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INTRODUCTION: The phase 2 RAMONA study demonstrated that second-line nivolumab ± ipilimumab immunotherapy was feasible and effective in older patients with advanced esophageal squamous cell cancer (ESCC). Here, we presented results from functional status (FS) and quality-of-life (QoL) analyses. MATERIALS AND METHODS: Patients aged ≥65 years with advanced ESCC and disease progression following first-line therapy were enrolled for study treatment with nivolumab ± ipilimumab. Geriatric assessments (GA) consisting of G8 and GoGo/SlowGo evaluation, and quality of life (QoL) assessments with EORTC QLQ-C30 questionnaires were conducted at baseline and during the treatment. A post hoc analysis was performed to compare therapy efficacy, toxicity, and QoL between age groups (≥70 years vs. <70 years) and functionality groups (G8 > 14 vs. ≤14 and GoGo vs. SlowGo). RESULTS: In 66 treated patients with a median age of 70.5 years, older patients had non-inferior overall survival and tumor response compared to younger patients, with no increased treatment-related adverse events. Fitter patients (G8 > 14, GoGo) had a clinically, yet not statistically significant, survival advantage than less fit patients (G8 ≤ 14, SlowGo) patients. Moreover, FS by G8 and GoGo/SlowGo significantly correlated with QoL. Overall, QoL was impaired at baseline but remained stable in all scales over the course of immunotherapy. DISCUSSION: The administration of nivolumab ± ipilimumab second-line immunotherapy in older patients with ESCC did not show age-dependent effects and maintained QoL. GA could identify functional deficits and limitations of QoL and should be implemented in the context of immunotherapy. CLINICALTRIALS: gov: NCT03416244.
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INTRODUCTION: WNT1-inducible signalling pathway protein 1 (WISP1) promotes progression of several tumor entities often correlating with worse prognosis. Here its expression regulation and role in the progression of chronic liver diseases (CLD) was investigated. METHODS: WISP1 expression was analyzed in human HCC datasets, in biopsies and serum samples and an HCC patient tissue microarray (TMA) including correlation to clinicopathological parameters. Spatial distribution of WISP1 expression was determined using RNAscope analysis. Regulation of WISP1 expression was investigated in cytokine-stimulated primary mouse hepatocytes (PMH) by array analysis and qRT-PCR. Outcome of WISP1 stimulation was analyzed by IncuCyte S3-live cell imaging, qRT-PCR, and immunoblotting in murine AML12 cells. RESULTS: In a TMA, high WISP1 expression was positively correlated with early HCC stages and male sex. Highest WISP1 expression levels were detected in patients with cirrhosis as compared to healthy individuals, patients with early fibrosis, and non-cirrhotic HCC in liver biopsies, expression datasets and serum samples. WISP1 transcripts were predominantly detected in hepatocytes of cirrhotic rather than tumorous liver tissue. High WISP1 expression was associated with better survival. In PMH, AML12 and HepaRG, WISP1 was identified as a specific TGF-ß1 target gene. Accordingly, expression levels of both cytokines positively correlated in human HCC patient samples. WISP1-stimulation induced the expression of Bcl-xL, PCNA and p21 in AML12 cells. CONCLUSIONS: WISP1 expression is induced by TGF-ß1 in hepatocytes and is associated with cirrhotic liver disease. We propose a crucial role of WISP1 in balancing pro- and anti-tumorigenic effects during premalignant stages of CLD.
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Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction, including autoimmune diseases, chronic infections, and cancer. Co-inhibitory "checkpoint molecules," such as programmed cell death protein-1, balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of natural killer (NK) cell recognition on T cells, we identified the immunoglobulin superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR) T cells. Unlike other checkpoint molecules, B7H6 mediated NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells were prevalent in the tissue and blood of several diseases, and their abundance in tumor tissue positively correlated with clinical response in a cohort of patients with immune checkpoint inhibitor-treated esophageal cancer. In humanized mouse models, NK cell surveillance via B7H6 limited the persistence and antitumor activity of CAR T cells, and its genetic deletion enhanced T cell proliferation and persistence. Together, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as a therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.
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Antígenos B7 , Células Asesinas Naturales , Linfocitos T , Humanos , Células Asesinas Naturales/inmunología , Animales , Ratones , Antígenos B7/inmunología , Linfocitos T/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Activación de Linfocitos/inmunología , Femenino , Neoplasias Esofágicas/inmunologíaRESUMEN
Transforming growth factor (TGF)-ß and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFßR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFß1 cytokine or TGFßR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFßR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFßR1 kinase inhibition abolished the cytostatic effects of TGFß1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFß1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFßR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFß signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.
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Antineoplásicos , Carcinoma Hepatocelular , Citostáticos , Neoplasias Hepáticas , Animales , Humanos , Ratones , Carcinoma Hepatocelular/patología , Proliferación Celular , Neoplasias Hepáticas/patología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Small bowel adenocarcinoma (SBA) remains a rare malignancy accounting for less than 5% of all the gastrointestinal tract cancers. However, only limited data and expert guidelines are available for this entity. As a result, treatment concepts are predominantly derived from colorectal cancer. METHODS: To substantiate data on the course of disease, diagnosis and treatment of SBA, we performed a population-based analysis from a Bavarian population of 2.2 million people. RESULTS: We identified 223 patients with SBA. Mean age at diagnosis was 67.8 years and patients were diagnosed rather late (34.5% UICC stage IV). Largest proportion of these patients were diagnosed with adenocarcinoma of the duodenum (132 patients, 59.2%) and most patients were diagnosed with late stage cancer, stage IV (70 patients, 31.4%). With respect to treatment, most patients underwent primary surgery (187 patients, 84.6%). Systemic therapy seemed to have an impact in UICC stage IV patients but not in UICC stage IIB or III. The 5-year survival rate was 29.0%. This was significantly less compared to colon cancer in the same cohort, which was 50.0%. Furthermore, median survival of patients with small bowel cancer was only 2.0 years (95% CI 1.4-2.5) compared to 4.9 years (95% CI 4.8-5.1) of patients with colon cancer. CONCLUSION: SBA showed a distinct epidemiology compared to colon cancer. Thus, data acquisition particularly on systemic treatment are paramount, with the objective to complement the available guidelines.
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Adenocarcinoma , Neoplasias del Colon , Neoplasias Duodenales , Neoplasias del Íleon , Neoplasias Intestinales , Neoplasias del Yeyuno , Humanos , Neoplasias Intestinales/epidemiología , Neoplasias Intestinales/terapia , Intestino Delgado/patología , Neoplasias del Íleon/patología , Neoplasias del Íleon/terapia , Neoplasias Duodenales/epidemiología , Neoplasias Duodenales/terapia , Neoplasias del Yeyuno/patología , Neoplasias del Yeyuno/terapia , Adenocarcinoma/epidemiología , Adenocarcinoma/terapia , Adenocarcinoma/diagnóstico , Neoplasias del Colon/patologíaRESUMEN
BACKGROUND: The overall survival of patients with advanced and refractory oesophageal squamous cell carcinoma, mostly aged 65 years and older, is poor. Treatment with PD-1 antibodies showed improved progression-free survival and overall survival. We assessed the safety and efficacy of combined nivolumab and ipilimumab therapy in this population. METHODS: This multicentre, open-label, phase 2 trial done in 32 sites in Germany included patients aged 65 years and older with oesophageal squamous cell carcinoma and disease progression or recurrence following first-line therapy. Patients were treated with nivolumab (240 mg fixed dose once every 2 weeks, intravenously) in the safety run-in phase and continued with nivolumab and ipilimumab (nivolumab 240 mg fixed dose once every 2 weeks and ipilimumab 1 mg/kg once every 6 weeks, intravenously). The primary endpoint was overall survival, which was compared with a historical cohort receiving standard chemotherapy in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT03416244. FINDINGS: Between March 2, 2018, and Aug 20, 2020, we screened 75 patients with advanced oesophageal squamous cell carcinoma. We enrolled 66 patients (50 [76%] men and 16 [24%] women; median age 70·5 years [IQR 67·0-76·0]), 44 (67%) of whom received combined nivolumab and ipilimumab therapy and 22 (33%) received nivolumab alone. Median overall survival time at the prespecified data cutoff was 7·2 months (95% CI 5·7-12·4) and significantly higher than in a historical cohort receiving standard chemotherapy (p=0·0063). The most common treatment-related adverse events were fatigue (12 [29%] of 42), nausea (11 [26%]), and diarrhoea (ten [24%]). Grade 3-5 treatment-related adverse events occurred in 13 (20%) of 66 patients. Treatment-related death occurred in one patient with bronchiolitis obliterans while on nivolumab and ipilimumab treatment. INTERPRETATION: Patients aged at least 65 years, with advanced oesophageal squamous cell carcinoma might benefit from combined nivolumab and ipilimumab therapy in second-line treatment. FUNDING: Bristol Myers Squibb.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Células Epiteliales , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Femenino , Humanos , Ipilimumab/efectos adversos , Masculino , Nivolumab/efectos adversosRESUMEN
OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.
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Colangitis Esclerosante , Silenciador del Gen , Cirrosis Hepática Biliar , Cirrosis Hepática , Oligonucleótidos Antisentido , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Colangitis Esclerosante/metabolismo , Colangitis Esclerosante/patología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/prevención & control , Cirrosis Hepática Biliar/metabolismo , Cirrosis Hepática Biliar/patología , Ratones , Ratones Noqueados , Regulación hacia Arriba , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND: Advanced esophageal squamous cell cancer (ESCC) is frequently diagnosed in elderly patients. The impact of 2nd line chemotherapy is poorly defined. Recent data demonstrated effectiveness of checkpoint inhibitors in different squamous cell carcinomas. Therefore, we assess combined nivolumab/ipilimumab as 2nd line therapy in elderly ESCC patients. METHODS: RAMONA is a multicenter open-label phase II trial. The primary objective is to demonstrate a significant survival benefit of nivolumab/ipilimumab in advanced ESCC compared to historical data of standard chemotherapy. Primary endpoint is therefore overall survival (OS). Major secondary objective is the evaluation of tolerability. Time to QoL deterioration will thus be determined as key secondary endpoint. Further secondary endpoints are tumor response, PFS and safety. We aim to recruit a total of n = 75 subjects that have to be > 65 years old. Eligibility is determined by the geriatric status (G8 screening and Deficit Accumulation Frailty Index (DAFI)). A safety assessment will be performed after a 3 cycle run-in phase of nivolumab (240 mg Q2W) to justify escalation for eligible patients to combined nivolumab (240 mg Q2W) and ipilimumab (1 mg/kg Q6W), while the other patients will remain on nivolumab only. RAMONA also includes translational research sub-studies to identify predictive biomarkers, including PD-1 and PD-L1 evaluation at different time points, establishment of organoid cultures and microbiome analyses for response prediction. DISCUSSION: The RAMONA trial aims to implement checkpoint inhibitors for elderly patients with advanced ESCC as second line therapy. Novel biomarkers for checkpoint-inhibitor response are analyzed in extensive translational sub-studies. TRIAL REGISTRATION: EudraCT Number: 2017-002056-86 ; NCT03416244 , registered: 31.1.2018.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias Esofágicas/tratamiento farmacológico , Ipilimumab/administración & dosificación , Nivolumab/administración & dosificación , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Humanos , Ipilimumab/uso terapéutico , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/metabolismo , Análisis de Supervivencia , Resultado del TratamientoAsunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Toxicología/métodos , Investigación Biomédica Traslacional/métodos , Animales , Diferenciación Celular/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Toxicología/tendencias , Investigación Biomédica Traslacional/tendenciasRESUMEN
TGF-ß1 is a major player in chronic liver diseases promoting fibrogenesis and tumorigenesis through various mechanisms. The expression and function of TGF-ß2 have not been investigated thoroughly in liver disease to date. In this paper, we provide evidence that TGF-ß2 expression correlates with fibrogenesis and liver cancer development.Using quantitative realtime PCR and ELISA, we show that TGF-ß2 mRNA expression and secretion increased in murine HSCs and hepatocytes over time in culture and were found in the human-derived HSC cell line LX-2. TGF-ß2 stimulation of the LX-2 cells led to upregulation of the TGF-ß receptors 1, 2, and 3, whereas TGF-ß1 treatment did not alter or decrease their expression. In liver regeneration and fibrosis upon CCl4 challenge, the transient increase of TGF-ß2 expression was accompanied by TGF-ß1 and collagen expression. In bile duct ligation-induced fibrosis, TGF-ß2 upregulation correlated with fibrotic markers and was more prominent than TGF-ß1 expression. Accordingly, MDR2-KO mice showed significant TGF-ß2 upregulation within 3 to 15 months but minor TGF-ß1 expression changes. In 5 of 8 hepatocellular carcinoma (HCC)/hepatoblastoma cell lines, relatively high TGF-ß2 expression and secretion were observed, with some cell lines even secreting more TGF-ß2 than TGF-ß1. TGF-ß2 was also upregulated in tumors of TGFα/cMyc and DEN-treated mice. The analysis of publically available microarray data of 13 human HCC collectives revealed considerable upregulation of TGF-ß2 as compared to normal liver.Our study demonstrates upregulation of TGF-ß2 in liver disease and suggests TGF-ß2 as a promising therapeutic target for tackling fibrosis and HCC.
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Hepatopatías/genética , Neoplasias Hepáticas/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta2/genética , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hepatopatías/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/farmacologíaRESUMEN
Transforming growth factor ß (TGF-ß) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-ß role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-ß inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-ß effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-ß-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-ß actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.
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Carcinoma Hepatocelular/genética , Proliferación Celular , Hepatocitos/metabolismo , Hepatopatías/genética , Neoplasias Hepáticas/genética , Proteína smad7/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Cultivadas , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Hepatopatías/metabolismo , Hepatopatías/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Persona de Mediana Edad , Análisis Multivariante , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína smad7/metabolismo , Análisis de Supervivencia , Factor de Crecimiento Transformador beta/farmacología , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-ß is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-ß effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-ß inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-ß and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-ß signatures correlated with better outcome in HCC patients. TGF-ß dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-ß and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-ß signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-ß signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-ß expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-ß sensitivity. Cell lines responsive to the TGF-ß cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-ß/Smad3 signaling are insensitive to TGF-ß dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-ß functions for the design of efficient TGF-ß directed therapy in liver cancer.
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Carcinoma Hepatocelular/patología , Ciclo Celular , Neoplasias Hepáticas/patología , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Bases , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Humanos , Neoplasias Hepáticas/metabolismo , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Transforming growth factor (TGF)-ß is a central regulator in chronic liver disease, contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver damage-induced levels of active TGF-ß enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Further evidence points to a decisive role of cytostatic and apoptotic functions mediated on hepatocytes, which is critical for the control of liver mass, with loss of TGF-ß activities resulting in hyperproliferative disorders and cancer. This concept is based on studies that describe a bipartite role of TGF-ß with tumor suppressor functions at early stages of liver damage and regeneration, whereas during cancer progression TGF-ß may turn from a tumor suppressor into a tumor promoter that exacerbates invasive and metastatic behavior. We have delineated this molecular switch of the pathway from cytostatic to tumor promoting in further detail and identify activation of survival signaling pathways in hepatocytes as a most critical requirement. Targeting the TGF-ß signaling pathway has been explored to inhibit liver disease progression. While interfering with TGF-ß signaling in various short-term animal models has demonstrated promising results, liver disease progression in humans is a process of decades with different phases in which TGF-ß or its targeting may have both beneficial and adverse outcomes. We emphasize that, in order to achieve therapeutic effects, targeting TGF-ß signaling in the right cell type at the right time is required.
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Carcinoma Hepatocelular/patología , Progresión de la Enfermedad , Neoplasias Hepáticas/patología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Apoptosis/fisiología , Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , RatasRESUMEN
AIM: To analyse the influence of Smad7, antagonist of transforming growth factor (TGF)-ß canonical signaling pathways on hepatic stellate cell (HSC) transdifferentiation in detail. METHODS: We systematically analysed genes regulated by TGF-ß/Smad7 in activated HSCs by microarray analysis and validated the results using real time polymerase chain reaction and Western blotting analysis. RESULTS: We identified 100 known and unknown targets underlying the regulation of Smad7 expression and delineated 8 gene ontology groups. Hk2, involved in glycolysis, was one of the most downregulated proteins, while BMP2, activator of the Smad1/5/8 pathway, was extremely upregulated by Smad7. However, BMP2 dependent Smad1 activation could be inhibited in vitro by Smad7 overexpression in HSCs. CONCLUSION: We conclude (1) the existence of a tight crosstalk of TGF-ß and BMP2 pathways in HSCs and (2) a Smad7 dependently decreased sugar metabolism ameliorates HSC activation probably by energy withdrawal.
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Proteína Morfogenética Ósea 2/metabolismo , Transdiferenciación Celular/fisiología , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/fisiología , Transducción de Señal/fisiología , Proteína smad7/metabolismo , Calicreínas de Tejido/metabolismo , Animales , Proteína Morfogenética Ósea 2/genética , Células Cultivadas , Femenino , Masculino , Análisis por Micromatrices , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteína smad7/genética , Calicreínas de Tejido/genéticaRESUMEN
BACKGROUND & AIMS: Adverse alcohol effects in the liver involve oxidative metabolism, fat deposition and release of fibrogenic mediators, including TGF-beta. The work presents an assessment of liver damaging cross-talk between ethanol and TGF-beta in hepatocytes. METHODS: To investigate TGF-beta effects on hepatocytes, microarray analyses were performed and validated by qRT-PCR, Western blot analysis and immunohistochemistry. The cellular state was determined by assessing lactate dehydrogenase, cellular glutathione, reactive oxygen species, lipid peroxidation and neutral lipid deposition. RNA interference was used for gene silencing in vitro. RESULTS: TGF-beta is induced in mouse livers after chronic ethanol insult, enhances ethanol induced oxidative stress and toxicity towards cultured hepatocytes plus induces lipid-, oxidative stress metabolism- and fibrogenesis-gene expression signatures. Interestingly, TGF-beta down-regulates alcohol metabolizing enzyme Adh1 mRNA in cultured hepatocytes and liver tissue from TGF-beta transgenic mice via the ALK5/Smad2/3 signalling branch, with Smad7 as a potent negative regulator. ADH1 deficiency is a determining factor for the increased lipid accumulation and Cyp2E1 dependent toxicity in liver cells upon alcohol challenge. Further, ADH1 expression was decreased during liver damage in an intragastric ethanol infusion mouse model. CONCLUSION: In the presence of ethanol, TGF-beta displays pro-steatotic action in hepatocytes via decreasing ADH1 expression. Low ADH1 levels are correlated with enhanced hepatocyte damage upon chronic alcohol consumption by favoring secondary metabolic pathways.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Regulación hacia Abajo/fisiología , Etanol/efectos adversos , Hepatocitos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Hepatocitos/patología , Metabolismo de los Lípidos/fisiología , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Proteína smad7/metabolismoRESUMEN
Alcohol is recognized as a direct hepatotoxin, but the precise molecular pathways that are important for the initiation and progression of alcohol-induced tissue injury are not completely understood. The current understanding of alcohol toxicity to organs suggests that alcohol initiates injury by generation of oxidative and nonoxidative ethanol metabolites and via translocation of gut-derived endotoxin. These processes lead to cellular injury and stimulation of the inflammatory responses mediated through a variety of molecules. With continuing alcohol abuse, the injury progresses through impairment of tissue regeneration and extracellular matrix (ECM) turnover, leading to fibrogenesis and cirrhosis. Several cell types are involved in this process, the predominant being stellate cells, macrophages, and parenchymal cells. In response to alcohol, growth factors and cytokines activate many signaling cascades that regulate fibrogenesis. This mini-review brings together research focusing on the underlying mechanisms of alcohol-mediated injury in a number of organs. It highlights the various processes and molecules that are likely involved in inflammation, immune modulation, susceptibility to infection, ECM turnover and fibrogenesis in the liver, pancreas, and lung triggered by alcohol abuse.
Asunto(s)
Alcoholismo/metabolismo , Matriz Extracelular/metabolismo , Transducción de Señal/fisiología , Alcoholismo/patología , Animales , Etanol/administración & dosificación , Etanol/metabolismo , Matriz Extracelular/patología , Humanos , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patologíaRESUMEN
Chronic alcohol consumption is a risk factor for the development of chronic liver disease. Ethanol exerts its detrimental effects by various means: Directly via toxic metabolites, and indirectly by affecting the gut barrier leading to elevated levels of endotoxins in the blood challenging the liver. These factors, together with the resulting inflammatory and profibrogenic cytokine production, drive the organ's response, characterized by activation of hepatic stellate cells. Recent evidence argues for other cell types besides hepatic stellate cells, including hepatocytes, as additional sources of fibroblasts producing extracellular matrix and to be responsible for scar formation. Besides mediating hepatocyte apoptosis, TGF-beta additionally induces fibroblastoid transdifferentiation. This process is accompanied with loss of epithelial marker proteins and upregulation of fibrosis related proteins. These findings challenge the current view of the passive role of hepatocytes in liver fibrosis. In line, hepatocyte-specific inhibition of the TGF-beta pathway prevents CCl4 induced liver injury. Hence, this review focuses on the interplay of TGF-beta and alcohol in chronic liver disease with special emphasis on the potential contribution of hepatocytes.
Asunto(s)
Etanol/farmacología , Hígado/efectos de los fármacos , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Depresores del Sistema Nervioso Central/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Modelos BiológicosRESUMEN
The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Péptidos/metabolismo , Simulación por Computador , Enlace de Hidrógeno , Canales de Translocación SECRESUMEN
Currently, hepatic stellate cells (HSC) are thought to be the major fibrotic precursor cells that transdifferentiate to fibrogenic, extracellular matrix producing myofibroblasts in inflammatory liver tissue upon transforming growth factor-beta (TGF-beta) signaling, whereas hepatocytes are thought to respond with apoptosis to this cytokine. Starting out from in vitro experiments with primary hepatocyte cultures and immortalized AML-12 cells, TGF-beta signaling in this cell type was assessed and apoptosis was found to be only a minor effect. Instead, hepatocytes undergo epithelial mesenchymal transition (EMT), a physiological process in embryogenesis and of relevance for cancerous cell transformation. In injured liver, however, this process contributes to the promotion of fibrosis. Already after a few days of culture, hepatocytes lose their epithelial honeycomb-like shape towards a fibroblast-like phenotype. We could demonstrate by microarray analysis that stimulation of hepatocytes with TGF-beta regulates the expression of genes involved in EMT and fibrosis. Among these were, for example, Snail, a known mediator of EMT, and connective tissue growth factor (CTGF), a strong inducer of fibrosis. In a mouse model, hepatocyte-specific overexpression of Smad7 was able to blunt a fibrogenic response after CCl(4) intoxication. These results emphasize the dynamic nature of liver fibrosis, challenge the paradigm of HSC as a crucial source of liver myofibroblasts and hint towards a prominent role for hepatocytes in liver fibrogenesis.
Asunto(s)
Transdiferenciación Celular/fisiología , Hepatocitos/citología , Cirrosis Hepática/etiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Células Epiteliales/citología , Humanos , Mesodermo/citologíaRESUMEN
Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.