RESUMO
Programmed cell death 1 ligand (PD-L1) blockade has been used therapeutically in the treatment of ovarian cancer, and potential combination treatment approaches are under investigation to improve the treatment response rate. The increased dependence on glutamine is widely observed in various type of tumors, including ovarian cancer. Kidney-type glutaminase (GLS), as one of the isotypes of glutaminase, is found to promote tumorigenesis. Here, we have demonstrated that the combined treatment with GLS inhibitor 968 and PD-L1 blockade enhances the immune response against ovarian cancer. Survival analysis using the Kaplan-Meier plotter dataset from ovarian cancer patients revealed that the expression level of GLS predicts poor survival and correlates with the immunosuppressive microenvironment of ovarian cancer. 968 inhibits the proliferation of ovarian cancer cells and enhances granzyme B secretion by CD8+ T cells as detected by XTT assay and flow cytometry, respectively. Furthermore, 968 enhances the apoptosis-inducing ability of CD8+ T cells toward cancer cells and improves the treatment effect of anti-PD-L1 in treating ovarian cancer as assessed by Annexin V apoptosis assay. In vivo studies demonstrated the prolonged overall survival upon combined treatment of 968 with anti-PD-L1 accompanied by increased granzyme B secretion by CD4+ and CD8+ T cells isolated from ovarian tumor xenografts. Additionally, 968 increases the infiltration of CD3+ T cells into tumors, possibly through enhancing the secretion of CXCL10 and CXCL11 by tumor cells. In conclusion, our findings provide a novel insight into ovarian cancer cells influence the immune system in the tumor microenvironment and highlight the potential clinical implication of combination of immune checkpoints with GLS inhibitor 968 in treating ovarian cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Benzofenantridinas/administração & dosagem , Glutaminase/genética , Inibidores de Checkpoint Imunológico/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzofenantridinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human papillomavirus (HPV) is the etiological agent of cervical cancer; however, the mechanisms underlying HPV-mediated carcinogenesis remain poorly understood. Here, we showed that nuclear receptor related-1 protein (Nurr1) was upregulated in primary cervical cancer tissue-derived spheroid cells and HPV-positive cell lines, and Nurr1 upregulation was correlated with cancer grade. Nurr1 promoted cell proliferation, migration, invasion, and anchorage-independent cell growth. In addition to its effect on cancer aggressiveness, Nurr1 enhanced the self-renewal ability of cells in vitro and in vivo, underscoring the importance of Nurr1 in maintaining the stemness of cancer stem-like cells (CSLCs). Mechanistically, Nurr1 independently activated the MEK/ERK and PI3K/Akt/mTOR signaling cascades. The MEK inhibitor trametinib (GSK) and PI3K/mTOR dual inhibitor dactolisib (BEZ) were shown to abrogate Nurr1-augmented tumorigenesis by upregulating p21 and p27 expression and by suppressing MMP9 and KLF4 expression. We provided further evidence that BEZ, but not GSK, could abolish Nurr1-enhanced radioresistance, suggesting its potential value for radiosensitizing CSLCs in the clinical setting. This study highlights the unprecedented roles of Nurr1 and elucidates mechanisms by which Nurr1 promotes tumor progression and radioresistance, providing a novel therapeutic strategy for cervical cancer treatment.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/patologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Tolerância a Radiação , Neoplasias do Colo do Útero/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/fisiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/radioterapia , Neoplasias do Colo do Útero/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nuclear receptor related-1 protein (Nurr1), coded by an early response gene, is involved in multiple cellular and physiological functions, including proliferation, survival, and self-renewal. Dysregulation of Nurr1 has been frequently observed in many cancers and is attributed to multiple transcriptional and post-transcriptional mechanisms. Besides, Nurr1 exhibits extensive crosstalk with many oncogenic and tumor suppressor molecules, which contribute to its potential pro-malignant behaviors. Furthermore, Nurr1 is a key player in attenuating antitumor immune responses. It not only potentiates immunosuppressive functions of regulatory T cells but also dampens the activity of cytotoxic T cells. The selective accessibility of chromatin by Nurr1 in T cells is closely associated with cell exhaustion and poor efficacy of cancer immunotherapy. In this review, we summarize the reported findings of Nurr1 in different malignancies, the mechanisms that regulate Nurr1 expression, and the downstream signaling pathways that Nurr1 employs to promote a wide range of malignant phenotypes. We also give an overview of the association between Nurr1 and antitumor immunity and discuss the inhibition of Nurr1 as a potential immunotherapeutic strategy.
RESUMO
BACKGROUND: CD109 was involved in the tumorigenesis and progression of various cancers via TGF-ß1 signalling and STAT3 activation. As CD109 is strongly expressed in cervical squamous cell carcinoma, this study was conducted to investigate its functional characteristics in cervical cancer. METHODS: CD109 expression was examined by immunohistochemistry (IHC) with cervical tissue microarray. The effects of CD109 expression were examined on migration, cell proliferation, spheroid formation and soft-agar colony-formation assay. Meanwhile, cervical cancer cell lines with high CD109 expression were chosen for the functional study using siRNA knockdown and CRISPR/Cas9 knockout. RESULTS: IHC demonstrated an upregulation of CD109 in the cell membrane of cervical squamous cell carcinoma. CD109( + ) cells isolated by flow-cytometric sorting displayed enhanced migration, cell proliferation, sphere-forming and anchorage-independent cell growth ability. In contrast, silencing of CD109 expression could reverse the in vitro and in vivo tumorigenic and aggressive properties. Furthermore, CD109 induced EGFR-mediated STAT3 phosphorylation known to be responsible for cell migration, proliferation and maintenance of CSC phenotype. CONCLUSION: Abundant CD109( + ) populations in cervical cancer cells potentially contributed to carcinogenesis and aggressiveness, whereas silencing of CD109 expression could reverse those properties. CD109 mediates cervical tumorigenicity and aggressiveness via CD109/EGFR/STAT3 signalling.
Assuntos
Antígenos CD/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias do Colo do Útero/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Sequência de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosforilação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
BACKGROUND: Ovarian cancer is characterised by frequent recurrence due to persistent presence of residual cancer stem cells (CSCs). Here, we identify and characterise tumour subsets from ascites-derived tumour cells with stemness, metastasis and metabolic switch properties and to delineate the involvement of pyruvate dehydrogenase kinase 4 (PDK4) in such process. METHODS: Ovarian cancer cells/cell lines derived from ascites were used for tumourspheres/ALDH+CD44+ subset isolation. The functional roles and downstream signalling of PDK4 were explored. Its association with clinical outcome of ovarian cancer was analysed. RESULTS: We demonstrated enhanced CSC characteristics of tumour cells derived from ovarian cancer ascites, concomitant with ALDH and CD44 subset enrichment and high PDK4 expression, compared to primary tumours. We further showed tumourspheres/ALDH+CD44+ subsets from ascites-derived tumour cells/cell lines with CSC properties and enhanced glycolysis. Clinically, PDK4 expression was correlated with aggressive features. Notably, blockade of PDK4 in tumourspheres/ALDH+CD44+ subsets led to inhibition of CSC characteristics, glycolysis and activation of STAT3/AKT/NF-κB/IL-8 (signal transducer and activator of transcription 3/protein kinases B/nuclear factor-κB/interleukin-8) signalling. Conversely, overexpression of PDK4 in ALDH-CD44- subsets exerted the opposite effects. CONCLUSION: Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastatic and metabolic switch properties via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling, suggesting PDK4 as a viable therapeutic molecular target for ovarian cancer management.
Assuntos
Interleucina-8/genética , Recidiva Local de Neoplasia/genética , Neoplasias Ovarianas/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Fator de Transcrição STAT3/genética , Aldeído Desidrogenase/genética , Ascite/metabolismo , Ascite/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores de Hialuronatos/genética , NF-kappa B/genética , Metástase Neoplásica , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Oncogênica v-akt/genética , Neoplasias Ovarianas/patologia , Receptores de Interleucina-8A/genética , Transdução de Sinais/genéticaRESUMO
A subpopulation of cells within tumors has been suggested to possess the ability to initiate tumorigenesis and contribute to resistance to cancer therapy. Identification and isolation of this subpopulation in cancer cells can be achieved by detecting specific cell-surface markers. In this study, flow cytometry analysis revealed an abundant CD71+ subpopulation in human papillomavirus (HPV)-positive cervical cancer cells, while limited CD71+ cells were detected in HPV-negative cervical cancer cells. Furthermore, ectopic expression of the HPV-E6 protein in HPV-negative C33A cells enriched the CD71+subpopulation. The CD71+ subpopulation isolated from the C33A cell line and an HPV-E6-overexpressing clone exhibited enhanced transforming ability, proliferation, and resistance to irradiation. In contrast, suppression of CD71 in HPV-positive SiHa cells and the HPV-E6-overexpressing stable clone inhibited spheroid formation and in vitro and in vivo tumorigenicity and sensitized cells to irradiation treatment. CRISPR/Cas9 knockout of CD71 in SiHa cells also produced similar inhibitory effects on tumorigenicity. Double knockout of CD71 and CD55 reversed the oncogenic properties of the HPV-E6-overexpressing clone. These findings suggest that the HPV-E6 protein enriches the subpopulation of CD71+cells in cervical cancer, which exhibit cancer stem-like cell properties and are resistant to irradiation treatment. Targeting the CD71+ subpopulation in cervical cancer cells with siRNAs or CRISPR/Cas9 may provide new insights for the development of novel therapeutic approaches for treating cervical cancer. IMPLICATIONS: We describe the enrichment of CD71+ population by HPV-E6 protein in cervical cancer cells that promotes cancer aggressiveness and resistance to irradiation treatment.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/patologia , Tolerância a Radiação , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Neoplasias do Colo do Útero/virologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/radioterapia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/radioterapiaRESUMO
Intraperitoneal metastasis is a common occurrence and is usually involved in the poor prognosis of ovarian cancer. Its specific metastatic pattern implies that certain indispensable microenvironmental factors secreted in the peritoneal cavity can direct metastatic ovarian cancer cells to permissive niches for secondary lesion formation. However, the underlying molecular mechanisms are ill defined. Herein, we report that GRO-α and IL-8 are predominately upregulated in culture media derived from either normal or cancerous omenta and are associated with increased ovarian cancer aggressiveness. Methods: OCM was established from culture medium of fresh human omental tissues. Primary and metastatic ovarian cancer cell lines were generated from human tumor tissues and verified by specific antibodies. The functional roles of GRO-α, IL-8, and their specific receptor CXCR2 were examined by neutralizing antibodies, shRNA gene knockdown, CRISPR/Cas9 gene knockout and pharmaceutical CXCR2 inhibitor SB225002. The oncogenic properties of ovarian cancer cells were examined by in vitro and in vivo mouse models. Results: Both GRO-α and IL-8 can activate TAK1/NFκB signaling via the CXCR2 receptor. Intriguingly, TAK1/NFκB signaling activity was higher in metastatic ovarian cancer cells; this higher activity makes them more susceptible to OCM-induced tumor aggressiveness. Treatment of ovarian cancer cells with GRO-α and IL-8 neutralizing antibodies or ablation of CXCR2 by shRNA gene knockdown, CRISPR/Cas9 gene knockout, or CXCR2 inhibitor SB225002 treatment significantly attenuated TAK1/NFκB signaling and decreased in vitro and in vivo oncogenic and metastatic potential, suggesting CXCR2 plays a key role in the GRO-α and IL-8-governed metastatic spreading of ovarian cancer cells in the intraperitoneal cavity. Conclusion: This study highlights the significance of GRO-α and IL-8 as the key chemokines in the peritoneal tumor microenvironment and suggests the utility of targeting their receptor CXCR2 as a potential target-based therapy for peritoneal metastases of ovarian cancer.
Assuntos
Quimiocina CXCL1/metabolismo , Interleucina-8/metabolismo , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos SCID , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Regulação para CimaRESUMO
Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.
Assuntos
Movimento Celular/genética , Proteína Adaptadora GRB7/genética , Inativação Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Interferência de RNA/fisiologia , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Accumulating evidence indicates that the human papillomavirus (HPV) E6 protein plays a crucial role in the development of cervical cancer. Subpopulations of cells that reside within tumours are responsible for tumour resistance to cancer therapy and recurrence. However, the identity of such cells residing in cervical cancer and their relationship with the HPV-E6 protein have not been identified. Here, we isolated sphere-forming cells, which showed self-renewal ability, from primary cervical tumours. Gene expression profiling revealed that cluster of differentiation (CD) 55 was upregulated in primary cervical cancer sphere cells. Flow-cytometric analysis detected abundant CD55(+) populations among a panel of HPV-positive cervical cancer cell lines, whereas few CD55(+) cells were found in HPV-negative cervical cancer and normal cervical epithelial cell lines. The CD55(+) subpopulation isolated from the C33A cell line showed significant sphere-forming ability and enhanced tumourigenicity, cell migration, and radioresistance. In contrast, the suppression of CD55 in HPV-positive CaSki cells inhibited tumourigenicity both in vitro and in vivo, and sensitized cells to radiation treatment. In addition, ectopic expression of the HPV-E6 protein in HPV-negative cervical cancer cells dramatically enriched the CD55(+) subpopulation. CRISPR/Cas9 knockout of CD55 in an HPV-E6-overexpressing stable clone abolished the tumourigenic effects of the HPV-E6 protein. Taken together, our data suggest that HPV-E6 protein expression enriches the CD55(+) population, which contributes to tumourigenicity and radioresistance in cervical cancer cells. Targeting CD55 via CRISPR/Cas9 may represent a novel avenue for developing new strategies and effective therapies for the treatment of cervical cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Antígenos CD55/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/virologia , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/virologia , Animais , Antígenos CD55/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Autorrenovação Celular , Feminino , Edição de Genes/métodos , Terapia Genética/métodos , Interações Hospedeiro-Patógeno , Humanos , Camundongos Nus , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Tolerância a Radiação , Proteínas Repressoras/genética , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer cells express both estrogen receptor α (ERα) and ERß, and hormonal therapy is an attractive treatment option because of its relatively few side effects. However, estrogen was previously shown to have opposite effects in tumors expressing ERα compared with ERß, indicating that the two receptor subtypes may have opposing effects. This may explain the modest response to nonselective estrogen inhibition in clinical practice. In this study, we aimed to investigate the effect of selectively targeting each ER subtype on ovarian cancer growth. Ovarian cancer cell lines SKOV3 and OV2008, expressing both ER subtypes, were treated with highly selective ER modulators. Sodium 3'-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay revealed that treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) (ERα antagonist) or 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) (ERß agonist) significantly suppressed cell growth in both cell lines. In contrast, 4,4',4â³-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) (ERα agonist) or 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]-pyrimidin-3-yl]phenol (PHTPP) (ERß antagonist) significantly enhanced cell growth. These results were confirmed on a xenograft model where SKOV3 cells were injected s.c. into ovariectomized mice. We observed that the average size of xenografts in both the DPN-treated group and the MPP-treated group was significantly smaller than that for the vehicle-treated group. In addition, we found that phospho-AKT expressions in SKOV3 cells were reduced by 80% after treatment with MPP and DPN, indicating that the AKT pathway was involved. The combined treatment with MPP and DPN had a synergistic effect in suppressing ovarian cancer cell growth. Our findings indicate that targeting ER subtypes may enhance the response to hormonal treatment in women with ovarian cancer.
Assuntos
Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Terapia de Alvo Molecular , Neoplasias Ovarianas/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Animais , Linhagem Celular Tumoral , Estradiol/farmacologia , Estradiol/uso terapêutico , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Moduladores Seletivos de Receptor Estrogênico/farmacologiaRESUMO
BACKGROUND: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase-activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation. AIMS: In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain. METHODS AND RESULTS: On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ-stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ-stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector-stable clone, suggesting that downregulation of RhoA activity in DLC2γ-stable clones inhibited cell cycle progression. In the DLC2γ-stable clone, the levels of Raf-1 and extracellular signal-regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ-GAP defective mutant-stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2-stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho-p70S6K and phospho-ERK1/2 were upregulated. CONCLUSION: Our data show that DLC2 inhibits the activity of Raf-1-ERK1/2-p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.
Assuntos
Carcinoma Hepatocelular/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular/genética , Proliferação de Células , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Paxilina/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais , Transfecção/métodosRESUMO
Identification of proteins that are involved in the sensitivity of radiotherapy of cancers is important to enhance the response to cancer treatment. Expression of TAp73 is associated with the sensitivity of radiotherapy in cervical cancer patients, suggesting it plays an important role in controlling radiosensitivity. Here, by using yeast two-hybrid system, we identify breast cancer-associated gene 3 (BCA3) as the first and novel protein interacting partner of TAp73. By coimmunoprecipitation and Western blot analysis, we confirm that TAp73 binds with and stabilizes BCA3 in cervical cancer cell line HeLa. Immunofluorescence staining indicates that BCA3 is localized in the cytoplasm and nucleus. Interestingly, when coexpressed with TAp73, BCA3 interacts and colocalizes with TAp73 at the mitochondria. Mutagenesis reveals that the oligomerization domain of TAp73 is responsible for the interaction with BCA3. Furthermore, BCA3 augments the transactivation activity of TAp73 on bax promoter and protein expression. In addition, the expression of BCA3 also increases the sensitivity of TAp73-transfected cells in response to gamma-irradiation-induced apoptosis. Western blot analysis also shows that TAp73 and BCA3 induce activation of caspase-7 and caspase-9. In summary, these findings suggested that BCA3 is a novel protein partner of TAp73, and they cooperate with each other to exert tumor-suppressive functions and sensitize the response of cervical cancer cells to radiotherapy.
Assuntos
Apoptose/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/radioterapia , Proteínas Adaptadoras de Transdução de Sinal , Caspase 7/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Feminino , Raios gama , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Tolerância a Radiação , Ativação Transcricional , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.
Assuntos
Neoplasias Hepáticas Experimentais/fisiopatologia , Proteínas Supressoras de Tumor/fisiologia , Tecido Adiposo/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Dietilnitrosamina/toxicidade , Feminino , Imunofluorescência , Neoplasias Hepáticas Experimentais/induzido quimicamente , Camundongos , Camundongos Knockout , Gravidez , Proteínas Supressoras de Tumor/genéticaRESUMO
Tensins are a new family of proteins that act as an important link among extracellular matrix, actin cytoskeleton, and signal transduction and have been implicated in human cancers. Tensin2 was initially identified in a search for new tensin family members that share extensive sequence homology with tensin1. Tensin2 was highly expressed in liver tissues. A recent study reported that one of the splicing variants of tensin2, variant 3, promotes cell migration. In the present study, we aimed to elucidate the role of variant 3 in hepatocarcinogenesis by assessing the expression of variant 3 mRNA in hepatocellular carcinoma (HCC) tissue and ectopically expressing variant 3 in HCC cell lines. Analysis of variant 3 expression in human HCC tissue revealed it was overexpressed in 46% (23/50) of tumor tissues as compared with the corresponding nontumorous livers. High expression of variant 3 was significantly associated with venous invasion (P = .037), tumor microsatellite formation (P = .022), and tumor nonencapsulation (P = .049). Our ectopic expression study showed that variant 3 significantly promoted the cell growth and motility of HCC cells. The clonal transfectants of variant 3 were more closely packed and resulted in a higher saturation density than in the control vector transfectants. Variant 3 expression also enhanced the proliferation rate in culture and in vivo tumorigenicity in nude mice. In conclusion, we reveal a novel role for variant 3 in the progression of HCC and suggest the feasibility of elevated variant 3 expression as a tumor progression marker for HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Movimento Celular/fisiologia , Neoplasias Hepáticas/patologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Biomarcadores Tumorais/genética , Linhagem Celular , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/genética , Invasividade Neoplásica , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/metabolismo , Tensinas , TransfecçãoRESUMO
Deleted in liver cancer (DLC1) is a candidate tumor suppressor gene recently isolated from human hepatocellular carcinoma. Structurally, DLC1 protein contains a conserved GTPase-activating protein for Rho family protein (RhoGAP) domain, which has been thought to regulate the activity of Rho family proteins. Previous studies indicated that DLC1 was frequently inactivated in cancer cells. In the present study, we aimed to characterize the tumor suppressor roles of DLC1 in hepatocellular carcinoma. We showed that DLC1 significantly inhibited cell proliferation, anchorage-independent growth, and in vivo tumorigenicity when stably expressed in hepatocellular carcinoma cells. Moreover, DLC1 expression greatly reduced the motility and invasiveness of hepatocellular carcinoma cells. With RhoGAP-deficient DLC1 mutant (DLC1-K714E), we showed that the RhoGAP activity was essential for DLC1-mediated tumor suppressor function. Furthermore, the 292- to 648-amino acid region and the steroidogenic acute regulatory related lipid transfer domain played an auxiliary role to RhoGAP and tumor suppressor function of DLC1. Taken together, our findings showed that DLC1 functions as a tumor suppressor in hepatocellular carcinoma and provide the first evidence to support the hypothesis that DLC1 suppresses cancer cell growth by negatively regulating the activity of Rho proteins.
Assuntos
Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Genes Supressores de Tumor , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Proteínas Supressoras de Tumor/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Adesão Celular/fisiologia , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Ativação Enzimática , Feminino , Proteínas Ativadoras de GTPase , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutagênese Sítio-Dirigida , Invasividade Neoplásica , Proteínas Supressoras de Tumor/biossínteseRESUMO
The deleted in liver cancer 2 (DLC2) gene, located at chromosome 13q12.3, is a recently identified tumor suppressor gene. The gene is frequently underexpressed in human hepatocellular carcinoma, and its chromosomal region shows frequent deletion. DLC2 encodes a unique RhoGTPase-activating protein (RhoGAP) specific for small RhoGTPases, RhoA, and Cdc42. With bioinformatic analysis, we have identified four different isoforms of DLC2, which we named DLC2alpha, DLC2beta, DLC2gamma, and DLC2delta. Three of the isoforms contain the RhoGAP domain, namely, DLC2alpha, DLC2beta, and DLC2gamma. Ectopic expression of these three isoforms in mouse fibroblasts showed cytoplasmic localization. Of interest, overexpression of these isoforms suppressed the lysophosphatidic acid-induced stress fiber formation in mouse fibroblasts and changed the morphology of the transfected cells from angular and spindle to round. Furthermore, the RhoA pull-down assay demonstrated a remarkable reduction in RhoA activity in the DLC2 transiently transfected cells. In contrast, cells transfected with inactive DLC2 GAP-mutant remained unchanged in cell morphology, actin stress fiber formation, and RhoA activity. HepG2 hepatoma cells stably transfected with the DLC2gamma isoform also changed to a round morphology, as in mouse fibroblasts. Of significance, these DLC2gamma stable transfectants showed marked suppression in cell proliferation, motility, and transformation, and there was a remarkable reduction in in vivo RhoA activity in these cells. These results suggest that DLC2 exhibits its tumor suppressor functions in vivo as a GAP specific for RhoA, exerting its effects in suppression of cytoskeleton reorganization, cell growth, cell migration, and transformation.
Assuntos
Transformação Celular Neoplásica , Isoformas de Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células 3T3 , Actinas/metabolismo , Animais , Movimento Celular , Proliferação de Células , Forma Celular , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Proteínas Supressoras de Tumor/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Hepatocellular carcinoma (HCC) is a major malignancy in many parts of the world, especially in Asia and Africa. Loss of heterozygosity (LOH) on the long arm of chromosome 13 has been reported in HCC. In search of tumor suppressor genes in this region, here we have identified DLC2 (for deleted in liver cancer 2) at 13q12.3 encoding a novel Rho family GTPase-activating protein (GAP). DLC2 mRNA is ubiquitously expressed in normal tissues but was significantly underexpressed in 18% (8/45) of human HCCs. DLC2 is homologous to DLC1, a previously identified tumor suppressor gene at 8p22-p21.3 frequently deleted in HCC. DLC2 encodes a novel protein with a RhoGAP domain, a SAM (sterile alpha motif) domain related to p73/p63, and a lipid-binding StAR-related lipid transfer (START) domain. Biochemical analysis indicates that DLC2 protein has GAP activity specific for small GTPases RhoA and Cdc42. Expression of the GAP domain of DLC2 sufficiently inhibits the Rho-mediated formation of actin stress fibers. Introduction of human DLC2 into mouse fibroblasts suppresses Ras signaling and Ras-induced cellular transformation in a GAP-dependent manner. Taken together, our findings suggest a role for DLC2 in growth suppression and hepatocarcinogenesis.