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Besides its mitochondria-based anti-apoptotic role, Bcl-xL also travels to the nucleus to promote cancer metastasis by upregulating global histone H3 trimethyl Lys4 (H3K4me3) and TGFß transcription. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates H3K4me3 modification are not understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced cell migration and metastasis in mouse models. Furthermore, knockout of CtBP2 suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFß mRNA upregulation as well as cell invasion. Moreover, cleavage under targets and release using nuclease (CUT&RUN) coupled with next generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor region of genes encoding TGFß and its signaling pathway in the cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1.
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We developed cProSite, a website that provides online genomics, proteomics, and phosphoproteomics analysis for the data of The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC). This tool focuses on comparisons and correlations between different proteins and mRNAs of tumors and normal tissues. Our website is designed with biologists and clinicians in mind, with a user-friendly environment and fast search engine. The search results of cProSite can be used for clinical data validation and provide useful strategic information to identify drug targets at proteomic, phosphoproteomic, or genomic levels. The site is available at http://cprosite.ccr.cancer.gov.
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mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.
Assuntos
Antineoplásicos/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Compostos de Boro/farmacologia , Compostos de Boro/uso terapêutico , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Glicina/análogos & derivados , Glicina/farmacologia , Glicina/uso terapêutico , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. METHODS: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. RESULTS: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. CONCLUSION: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.
Assuntos
Caveolina 1/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipase C delta/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Proteínas de Transporte , Caveolina 1/química , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas Ativadoras de GTPase/genética , Humanos , Modelos Moleculares , Mutação , Fosfolipase C delta/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/genéticaRESUMO
The tyrosine phosphatase SHP2 is oncogenic in cancers driven by receptor-tyrosine-kinases, and SHP2 inhibition reduces tumor growth. Here, we report that SHP2 is an essential promoter of endothelial cell survival and growth in the remodeling tumor vasculature. Using genetic and chemical approaches to inhibit SHP2 activity in endothelial cells, we show that SHP2 inhibits pro-apoptotic STAT3 and stimulates proliferative ERK1/2 signaling. Systemic SHP2 inhibition in mice bearing tumor types selected for SHP2-independent tumor cell growth promotes degeneration of the tumor vasculature and blood extravasation; reduces tumor vascularity and blood perfusion; and increases tumor necrosis. Reduction of tumor growth ensues, independent of SHP2 targeting in the tumor cells, blocking immune checkpoints, or recruiting macrophages. We also show that inhibiting the Angiopoietin/TIE2/AKT cascade magnifies the vascular and anti-tumor effects of SHP2 inhibition by blocking tumor endothelial AKT signaling, not a target of SHP2. Since the SHP2 and Ang2/TIE2 pathways are active in vascular endothelial cells of human melanoma and colon carcinoma, SHP2 inhibitors alone or with Ang2/TIE2 inhibitors hold promise to effectively target the tumor endothelium.
Assuntos
Neoplasias , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Animais , Células Endoteliais/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores Proteína Tirosina Quinases , Transdução de SinaisRESUMO
Pancreatic cancer has the lowest survival rate out of all types of cancer. Pancreatic cancer patients are often diagnosed at advanced stages, hence an urgent need for a better therapeutic development of this devastating disease. Receptor for hyaluronan-mediated motility (RHAMM), not expressed in adult normal pancreas, has been suggested as a prognostic factor and a potential therapeutic target for pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET). In this study, we initially sought to determine whether genetic deletion of RHAMM would slow down pancreatic cancer progression using Rhamm-/- mice. However, we found that Rhamm-/- mice expressed a truncated HMMRΔexon8-16 protein at higher abundance levels than wild-type RHAMM. While HMMRΔexon8-16 did not enable malignant progression of pancreatic intraepithelial neoplasia in p48-Cre; LSL-KRASG12D mice, it accelerated the formation of invasive PDAC and shortened the survival of p48-Cre; LSL-KRASG12D mice with heterozygous p53 knockout. KrasG12D PDAC mice with homozygous p53 knockout mice died around 10 weeks, and the effect of HMMRΔexon8-16 was not apparent in these short lifespan mice. In addition, HMMRΔexon8-16 shortened the survival of PNET-bearing RIP-Tag mice, which had inactivated p53. In our analysis of TCGA dataset, pancreatic cancer patients with mutant TP53 or loss of one copy of TP53 had higher RHAMM expression, which, combined, predicted worse outcomes. Taken together, by collaborating with dysfunctional p53, high levels of HMMRΔexon8-16 , which lacks the centrosome targeting domain and degrons for interaction with the Anaphase-Promoting Complex (APC), accelerated pancreatic cancer progression.
Assuntos
Proteínas da Matriz Extracelular/genética , Receptores de Hialuronatos/genética , Neoplasias Pancreáticas/genética , Proteína Supressora de Tumor p53/genética , Animais , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologiaRESUMO
In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity. SIGNIFICANCE: These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.
Assuntos
Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Humanos , Mutação de Sentido Incorreto , Mutação PuntualRESUMO
Endometrial cancer is one of the most common gynecologic malignancies worldwide. Only 2 agents have been approved by Food and Drug Administration for endometrial cancer since 1971. There is a need to identify molecular targets to treat advanced endometrial cancer. The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various types of cancer. Here, we aimed to determine the clinical significance of RHAMM expression in endometrial cancer. Two hundred twenty-five cases of endometrial cancer, including serous and endometrioid types, and 8 cases of normal endometrium were used for studying RHAMM protein levels. The Cancer Genome Atlas database was also queried for RHAMM mRNA expression in endometrial cancer. Increased expression of RHAMM protein was seen in endometrial cancer compared with no or weak expression in normal endometrium. RHAMM expression positively correlated with tumor grade. RHAMM expression was significantly increased in endometrial serous carcinomas, which are high-grade, aggressive types of endometrial cancer, compared with the relatively less aggressive endometrioid carcinomas. RHAMM expression also correlated with the presence of lymphovascular invasion. RHAMM mRNA expression correlated with decreased survival in The Cancer Genome Atlas cohort. Therefore, increased RHAMM expression in endometrial cancer is associated with high-grade tumors and is indicative of more aggressive behavior. These findings suggest RHAMM as a prognostic factor in endometrial cancer and as a potential therapeutic target in advanced endometrial cancer for future studies.
Assuntos
Carcinoma Endometrioide/metabolismo , Neoplasias do Endométrio/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Receptores de Hialuronatos/metabolismo , Biomarcadores Tumorais/metabolismo , Biópsia , Carcinoma Endometrioide/mortalidade , Carcinoma Endometrioide/patologia , Carcinoma Endometrioide/secundário , Estudos de Coortes , Bases de Dados Factuais , Progressão da Doença , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/mortalidade , Neoplasias do Endométrio/patologia , Feminino , Humanos , Receptores de Hialuronatos/genética , Metástase Linfática , Gradação de Tumores , Prognóstico , Regulação para CimaRESUMO
Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.
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Autofagia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Terapia de Alvo Molecular , Receptores da Família Eph/metabolismo , Transdução de Sinais , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Efrina-B2/metabolismo , Feminino , Inativação Gênica/efeitos dos fármacos , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de SobrevidaRESUMO
Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene deleted in many cancers, including angiosarcoma, an aggressive malignancy of endothelial cell derivation. DLC1-deficiency in primary endothelial cells causes the loss of cell contact inhibition of growth through incompletely defined mechanisms. We report that DLC1 is a regulator of YAP, a transcriptional coactivator of proliferation-promoting and tumor-promoting genes; when confluent, active/nuclear YAP was significantly more abundant in DLC1-deficient endothelial cells compared with control cells. We also found that YAP is a required effector of the loss of cell contact inhibition of growth manifested by DLC1-deficient endothelial cells, as the silencing of YAP prevents this loss. Consistently, human angiosarcomas specimens contained a significantly greater proportion of DLC1- tumor cells with nuclear YAP compared with the DLC1+ normal cells in the adjacent tissue. Verteporfin, an inhibitor of YAP, significantly reduced angiosarcoma growth in mice. These results identify YAP as a previously unrecognized effector of DLC1 deficiency-associated loss of cell contact growth inhibition in endothelial cells and a potential therapeutic target in angiosarcoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/patologia , Inibição de Contato , Células Endoteliais/patologia , Proteínas Ativadoras de GTPase/metabolismo , Hemangiossarcoma/patologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Endoteliais/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Hemangiossarcoma/genética , Hemangiossarcoma/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
SRC and ERK kinases control many cell biological processes that promote tumorigenesis by altering the activity of oncogenic and tumor suppressor proteins. We identify here a physiological interaction between DLC1, a focal adhesion protein and tumor suppressor, with SRC and ERK. The tumor suppressor function of DLC1 is attenuated by phosphorylation of tyrosines Y451 and Y701 by SRC, which down-regulates DLC1's tensin-binding and Rho-GAP activities. ERK1/2 phosphorylate DLC1 on serine S129, which increases both the binding of SRC to DLC1 and SRC-dependent phosphorylation of DLC1. SRC inhibitors exhibit potent antitumor activity in a DLC1-positive transgenic cancer model and a DLC1-positive tumor xenograft model, due to reactivation of the tumor suppressor activities of DLC1. Combined treatment of DLC1-positive tumors with SRC plus AKT inhibitors has even greater antitumor activity. Together, these findings indicate cooperation between the SRC, ERK1/2, and AKT kinases to reduce DLC1 Rho-GAP and tumor suppressor activities in cancer cells, which can be reactivated by the kinase inhibitors.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/genética , Quinases da Família src/genéticaRESUMO
The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers. We previously screened genes upregulated in human hepatocellular carcinomas for their metastatic function in a mouse model of pancreatic neuroendocrine tumor (PNET) and identified that human RHAMMB promoted liver metastasis. It was unknown whether RHAMMB is upregulated in pancreatic cancer or contributes to its progression. In this study, we found that RHAMM protein was frequently upregulated in human PNETs. We investigated alternative splicing isoforms, RHAMMA and RHAMMB, by RNA-Seq analysis of primary PNETs and liver metastases. RHAMMB, but not RHAMMA, was significantly upregulated in liver metastases. RHAMMB was crucial for in vivo metastatic capacity of mouse and human PNETs. RHAMMA, carrying an extra 15-amino acid-stretch, did not promote metastasis in spontaneous and experimental metastasis mouse models. Moreover, RHAMMB was substantially higher than RHAMMA in pancreatic ductal adenocarcinoma (PDAC). RHAMMB, but not RHAMMA, correlated with both higher EGFR expression and poorer survival of PDAC patients. Knockdown of EGFR abolished RHAMMB-driven PNET metastasis. Altogether, our findings suggest a clinically relevant function of RHAMMB, but not RHAMMA, in promoting PNET metastasis in part through EGFR signaling. RHAMMB can thus serve as a prognostic factor for pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/patologia , Proteínas da Matriz Extracelular/genética , Receptores de Hialuronatos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Regulação para Cima , Processamento Alternativo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Receptores ErbB/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Prognóstico , Transdução de Sinais , Análise de SobrevidaRESUMO
OBJECTIVES: In accordance with the Precision Medicine Initiative, new treatment strategies for head and neck squamous cell carcinoma (HNSCC) are needed to yield better therapeutic outcomes. The purpose of this study was to establish and validate chimeric antigen receptor (CAR)-T cells targets in HNSCC. METHODS: Putative CAR-T antigens were identified in The Cancer Genome Atlas database. To validate antigen suitability, quantitative RT-PCR, flow cytometry, and immunofluorescent staining were performed. A retroviral human CD70 CAR construct, using truncated CD27 conjugated with 4-1BB and CD3-zeta costimulatory molecules, was used to transduce activated human T cells to generate CD70 CAR-T cells. Cell-based cytotoxicity and cytokine ELISAs were used to measure efficacy of killing. RESULTS: Nine potential CAR-T targets (CD276, EGFR, MICA, MICB, MAGE-A4, FAP, EPCAM, CD70, B4GALNT1) were identified based on their high expression in tumors compared to flanking control tissues. CD70 was selected for further proof-of-principle analysis based on its differential expression in several tumor subtypes, and showed substantial heterogeneity in individual tumors analyzed. Cell surface CD70 protein and CD70 mRNA were detected from low to high levels in established HNSCC cancer cell lines. CD70 was highly expressed in 4 of 21 tumor biopsies (19%), and 3 of 4 specimens showed strong CD70 expression on the tumor cell surface. CD70-specific CAR-T cells were generated and further demonstrated to recognize and kill CD70-positive HNSCC cells efficiently, but not CD70-negative cancer cells. CONCLUSION: CD70-specific CAR-T cells specifically recognized and efficiently eliminated CD70-positive HNSCC cells. This study provides the basis for further investigation into CD70 and other CAR-T targets.
Assuntos
Ligante CD27/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Linfócitos T/imunologia , HumanosRESUMO
Background: Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. Methods: We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. Results: We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). Conclusions: These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.
Assuntos
Biomarcadores Tumorais/metabolismo , Clusterina/metabolismo , Inibição de Contato , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica , Hemangiossarcoma/patologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Biomarcadores Tumorais/genética , Ciclo Celular , Movimento Celular , Proliferação de Células , Clusterina/genética , Progressão da Doença , Feminino , Proteínas Ativadoras de GTPase/genética , Hemangiossarcoma/genética , Hemangiossarcoma/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Prognóstico , Transdução de Sinais , Células Tumorais Cultivadas , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
We report several receptor tyrosine kinase (RTK) ligands increase RhoA-guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.
Assuntos
Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sítios de Ligação , Linhagem Celular , Movimento Celular , Células Epiteliais/ultraestrutura , Fibroblastos/ultraestrutura , Adesões Focais/ultraestrutura , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Hidrólise , Cristalino , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
OBJECTIVE: Malignant transformation of oral premalignant lesions is the key process in the progression to oral squamous cell carcinoma (OSCC). Previously, we identified miR-7 and miR-21 as candidate oncogenes and miR-375 and miR-494 as candidate tumor suppressors in OSCC. We aim to evaluate these microRNAs as biomarkers of malignant transformation in oral premalignant lesions. STUDY DESIGN: Formalin-fixed, paraffin-embedded samples from progressive premalignant lesions and paired sequential OSCC tumors at the same site were obtained from same patients (n = 31). Total RNA was extracted and analyzed for microRNA levels using real-time polymerase chain reaction. RESULTS: MiR-375 expression in progressive lesions was clearly lower than in nonprogressive control lesions (average eightfold difference, P = .0004). Furthermore, the expression of miR-375 decreased significantly after the progression from premalignant lesion to OSCC (P < .0001). Receiver operating characteristic curve analysis revealed that miR-375 was able to differentiate between progressive and nonprogressive premalignant lesions (P < .0001). CONCLUSIONS: MiR-375 downregulation in oral premalignant lesions is associated with a higher risk of malignant transformation.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers, but its role in primary and metastatic non-small cell lung carcinoma (NSCLC) remains to be determined. Here, we investigate the clinical relevance of RHAMM expression in NSCLC. RHAMM protein expression correlates with histological differentiation stages and extent of the primary tumor (T stages) in 156 patients with primary NSCLC. Importantly, while focal RHAMM staining pattern is present in 57% of primary NSCLC, intense RHAMM protein expression is present in 96% of metastatic NSCLC cases. In a publicly available database, The Cancer Genome Atlas (TCGA), RHAMM mRNA expression is 12- and 10-fold higher in lung adenocarcinoma and squamous lung carcinoma than in matched normal lung tissues, respectively. RHAMM mRNA expression correlates with stages of differentiation and inferior survival in more than 400 cases of lung adenocarcinoma in the Director's Challenge cohort. Of 4 RHAMM splice variants, RHAMMv3 (also known as RHAMMB) is the dominant variant in NSCLC. Moreover, shRNA-mediated knockdown of RHAMM reduced the migratory ability of two lung adenocarcinoma cell lines, H1975 and H3255. Taken together, RHAMM, most likely RHAMMv3 (RHAMMB), can serve as a prognostic factor for lung adenocarcinomas and a potential therapeutic target in NSCLC to inhibit tumor migration.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/metabolismo , Idoso , Diferenciação Celular , Movimento Celular , Estudos de Coortes , Biologia Computacional , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno/metabolismo , Análise Serial de Tecidos , Resultado do TratamentoRESUMO
The RHO family of RAS-related GTPases in tumors may be activated by reduced levels of RHO GTPase accelerating proteins (GAPs). One common mechanism is decreased expression of one or more members of the Deleted in Liver Cancer (DLC) family of Rho-GAPs, which comprises three closely related genes (DLC1, DLC2, and DLC3) that are down-regulated in a wide range of malignancies. Here we have studied their comparative biological activity in cultured cells and used publicly available datasets to examine their mRNA expression patterns in normal and cancer tissues, and to explore their relationship to cancer phenotypes and survival outcomes. In The Cancer Genome Atlas (TCGA) database, DLC1 expression predominated in normal lung, breast, and liver, but not in colorectum. Conversely, reduced DLC1 expression predominated in lung squamous cell carcinoma (LSC), lung adenocarcinoma (LAD), breast cancer, and hepatocellular carcinoma (HCC), but not in colorectal cancer. Reduced DLC1 expression was frequently associated with promoter methylation in LSC and LAD, while DLC1 copy number loss was frequent in HCC. DLC1 expression was higher in TCGA LAD patients who remained cancer-free, while low DLC1 had a poorer prognosis than low DLC2 or low DLC3 in a more completely annotated database. The poorest prognosis was associated with low expression of both DLC1 and DLC2 (P < 0.0001). In cultured cells, the three genes induced a similar reduction of Rho-GTP and cell migration. We conclude that DLC1 is the predominant family member expressed in several normal tissues, and its expression is preferentially reduced in common cancers at these sites.
Assuntos
Proteínas Ativadoras de GTPase/genética , Neoplasias/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular , Movimento Celular , Metilação de DNA , Regulação para Baixo , Proteínas Ativadoras de GTPase/análise , Proteínas Ativadoras de GTPase/fisiologia , Genes p53 , Humanos , Mutação , Neoplasias/patologia , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/fisiologiaRESUMO
DLC1 is a tumor suppressor protein whose full activity depends on its presence at focal adhesions, its Rho-GTPase activating protein (Rho-GAP) function, and its ability to bind several ligands, including tensin and talin. However, the mechanisms that regulate and coordinate these activities remain poorly understood. Here we identify CDK5, a predominantly cytoplasmic serine/threonine kinase, as an important regulator of DLC1 functions. The CDK5 kinase phosphorylates four serines in DLC1 located N-terminal to the Rho-GAP domain. When not phosphorylated, this N-terminal region functions as an autoinhibitory domain that places DLC1 in a closed, inactive conformation by efficiently binding to the Rho-GAP domain. CDK5 phosphorylation reduces this binding and orchestrates the coordinate activation DLC1, including its localization to focal adhesions, its Rho-GAP activity, and its ability to bind tensin and talin. In cancer, these anti-oncogenic effects of CDK5 can provide selective pressure for the down-regulation of DLC1, which occurs frequently in tumors, and can contribute to the pro-oncogenic activity of CDK5 in lung adenocarcinoma.
Assuntos
Quinase 5 Dependente de Ciclina/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Myeloid cells that orchestrate malignant progression in the tumor microenvironment offer targets for a generalized strategy to attack solid tumors. Through an analysis of tumor microenvironments, we explored an experimental model of lung cancer that uncovered a network of Dll4/Notch/TGF-ß1 signals that links myeloid cells to cancer progression. Myeloid cells attracted to the tumor microenvironment by the tumor-derived cytokines CCL2 and M-CSF expressed increased levels of the Notch ligand Dll4, thereby activating Notch signaling in the tumor cells and amplifying tumor-intrinsic Notch activation. Heightened Dll4/Notch signaling in tumor cells magnified TGF-ß-induced pSMAD2/3 signaling and was required to sustain TGF-ß-induced tumor cell growth. Conversely, Notch blockade reduced TGF-ß signaling and limited lung carcinoma tumor progression. Corroborating these findings, by interrogating RNAseq results from tumor and adjacent normal tissue in clinical specimens of human head and neck squamous carcinoma, we found evidence that TGF-ß/Notch crosstalk contributed to progression. In summary, the myeloid cell-carcinoma signaling network we describe uncovers novel mechanistic links between the tumor microenvironment and tumor growth, highlighting new opportunities to target tumors where this network is active.