Nischarin and ShcD interaction:  A novel mechanism to control cell migration and affecting patients’ overall survival


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Samrein Ahmed1
1University of Sharjah

Abstract

Background

The melanoma associated Shc adaptor, ShcD, was found to promote cell motility via both MAPK-dependent and-independent pathways. ShcD-mediated cell migration was found to be via recruiting Grb2, which is known promoter to MAPK pathway. Mouse embryonic cDNA library was screened by yeast two hybrid assays to identify ShcD interacting partners that facilitate ShcD migratory role by MAPK-independent pathway. A novel negative regulator to cell motility, Nischarin was found to interact with ShcD-CH2 domain.

In this study, it was aimed to investigate Nischarin and ShcD interaction in mammalian cells as well as to determine their functional impact on cell migration in MCF7 breast cancer cell line.

Method

Yeast two-hybrid screens were employed to investigate the exact Nischarin sequence required for ShcD interaction, co-immunoprecipitation assays and confocal microscopy were used to study the interaction between ShcD and Nischarin in mammalian cells. The impact of the ShcD and Nischarin association in MCF7 was tested by western blotting as well as by wound migration assays and transwell assays. In silico analysis was performed to study the impact of Nischarin/ShcD interaction on patients’ overall survival.

Results

Using a yeast two-hybrid assay, it was revealed that the Nischarin coiled-coil region was not sufficient to mediate Nischarin and ShcD binding. Nischarin/ShcD interaction was confirmed in both cell lines by employing co-immunoprecipitation technique as well as by fluorescence microscope in MCF7 cell line. The co-expression of Nischarin and ShcD demonstrated an inhibitory effect on phopho-ERK and phopsho-LIMK. Utilizing transwell and wound healing assays, we were able to elucidate that Nischarin prohibits ShcD migratory abilities. A brief in silico analysis from breast cancer patients’ data was performed to elucidate the effect of the Nischarin/ShcD co-expression on the overall survival. Patients with high expression of both proteins had a better survival than those with only ShcD overexpression.

Conclusion

Collectively, we revealed for the first time that the novel protein Nischarin exclusively interacts with ShcD and not with other Shc proteins. Interestingly, our data revealed that Nischarin tumour suppression abilities overcome ShcD-mediated cell migration when they are concomitantly expressed.