Mutation in the BRCA1 gene is associated with increased risk for hereditary breast and ovarian cancers. In sporadic ovarian tumors, BRCA1 dysfunction is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein. Our group has previously reported that BRCA1 proteins, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins, bind the sole SUMO E2-conjugating enzyme Ubc9. In this study, we examined the result of altered Ubc9 binding and knockdown on the sub-cellular localization and growth inhibitory function of BRCA1 proteins in ovarian cancer cells. Using live imaging of YFP, RFP-tagged BRCA1 and BRCA1a proteins, our results show enhanced cytoplasmic localization of K109R and C61G mutant BRCA1 proteins in ES-2, NIHOVCAR3 and UWB 1.289 ovarian cancer cells. Down-regulation of Ubc9 in ovarian cancer cells using Ubc9 siRNA resulted in cytoplasmic localization of BRCA1 and BRCA1a proteins. These mutant BRCA1a proteins were impaired in their capacity to inhibit growth of ES-2 ovarian cancer cells. Several ovarian cancer cells, including a BRCA1-null ovarian cancer cell line, showed higher levels of expression of Ubc9. This is the first study demonstrating the physiological link between loss of Ubc9 binding and loss of growth suppression of disease-associated mutant BRCA1a proteins in ovarian cancer cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of ovarian cancers.

Background

Overexpression of STYK1, a putative serine/threonine and tyrosine receptor protein kinase has been shown to confer tumorigenicity and metastatic potential to normal cells injected into nude mice. Mutation of a tyrosine residue in the catalytic STYK1 domain attenuates the tumorigenic potential of tumor cells in vivo, collectively, suggesting an oncogenic role for STYK1.

Methods

To investigate the role of STYK1 expression in ovarian cancer, a panel of normal, benign, and ovarian cancer tissues was evaluated for STYK1 immunoreactivity using STYK1 antibodies. In addition, mRNA levels were measured by reverse transcription PCR and real-time PCR of estrogen receptors, GPR30 and STYK1 following treatment of ovarian cell lines with estrogen or G1, a GPR30 agonist, as well as western analysis.

Results

Our data showed higher expression of STYK1 in cancer tissues versus normal or benign. Only normal or benign, and one cancer tissue were STYK1-negative. Moreover, benign and ovarian cancer cell lines expressed STYK1 as determined by RT-PCR. Estradiol treatment of these cells resulted in up- and down-regulation of STYK1 despite estrogen receptor status; whereas G-1, a GPR30-specific agonist, increased STYK1 mRNA levels higher than that of estradiol.

Conclusion

We conclude that STYK1 is expressed in ovarian cancer and is regulated by estrogen through a GPR30 hormone-signaling pathway, to the exclusion of estrogen receptor-alpha.