ABSTRACT
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.
Subject(s)
Cell Communication , Liver , YAP-Signaling Proteins , Animals , Mice , Cell Communication/genetics , Endothelial Cells , Hepatocytes , Ligands , Liver/metabolism , YAP-Signaling Proteins/genetics , YAP-Signaling Proteins/metabolismABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing , Carcinoma, Hepatocellular , Liver Neoplasms , Nuclear Proteins , Transcription Factors , YAP-Signaling Proteins , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , YAP-Signaling Proteins/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Proto-Oncogene Mas , Cell Line, Tumor , Protein Binding , MAP Kinase Signaling System , Gene Expression Regulation, Neoplastic , Signal TransductionABSTRACT
BACKGROUND: Overexpression and nuclear enrichment of the oncogene yes-associated protein (YAP) cause tumor initiation and support tumor progression in human hepatocellular carcinoma (HCC) via cell autonomous mechanisms. However, how YAP expression in tumor cells affects intercellular communication within the tumor microenvironment is not well understood. METHODS: To investigate how tumor cell-derived YAP is changing the paracrine communication network between tumor cells and non-neoplastic cells in hepatocarcinogenesis, the expression and secretion of cytokines, growth factors and chemokines were analyzed in transgenic mice with liver-specific and inducible expression of constitutively active YAP (YAPS127A). Transcriptomic and proteomic analyses were performed using primary isolated hepatocytes and blood plasma. In vitro, RNAinterference (RNAi), expression profiling, functional analyses and chromatin immunoprecipitation (ChIP) analyses of YAP and the transcription factor TEA domain transcription factor 4 (TEAD4) were performed using immortalized cell lines. Findings were confirmed in cohorts of HCC patients at the transcript and protein levels. RESULTS: YAP overexpression induced the expression and secretion of many paracrine-acting factors with potential impact on tumorous or non-neoplastic cells (e.g. plasminogen activator inhibitor-1 (PAI-1), C-X-C motif chemokine ligand 13 (CXCL13), CXCL16). Expression analyses of human HCC patients showed an overexpression of PAI-1 in human HCC tissues and a correlation with poor overall survival as well as early cancer recurrence. PAI-1 statistically correlated with genes typically induced by YAP, such as connective tissue growth factor (CTGF) and cysteine rich angiogenic inducer 61 (CYR61) or YAP-dependent gene signatures (CIN4/25). In vitro, YAP inhibition diminished the expression and secretion of PAI-1 in murine and human liver cancer cell lines. PAI-1 affected the expression of genes involved in cellular senescence and oncogene-induced senescence was confirmed in YAPS127A transgenic mice. Silencing of TEAD4 as well as treatment with the YAP/TEAD interfering substance Verteporfin reduced PAI-1 expression. ChIP analyses confirmed the binding of YAP and TEAD4 to the gene promoter of PAI-1 (SERPINE1). CONCLUSIONS: These results demonstrate that the oncogene YAP changes the secretome response of hepatocytes and hepatocyte-derived tumor cells. In this context, the secreted protein PAI-1 is transcriptionally regulated by YAP in hepatocarcinogenesis. Perturbation of these YAP-dependent communication hubs including PAI-1 may represent a promising pharmacological approach in tumors with YAP overexpression. Video abstract.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Plasminogen Activator Inhibitor 1/metabolism , Proteome/metabolism , Transcription, Genetic , Animals , Carcinogenesis/pathology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cellular Senescence/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Hepatocytes/metabolism , Liver Neoplasms/pathology , Mice, Transgenic , Muscle Proteins/metabolism , Phenotype , Plasminogen Activator Inhibitor 1/genetics , Prognosis , Promoter Regions, Genetic/genetics , TEA Domain Transcription Factors , Transcription Factors/metabolism , YAP-Signaling ProteinsABSTRACT
BACKGROUND & AIMS: Many different types of cancer cells have chromosome instability. The hippo pathway leads to phosphorylation of the transcriptional activator yes-associated protein 1 (YAP1, YAP), which regulates proliferation and has been associated with the development of liver cancer. We investigated the effects of hippo signaling via YAP on chromosome stability and hepatocarcinogenesis in humans and mice. METHODS: We analyzed transcriptome data from 242 patients with hepatocellular carcinoma (HCC) to search for gene signatures associated with chromosomal instability (CIN); we investigated associations with overall survival time and cancer recurrence using Kaplan-Meier curves. We analyzed changes in expression of these signature genes, at mRNA and protein levels, after small interfering RNA-mediated silencing of YAP in Sk-Hep1, SNU182, HepG2, or pancreatic cancer cells, as well as incubation with thiostrepton (an inhibitor of forkhead box M1 [FOXM1]) or verteporfin (inhibitor of the interaction between YAP and TEA domain transcription factor 4 [TEAD4]). We performed co-immunoprecipitation and chromatin immunoprecipitation experiments. We collected liver tissues from mice that express a constitutively active form of YAP (YAPS127A) and analyzed gene expression signatures and histomorphologic parameters associated with chromosomal instability. Mice were given injections of thiostrepton and livers were collected and analyzed by immunoblotting, immunohistochemistry, histology, and real-time polymerase chain reaction. We performed immunohistochemical analyses on tissue microarrays of 105 HCCs and 7 nontumor liver tissues. RESULTS: Gene expression patterns associated with chromosome instability, called CIN25 and CIN70, were detected in HCCs from patients with shorter survival time or early cancer recurrence. TEAD4 and YAP were required for CIN25 and CIN70 signature expression via induction and binding of FOXM1. Disrupting the interaction between YAP and TEAD4 with verteporfin, or inhibiting FOXM1 with thiostrepton, reduced the chromosome instability gene expression patterns. Hyperplastic livers and tumors from YAPS127A mice had increased CIN25 and CIN70 gene expression patterns, aneuploidy, and defects in mitosis. Injection of YAPS127A mice with thiostrepton reduced liver overgrowth and signs of chromosomal instability. In human HCC tissues, high levels of nuclear YAP correlated with increased chromosome instability gene expression patterns and aneuploidy. CONCLUSIONS: By analyzing cell lines, genetically modified mice, and HCC tissues, we found that YAP cooperates with FOXM1 to contribute to chromosome instability. Agents that disrupt this pathway might be developed as treatments for liver cancer. Transcriptome data are available in the Gene Expression Omnibus public database (accession numbers: GSE32597 and GSE73396).
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Carcinoma, Hepatocellular/genetics , Chromosomal Instability , Forkhead Box Protein M1/genetics , Liver Neoplasms/genetics , Phosphoproteins/genetics , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , DNA-Binding Proteins/metabolism , Disease Models, Animal , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Hep G2 Cells , Humans , Kaplan-Meier Estimate , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice, Inbred C57BL , Mice, Transgenic , Muscle Proteins/metabolism , Phenotype , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Porphyrins/pharmacology , Prognosis , RNA Interference , Signal Transduction , TEA Domain Transcription Factors , Thiostrepton/pharmacology , Time Factors , Tissue Array Analysis , Transcription Factors/metabolism , Transcriptome , Transfection , Verteporfin , YAP-Signaling ProteinsABSTRACT
UNLABELLED: Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. CONCLUSION: These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.
Subject(s)
Carcinoma, Hepatocellular/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Liver Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Caspase 3/metabolism , Caspase 7/metabolism , Female , Humans , Male , Protein Stability , RNA-Binding ProteinsABSTRACT
UNLABELLED: The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. CONCLUSION: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression.
Subject(s)
Carcinoma, Hepatocellular/physiopathology , Cell Differentiation/physiology , Cell Movement/physiology , Cell Proliferation/physiology , DNA Helicases/physiology , DNA-Binding Proteins/physiology , Liver Neoplasms/physiopathology , RNA-Binding Proteins/physiology , Repressor Proteins/physiology , Animals , Carcinoma, Hepatocellular/pathology , DNA Helicases/drug effects , DNA Helicases/genetics , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , Exons/genetics , Humans , In Vitro Techniques , Liver Neoplasms/pathology , Mice, SCID , Mice, Transgenic , Protein Isoforms/genetics , RNA Splicing Factors , RNA, Small Interfering/pharmacology , RNA-Binding Proteins/drug effects , RNA-Binding Proteins/genetics , Repressor Proteins/drug effects , Repressor Proteins/genetics , Transcription Factor DP1/physiology , Transplantation, HeterologousABSTRACT
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.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Mechanotransduction, Cellular , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling Proteins , Humans , Actin Cytoskeleton/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Trans-Activators/metabolism , Transcription Factors/metabolismABSTRACT
Stability of many tumor-relevant proteins is partly mediated by E3 ligases, which determine substrate specificity within the ubiquitin system. Recent data demonstrated that increased nuclear expression of the E3 ligase seven in absentia homologue (SIAH)-1 in human hepatocarcinogenesis supports tumor cell proliferation and migration. To define whether closely related SIAH-2 synergizes with protumorigenic SIAH-1, we systematically analyzed expression, localization and functional relevance of SIAH-2 in human hepatocellular carcinoma (HCC). Nuclear accumulation of SIAH-2 is detectable in more than 60% of all HCCs and correlates with tumor progression, cell proliferation and distant metastasis. An inverse correlation between nuclear SIAH-1 and SIAH-2 was detected, suggesting independent mechanisms for nuclear enrichment. Inhibition of nuclear SIAH-2 by RNAi in HCC cell lines reduced proliferation as well as lateral tumor cell motility and transmigration; however, combined knock down of both SIAH-1 and SIAH-2 did not further amplify biological effects compared to single gene inhibition. Reduction of SIAH-2 expression sensitizes HCC cells to the treatment with different cytostatic drugs, demonstrating that SIAH-2-targeting approaches may increase the response of HCC cells to conventional chemotherapy. Together, these data show that SIAH-2--as described for SIAH-1--accumulates in nuclei of HCC cells where it supports tumor growth and tumor cell dissemination. Because the nuclear pattern of SIAH-2 differs in HCC tissues from the SIAH-1 pattern and because the inactivation of SIAH-2 is not compensated by SIAH-1, the specific inhibition of SIAH-2 (especially in combination with other drugs) represents a promising therapeutic strategy for HCC.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement , Cell Nucleus/metabolism , Cell Proliferation , Humans , Liver Neoplasms/genetics , Neoplasm Metastasis , Nuclear Proteins/genetics , RNA Interference , RNA, Small Interfering , Ubiquitin-Protein Ligases/geneticsABSTRACT
BACKGROUND & AIMS: Differential expression of tumor-relevant proteins based on aberrant proteasomal degradation may contribute to human (hepato)carcinogenesis. Recently, we identified the E3 ubiquitin ligase seven in absentia homolog (SIAH)-1 as frequently dysregulated in human hepatocellular carcinoma (HCC). We therefore systematically analyzed the expression, functional relevance, as well as possible downstream effectors of SIAH-1 in human liver carcinogenesis. METHODS: SIAH-1 expression was analyzed at the transcript and protein levels in human hepatocarcinogenesis and in HCC cells. Proliferation, apoptosis, and migration of different HCC cell lines were examined after siRNA-mediated inhibition of SIAH-1. In order to identify downstream effectors that mediate SIAH-1 effects, correlative analyses of protein expression profiles were performed. RESULTS: In HCC tissues both reduction of cytoplasmic SIAH-1 and especially its nuclear accumulation positively correlated with HCC progression. RNA interference revealed that nuclear expression of SIAH-1 predominantly supported HCC cell proliferation and migration while only moderately affecting anti-apoptosis. In de-differentiated human HCCs, nuclear SIAH-1 accumulation significantly correlated with the expression of the transcription factor far-upstream element (FUSE)-binding protein (FBP)-3. In vitro, SIAH-1 positively and indirectly regulated FBP-3 which itself primarily supported HCC cell proliferation. Indeed, high level expression of FBP-3 in human HCCs significantly correlated with reduced overall survival of patients. CONCLUSIONS: Nuclear accumulation of the E3 ubiquitin ligase SIAH-1 supports different pro-tumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis. It promotes HCC cell proliferation by at least partly employing the transcription factor FBP-3. Therefore, interference with SIAH-1 activity represents a promising approach to suppress HCC growth.
Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Transformation, Neoplastic/metabolism , Liver Neoplasms/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Apoptosis , Carcinoma, Hepatocellular/enzymology , Cell Line, Tumor , Cell Movement , Cell Nucleus/metabolism , Cell Proliferation , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Liver Neoplasms/enzymology , Nuclear Proteins/antagonists & inhibitors , RNA, Small Interfering/genetics , Statistics, Nonparametric , Transcription Factors/metabolism , Transfection , Ubiquitin-Protein Ligases/antagonists & inhibitorsABSTRACT
UNLABELLED: Microtubule-dependent effects are partly regulated by factors that coordinate polymer dynamics such as the microtubule-destabilizing protein stathmin (oncoprotein 18). In cancer cells, increased microtubule turnover affects cell morphology and cellular processes that rely on microtubule dynamics such as mitosis and migration. However, the molecular mechanisms deregulating modifiers of microtubule activity in human hepatocarcinogenesis are poorly understood. Based on profiling data of human hepatocellular carcinoma (HCC), we identified far upstream element binding proteins (FBPs) as significantly coregulated with stathmin. Coordinated overexpression of two FBP family members (FBP-1 and FBP-2) in >70% of all analyzed human HCCs significantly correlated with poor patient survival. In vitro, FBP-1 predominantly induced tumor cell proliferation, while FBP-2 primarily supported migration in different HCC cell lines. Surprisingly, reduction of FBP-2 levels was associated with elevated FBP-1 expression, suggesting a regulatory interplay of FBP family members that functionally discriminate between cell division and mobility. Expression of FBP-1 correlated with stathmin expression in HCC tissues and inhibition of FBP-1 but not of FBP-2 drastically reduced stathmin at the transcript and protein levels. In contrast, further overexpression of FBP-1 did not affect stathmin bioavailability. Accordingly, analyzing nuclear and cytoplasmic areas of HCC cells revealed that reduced FBP-1 levels affected cell morphology and were associated with a less malignant phenotype. CONCLUSION: The coordinated activation of FBP-1 and FBP-2 represents a novel and frequent pro-tumorigenic mechanism promoting proliferation (tumor growth) and motility (dissemination) of human liver cancer cells. FBPs promote tumor-relevant functions by at least partly employing the microtubule-destabilizing factor stathmin and represent a new potential target structure for HCC treatment.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Movement/physiology , Cell Proliferation , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Apoptosis/physiology , Cell Division/physiology , Cell Line, Tumor , Cell Survival/physiology , Humans , Microtubules/metabolism , RNA-Binding Proteins , Stathmin/metabolismABSTRACT
The Hippo pathway effectors yes-associated protein (YAP) and WW domain containing transcription regulator 1 (TAZ/WWTR1) support tumor initiation and progression in various cancer entities including hepatocellular carcinoma (HCC). However, to which extent YAP and TAZ contribute to liver tumorigenesis via common and exclusive molecular mechanisms is poorly understood. RNAinterference (RNAi) experiments illustrate that YAP and TAZ individually support HCC cell viability and migration, while for invasion additive effects were observed. Comprehensive expression profiling revealed partly overlapping YAP/TAZ target genes as well as exclusively regulated genes. Integrin-αV (ITGAV) is a novel TAZ-specific target gene, whose overexpression in human HCC patients correlates with poor clinical outcome, TAZ expression in HCCs, and the abundance of YAP/TAZ target genes. Functionally, ITGAV contributes to actin stress fiber assembly, tumor cell migration and invasion. Perturbation of ITGAV diminishes actin fiber formation and nuclear YAP/TAZ protein levels. We describe a novel Hippo downstream mechanism in HCC cells, which is regulated by TAZ and ITGAV and that feedbacks on YAP/TAZ activity. This mechanism may represent a therapeutic target structure since it contributes to signal amplification of oncogenic YAP/TAZ in hepatocarcinogenesis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carcinoma, Hepatocellular/genetics , Feedback, Physiological , Integrin alphaV/genetics , Liver Neoplasms/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Carcinogenesis/genetics , Carcinogenesis/pathology , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement , Cohort Studies , Disease-Free Survival , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Hippo Signaling Pathway , Humans , Integrin alphaV/metabolism , Liver/pathology , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Neoplasm Invasiveness , Prognosis , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Signal Transduction/genetics , Stress Fibers/metabolism , Stress Fibers/pathology , Tissue Array Analysis , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling ProteinsABSTRACT
UNLABELLED: The protumorigenic insulin-like growth factor (IGF)-II is highly expressed in a significant fraction of human hepatocellular carcinomas (HCC). However, a functional dissection that clarifies the contribution of IGF-II-binding receptors in tumor progression and a respective molecular characterization of IGF-II signaling has not been performed. Therefore, expression of IGF-II and its receptors IGF-receptor type I (IGF-IR) and insulin receptor (IR) was efficiently blocked using small interfering RNA (siRNA) in HCC cells. Despite functional IR-signaling, oncogenic IGF-II effects such as tumor cell viability, proliferation, and anti-apoptosis were solely transmitted by IGF-IR. Although IGF-II signaling was previously not described in the context of HCC cell migration, the IGF-II-dependent expression profile displayed a high percentage of genes involved in cell motility and adhesion. Indeed, IGF-II overexpression promoted HCC cell migration, especially in synergy with hepatocyte growth factor (HGF). The therapeutic relevance of IGF-II/IGF-IR signaling was tested in vitro and in a murine xenograft transplantation model using the IGF-IR inhibitor picropodophyllin (PPP). IGF-IR inhibition by small molecule treatment efficiently reduced IGF-II-dependent signaling and all protumorigenic properties of the IGF-II/IGF-IR pathway. CONCLUSION: In human HCC cells, IGF-IR but not IR is involved in oncogenic IGF-II signaling. Autocrine stimulation of IGF-II induces HCC motility by integration of paracrine signals for full malignant competence. Thus, activation of IGF-II/IGF-IR signaling is likely a progression switch selected by function that promotes tumor cell dissemination and aggressive tumor behavior.
Subject(s)
Autocrine Communication , Carcinoma, Hepatocellular/physiopathology , Cell Movement , Insulin-Like Growth Factor II/metabolism , Liver Neoplasms/physiopathology , Receptor, IGF Type 1/metabolism , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Movement/drug effects , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Neoplasm Invasiveness , Neoplasm Transplantation , Podophyllotoxin/analogs & derivatives , Podophyllotoxin/pharmacology , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Transplantation, Heterologous , Tumor Cells, Cultured , Up-RegulationABSTRACT
In the liver tumor necrosis factor (TNF)-induced signaling critically regulates the immune response of non-parenchymal cells as well as proliferation and apoptosis of hepatocytes via activation of the NF-κB and JNK pathways. Especially, the induction of negative feedback regulators, such as IκBα and A20 is responsible for the dynamic and time-restricted response of these important pathways. However, the precise mechanisms responsible for different TNF-induced phenotypes under physiological stimulation conditions are not completely understood so far. In addition, it is not known if varying TNF concentrations may differentially affect the desensitization properties of both pathways. By using computational modeling, we first showed that TNF-induced activation and downstream signaling is qualitatively comparable between primary mouse hepatocytes and immortalized hepatocellular carcinoma (HCC) cells. In order to define physiologically relevant TNF levels, which allow for an adjustable and dynamic NF-κB/JNK pathway response in parenchymal liver cells, a range of cytokine concentrations was defined that led to gradual pathway responses in HCC cells (1-5 ng/ml). Repeated stimulations with low (1 ng/ml), medium (2.5 ng/ml) and high (5 ng/ml) TNF amounts demonstrated that JNK signaling was still active at cytokine concentrations, which led to dampened NF-κB signaling illustrating differential pathway responsiveness depending on TNF input dynamics. SiRNA-mediated inhibition of the negative feedback regulator A20 (syn. TNFAIP3) or its overexpression did not significantly affect the NF-κB response. In contrast, A20 silencing increased the JNK response, while its overexpression dampened JNK phosphorylation. In addition, the A20 knockdown sensitized hepatocellular cells to TNF-induced cleavage and activity of the effector caspase-3. In conclusion, a mathematical model-based approach shows that the TNF-induced pathway responses are qualitatively comparable in primary and immortalized mouse hepatocytes. The cytokine amount defines the pathway responsiveness under repeated treatment conditions with NF-κB signaling being dampened 'earlier' than JNK. A20 appears to be the molecular switch discriminating between NF-κB and JNK signaling when stimulating with varying physiological cytokine concentrations.
ABSTRACT
Gut-derived bacterial lipopolysaccharides (LPS) stimulate the secretion of tumour necrosis factor (TNF) from liver macrophages (MCs), liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), which control the acute phase response in hepatocytes through activation of the NF-κB pathway. The individual and cooperative impact of nonparenchymal cells on this clinically relevant response has not been analysed in detail due to technical limitations. To gain an integrative view on this complex inter- and intracellular communication, we combined a multiscale mathematical model with quantitative, time-resolved experimental data of different primary murine liver cell types. We established a computational model for TNF-induced NF-κB signalling in hepatocytes, accurately describing dose-responsiveness for physiologically relevant cytokine concentrations. TNF secretion profiles were quantitatively measured for all nonparenchymal cell types upon LPS stimulation. This novel approach allowed the analysis of individual and collective paracrine TNF-mediated NF-κB induction in hepatocytes, revealing strongest effects of MCs and LSECs on hepatocellular NF-κB signalling. Simulations suggest that both cell types act together to maximize the NF-κB pathway response induced by low LPS concentrations (0.1 and 1 ng/mL). Higher LPS concentrations (≥ 5 ng/mL) induced sufficient TNF levels from MCs or LSECs to induce a strong and nonadjustable pathway response. Importantly, these simulations also revealed that the initial cytokine secretion (1-2 h after stimulation) rather than final TNF level (10 h after stimulation) defines the hepatocellular NF-κB response. This raises the question whether the current experimental standard of single high-dose cytokine administration is suitable to mimic in vivo cytokine exposure. DATABASE: The computational models described in this manuscript are available in the JWS database via the following link: https://jjj.bio.vu.nl/database/beuke.
Subject(s)
Hepatocytes/metabolism , Lipopolysaccharides/metabolism , Liver/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Lipopolysaccharides/administration & dosage , Macrophages/drug effects , Macrophages/metabolism , Mice , Paracrine Communication/drug effects , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The cytokine tumor necrosis factor-alpha (TNFα) is one of the key factors during the priming phase of liver regeneration as well as in hepatocarcinogenesis. TNFα activates the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) signaling pathway and contributes to the conversion of quiescent hepatocytes to activated hepatocytes that are able to proliferate in response to growth factor stimulation. Different mathematical models have been previously established for TNFα/NFκB signaling in the context of tumor cells. Combining these mathematical models with time-resolved measurements of expression and phosphorylation of TNFα/NFκB pathway constituents in primary mouse hepatocytes revealed that an additional phosphorylation step of the NFκB isoform p65 has to be considered in the mathematical model in order to sufficiently describe the dynamics of pathway activation in the primary cells. Also, we addressed the role of basal protein turnover by experimentally measuring the degradation rate of pivotal players in the absence of TNFα and including this information in the model. To elucidate the impact of variations in the protein degradation rates on TNFα/NFκB signaling on the overall dynamic behavior we used global sensitivity analysis that accounts for parameter uncertainties and showed that degradation and translation of p65 had a major impact on the amplitude and the integral of p65 phosphorylation. Finally, our mathematical model of TNFα/NFκB signaling was able to predict the time-course of the complex formation of p65 and of the inhibitor of NFκB (IκB) in primary mouse hepatocytes, which was experimentally verified. Hence, we here present a mathematical model for TNFα/NFκB signaling in primary mouse hepatocytes that provides an important basis to quantitatively disentangle the complex interplay of multiple factors in liver regeneration and tumorigenesis.
ABSTRACT
Keratinocyte migration is essential for the rapid closure of the epidermis in the process of wound healing. Mesenchymal cell-derived hepatocyte growth factor (HGF) is a central regulator of this process. However, the molecular mechanisms and relevant genes that facilitate this cellular response are still poorly defined. We used heterologous cocultures combining primary human keratinocytes and genetically modified murine fibroblasts to identify key factors mediating HGF-induced epidermal cell migration. The absence of c-Jun activity in fibroblasts completely abolished the expression of HGF in these cells and consequently altered the behavior of keratinocytes. Time-resolved expression series of keratinocytes stimulated with HGF disclosed target genes regulating HGF-dependent motility. In addition to well-established HGF-dependent wound healing-associated genes, carcinoembryogenic antigen-related cell adhesion molecule (CEACAM)-1 and the urokinase plasminogen activator (uPA)/uPA-receptor (uPAR) pathway were identified as possible mediators in HGF-induced keratinocyte migration. The functional relevance of CEACAM-1 and uPA/uPAR on epidermal cell motility was demonstrated using the HaCaT cell culture model. In conclusion, the distinct spatiotemporal regulation of genes by HGF is essential for proper epidermal cell migration in cutaneous wound healing.
Subject(s)
Antigens, CD/metabolism , Cell Adhesion Molecules/metabolism , Cell Movement/physiology , Hepatocyte Growth Factor/metabolism , Keratinocytes/metabolism , Receptors, Urokinase Plasminogen Activator/metabolism , Transcription Factor AP-1/metabolism , Animals , Cell Line , Coculture Techniques , Fibroblasts/cytology , Fibroblasts/metabolism , Hepatocyte Growth Factor/genetics , Humans , Keratinocytes/cytology , Mice , Mice, Knockout , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction/physiology , Wound Healing/physiologyABSTRACT
Dynamic instability of the microtubule network modulates processes such as cell division and motility, as well as cellular morphology. Overexpression of the microtubule-destabilizing phosphoprotein stathmin is frequent in human malignancies and represents a promising therapeutic target. Although stathmin inhibition gives rise to antineoplastic effects, additional and functionally redundant microtubule-interacting proteins may attenuate the efficiency of this therapeutic approach. We have systematically analyzed the expression and potential protumorigenic effects of stathmin family members in human non-small cell lung cancer (NSCLC). Both stathmin and stathmin-like 3 (SCLIP) were overexpressed in adenocarcinoma as well as squamous cell carcinoma (SCC) tissues and induced tumor cell proliferation, migration, and matrix invasion in respective cell lines. Accordingly, reduced stathmin and SCLIP levels affected cell morphology and were associated with a less malignant phenotype. Combined inhibition of both factors caused additive effects on tumor cell motility, indicating partial functional redundancy. Because stathmin and SCLIP expression significantly correlated in NSCLC tissues, we searched for common upstream regulators and identified the far upstream sequence element-binding protein-1 (FBP-1) as a pivotal inducer of several stathmin family members. Our results indicate that the coordinated overexpression of microtubule-destabilizing factors by FBP-1 is a critical step to facilitate microtubule dynamics and subsequently increases proliferation and motility of tumor cells.