Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To define the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based timelapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.
Serous ovarian cancer (SOC) accounts for >50% of all epithelial ovarian cancers. However, patients with SOC present with various degrees of response to platinum-based chemotherapy and, thus, their survival may differ. The present study aimed to identify the candidate genes involved in the carcinogenesis and drug resistance of SOC by analyzing the microarray datasets GDS1381 and GDS3592. GDS1381 and GDS3592 were downloaded from the Gene Expression Omnibus database (). A total of 219 differentially expressed genes (DEGs) were identified. Potential genes that may predict the response to carboplatin and, thus, the prognosis of SOC were analyzed. The enriched functions and pathways of DEGs included extracellular region, extracellular space and extracellular exosome, among others. Upon screening the upregulated and downregulated genes on the connectivity map, 10 small-molecule drugs were identified that may be helpful in improving drug sensitivity in patients with ovarian cancer. A total of 30 hub genes were screened for further analysis after constructing the protein-to-protein interaction network. Through survival analysis, comparison of genes across numerous analyses, and immunohistochemistry, GNAI1, non-structural maintenance of chromosomes (non-SMC) condensin I complex subunit H (NCAPH), matrix metallopeptidase 9 (MMP9), aurora kinase A (AURKA) and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) were identified as the key molecules that may be involved in the carcinogenesis and carboplatin resistance of SOC. In conclusion, GNAI1, NCAPH, MMP9, AURKA and EZH2 should be examined in further studies for the possibility of their participation in the carcinogenesis and carboplatin response of SOC.
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.
Type I epithelial ovarian cancer (EOC) is primarily resistant to platinum-based chemotherapies and needs novel therapeutics. Given the aberrant Wnt activation in type I EOC and the involvement of Dapper1 Antagonist of Catenin-1 (DACT1) in Wnt signalling, the role of DACT1 in tumourigenesis of type I EOC was evaluated. Firstly, all tested EOC cell lines and primary EOC tissues, especially type I EOC, were observed to have significantly lower DACT1 expression than normal controls. Next, 3AO cells, which arise from a patient with primary mucinous EOC and express low endogenous levels of DACT1, were transfected with a lentivirus carrying full-length DACT1 (3AO-DACT1), grew slower and formed smaller tumours in nude mice compared to 3AO-NC. Furthermore, 3AO-DACT1 had lower levels of key mediators of canonical Wnt signalling, Dvl2 and β-catenin, GSK-3β with phosphorylated Ser9, and the Wnt/β-catenin target genes, with significantly lower nuclear β-catenin levels. Additionally, 3AO-DACT which contained higher levels of lipidated LC3 (LC3-II) and Beclin1, but lower levels of p62/SQSTM1, were more sensitive to cis-platinum. And chloroquine partially rescued its cis-platinum resistance. We identified DACT1 as a negative regulator in type I EOC, protecting against malignant expansion by inhibiting canonical Wnt signalling and cis-platinum resistance by regulating autophagy.
Background:Integrin b1 subunit and its downstream molecule, focal adhesion kinase (FAK), have been demonstrated to be indispensible to the promotion of cell proliferation and survival and anti-apoptosis in cardiomyocytes via activation of their downstream pro-survival signaling molecule, AKT. As a component of the integrin pathway, Dock180 (dedicator of cytokinesis 1) protein is also thought to be involved in the promotion of cell proliferation and survival and anti-apoptosis in the H9C2 cardiomyocytes. Material/Methods:Rat-derived H9C2 cardiomyocytes were transfected with pCXN2-flag-hDock180, a human Dock180 overexpression eukaryotic recombinant plasmid. The rat and human Dock180 mRNA and protein expression, apoptosis and cell proliferation and survival were analyzed in the H9C2 cardiomyocytes treated with either hypoxia/reoxygenation (H/R) or not, respectively. Results:Human Dock180 mRNA overexpression could significantly increase the Dock180 protein expression in the H9C2 cardiomyocytes, no matter whether treated with H/R or not. Dock180 overexpression could promote the cell proliferation and survival and anti-apoptosis, and relieve the cell proliferative and survival inhibition and apoptosis induced by H/R in the H9C2 cardiomyocytes via activation of its downstream pro-survival signaling molecule AKT. Conclusions: Dock180 could act as a pro-survival molecule in H9C2 cardiomyocytes via activation of its downstream prosurvival signaling molecule, AKT.key words: Dock180 apoptosis cell proliferation cardiomyocyte H9C2
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