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Yoshihiko Murata
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P2.14 - Targeted Therapy (ID 183)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Targeted Therapy
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.14-22 - Loss of Ect2 Expression Impairs Cell-Matrix Adhesion and FAK/Src Signaling in Lung Adenocarcinoma Cells (ID 192)
10:15 - 18:15 | Author(s): Yoshihiko Murata
- Abstract
Background
Cell adhesion is a crucial step in cancer development and progression. We have previously demonstrated that epithelial cell transforming sequence 2 (Ect2), a guanine nucleotide exchange factor for the Rho family small GTPases Rac1, RhoA, and Cdc42, is overexpressed in early invasive adenocarcinoma. We subsequently showed that suppression of Ect2 significantly reduces cell growth, migration, and invasion of lung adenocarcinoma cells. In the present study, we investigated the potential role of Ect2 on cell-matrix adhesion and adhesion signaling complex in lung adenocarcinoma.
Method
Cell attachment assay was used to assess the viability of attached Calu-3 and PC-9 cells after suppression of Ect2 using small interfering RNA (siRNA). Adhesion of cells to extracellular matrix (ECM) was examined by adhesion assay, and changes in cell morphology were observed by immunofluorescence. RT-PCR and Western blotting analysis were conducted to examine the effects of Ect2 suppression on molecules involved in the adhesion cascade in Calu-3 and NCI-H2342 cells. To evaluate the effect of Ect2 on adhesion complex formation, immunoprecipitation was performed after treatment with siRNA-Ect2 in Calu-3 cells.
Result
We found that suppression of Ect2 significantly reduced the viability of attached cells. Furthermore, PC-9 and Calu-3 cells transfected with siRNA-Ect2 showed markedly decreased adhesion to collagen I and fibronectin. In terms of morphological changes, Calu-3 and PC-9 cells showed non-cohesive growth and a clear rounded shape after siRNA-Ect2 treatment. To investigate the underlying molecular mechanism, we examined the levels of expression of several proteins that are directly associated with cancer cell adhesion. We found that focal adhesion kinase (FAK), a key regulator of cell adhesion, was markedly down-regulated at both the mRNA and protein levels after treatment of the lung adenocarcinoma cells with siRNA-Ect2. Consistently, the levels of expression of molecules involved in the adhesion cascade, including integrin β1, paxillin, p-Src (Tyr416), p130Cas, and Crk, were further decreased in siRNA-Ect2. Since FAK/Src signaling can be activated through interaction the adhesion molecules on the cell surface, we speculate that these interactions might be affected by Ect2. Our data showed that Ect2 suppression impairs FAK interaction with Src, integrin β1, and paxillin in lung adenocarcinoma cells.
Conclusion
We have obtained novel data suggesting that Ect2 suppression leads to attenuation of cell adhesion to ECM, with a consequent impact on adhesion complex formation. These findings further demonstrate that Ect2 plays an essential role in the pathological steps of lung adenocarcinoma progression, and could be a potential molecular target for therapy.
