (HCC) cells knocked out for DEPDC5 by using the CRISPR/Cas9 system, and elucidated that cell viability of the DEPDC5 knockout (DEPDC5-KO) cells was higher than that of the DEPDC5 wild-type (DEPDC5-WT) under leucine starvation. Considering that autophagy deficiency might be involved in acquired resistance to leucine deprivation, we observed reduction of LC3-II followed by accumulation of p62 in the DEPDC5-KO, which induced reactive oxygen species (ROS) tolerance. DEPDC5 overexpression suppressed cell proliferation and tumorigenicity in immunocompromised mice, and triggered p62 degradation with increased ROS susceptibility. In clinical specimens of HCC patients, decreased expression of DEPDC5 was positively correlated with p62 overexpression, and the progression-free (PFS) and overall survival (OS) were worse in the DEPDC5-negative cases than in the DEPDC5-positive. Moreover, multivariate analysis demonstrated DEPDC5 was an independent prognostic factor for both PFS and OS. Thus, DEPDC5 inactivation enhanced ROS resistance in HCC under the leucine-depleted conditions of chronic liver disease, contributing to poor patient outcome. It could be a potential target for cancer therapy with oxidative stress control.