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The Hippo pathway transducers yes-associated protein (YAP) and WW-domain containing transcription regulator 1 (WWTR1/TAZ) are key regulators of liver tumorigenesis, promoting tumor formation and progression. Although the first inhibitors are in clinical trials, targeting the relevant upstream regulators of YAP/TAZ activity could prove equally beneficial. To identify regulators of YAP/TAZ activity in hepatocarcinoma (HCC) cells, we carried out a proximity labelling approach (BioID) coupled with mass spectrometry. We verified CRK-like proto-oncogene adaptor protein (CRKL) as a new YAP-exclusive interaction partner. CRKL is highly expressed in HCC patients, and its expression is associated with YAP activity as well as poor survival prognosis. In vitro experiments demonstrated CRKL-dependent cell survival and the loss of YAP binding induced through actin disruption. Moreover, we delineated the activation of the JNK/JUN pathway by CRKL, which promoted YAP transcription. Our data illustrate that CRKL not only promoted YAP activity through its binding but also through the induction of YAP transcription by JNK/JUN activation. This emphasizes the potential use of targeting the JNK/JUN pathway to suppress YAP expression in HCC patients.
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Proteínas Adaptadoras Transductoras de Señales , Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Nucleares , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Señalizadoras YAP/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proto-Oncogenes Mas , Línea Celular Tumoral , Unión Proteica , Sistema de Señalización de MAP Quinasas , Regulación Neoplásica de la Expresión Génica , Transducción de SeñalRESUMEN
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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BACKGROUND: Somatic copy number alterations are a hallmark of cancer that offer unique opportunities for therapeutic exploitation. Here, we focused on the identification of specific vulnerabilities for tumors harboring chromosome 8p deletions. METHODS: We developed and applied an integrative analysis of The Cancer Genome Atlas (TCGA), the Cancer Dependency Map (DepMap), and the Cancer Cell Line Encyclopedia to identify chromosome 8p-specific vulnerabilities. We employ orthogonal gene targeting strategies, both in vitro and in vivo, including short hairpin RNA-mediated gene knockdown and CRISPR/Cas9-mediated gene knockout to validate vulnerabilities. RESULTS: We identified SLC25A28 (also known as MFRN2), as a specific vulnerability for tumors harboring chromosome 8p deletions. We demonstrate that vulnerability towards MFRN2 loss is dictated by the expression of its paralog, SLC25A37 (also known as MFRN1), which resides on chromosome 8p. In line with their function as mitochondrial iron transporters, MFRN1/2 paralog protein deficiency profoundly impaired mitochondrial respiration, induced global depletion of iron-sulfur cluster proteins, and resulted in DNA-damage and cell death. MFRN2 depletion in MFRN1-deficient tumors led to impaired growth and even tumor eradication in preclinical mouse xenograft experiments, highlighting its therapeutic potential. CONCLUSIONS: Our data reveal MFRN2 as a therapeutic target of chromosome 8p deleted cancers and nominate MFNR1 as the complimentary biomarker for MFRN2-directed therapies.
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Deleción Cromosómica , Cromosomas Humanos Par 8 , Neoplasias , Humanos , Cromosomas Humanos Par 8/genética , Animales , Ratones , Neoplasias/genética , Línea Celular Tumoral , Mutaciones Letales Sintéticas , Mitocondrias/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Regulación Neoplásica de la Expresión Génica , Variaciones en el Número de Copia de ADNRESUMEN
AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a diagnosis of exclusion that can pose a challenge to the pathologist despite thorough clinical workup. Although several immunohistochemical markers have been proposed for iCCA, none of them reached clinical practice. We here assessed the combined usage of two promising diagnostic approaches, albumin in situ hybridisation (Alb-ISH) and C reactive protein (CRP) immunohistochemistry, for distinguishing iCCA from other adenocarcinoma primaries. METHODS: We conducted Alb-ISH and CRP immunohistochemistry in a large European iCCA cohort (n=153) and compared the results with a spectrum of other glandular adenocarcinomas of different origin (n=885). In addition, we correlated expression patterns with clinicopathological information and mutation data. RESULTS: Alb-ISH was highly specific for iCCA (specificity 98.8%) with almost complete negativity in perihilar CCA and only rare positives among other adenocarcinomas (sensitivity 69.5%). CRP identified the vast majority of iCCA cases (sensitivity 84.1%) at a lower specificity of 86.4%. Strikingly, the combination of CRP and Alb-ISH boosted the diagnostic sensitivity to 88.0% while retaining a considerable specificity of 86.1%. Alb-ISH significantly correlated with CRP expression, specific tumour morphologies and small or large duct iCCA subtypes. Neither Alb-ISH nor CRP was associated with iCCA patient survival. 16 of 17 recurrent mutations in either IDH1, IDH2 and FGFR2 affected Alb-ISH positive cases, while the only KRAS mutation corresponded to an Alb-ISH negative case. CONCLUSIONS: In conclusion, we propose a sequential diagnostic approach for iCCA, integrating CRP immunohistochemistry and Alb-ISH. This may improve the accuracy of CCA classification and pave the way towards a molecular-guided CCA classification.
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BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.
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Factores Despolimerizantes de la Actina , Carcinoma Hepatocelular , Movimiento Celular , Citoesqueleto , Neoplasias Hepáticas , Invasividad Neoplásica , Paxillin , Transducción de Señal , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Humanos , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Paxillin/metabolismo , Ratones , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Fosforilación , Hepacivirus , Línea Celular Tumoral , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Células Hep G2 , Hepatitis C/patología , Hepatitis C/metabolismo , Hepatitis C/virología , Interferencia de ARNRESUMEN
INTRODUCTION: The Hippo pathway and its transcriptional effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are targets for cancer therapy. It is important to determine if the activation of one factor compensates for the inhibition of the other. Moreover, it is unknown if YAP/TAZ-directed perturbation affects cell-cell communication of non-malignant liver cells. MATERIALS AND METHODS: To investigate liver-specific phenotypes caused by YAP and TAZ inactivation, we generated mice with hepatocyte (HC) and biliary epithelial cell (BEC)-specific deletions for both factors (YAPKO, TAZKO and double knock-out (DKO)). Immunohistochemistry, single-cell sequencing, and proteomics were used to analyze liver tissues and serum. RESULTS: The loss of BECs, liver fibrosis, and necrosis characterized livers from YAPKO and DKO mice. This phenotype was weakened in DKO tissues compared to specimens from YAPKO animals. After depletion of YAP in HCs and BECs, YAP expression was induced in non-parenchymal cells (NPCs) in a cholestasis-independent manner. YAP positivity was detected in subgroups of Kupffer cells (KCs) and endothelial cells (ECs). The secretion of pro-inflammatory chemokines and cytokines such as C-X-C motif chemokine ligand 11 (CXCL11), fms-related receptor tyrosine kinase 3 ligand (FLT3L), and soluble intercellular adhesion molecule-1 (ICAM1) was increased in the serum of YAPKO animals. YAP activation in NPCs could contribute to inflammation via TEA domain transcription factor (TEAD)-dependent transcriptional regulation of secreted factors. CONCLUSION: YAP inactivation in HCs and BECs causes liver damage, and concomitant TAZ deletion does not enhance but reduces this phenotype. Additionally, we present a new mechanism by which YAP contributes to cell-cell communication originating from NPCs.
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Comunicación Celular , Hígado , Proteínas Señalizadoras YAP , Animales , Ratones , Comunicación Celular/genética , Células Endoteliales , Hepatocitos , Ligandos , Hígado/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Dysregulation of cholesterol homeostasis is implicated in the development and progression of hepatocellular carcinoma (HCC) that is characterized by intrahepatic and early extrahepatic metastases. A better understanding of the underlying mechanisms regulating cholesterol metabolism in HCC could help identify strategies to circumvent the aggressive phenotype. Here, we found that high expression of intracellular SPARC (secreted protein acidic and rich in cysteine) was significantly associated with elevated cholesterol levels and an enhanced invasive phenotype in HCC. SPARC potentiated cholesterol accumulation in HCC cells during tumor progression by stabilizing the ApoE protein. Mechanistically, SPARC competitively bound to ApoE, impairing its interaction with the E3 ligase tripartite motif containing 21 (TRIM21) and preventing its ubiquitylation and subsequent degradation. ApoE accumulation led to cholesterol enrichment in HCC cells, stimulating PI3K-AKT signaling and inducing epithelial-mesenchymal transition (EMT). Importantly, sorafenib-resistant HCC cells were characterized by increased expression of intracellular SPARC, elevated cholesterol levels, and enhanced invasive capacity. Inhibiting SPARC expression or reducing cholesterol levels enhanced the sensitivity of HCC cells to sorafenib treatment. Together, these findings unveil interplay between SPARC and cholesterol homeostasis. Targeting SPARC-triggered cholesterol-dependent oncogenic signaling is a potential therapeutic strategy for advanced HCC. SIGNIFICANCE: Intracellular SPARC boosts cholesterol availability to fuel invasion and drug resistance in hepatocellular carcinoma, providing a rational approach to improve the treatment of advanced liver cancer.
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Apolipoproteínas E , Carcinoma Hepatocelular , Colesterol , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Neoplasias Hepáticas , Invasividad Neoplásica , Osteonectina , Sorafenib , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Osteonectina/metabolismo , Osteonectina/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Humanos , Sorafenib/farmacología , Colesterol/metabolismo , Animales , Ratones , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Línea Celular Tumoral , Ratones Desnudos , Masculino , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The transcriptional co-activators of the Hippo pathway, YAP and TAZ, are regulated by mechanotransduction, which depends on dynamic actin cytoskeleton remodeling. Here, we identified SEPTIN10 as a novel cytoskeletal protein, which is transcriptionally regulated by YAP/TAZ and whose overexpression correlates with poor survival and vascular invasion in hepatocellular carcinoma (HCC) patients. Functional characterization demonstrated that SEPTIN10 promotes YAP/TAZ-dependent cell viability, migration and invasion of liver cancer cells. Mechanistically, SEPTIN10 interacts with actin and microtubule filaments supporting actin stress fiber formation and intracellular tension through binding to CAPZA2 while concurrently inhibiting microtubule polymerization through the blockage of MAP4 function. This functional antagonism is important for cytoskeleton-dependent feedback activation of YAP/TAZ, as microtubule depolymerization induces actin stress fiber formation and subsequently YAP/TAZ activity. Importantly, the crosstalk between microfilaments and microtubules is mediated by SEPTIN10 as its loss abrogates actin stress fiber formation after microtubule disruption. Together, the YAP/TAZ target gene SEPTIN10 controls the dynamic interplay between actin and microtubule filaments, which feeds back on Hippo pathway activity in HCC cells and thus acts as molecular switch with impact on oncogenic signaling and cancer cell biology.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Mecanotransducción Celular , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Humanos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.
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BACKGROUND: Adherens junctions (AJs) facilitate cell-cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α-catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α-catenin contributes to liver cancer formation. METHODS: TCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α-catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α-catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation. RESULTS: α-catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α-catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α-catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α-catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α-catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α-catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α-catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α-catenin. CONCLUSION: Migration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α-catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.
Cellcell contact in epithelial cells is important for cell polarity, cellular compartmentalisation, as well as tissue architecture during development, homeostasis, and regeneration of adult tissues in metazoans. In this context, adherens junctions (AJs) mechanically sense cell contact information with direct impact on cytoskeletal remodelling, the regulation of signalling pathways, and eventually cell biology. Indeed, the loss of cellcell contact and cellular polarity are key features in human carcinogenesis and important pathological parameters for the identification of many epithelial tumors.We demonstrate in this study, that overexpression of the AJ constituent αcatenin is frequently observed in human hepatocellular carcinoma (HCC). αcatenin supports HCC cell proliferation and migration. Together with the oncogene AKT, αcatenin moderately facilitates tumor initiation in mouse livers. Using mass spectrometry, we identified several new αcatenin interaction partners in the cytosol of liver cancer cells, including the cytokinesis regulator centrosomal protein 55 (CEP55). CEP55 mediates pro-migratory effects and its overexpression in HCC cells is controlled by two molecular mechanisms: αcatenin-dependent protein stabilization and transcriptional induction by the TEA domain transcription factors (TEADs)/forkhead box M1 (FoxM1)/yes-associated protein (YAP) complex.In summary, we here describe a new mechanism how changes in cellcell contact support liver cancer formation and progression. This study demonstrates that dysregulation of the AJ component αcatenin contributes to liver carcinogenesis via distinct molecular mechanisms. Video Abstract.
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Adenocarcinoma , Carcinoma Hepatocelular , Proteínas de Ciclo Celular , Neoplasias del Colon , Neoplasias Hepáticas , Animales , Humanos , Ratones , alfa Catenina , Línea Celular , Movimiento Celular , Recurrencia Local de Neoplasia , Proteínas Proto-Oncogénicas c-aktRESUMEN
The Hippo signaling pathway controls cell proliferation and tissue regeneration via its transcriptional effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). The canonical pathway topology is characterized by sequential phosphorylation of kinases in the cytoplasm that defines the subcellular localization of YAP and TAZ. However, the molecular mechanisms controlling the nuclear/cytoplasmic shuttling dynamics of both factors under physiological and tissue-damaging conditions are poorly understood. By implementing experimental in vitro data, partial differential equation modeling, as well as automated image analysis, we demonstrate that nuclear phosphorylation contributes to differences between YAP and TAZ localization in the nucleus and cytoplasm. Treatment of hepatocyte-derived cells with hepatotoxic acetaminophen (APAP) induces a biphasic protein phosphorylation eventually leading to nuclear protein enrichment of YAP but not TAZ. APAP-dependent regulation of nuclear/cytoplasmic YAP shuttling is not an unspecific cellular response but relies on the sequential induction of reactive oxygen species (ROS), RAC-alpha serine/threonine-protein kinase (AKT, synonym: protein kinase B), as well as elevated nuclear interaction between YAP and AKT. Mouse experiments confirm this sequence of events illustrated by the expression of ROS-, AKT-, and YAP-specific gene signatures upon APAP administration. In summary, our data illustrate the importance of nuclear processes in the regulation of Hippo pathway activity. YAP and TAZ exhibit different shuttling dynamics, which explains distinct cellular responses of both factors under physiological and tissue-damaging conditions.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Proteínas Proto-Oncogénicas c-akt , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosforilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Acetaminofén/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Señalizadoras YAP , Proteínas Nucleares/metabolismo , Treonina/metabolismo , Serina/metabolismoRESUMEN
Cell-cell junctions are pivotal for embryogenesis and tissue homeostasis but also play a major role in tumorigenesis, tumor invasion, and metastasis. E-cadherin (CDH1) and N-cadherin (CDH2) are two adherens junction's transmembrane glycoproteins with tissue-specific expression patterns in epithelial and neural/mesenchymal cells. Aberrant expression has been implicated in the process of epithelial-mesenchymal transition (EMT) in malignant tumors. We could hitherto demonstrate cis-E:N-cadherin heterodimer in endoderm-derived cells. Using immunoprecipitation in cultured cells of the line PLC as well as in human hepatocellular carcinoma (HCC)-lysates, we isolated E-N-cadherin heterodimers in a complex with the plaque proteins α- and ß-catenin, plakoglobin, and vinculin. In confocal laser scanning microscopy, E-cadherin co-localized with N-cadherin at the basolateral membrane of normal hepatocytes, hepatocellular adenoma (HCA), and in most cases of HCC. In addition, we analyzed E- and N-cadherin expression via immunohistochemistry in a large cohort of 868 HCCs from 570 patients, 25 HCA, and respective non-neoplastic liver tissue, and correlated our results with multiple prognostic markers. While E- or N-cadherin were similarly expressed in tumor sites with vascular invasion or HCC metastases, HCC with vascular encapsulated tumor clusters (VETC) displayed slightly reduced E-cadherin, and slightly increased N-cadherin expression. Analyzing The Cancer Genome Atlas patient cohort, we found that reduced mRNA levels of CDH1, but not CDH2 were significantly associated with unfavorable prognosis; however, in multivariate analysis, CDH1 did not correlate with prognosis. In summary, E- and N-cadherin are specific markers for hepatocytes and derived HCA and HCC. E:N-cadherin heterodimers are constitutively expressed in the hepatocytic lineage and only slightly altered in malignant progression, thereby not complying with the concept of EMT.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Carcinoma Hepatocelular/patología , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/patología , Multimerización de ProteínaRESUMEN
Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , ARN Largo no Codificante , Carcinogénesis/patología , Carcinoma Hepatocelular/patología , Proliferación Celular/genética , Humanos , Neoplasias Hepáticas/patología , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.
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The major tumor suppressor P53 (TP53) acts primarily as a transcription factor by activating or repressing subsets of its numerous target genes, resulting in different cellular outcomes (e.g., cell cycle arrest, apoptosis and senescence). P53-dependent gene regulation is linked to several aspects of chromatin remodeling; however, regulation of chromatin-modifying enzymes by P53 is poorly understood in hepatocarcinogenesis. Herein, we identified Helicase, lymphoid specific (HELLS), a major epigenetic regulator in liver cancer, as a strong and selective P53 repression target within the SNF2-like helicase family. The underlying regulatory mechanism involved P53-dependent induction of P21 (CDKN1A), leading to repression of Forkhead Box Protein M1 (FOXM1) that in turn resulted in downregulation of HELLS expression. Supporting our in vitro data, we found higher expression of HELLS in murine HCCs arising in a Trp53-/- background compared to Trp53+/+ HCCs as well as a strong and highly significant correlation between HELLS and FOXM1 expression in different HCC patient cohorts. Our data suggest that functional or mutational inactivation of P53 substantially contributes to overexpression of HELLS in HCC patients and indicates a previously unstudied aspect of P53's ability to suppress liver cancer formation.
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Induction of neoangiogenesis is a hallmark feature during disease progression of hepatocellular carcinoma (HCC). Antiangiogenetic compounds represent a mainstay of therapeutic approaches; however, development of chemoresistance is observed in the majority of patients. Recent findings suggest that tumor-initiating cells (TICs) may play a key role in acquisition of resistance, but the exact relevance for HCC in this process remains to be defined. Primary and established hepatoma cell lines were exposed to long-term sorafenib treatment to model acquisition of resistance. Treatment effects on TICs were estimated by sphere-forming capacity in vitro, tumorigenicity in vivo, and flow cytometry. Adaptive molecular changes were assessed by whole transcriptome analyses. Compensatory mechanisms of resistance were identified and directly evaluated. Sustained antiproliferative effect following sorafenib treatment was observed in three of six HCC cell lines and was followed by rapid regrowth, thereby mimicking responses observed in patients. Resistant cells showed induction in sphere forming in vitro and tumor-initiating capacity in vivo as well as increased number of side population and epithelial cell adhesion molecule-positive cells. Conversely, sensitive cell lines showed consistent reduction of TIC properties. Gene sets associated with resistance and poor prognosis, including Hippo/yes-associated protein (YAP), were identified. Western blot and immunohistochemistry confirmed increased levels of YAP. Combined treatment of sorafenib and specific YAP inhibitor consistently revealed synergistic antioncogenic effects in resistant cell lines. Conclusion: Resistance to antiangiogenic therapy might be driven by transient expansion of TICs and activation of compensatory pro-oncogenic signaling pathways, including YAP. Specific targeting of TICs might be an effective therapeutic strategy to overcome resistance in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Sorafenib/farmacología , Proteínas Señalizadoras YAPRESUMEN
OBJECTIVE: Large-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer. DESIGN: Genetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted. RESULTS: KDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition. CONCLUSION: KDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition.
Asunto(s)
Histona Demetilasas/metabolismo , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animales , Epigénesis Genética , Histona Demetilasas/genética , Histonas/genética , Neoplasias Hepáticas/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Neoplasias Pancreáticas/genética , Neoplasias PancreáticasRESUMEN
BACKGROUND: Activation of the oncogene yes-associated protein (YAP) is frequently detected in intrahepatic cholangiocarcinoma (iCCA); however, the expression pattern and the functional impact of its paralogue WW domain-containing transcription regulator 1 (WWTR1; synonym: TAZ) are not well described in different CCA subtypes. METHODS: Immunohistochemical analysis of YAP and TAZ in iCCA and extrahepatic CCA (eCCA) cohorts was performed. YAP/TAZ shuttling and their functional impact on CCA cell lines were investigated. Target genes expression after combined YAP/TAZ inhibition was analyzed. RESULTS: Immunohistochemical analysis of iCCA and eCCA revealed YAP or TAZ positivity in up to 49.2%; however, oncogene co-expression was less frequent (up to 23%). In contrast, both proteins were jointly detectable in most CCA cell lines and showed nuclear/cytoplasmic shuttling in a cell density-dependent manner. Next to the pro-proliferative function of YAP/TAZ, both transcriptional co-activators cooperated in the regulation of a gene signature that indicated the presence of chromosomal instability (CIN). A correlation between YAP and the CIN marker phospho-H2A histone family member X (pH2AX) was particularly observed in tissues from iCCA and distal CCA (dCCA). The presence of the CIN genes in about 25% of iCCA was statistically associated with worse prognosis. CONCLUSIONS: YAP and TAZ activation is not uncoupled from cell density in CCA cells and both factors cooperatively contribute to proliferation and expression of CIN-associated genes. The corresponding group of CCA patients is characterized by CIN and may benefit from YAP/TAZ-directed therapies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de los Conductos Biliares/genética , Colangiocarcinoma/genética , Inestabilidad Cromosómica/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Conductos Biliares Extrahepáticos , Conductos Biliares Intrahepáticos , Recuento de Células , Línea Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Histonas/metabolismo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Pronóstico , Análisis de Matrices Tisulares , Factores de Transcripción/antagonistas & inhibidores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND & AIM: The development of hepatocellular carcinoma (HCC) is associated with the formation of communication networks leading to the recruitment of disease-modifying macrophages. However, how oncogenes in tumour cells control paracrine communication is not fully understood. METHODS: Transgenic mice with liver-specific expression of the constitutively active yes-associated protein (YAPS127A ) or an orthotopic implantation model served as tumour models. FACS-sorted F4/80+ /CD11bdim /CD146- /retinoid- macrophages from healthy and tumour-bearing livers were used for transcriptomic profiling. Expression data of 242 human HCCs and a tissue microarray consisting of 91 HCCs and seven liver tissues were analyzed. RESULTS: Screening of primary tumour cells expressing YAPS127A identified CC chemokine ligand 2 (Ccl2) as a macrophage chemoattractant, whose expression was regulated in a YAP/TEA domain family member 4 (TEAD4)-dependent manner. Ccl2 expression was associated with a loss of Kupffer cells (KCs) and an increase in immature macrophages (Mɸimm ) in hepatocarcinogenesis. Recruited Mɸimm were characterized by a lack of functional polarization (M0 signature) and high expression of the Ccl2 receptors C-C motif chemokine receptor 2 (Ccr2), C-X3-C motif chemokine receptor 1 (Cx3cr1) and pro-angiogenic platelet-derived growth factors (Pdgfa/Pdgfb). Mɸimm formed cellular clusters in the perivascular space, which correlated with vascular morphometric changes indicative for angiogenesis. In human HCCs, the M0 signature served as an identifier for poor clinical outcome and CCL2 correlated with YAP expression and vascular network formation. CONCLUSIONS: In conclusion, YAP/TEAD4-regulated Ccl2 associates with perivascular recruitment of unpolarized Mɸimm and may contribute to a proangiogenic microenvironment in liver cancer.
Asunto(s)
Carcinoma Hepatocelular , Quimiocina CCL2 , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular , Quimiocina CCL2/metabolismo , Humanos , Macrófagos del Hígado/metabolismo , Ligandos , Neoplasias Hepáticas/patología , Macrófagos/metabolismo , Ratones , Receptores CCR2/genética , Receptores CCR2/metabolismo , Factores de Transcripción , Microambiente Tumoral , Remodelación Vascular , Proteínas Señalizadoras YAPRESUMEN
The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET-expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. SIGNIFICANCE: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5502/F1.large.jpg.