Abstract/Summary

Metastasis is a multistep process which involves the tumour cells invasion through extracellular matrix (ECM) barriers and finding new spaces for colonization and proliferation. Focal adhesions (FAs) are important structures in facilitating cell migration and invasion. FAs can be modified through post-translational modifications (PTMs) and one of them is SUMOylation. In this study, MDA-MB-231 breast carcinoma and U2OS osteosarcoma cells have been used to study the regulation of FAs in the cancer cells and a regulatory mechanism of SUMOylation has been proposed. Later, the platelets and a megakaryocytelike cell line (CMK11-5 cells) were used. Firstly, a SUMOylation inhibitor ginkgolic acid (GA) was used in MDA-MB-231 cells, which significantly increased the mean number, size and turnover time of FAs; GA at 25, 50 and 100µM significantly decreased the speed of cell migration in MDA-MB-231 cells after 24 hours. Ubc9 (SUMO-conjugating enzyme) siRNA was used in MDA-MB-231 cells to knock down Ubc9, which also significantly increased the mean number and size of FAs; the combination of GA and Ubc9 siRNA did not further increase the mean number and size of FAs. 25nM Ubc9 siRNA reduced the speed of cell migration after 25 and 48 hours. All SUMOylation inhibitors including ginkgolic acid, 2-D08 and gossypetin significantly increased the FAs mean number or size in U2OS cells and in MDA-MB-231 cells. Bioinformatics software, such as SUMOplot and GPS SUMO predicted that talin and vinculin were SUMOylated. Three different immunoprecipitation methods (IP) have been developed as endogenous IP, HA-tagged SUMO-2 IP and using a SUMO-VIVATM binding assay. The IP and western blotting showed that talin could be SUMOylated in both MDA-MB-231 and U2OS cells; vinculin SUMOylation was determined in MDA-MB-231 cells and in the coimmunoprecipitation experiments (co-IP). The effects of inhibiting SUMOylation on FA cleavage were further investigated in the IP and co-IP experiments in both cancer cells. For example, in the IP and co-IP experiments, talin appeared at the intact molecular weight 250kDa (230kDa using different antibodies) as well as 47kDa and other molecular weights (such as 220kDa and 100kDa) during inhibitor treatments, suggesting that talin could be cleaved and the inhibitor treatment either inhibited the intact talin protein or the cleaved products of talin. A Mass Spectrometry study was conducted to identify and confirm the previously SUMOylated proteins in all three cell lines: MDA-MB-231, CMK11-5 and platelets; talin SUMOylation was identified in all three cell lines. Furthermore, fila.min-1 was identified and confirmed in the experimental conditions.