ABSTRACT
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Subject(s)
Antineoplastic Agents , Drug Discovery , Enzyme Inhibitors , Microsatellite Instability , Neoplasms , Synthetic Lethal Mutations , Werner Syndrome Helicase , Animals , Female , Humans , Mice , Administration, Oral , Allosteric Regulation/drug effects , Antineoplastic Agents/adverse effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Clinical Trials as Topic , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Damage/drug effects , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Mice, Nude , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/metabolism , Protein Domains , Reproducibility of Results , Suppression, Genetic , Synthetic Lethal Mutations/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Werner Syndrome Helicase/antagonists & inhibitors , Werner Syndrome Helicase/genetics , Werner Syndrome Helicase/metabolism , Xenograft Model Antitumor AssaysABSTRACT
One important prerequisite for developing a therapeutic monoclonal antibody is to evaluate its in vivo efficacy. We tested the therapeutic potential of an anti-CD96 antibody alone or in combination with an anti-PD-1 antibody in a mouse colon cancer model. Early anti-PD-1 treatment significantly decreased tumor growth and the combination with anti-CD96 further increased the therapeutic benefit, while anti-CD96 treatment alone had no effect. In late therapeutic settings, the treatment combination resulted in enhanced CD8+ T cell infiltration of tumors and an increased CD8/Treg ratio. Measured anti-PD-1 concentrations were as expected in animals treated with anti-PD-1 alone, but lower at later time points in animals receiving combination treatment. Moreover, anti-CD96 concentrations dropped dramatically after 10 days and were undetectable thereafter in most animals due to the occurrence of anti-drug antibodies that were increasing antibody clearance. Comparison of the anti-PD-1 concentrations with tumor growth showed that higher antibody concentrations in plasma correlated with better therapeutic efficacy. The therapeutic effect of anti-CD96 treatment could not be evaluated, because plasma concentrations were too low. Our findings strongly support the notion of measuring both plasma concentration and anti-drug antibody formation throughout in vivo studies, in order to interpret pharmacodynamic data correctly.
ABSTRACT
Upregulation of inhibitory receptors, such as lymphocyte activation gene-3 (LAG-3), may limit the antitumor activity of therapeutic antibodies targeting the programmed cell death protein-1 (PD-1) pathway. We describe the binding properties of ezabenlimab, an anti-human PD-1 antibody, and BI 754111, an anti-human LAG-3 antibody, and assess their activity alone and in combination. Ezabenlimab bound with high affinity to human PD-1 (KD = 6 nM) and blocked the interaction of PD-1 with PD-L1 and PD-L2. Ezabenlimab dose-dependently increased interferon-γ secretion in human T cells expressing PD-1 in co-culture with PD-L1-expressing dendritic cells. Administration of ezabenlimab to human PD-1 knock-in mice dose-dependently inhibited growth of MC38 tumors. To reduce immunogenicity, ezabenlimab was reformatted from a human IgG4 to a chimeric variant with a mouse IgG1 backbone (BI 905725) for further in vivo studies. Combining BI 905725 with anti-mouse LAG-3 antibodies improved antitumor activity versus BI 905725 monotherapy in the MC38 tumor model. We generated BI 754111, which bound with high affinity to human LAG-3 and prevented LAG-3 interaction with its ligand, major histocompatibility complex class II. In an in vitro model of antigen-experienced memory T cells expressing PD-1 and LAG-3, interferon-γ secretion increased by an average 1.8-fold versus isotype control (p = 0.027) with BI 754111 monotherapy, 6.9-fold (p < 0.0001) with ezabenlimab monotherapy and 13.2-fold (p < 0.0001) with BI 754111 plus ezabenlimab. Overall, ezabenlimab and BI 754111 bound to their respective targets with high affinity and prevented ligand binding. Combining ezabenlimab with BI 754111 enhanced in vitro activity versus monotherapy, supporting clinical investigation of this combination (NCT03156114; NCT03433898).
Subject(s)
B7-H1 Antigen , Programmed Cell Death 1 Receptor , Animals , Antibodies, Blocking , Antibodies, Monoclonal/pharmacology , Clinical Studies as Topic , Immune Checkpoint Inhibitors , Interferon-gamma , Ligands , MiceABSTRACT
MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.
Subject(s)
CRISPR-Cas Systems , Neoplasms/pathology , Proteolysis , Proto-Oncogene Proteins c-myc/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Apoptosis , Humans , Neoplasms/genetics , Neoplasms/metabolism , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/geneticsABSTRACT
Flow cytometry enables the measurement of single cells in a flowing system. Heterogeneous mixtures of cells or particles can be analyzed with respect to their morphology, surface and intracellular protein expression, DNA content, and cellular physiology at high speed and purity. A series of key technical developments and improvements in flow cytometry hardware, software, and dye chemistry made it possible to measure more than 20 parameters simultaneously. Here, we provide a stepwise protocol for the preparation of single cell suspension samples from different murine lymphoid or tumor tissues and a detailed description of a 17-color polychromatic flow cytometry analysis of tumor-infiltrating leukocytes.
Subject(s)
Flow Cytometry/methods , Immunophenotyping/methods , Animals , Blood Cells/cytology , Blood Cells/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Cell Separation/methods , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Neoplasms/immunology , Single-Cell Analysis/methods , Spleen/cytology , Spleen/immunologyABSTRACT
Great strides in gene discovery have been made using a multitude of methods to associate phenotypes with genetic variants, but there still remains a substantial gap between observed symptoms and identified genetic defects. Herein, we use the convergence of various genetic and genomic techniques to investigate the underpinnings of a constellation of phenotypes that include prostate cancer (PCa) and sensorineural hearing loss (SNHL) in a human subject. Through interrogation of the subject's de novo, germline, balanced chromosomal translocation, we first identify a correlation between his disorders and a poorly annotated gene known as lipid droplet associated hydrolase (LDAH). Using data repositories of both germline and somatic variants, we identify convergent genomic evidence that substantiates a correlation between loss of LDAH and PCa. This correlation is validated through both in vitro and in vivo models that show loss of LDAH results in increased risk of PCa and, to a lesser extent, SNHL. By leveraging convergent evidence in emerging genomic data, we hypothesize that loss of LDAH is involved in PCa and other phenotypes observed in support of a genotype-phenotype association in an n-of-one human subject.
Subject(s)
Hearing Loss, Sensorineural/genetics , Prostatic Neoplasms/genetics , Serine Proteases/genetics , Translocation, Genetic/genetics , Adult , Aged , Animals , Genome-Wide Association Study , Germ Cells/pathology , Hearing Loss, Sensorineural/pathology , Humans , Male , Mice , Mice, Knockout , Phenotype , Prostatic Neoplasms/pathologyABSTRACT
Multiple immune-cell types can infiltrate tumors and promote progression and metastasis through different mechanisms, including immunosuppression. How distinct genetic alterations in tumors affect the composition of the immune landscape is currently unclear. Here, we characterized the immune-cell composition of prostate cancers driven by the loss of the critical tumor suppressor gene Pten, either alone or in combination with the loss of Trp53, Zbtb7a or Pml. We observed a striking quantitative and qualitative heterogeneity that was directly dependent on the specific genetic events in the tumor and ranged from 'cold', noninflamed tumors to massively infiltrated landscapes-results with important therapeutic implications. Further, we showed these qualitative differences in transcriptomic analysis of human prostate cancer samples. These data suggest that patient stratification on the basis of integrated genotypic-immunophenotypic analyses may be necessary for successful clinical trials and tailored precision immunological therapies.
Subject(s)
DNA-Binding Proteins/genetics , PTEN Phosphohydrolase/genetics , Prostatic Neoplasms/immunology , Transcription Factors/genetics , Tumor Suppressor Protein p53/genetics , Animals , B-Lymphocytes/immunology , DNA-Binding Proteins/immunology , Disease Models, Animal , Female , Gene Expression Regulation, Neoplastic , Genetic Heterogeneity , Genotype , Humans , Immunotherapy , Lymphocytes, Tumor-Infiltrating/immunology , Macrophages/immunology , Male , Mice , Mice, Knockout , Myeloid Cells/immunology , PTEN Phosphohydrolase/immunology , Promyelocytic Leukemia Protein/genetics , Promyelocytic Leukemia Protein/immunology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , T-Lymphocytes/immunology , Transcription Factors/immunology , Transcriptome/genetics , Transcriptome/immunology , Tumor Suppressor Protein p53/immunologyABSTRACT
Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo "CDS" or "3' UTR" develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , Proto-Oncogene Proteins B-raf/genetics , Pseudogenes , RNA/metabolism , Animals , Base Sequence , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice , Molecular Sequence Data , Proto-Oncogene Proteins B-raf/metabolismABSTRACT
A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.
Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Leukemic , Gene Expression Regulation, Neoplastic , Leukemia, Promyelocytic, Acute/metabolism , Peptidylprolyl Isomerase/genetics , Tretinoin/metabolism , Animals , Antineoplastic Agents/chemistry , Breast Neoplasms/genetics , Catalysis , Catalytic Domain , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Fibroblasts/metabolism , HEK293 Cells , Humans , Leukemia, Promyelocytic, Acute/genetics , MCF-7 Cells , Mice , Mice, Inbred BALB C , Mice, Knockout , NIMA-Interacting Peptidylprolyl Isomerase , Neoplasm Transplantation , Phosphates/chemistry , Phosphorylation , Proline/chemistry , Triple Negative Breast Neoplasms/metabolismABSTRACT
Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/enzymology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Pteridines/therapeutic use , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Female , HeLa Cells , Humans , Mice , Mice, Nude , Mice, SCID , Mice, Transgenic , Protein Kinase Inhibitors/pharmacology , Pteridines/pharmacology , Treatment Outcome , Xenograft Model Antitumor Assays/methods , Polo-Like Kinase 1ABSTRACT
Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2(R140Q) in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2(R140Q) can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.
Subject(s)
Carcinogenesis/pathology , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/pathology , Mutation/genetics , Oncogenes , Animals , Bone Marrow/pathology , Carcinogenesis/genetics , Cell Differentiation , Cell Proliferation , Cell Transformation, Neoplastic/pathology , Disease Models, Animal , Erythroid Cells/metabolism , Erythroid Cells/pathology , Hematopoiesis , Hematopoietic Stem Cells/pathology , Homeodomain Proteins/metabolism , Isocitrate Dehydrogenase/metabolism , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Ecotropic Viral Integration Site 1 Protein , Neoplasm Proteins/metabolism , Spleen/pathology , Transcription, Genetic , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Cytoplasmic nucleophosmin (NPMc(+)) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.
Subject(s)
Cytoplasm/metabolism , Leukemia, Myeloid, Acute/genetics , Mutation , Nuclear Proteins/genetics , fms-Like Tyrosine Kinase 3/genetics , Alleles , Animals , Gene Duplication , Loss of Heterozygosity , Mice , Mice, Transgenic , Nuclear Proteins/metabolism , Nucleophosmin , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Increasing evidence points to an important role for the ribosome in the regulation of biological processes and as a target for deregulation in disease. Here, we describe a SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry approach to probing mammalian riboproteomes. Using a panel of cell lines, as well as genetic and pharmacological perturbations, we obtained a comparative characterization of the cellular riboproteome. This analysis identified a set of riboproteome components, consisting of a diverse array of proteins with a strong enrichment for RNA-binding proteins. Importantly, this global analysis uncovers a high incidence of genetic alterations to riboproteome components in cancer, with a distinct bias toward genetic amplification. We further validated association with polyribosomes for several riboproteome components and demonstrate that enrichment at the riboproteome can depend on cell type, genetics, or cellular stimulus. Our results have important implications for the understanding of how ribosomes function and provide a platform for uncovering regulators of translation.
Subject(s)
Prostatic Neoplasms/metabolism , Proteome/metabolism , Proteomics/methods , Ribosomes/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Gene Amplification , Humans , Male , Mammals , Mass Spectrometry , Prostatic Neoplasms/genetics , Proteome/genetics , Ribosomes/genetics , TranscriptomeABSTRACT
In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology.
Subject(s)
Embryonic Stem Cells/physiology , Gene Expression , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , Epithelial Cells/metabolism , Female , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/geneticsABSTRACT
The mammalian target of rapamycin (mTOR) protein kinase regulates a wide variety of cellular processes, including protein synthesis, yet the downstream translational program under the control of mTOR is poorly understood. Two recent studies by Hsieh et al. and Thoreen et al. now start to address this issue, and uncover a subset of genes translationally regulated by oncogenic mTOR signaling that may contribute to tumorigenesis.
Subject(s)
TOR Serine-Threonine Kinases/metabolism , Animals , Down-Regulation/drug effects , Eukaryotic Initiation Factors/metabolism , Humans , Indoles/pharmacology , Mice , Naphthyridines/pharmacology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , RNA, Messenger/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
UNLABELLED: The mitogen-inducible gene-6 (mig-6) is a multi-adaptor protein implicated in the regulation of the HER family of receptor tyrosine kinases. We have reported recently that mig-6 is a negative regulator of epidermal growth factor receptor (EGFR)-dependent skin morphogenesis and tumor formation in vivo. In the liver, ablation of mig-6 leads to an increase in EGFR protein levels, suggesting that mig-6 is a negative regulator of EGFR function. In line with this observation, primary hepatocytes isolated from mig-6 knockout and wild-type control mice display sustained mitogenic signaling in response to EGF. In order to explore the role of mig-6 in the liver in vivo, we analyzed liver regeneration in mig-6 knockout and wild-type control mice. Interestingly, mig-6 knockout mice display enhanced hepatocyte proliferation in the initial phases after partial hepatectomy. This phenotype correlates with activation of endogenous EGFR signaling, predominantly through the protein kinase B pathway. In addition, mig-6 is an endogenous inhibitor of EGFR signaling and EGF-induced tumor cell migration in human liver cancer cell lines. Moreover, mig-6 is down-regulated in human hepatocellular carcinoma and this correlates with increased EGFR expression. CONCLUSION: Our data implicate mig-6 as a regulator of EGFR activity in hepatocytes and as a suppressor of EGFR signaling in human liver cancer.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Carcinoma, Hepatocellular/pathology , ErbB Receptors/physiology , Hepatocytes/physiology , Liver Neoplasms/pathology , Signal Transduction/physiology , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Extracellular Signal-Regulated MAP Kinases/physiology , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BL , Tumor Suppressor ProteinsABSTRACT
We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF.
Subject(s)
Actins/physiology , Adaptor Proteins, Signal Transducing/physiology , ErbB Receptors/metabolism , MAP Kinase Signaling System , Membrane Transport Proteins/physiology , Myelin Proteins/physiology , Proteolipids/physiology , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Cell Proliferation , Cycloheximide/pharmacology , Cytochalasin D/pharmacology , Gene Expression Profiling , Gene Expression Regulation , Humans , Intracellular Signaling Peptides and Proteins , Ligands , MAP Kinase Signaling System/drug effects , Mice , Myelin and Lymphocyte-Associated Proteolipid Proteins , Nucleic Acid Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Thiazolidines/pharmacology , Tumor Suppressor ProteinsABSTRACT
PURPOSE: The epidermal growth factor receptor family member HER3 is overexpressed in diverse human cancers and has been associated with poor prognosis in breast, lung, and ovarian cancer. However, the relevance of HER3 with regard to its prognostic significance and function in primary melanoma and metastases remains largely elusive. EXPERIMENTAL DESIGN: HER3 protein expression was analyzed immunohistochemically using tissue microarrays of 130 primary melanoma and 87 metastases relative to established clinical variables. The possibility of an influence of HER3 on melanoma cell proliferation, migration, invasion, and chemotherapy-induced apoptosis was studied in human melanoma cell lines. RESULTS: We show that HER3 is frequently expressed in malignant melanoma and metastases at elevated levels. High HER3 expression may serve as a prognostic marker because it correlates with cell proliferation, tumor progression, and reduced patient survival. Suppression of HER3 expression by RNA interference reduces melanoma cell proliferation, migration, and invasion in vitro. In addition, down-regulation of HER3 synergistically enhances dacarbazine-induced apoptosis. Moreover, monoclonal antibodies specific for the extracellular portion of HER3 efficiently block heregulin-induced proliferation, migration, and invasion of melanoma cell lines. CONCLUSION: Our results provide novel insights into the role of HER3 in melanoma and point out new possibilities for therapeutic intervention.
Subject(s)
Biomarkers, Tumor/analysis , Melanoma/metabolism , Melanoma/pathology , Receptor, ErbB-3/biosynthesis , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Apoptosis/physiology , Blotting, Western , Cell Line, Tumor , Cell Movement/physiology , Cell Proliferation , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Melanoma/mortality , Middle Aged , Prognosis , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Skin Neoplasms/mortality , Tissue Array AnalysisABSTRACT
PURPOSE: The receptor tyrosine kinase Axl has recently been identified as a critical element in the invasive properties of glioma cell lines. However, the effect of Axl and its ligand growth arrest--specific gene 6 (Gas6) in human gliomas is still unknown. EXPERIMENTAL DESIGN: Axl and Gas6 expression was studied in 42 fresh-frozen and 79 paraffin-embedded glioma specimens by means of reverse transcription-PCR and immunohistochemistry. The prognostic value of Axl and Gas6 expression was evaluated using a population-based tissue microarray derived from a cohort of 55 glioblastoma multiforme (GBM) patients. RESULTS: Axl and Gas6 were detectable in gliomas of malignancy grades WHO 2 to 4. Moderate to high Axl mRNA expression was found in 61%, Axl protein in 55%, Gas6 mRNA in 81%, and Gas6 protein in 74% of GBM samples, respectively. GBM patients with high Axl expression and Axl/Gas6 coexpression showed a significantly shorter time to tumor progression and an association with poorer overall survival. Comparative immunohistochemical studies showed that Axl staining was most pronounced in glioma cells of pseudopalisades and reactive astrocytes. Additionally, Axl/Gas6 coexpression was observed in glioma cells and tumor vessels. In contrast, Axl staining was not detectable in nonneoplastic brain tissue and Gas6 was strongly expressed in neurons. CONCLUSIONS: In human gliomas, Axl and Gas6 are frequently overexpressed in both glioma and vascular cells and predict poor prognosis in GBM patients. Our results indicate that specific targeting of the Axl/Gas6 signaling pathway may represent a potential new approach for glioma treatment.