Project description:Cancer cells interact with endothelial cells during the process of metastatic spreading. Here, we use a small interfering RNA screen targeting Rho GTPases in cancer cells to identify Cdc42 as a critical regulator of cancer cell-endothelial cell interactions and transendothelial migration. We find that Cdc42 regulates ?1 integrin expression at the transcriptional level via the transcription factor serum response factor (SRF). ?1 integrin is the main target for Cdc42-mediating interaction of cancer cells with endothelial cells and the underlying extracellular matrix, as exogenous ?1 integrin expression was sufficient to rescue the Cdc42-silencing phenotype. We show that Cdc42 was required in vivo for cancer cell spreading and protrusion extension along blood vessels and retention in the lungs. Interestingly, transient Cdc42 depletion was sufficient to decrease experimental lung metastases, which suggests that its role in endothelial attachment is important for metastasis. By identifying ?1 integrin as a transcriptional target of Cdc42, our results provide new insight into Cdc42 function.
Project description:Patients with non-small cell lung cancer (NSCLC) treated with EGFR-tyrosine kinase inhibitors (TKIs) ultimately develop drug resistance and metastasis. Therefore, there is a need to identify the underlying mechanisms of resistance to EGFR-TKIs. In the present study, colony formation and MTT assays were performed to investigate cell viability following treatment with icotinib. Gene Expression Omnibus datasets were used to identify genes associated with resistance. Wound healing and Transwell assays were used to detect cell migration and invasion with icotinib treatment and integrin α5-knockdown. The expression levels of integrin α5 and downstream genes were detected using western blotting. Stable icotinib-resistant (IcoR) cell lines (827/IcoR and PC9/IcoR) were established that showed enhanced malignant properties compared with parental cells (HCC827 and PC9). Furthermore, the resistant cell lines were resistant to icotinib in terms of proliferation, migration and invasion. The enrichment of function and signaling pathways analysis showed that integrin α5-upregulation was associated with the development of icotinib resistance. The knockdown of integrin α5 attenuated the migration and invasion capability of the resistant cells. Moreover, a combination of icotinib and integrin α5 siRNA significantly inhibited migration and partly restored icotinib sensitivity in IcoR cells. The expression levels of phosphorylated (p)-focal adhesion kinase (FAK), p-STAT3 and p-AKT decreased after knockdown of integrin α5, suggesting that FAK/STAT3/AKT signaling had a notable effect on the resistant cells. The present study revealed that the integrin α5/FAK/STAT3/AKT signaling pathway promoted icotinib resistance and malignancy in IcoR NSCLC cells. This signaling pathway may provide promising targets against acquired resistance to EGFR-TKI in patients with NSCLC.
Project description:MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (?5, ?1, ?3, ?4 and ?5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin ?4. The co-localization of MUC5AC and integrin ?4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin ?4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin ?4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.
Project description:MicroRNA 130b (miR-130b) is significantly dysregulated in various human tumor types. In this study, using a microarray assay, we characterized the upregulation of miR-130b expression in colorectal cancer (CRC) specimens. However, there is limited knowledge about the roles of aberrant miR-130b expression in CRC. Our studies in CRC cells demonstrated that miR-130b significantly decreases cell migration and invasion, but it has no evidently effects on cell proliferation and apoptosis. In the overexpression miR-130b CRC cells and the CRC specimens, we observed a decreased level of integrin β1 protein, which is considered as a key molecule involved in cell motility. The targeting of the 3'-UTR region of integrin β1 gene by miR-130b was revealed using a luciferase reporter assay. The regulation of integrin β1 by miR-130b was further shown using the miR-130b mimics and the inhibitor of miR-130b. The impaired motility of the miR-130b overexpression cells is recovered partly by the expression of integrin β1 lacking the 3'-UTR. Additionally, the knockdown of integrin β1 also gives rise to a decrease in cell migration and invasion, which is similar to the impeded motility due to overexpression of miR-130b in CRC cells. Furthermore, the inverse expressions of miR-130b and integrin β1 were observed in CRC specimens. In summary, these data demonstrate that miR-130b downregulates its target-integrin β1, leading to the impaired migration and invasion of CRC cells.
Project description:The role and the mechanisms by which β1 integrins regulate the survival and chemoresistance of T cell acute lymphoblastic leukemia (T-ALL) still are poorly addressed. In this study, we demonstrate in T-ALL cell lines and primary blasts, that engagement of α2β1 integrin with its ligand collagen I (ColI), reduces doxorubicin-induced apoptosis, whereas fibronectin (Fn) had no effect. ColI but not Fn inhibited doxorubicin-induced mitochondrial depolarization, cytochrome c release, and activation of caspase-9 and -3. ColI but not Fn also prevented doxorubicin from down-regulating the levels of the prosurvival Bcl-2 protein family member Mcl-1. The effect of ColI on Mcl-1 occurred through the inhibition of doxorubicin-induced activation of c-Jun N-terminal kinase (JNK). Mcl-1 knockdown experiments showed that the maintenance of Mcl-1 levels is essential for ColI-mediated T-ALL cell survival. Furthermore, activation of MAPK/ERK, but not PI3K/AKT, is required for ColI-mediated inhibition of doxorubicin-induced JNK activation and apoptosis and for ColI-mediated maintenance of Mcl-1 levels. Thus, our study identifies α2β1 integrin as an important survival pathway in drug-induced apoptosis of T-ALL cells and suggests that its activation can contribute to the generation of drug resistance.
Project description:The lethality of pancreatic adenocarcinoma stems from an elevated incidence of tumor cell invasion and metastasis that are mediated by mechanisms not yet understood. Recent studies indicate that the proinvasive integrin alpha 6 beta 4 is highly upregulated in pancreatic adenocarcinomas. To assess the importance of this integrin in pancreatic cancer cell migration and invasion, cell lines were screened for integrin alpha 6 beta 4 expression by immunoblotting and fluorescence-activated cell sorting and their ability to migrate and invade toward hepatocyte growth factor (HGF). We found that cell surface expression of the alpha 6 beta 4 integrin correlated with the cells' ability to migrate and invade toward HGF. When cells expressing high levels of integrin alpha 6 beta 4 were treated with small interfering RNA targeting alpha 6 or beta 4 integrin subunits, we observed a reduction in cell migration and invasion. Furthermore, the activity of the small GTPase Rac1 was stimulated by alpha 6 beta 4 integrin expression and was necessary for HGF-stimulated chemotaxis. We discovered that expression of the Rac-specific nucleotide exchange factor, Tiam1 (T-lymphoma invasion and metastasis), was upregulated in cells overexpressing the integrin alpha 6 beta 4 and required for the elevated Rac1 activity in these cells. We conclude that the integrin alpha 6 beta 4 promotes the migratory and invasive phenotype of pancreatic carcinoma cells through the Tiam1-Rac1 pathway in part through the upregulation of Tiam1.
Project description:Endoplasmic reticulum disulphide oxidase 1? (ERO1?) is an oxidase localized in the endoplasmic reticulum that plays a role in the formation of disulphide bonds of secreted and cell-surface proteins. We previously showed that ERO1? is overexpressed in various types of cancer and we further identified ERO1? expression as a novel factor related to poor prognosis in cancer. However, the biological functions of ERO1? in cancer remain unclear. Here, we investigated the cell biological roles of ERO1? in the human colon-cancer cell line HCT116. ERO1? knockout (KO) by using CRISPR/Cas9 resulted in decreased tumourigenicity in vivo and reduced cell proliferation only under hypoxia in vitro, which suggested that ERO1? promotes cancer progression specifically in a low-oxygen environment. Thus, we evaluated the function of ERO1? in cell proliferation under hypoxia, and found that under hypoxic conditions, ERO1? KO resulted in a contact-inhibited morphology and diminished motility of cells. We further showed that ERO1? KO induced a change in integrin-?1 glycosylation and thus an attenuation of cell-surface integrin-?1 expression, which resulted in the aforementioned phenotype. Our study has established a previously unrecognized link between ERO1? expression and integrin activation, and thus provides new evidence for the effectiveness of ERO1?-targeted therapy for colorectal carcinoma.
Project description:SMAD family member 1 (Smad1) have been involved in metastatic progression of many cancer types. However, the detailed molecular signalling pathway underlying the regulatory link between Smad1 and metastasis remains elusive. Here, we demonstrate that Smad1 promotes migration of colorectal cancer (CRC) cells by inducing Snail and Ajuba expression simultaneously, but no apparent effect on Twist1 expression. Remarkably, E-cadherin, the best known Snail/Ajuba target gene is downregulated by Smad1 expression. Further, depletion of Ajuba in HCT116 cells significantly dampens the cell migration capability induced by Smad1 overexpression, suggesting that Ajuba is required for Smad1 to induce cell migration. Moreover, clinical analysis shows a significant positive correlation between the level of Smad1 and Ajuba in CRC samples. Together, our data provides the first evidence of the regulatory network of Smad1/Snail/Ajuba axis in CRC migration, suggesting that Smad1 and Ajuba are potential new therapeutic targets and prognostic factors for CRC.
Project description:Arecoline is the primary alkaloid in betel nuts, which are known as a risk factor for oral submucosal fibrosis and oral cancer. Lung cancer is a severe type of carcinoma with high cell motility that is difficult to treat. However, the detailed mechanisms of the correlation between Arecoline and lung cancer are not fully understood. Here, we investigated the effect of Arecoline on migration in lung cancer cell lines and its potential mechanism through the muscarinic acetylcholine receptor 3 (mAChR3)-triggered EGFR/Src/FAK pathway. Our results indicate that different concentrations of Arecoline treatment (10 µM, 20 µM, and 40 µM) significantly increased the cell migration ability in A549 and CL1-0 cells and promoted the formation of the filamentous actin (F-actin) cytoskeleton, which is a crucial element for cell migration. However, migration of H460, CL1-5, and H520 cell lines, which have a higher migration ability, was not affected by Arecoline treatment. The EGFR/c-Src/Fak pathway, which is responsible for cell migration, was activated by Arecoline treatment, and a decreased expression level of E-cadherin, which is an epithelial marker, was observed in Arecoline-treated cell lines. Blockade of the EGFR/c-Src/Fak pathway with the inhibitors of EGFR (Gefitinib) or c-Src (Dasatinib) significantly prevented Arecoline-promoted migration in A549 cells. Gefitinib or Dasatinib treatment significantly disrupted the Arecoline-induced localization of phospho-Y576-Fak during focal adhesion in A549 cells. Interestingly, Arecoline-promoted migration in A549 cells was blocked by a specific mAChR3 inhibitor (4-DAMP) or a neutralizing antibody of matrix metalloproteinase (MMP7 or Matrilysin). Taken together, our findings suggest that mAChR3 might play an essential role in Arecoline-promoted EGFR/c-Src/Fak activation and migration in an A549 lung cancer cell line.