RESUMO
Complete resection is pivotal to improve survival to epithelial ovarian cancer (EOC). Crk SH3-domain-binding guanine nucleotide-releasing factor (C3G) is involved in multiple signaling pathways and it has opposite roles in different cancers. The present study aimed to identify C3G expression in ovarian tissue samples from patients with EOC and to explore its association with tumor grade. Eighty-seven archival paraffin-embedded, formalin-fixed, ovarian cancer tissues with serous histology were stained for C3G by immunohistochemistry. To evaluate the contribution of C3G to Rap1 activity, 36 patients with serous ovarian cancer (SOC) were investigated. Additionally, C3G was knocked down in SKOV3 and HEY cells. C3G regulated Rap1 activity and high Rap1 activity was correlated with poor differentiation, advanced FIGO stage, and unsuccessful cytoreductive surgery of SOC. Knockdown of C3G suppressed cell invasion, intravasation and extravasation, and reduced Rap1 activity and secretion of matrix metalloproteinase (MMP)-2 and MMP-9. C3G-mediated activation of Rap1 could direct the tumor pattern of human SOC by promoting the secretion of MMP-2 and MMP-9. These results suggest that C3G is involved in the metastatic spread of EOC.
Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/enzimologia , Neoplasias Ovarianas/enzimologia , Neoplasias Peritoneais/enzimologia , Proteínas de Ligação a Telômeros/metabolismo , Animais , Intervalo Livre de Doença , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Metaloproteinase 9 da Matriz , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/mortalidade , Neoplasias Peritoneais/secundário , Complexo ShelterinaRESUMO
OBJECTIVE: Engulfment and cell motility 1 (Elmo1) has been reported to cooperate with dedicator of cytokinesis 1 (Dock180) and to be linked to the invasive phenotype of cancer cells through activating small G-protein Rac. We aimed to study the role of Elmo1 in the malignant migration of ovarian cancer. METHODS: Engulfment and cell motility 1 expression was evaluated in specimens from 93 patients with serous ovarian cancer (SOC) by immunohistochemical staining. Next, Elmo1-RNAi cells were established by validated small interference RNAs. Cell proliferation and cell motility were observed and compared with Dock180-RNAi cells. To confirm their synergetic contribution to forming focal adhesion and activating Rac1, Rac1-GTP level was measured by GST pull-down assay and immunofluorescence was used to observe focal adhesion formation both in Elmo1-RNAi and Dock180-RNAi cells. RESULTS: Engulfment and cell motility 1 was mainly overexpressed in high-grade SOC tissues. Western blot analysis demonstrated that both Elmo1 and Dock180 expressions were hampered in Elmo1-RNAi cells. Compared with the negative control, decreased colony formation and cell invasion were observed in Elmo1-RNAi cells and Dock180-RNAi cells. Consistently, both exhibited reduced Rac1-GTP level and inhibited focal adhesion formation. CONCLUSIONS: Engulfment and cell motility 1 presents with synergetic action in helping Dock180 to activate Rac1 and promote cell motility, and thus promote untoward expansion and aggressiveness of SOC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Adesão Celular , Ciclo Celular , Proliferação de Células , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Citometria de Fluxo , Imunofluorescência , Guanosina Trifosfato/metabolismo , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Adaptor protein Crk and CrkL were thought to be closely related because both consist of one SH2 and two SH3 domains and share 60% homology with the highest identity within their functional domains. Their functions were most presumed to be in part, if not all, redundant. And both were suggested to be implicated in carcinogenesis. In this study, both Crk and CrkL presented with much higher expression in ovarian cancer tissues than those in normal and benign ovarian tissues. However, in contrast with CrkL, high Crk expression displayed close association with advanced stages and high-grade diseases. Furthermore, the differential binding selectivity of Crk and CrkL to their downstream partners Dock 180 and C3G was demonstrated in ovarian cancer cell line SKOV3 through coimmunoprecipitation. Additionally, Crk-knockdown cells presented with changed morphology, reduced growth, and cell invasion but remained viable. In contrast, all CrkL-knockdown cells could not survive over time, gradually detaching from the bottom of plastic dish. In conclusion, these two highly homologous proteins hold features that allow for the differential association with each binding molecules, thereby activating different signaling pathways and being involved in diverse roles in ovarian cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Bases , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-crk/genéticaRESUMO
Small GTPases, particularly the Rho family, are key regulators of cell motility and migration. Dock180 was well known for the main target of signal adaptor protein Crk and acted as a guanine-nucleotide exchange factor for small GTPase Rac1. In the present study, Dock180 was found to combine primarily with CrkI other than CrkII, and its association with Elmo1 was also demonstrated in ovarian cancer cell SKOV3. To evaluate the role of Dock180 in human ovarian cancer cell, we performed RNAi-mediated knockdown of Dock180 in SKOV3 cells using small interfering RNA expression vector. In Dock180 knockdown cells, we found that Elmo1 expression and Rac1 activity were decreased simultaneously. By contrast, the expressions of both another Crk-combining molecule C3G and Rap1 activity were observed to increase obviously. Accordingly, all Dock180 knockdown cells present with evident change in cell morphology, reduced cell proliferation, and attenuated cell migration. Taken together, these results suggest that signal transfer of Crk/Dock180/Rac1 is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion, and of human ovarian cancer cell line SKOV3.
