RESUMO
In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.
Assuntos
Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Animais , Humanos , Camundongos , Células Estreladas do Pâncreas/metabolismo , Fenótipo , Proteína Quinase C/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.
Assuntos
Processamento Alternativo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Processamento Alternativo/imunologia , Animais , Quimiocina CCL2/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Evasão da Resposta Imune/genética , Fígado/imunologia , Fígado/lesões , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Processamento de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.
Assuntos
Mesoderma/metabolismo , Proteína Quinase C/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Genes Reporter , Coração/crescimento & desenvolvimento , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Miocárdio/metabolismo , Miocárdio/patologia , Proteína Quinase C/deficiência , Proteína Quinase C/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Hepatitis B virus X protein (HBx) is involved in the development of hepatocellular carcinoma (HCC). The HBx sequence is a preferential site of integration into the human genome, leading to the formation of C-terminal-truncated HBx proteins (Ct-HBx). We previously reported that Ct-HBx proteins were able to potentiate cell transformation in vitro. Our present goal was to compare the ability of Ct-HBx and full-length HBx (FL-HBx) proteins to develop or enhance HCC in transgenic mice. In the absence of treatment, neither Ct-HBx- nor FL-HBx-transgenic mice developed HCC. In young mice treated with diethylnitrosamine (DEN) at 8 months of age, a significantly higher incidence and number of liver lesions were observed in Ct-HBx mice than in FL-HBx and control mice. The earlier development of tumours in Ct-HBx-transgenic mice was associated with increased liver inflammation. At 10 months, macroscopic and microscopic analyses showed that, statistically, FL-HBx mice developed more liver lesions with a larger surface area than control mice. Furthermore, during DEN-induced initiation of HCC, Ct-HBx- and FL-HBx-transgenic mice showed higher expression of IL-6, TNF-α and IL-1ß transcripts, activation of STAT3, ERK and JNK proteins and an increase in cell apoptosis. In conclusion, in DEN-treated transgenic mice, the expression of Ct-HBx protein causes a more rapid onset of HCC than does FL-HBx protein. HBV genome integration leading to the expression of a truncated form of HBx protein may therefore facilitate HCC development in chronically infected patients.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/patogenicidade , Neoplasias Hepáticas/virologia , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Carcinoma Hepatocelular/induzido quimicamente , Proliferação de Células , Dietilnitrosamina/toxicidade , Regulação Viral da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/fisiologia , Hepatócitos/virologia , Neoplasias Hepáticas/induzido quimicamente , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Virais Reguladoras e AcessóriasRESUMO
The prevalence of head and neck squamous cell carcinoma (HNSCC) related to human papillomavirus (HPV) is increasing, unlike tobacco- and alcohol-associated cancers. To gain a clearer understanding of the molecular mechanisms implicated in HNSCC, depending on the presence or not of a viral sequence, we investigated the expression of proteins detected in the tumor regions of HNSCC patients. Twenty-two untreated HNSCC patients were selected according to the presence of HPV-16. For six patients, tumor and controlateral healthy tissues were tested for viral detection before quantitative proteomic analysis. After confirmation by Western blot, proteins were connected into a network, leading to investigate interleukin-6 (IL-6) by immunocytochemistry and ELISA. 41 ± 5% of proteins quantified by proteomics were differentially expressed in tumor compared with healthy regions. Among them, 36 proteins were retained as modulated in HPV-16 positive or negative tumors, including cytokeratins, tubulins, annexin A1, and serpin B1. Network analysis suggested a central role of IL-6, confirmed by overexpression of IL-6 in tumor tissues as in sera of HPV-negative HNSCC compared with HPV-16-positive tumors. This modulation may contribute to the survival and proliferation of cancer cells, although it was not related to tumor stage or to the level of HPV-16 DNA.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Interleucina-6/metabolismo , Infecções por Papillomavirus/metabolismo , Idoso , Sequência de Bases , Western Blotting , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/virologia , Estudos de Casos e Controles , Estudos de Coortes , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Neoplasias de Cabeça e Pescoço/complicações , Neoplasias de Cabeça e Pescoço/virologia , Papillomavirus Humano 16/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Carga ViralRESUMO
Hepatocellular carcinoma (HCC) is the most frequent form of liver cancer worldwide, and represents the third cause of death. While epidemiological studies have clearly established that hepatitis B virus (HBV) infection is a major risk factor for the development of HCC, the molecular mechanisms underlying virally-induced tumourigenesis are not fully understood. The transcriptional regulatory HBx protein has been described as a multifunctional protein exhibiting numerous activities affecting gene transcription, intracellular signal transduction, cell proliferation, apoptosis and DNA repair. While any or all of the multiple activities of HBx could contribute to hepato-carcinogenesis, HBx is not considered as an oncogene. HBx rather acts as a co-factor of carcinogenesis, through the up-regulation of a large number of cellular genes involved in oncogenesis, proliferation, inflammation and immune response. In this review, we will summarize the current knowledge on the mechanisms involving HBx protein in liver carcinogenesis.
RESUMO
BACKGROUND & AIMS: Conflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatectomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH. METHODS: PH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutralizing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration. RESULTS: Cell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phosphorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neutralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor. CONCLUSIONS: HBx is able to induce delayed hepatocyte proliferation after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microenvironment, HBx may participate in cell cycle deregulation and progression of liver disease.