Net1A is a RhoA specific guanine nucleotide exchange factor that promotes breast cancer cell motility when it relocates from the nucleus into the cytosol. We have recently found that its relocation is regulated by the Src tyrosine kinase, an oncoprotein, which stimulates phosphorylation of Net1A on Y373, leading to Net1A relocalization into the cytosol. We have also found that the nuclear exportin CRM1 is required for cytosolic relocalization of Net1A and that phosphorylation of Net1A on S52A by JNK also promotes Net1A accumulation in the cytosol. The purpose of this project was to determine whether inhibitors of Src, JNK, or CRM1 can synergistically block Net1A cytosolic relocalization and inhibit cancer cell motility. This was accomplished by transfecting MCF7 cells with Net1A and treating them with the inhibitors at various concentrations before exposure to the stimulant EGF. We used immunofluorescence (IF) to visualize and quantify the localization of Net1A, and cell migration assays to assess cell motility. Cells were pre-treated with the JNK inhibitor at 10 µM for 30 minutes and then stimulated with EGF for 15 minutes. Net1A cytosolic localization was significantly lower in cells treated with the JNK inhibitor. For the migration assay, there were no significant differences in cell motility with the JNK inhibitor. However, there was a trend towards reduced motility in cells treated with the JNK inhibitor. Further experiments included assessment of dose dependency for inhibition of Net1A cytosolic localization by JNK and CRM1 inhibitors, and combining these two inhibitors to look for a synergistic effect. Overall, this work demonstrated that both JNK and CRM1 inhibition block Net1A cytosolic localization and laid the groundwork for testing for synergy between these inhibitors.

Net1A inhibitors: Looking for synergy

255th National Meeting (2018)

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