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1.
J Hepatol ; 75(4): 840-847, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34004216

RESUMO

BACKGROUND & AIMS: Loss of serum HBsAg is a hallmark of spontaneous and therapy induced resolution of HBV infection, since it generally reflects a profound decrease in viral replication. However, integrated HBV DNA can contribute to HBsAg expression independent of viral replication. The relative contributions of these sources of HBsAg are not well understood. Specifically, it is not known whether actively transcribed HBV integration could spread throughout the entire liver. METHODS: The relative distribution of HBsAg and HBV RNA in liver biopsy tissue from HBeAg-negative (HBe-) patients was analyzed by immunohistochemistry and in situ hybridization (ISH), respectively. Frozen biopsy tissue was used for molecular analysis of intrahepatic viral RNA, virus-host chimeric transcripts and viral DNA. RESULTS: Immunohistochemistry and ISH analysis revealed HBsAg and HBV RNA positivity in virtually all hepatocytes in the liver of some HBe- patients despite very low viremia. Reverse transcription quantitative PCR and RNA-sequencing analysis confirmed high expression levels of HBV envelope-encoding RNAs. However, the amount of viral transcriptional template (covalently closed circular (ccc)DNA) was too low to support this ubiquitous HBV RNA expression. In contrast, levels of total cellular HBV DNA were consistent with ubiquitous HBV integration. Finally, RNA-sequencing revealed the presence of many HBV-host chimeric transcripts with the potential for HBsAg expression. CONCLUSIONS: Transcriptionally active HBV integration can extend to the entire liver in some HBe- patients. This can lead to ubiquitous HBsAg expression independent of HBV replication. In such patients, HBsAg is probably not a clinically useful surrogate marker for viral resolution or functional cure. LAY SUMMARY: Loss of serum hepatitis B surface antigen (HBsAg) indicates resolution of HBV infection. However, integrated HBV DNA can contribute to HBsAg production independently of viral replication. We investigated the extent of HBsAg-producing viral integration in the livers of patients with low serum viral loads. Our findings suggest that transcriptionally active HBV integration can extend to the entire liver in some patients, questioning the clinical utility of HBsAg as a surrogate marker for viral replication.


Assuntos
DNA Viral/análise , Anticorpos Anti-Hepatite B/análise , Hepatite B/sangue , Carga Viral/estatística & dados numéricos , Adulto , DNA Viral/sangue , Feminino , Hepatite B/fisiopatologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Masculino , Pessoa de Meia-Idade , Carga Viral/métodos
2.
Gut ; 67(5): 953-962, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28159835

RESUMO

BACKGROUND AND AIMS: HCV infection is a leading risk factor of hepatocellular carcinoma (HCC). However, even after viral clearance, HCC risk remains elevated. HCV perturbs host cell signalling to maintain infection, and derailed signalling circuitry is a key driver of carcinogenesis. Since protein phosphatases are regulators of signalling events, we aimed to identify phosphatases that respond to HCV infection with relevance for hepatocarcinogenesis. METHODS: We assessed mRNA and microRNA (miRNA) expression profiles in primary human hepatocytes, liver biopsies and resections of patients with HCC, and analysed microarray and RNA-seq data from paired liver biopsies of patients with HCC. We revealed changes in transcriptional networks through gene set enrichment analysis and correlated phosphatase expression levels to patient survival and tumour recurrence. RESULTS: We demonstrate that tumour suppressor protein tyrosine phosphatase receptor delta (PTPRD) is impaired by HCV infection in vivo and in HCC lesions of paired liver biopsies independent from tissue inflammation or fibrosis. In liver tissue adjacent to tumour, high PTPRD levels are associated with a dampened transcriptional activity of STAT3, an increase of patient survival from HCC and reduced tumour recurrence after surgical resection. We identified miR-135a-5p as a mechanistic regulator of hepatic PTPRD expression in patients with HCV. CONCLUSIONS: We previously demonstrated that STAT3 is required for HCV infection. We conclude that HCV promotes a STAT3 transcriptional programme in the liver of patients by suppressing its regulator PTPRD via upregulation of miR-135a-5p. Our results show the existence of a perturbed PTPRD-STAT3 axis potentially driving malignant progression of HCV-associated liver disease.


Assuntos
Carcinoma Hepatocelular/metabolismo , Hepacivirus/patogenicidade , Hepatite C/complicações , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinogênese/metabolismo , Carcinoma Hepatocelular/virologia , Regulação para Baixo , Feminino , Hepatócitos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Fígado/patologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
3.
J Gastroenterol Hepatol ; 32(8): 1480-1486, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28052383

RESUMO

BACKGROUND AND AIM: Most patients with a hepatocellular carcinoma (HCC) have an underlying chronic liver inflammation, which causes a continuous damage leading to liver cirrhosis and eventually HCC. However, only a minority of cirrhotic patients develop HCC. To assess a possible differential impact of liver inflammation in patients developing HCC versus patients remaining tumor-free, we designed a longitudinal study and analysed liver tissue of the same patients (n = 33) at two points in time: once when no HCC was present and once several years later when an HCC was present. As a control group, we followed cirrhotic patients (n = 37) remaining tumor-free over a similar time frame. METHODS: We analysed cell damage and senescence of hepatocytes by measuring γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, nuclear size, and telomere length. RESULTS: γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, in the first liver biopsy was similar in patients developing HCC later on and cirrhotic patients remaining tumor free. In contrast, γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, was significantly higher in the second non-tumoral liver biopsy of HCC patients than in the control patients. Consequently, the individual increase in γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, from the first biopsy to the second biopsy was significantly higher in patients developing HCC than in patients remaining tumor free. In addition, changes in nuclear size and telomere length revealed a more pronounced cell aging in patients developing HCC than in patients remaining tumor free. CONCLUSIONS: Hepatocytes from patients developing HCC go through more pronounced cell damage and senescence in contrast to cirrhotic patients remaining tumor free.


Assuntos
Carcinoma Hepatocelular/patologia , Senescência Celular , Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Fígado/patologia , Adulto , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/genética , Tamanho do Núcleo Celular , Senescência Celular/genética , Feminino , Expressão Gênica , Histonas , Humanos , Inflamação , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/genética , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Homeostase do Telômero
4.
Hepatology ; 59(6): 2121-30, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24122862

RESUMO

UNLABELLED: Approximately 50% of patients with chronic hepatitis C (CHC) have ongoing expression of interferon stimulated genes (ISGs) in the liver. It is unclear why this endogenous antiviral response is inefficient in eradicating the infection. Several viral escape strategies have been identified in vitro, including inhibition of interferon (IFN) induction and ISG messenger RNA (mRNA) translation. The in vivo relevance of these mechanisms is unknown, because reliable methods to identify hepatitis C virus (HCV)-infected cells in human liver are lacking. We developed a highly sensitive in situ hybridization (ISH) system capable of HCV RNA and ISG mRNA detection in human liver biopsies and applied it to study the interaction of HCV with the endogenous IFN system. We simultaneously monitored HCV RNA and ISG mRNA using HCV isolate- and ISG mRNA-specific probes in liver biopsy sections from 18 CHC patients. The signals were quantified at the single-cell resolution in a series of random high-power fields. The proportion of infected hepatocytes ranged from 1%-54% and correlated with viral load, but not with HCV genotype or ISG expression. Infected cells occurred in clusters, pointing to cell-to-cell spread as the predominant mode of HCV transmission. ISG mRNAs were readily detected in HCV-infected cells, challenging previously proposed mechanisms of viral interference with the immune system. Conversely, infected cells and neighboring cells showed increased ISG mRNA levels, demonstrating that the stimulus driving ISG expression originates from HCV-infected hepatocytes. CONCLUSION: HCV infection in human hepatocytes during CHC does not efficiently interfere with IFN induction, IFN signaling, or transcription of ISG mRNA.


Assuntos
Regulação Viral da Expressão Gênica , Hepatite C/virologia , Interferons/fisiologia , Fígado/virologia , Hepatite C/genética , Hepatite C/metabolismo , Hepatócitos/virologia , Humanos , Hibridização In Situ , Fígado/metabolismo , RNA Viral/genética , Carga Viral/genética
5.
Carcinogenesis ; 35(1): 114-22, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23901063

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most HCCs develop in cirrhotic livers. Alcoholic liver disease, chronic hepatitis B and chronic hepatitis C are the most common underlying liver diseases. Hepatitis C virus (HCV)-specific mechanisms that contribute to HCC are presently unknown. Transgenic expression of HCV proteins in the mouse liver induces an overexpression of the protein phosphatase 2A catalytic subunit (PP2Ac). We have previously reported that HCV-induced PP2Ac overexpression modulates histone methylation and acetylation and inhibits DNA damage repair. In this study, we analyze tumor formation and gene expression using HCV transgenic mice that overexpress PP2Ac and liver tissues from patients with HCC. We demonstrate that PP2Ac overexpression interferes with p53-induced apoptosis. Injection of the carcinogen, diethylnitrosamine, induced significantly more and larger liver tumors in HCV transgenic mice that overexpress PP2Ac compared with control mice. In human liver biopsies from patients with HCC, PP2Ac expression was significantly higher in HCC tissue compared with non-tumorous liver tissue from the same patients. Our findings demonstrate an important role of PP2Ac overexpression in liver carcinogenesis and provide insights into the molecular pathogenesis of HCV-induced HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteína Fosfatase 2/metabolismo , Animais , Biópsia , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/enzimologia , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Etoposídeo/análogos & derivados , Etoposídeo/farmacologia , Regulação Enzimológica da Expressão Gênica , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite B Crônica/enzimologia , Hepatite B Crônica/patologia , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos Organofosforados/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Int J Cancer ; 134(11): 2583-94, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24214914

RESUMO

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-ß newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.


Assuntos
Adesão Celular , Comunicação Celular , Membrana Celular/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Células-Tronco Mesenquimais/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose , Western Blotting , Medula Óssea/metabolismo , Medula Óssea/patologia , Caderinas/genética , Caderinas/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocinas/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/metabolismo , Fator de Crescimento Transformador beta/genética
7.
Hepatol Commun ; 6(6): 1467-1481, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35132819

RESUMO

Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Estudos Longitudinais , MicroRNAs/genética , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética
8.
Cell Rep ; 40(7): 111181, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977490

RESUMO

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.


Assuntos
Glicoesfingolipídeos , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Gangliosídeos/metabolismo , Globosídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Transdução de Sinais
9.
Int J Cancer ; 128(11): 2663-72, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20715106

RESUMO

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan-Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearman's rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6-0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7-0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3-0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64- HLA-DR-myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.


Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Imunidade Celular/imunologia , Linfócitos do Interstício Tumoral/imunologia , Células Mieloides/metabolismo , Receptores de IgG/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/secundário , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Células Matadoras Naturais , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Invasividade Neoplásica , Prognóstico , Análise Serial de Tecidos
10.
J Exp Med ; 218(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34398180

RESUMO

Several RNA viruses can establish life-long persistent infection in mammalian hosts, but the fate of individual virus-infected cells remains undefined. Here we used Cre recombinase-encoding lymphocytic choriomeningitis virus to establish persistent infection in fluorescent cell fate reporter mice. Virus-infected hepatocytes underwent spontaneous noncytolytic viral clearance independently of type I or type II interferon signaling or adaptive immunity. Viral clearance was accompanied by persistent transcriptomic footprints related to proliferation and extracellular matrix remodeling, immune responses, and metabolism. Substantial overlap with persistent epigenetic alterations in HCV-cured patients suggested a universal RNA virus-induced transcriptomic footprint. Cell-intrinsic clearance occurred in cell culture, too, with sequential infection, reinfection cycles separated by a period of relative refractoriness to infection. Our study reveals that systemic persistence of a prototypic noncytolytic RNA virus depends on continuous spread and reinfection. Yet undefined cell-intrinsic mechanisms prevent viral persistence at the single-cell level but give way to profound transcriptomic alterations in virus-cleared cells.


Assuntos
Infecções por Arenaviridae/genética , Infecções por Arenaviridae/virologia , Hepatócitos/virologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Imunidade Adaptativa , Animais , Infecções por Arenaviridae/patologia , Chlorocebus aethiops , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Interferons/metabolismo , Vírus da Coriomeningite Linfocítica/genética , Camundongos Transgênicos , Reinfecção , Análise de Célula Única , Células Vero , Carga Viral , Proteínas Virais/metabolismo
11.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33158978

RESUMO

Compared with the ubiquitous expression of type I (IFNα and IFNß) interferon receptors, type III (IFNλ) interferon receptors are mainly expressed in epithelial cells of mucosal barriers of the of the intestine and respiratory tract. Consequently, IFNλs are important for innate pathogen defense in the lung and intestine. IFNλs also determine the outcome of hepatitis C virus (HCV) infections, with IFNλ4 inhibiting spontaneous clearance of HCV. Because viral clearance is dependent on T cells, we explored if IFNλs can directly bind to and regulate human T cells. We found that human B cells and CD8+ T cells express the IFNλ receptor and respond to IFNλs, including IFNλ4. IFNλs were not inhibitors but weak stimulators of B- and T-cell responses. Furthermore, IFNλ4 showed neither synergistic nor antagonistic effects in co-stimulatory experiments with IFNλ1 or IFNα. Multidimensional flow cytometry of cells from liver biopsies of hepatitis patients from IFNλ4-producers showed accumulation of activated CD8+ T cells with a central memory-like phenotype. In contrast, CD8+ T cells with a senescent/exhausted phenotype were more abundant in IFNλ4-non-producers. It remains to be elucidated how IFNλ4 promotes CD8 T-cell responses and inhibits the host immunity to HCV infections.


Assuntos
Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Hepacivirus , Hepatite C/sangue , Interleucinas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adolescente , Adulto , Idoso , Doadores de Sangue , Feminino , Hepatite C/patologia , Hepatite C/virologia , Humanos , Interferon-alfa/farmacologia , Interferons/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores de Interferon/metabolismo , Adulto Jovem
12.
Elife ; 92020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33026975

RESUMO

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.


Assuntos
Colágeno/metabolismo , Integrina alfa2/metabolismo , Metástase Neoplásica/fisiopatologia , Omento/fisiopatologia , Peritônio/fisiopatologia , Animais , Carcinoma Epitelial do Ovário/metabolismo , Linhagem Celular Tumoral , Feminino , Camundongos , Peixe-Zebra
13.
PLoS One ; 14(8): e0221762, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31465481

RESUMO

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known. METHODS: In the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles. RESULTS: Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues. CONCLUSIONS: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.


Assuntos
Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/patologia , Animais , Biomarcadores/metabolismo , Biópsia , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Reprogramação Celular , Análise por Conglomerados , Fibroblastos/citologia , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos SCID , Análise de Componente Principal , Fatores de Transcrição/metabolismo , Transcrição Gênica
14.
Hepatol Commun ; 3(7): 971-986, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31334445

RESUMO

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Treatment options for patients with advanced-stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient-derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma-c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor-associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.

15.
Sci Rep ; 8(1): 15331, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333571

RESUMO

Macrophages have been recognized as key players in non-alcoholic fatty liver disease (NAFLD). Our aim was to assess whether pharmacological attenuation of macrophages can be achieved by imatinib, an anti-leukemia drug with known anti-inflammatory and anti-diabetic properties, and how this impacts on NAFLD. We analyzed the pro- and anti-inflammatory gene expression of murine macrophages and human monocytes in vitro in the presence or absence of imatinib. In a time-resolved study, we characterized metabolic disease manifestations such as hepatic steatosis, systemic and adipose tissue inflammation as well as lipid and glucose metabolism in obese mice at one and three months of imatinib treatment. Our results showed that imatinib lowered pro-inflammatory markers in murine macrophages and human monocytes in vitro. In obese mice, imatinib reduced TNFα-gene expression in peritoneal and liver macrophages and systemic lipid levels at one month. This was followed by decreased hepatic steatosis, systemic and adipose tissue inflammation and increased insulin sensitivity after three months. As the transcription factor sterol regulatory element-binding protein (SREBP) links lipid metabolism to the innate immune response, we assessed the gene expression of SREBPs and their target genes, which was indeed downregulated in the liver and partially in peritoneal macrophages. In conclusion, targeting both inflammatory and lipogenic pathways in macrophages and liver as shown by imatinib could represent an attractive novel therapeutic strategy for patients with NAFLD.


Assuntos
Mesilato de Imatinib/farmacologia , Inflamação/prevenção & controle , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/metabolismo , Lipogênese/genética , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
16.
Cell Rep ; 24(5): 1363-1376, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30067989

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Organoides/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Técnicas de Cultura de Tecidos/métodos
17.
Methods Mol Biol ; 1540: 119-134, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27975312

RESUMO

As soon as HBV was identified, HBV localization at the cellular level became instrumental in studying HBV infection. Multiple methodologies for detection of viral antigens and nucleic acids at the cellular level, not only in patient derived liver biopsy samples, but also in many other experimental systems, have been developed over the years. Recently, the development of highly sensitive and specific in situ hybridization systems enabled detection of cellular mRNAs at the single copy level. Adaptation of such a system (ViewRNA ISH Tissue Assay; Affymetrix, Santa Clara, CA, USA) for the detection of viral RNAs allowed us to reliably identify hepatitis C virus RNA positive cells in the liver of HCV infected patients. Similarly, this protocol enabled detection of very rare HBV positive cells in liver biopsy tissue. Here, we now describe the specifics of the protocol for detection of HBV RNA using the ViewRNA ISH system. The protocol focuses on probe set design, sample preparation and data interpretation for accurate HBV RNA detection in liver tissue samples. The methodology is straightforward and will be very useful in the study of basic HBV virology as well as in clinical applications.


Assuntos
Vírus da Hepatite B/genética , Hepatite B/virologia , Hibridização In Situ , Fígado/virologia , RNA Viral , Animais , Biópsia , Hepatite B/patologia , Humanos , Hibridização In Situ/métodos , Fígado/patologia , Sondas Moleculares , Sensibilidade e Especificidade
18.
Oncotarget ; 8(1): 1725-1736, 2017 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-27965457

RESUMO

Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Hipóxia Celular/fisiologia , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fluoruracila/farmacologia , Necrose/patologia , Esferoides Celulares/fisiologia , Animais , Neoplasias Colorretais/tratamento farmacológico , Perfilação da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oxigênio/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Neoplasia ; 18(12): 724-731, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27855356

RESUMO

BACKGROUND: HMGA1 is a non-histone nuclear protein that regulates cellular proliferation, invasion and apoptosis and is overexpressed in many carcinomas. In this study we sought to explore the expression of HMGA1 in HCCs and cirrhotic tissues, and its effect in in vitro models. METHODS: We evaluated HMGA1 expression using gene expression microarrays (59 HCCs, of which 37 were matched with their corresponding cirrhotic tissue and 5 normal liver donors) and tissue microarray (192 HCCs, 108 cirrhotic tissues and 79 normal liver samples). HMGA1 expression was correlated with clinicopathologic features and patient outcome. Four liver cancer cell lines with stable induced or knockdown expression of HMGA1 were characterized using in vitro assays, including proliferation, migration and anchorage-independent growth. RESULTS: HMGA1 expression increased monotonically from normal liver tissues to cirrhotic tissue to HCC (P<.01) and was associated with Edmondson grade (P<.01). Overall, 51% and 42% of HCCs and cirrhotic tissues expressed HMGA1, respectively. Patients with HMGA1-positive HCCs had earlier disease progression and worse overall survival. Forced expression of HMGA1 in liver cancer models resulted in increased cell growth and migration, and vice versa. Soft agar assay showed that forced expression of HMGA1 led to increased foci formation, suggesting an oncogenic role of HMGA1 in hepatocarcinogenesis. CONCLUSIONS: HMGA1 is frequently expressed in cirrhotic tissues and HCCs and its expression is associated with high Edmondson grade and worse prognosis in HCC. Our results suggest that HMGA1 may act as oncogenic driver of progression, implicating it in tumor growth and migration potential in liver carcinogenesis.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Expressão Gênica , Proteínas HMGA/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Biomarcadores Tumorais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Nat Cell Biol ; 18(5): 467-79, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27088858

RESUMO

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/ß-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/ß-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/ß-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/ß-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/ß-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.


Assuntos
Fígado/citologia , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula , Proliferação de Células , Citocromo P-450 CYP2E1/metabolismo , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Homeostase , Antígeno Ki-67/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Regeneração Hepática , Tamanho do Órgão , Transdução de Sinais , beta-Galactosidase/metabolismo
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