Abstract
Successful therapies for patients with breast cancer often lose their initial effectiveness. Thus, identifying new molecular targets is a constant goal in the fight against breast cancer. Gpn3 is a protein required for RNA polymerase II nuclear targeting in both yeast and human cells. We investigated here the effect of suppressing Gpn3 expression on cell proliferation in a progression series of isogenic cell lines derived from the nontumorigenic MCF-10A breast cells that recapitulate different stages of breast carcinogenesis. Gpn3 protein levels were comparable in all malignant derivatives of the nontumorigenic MCF-10A cells. shRNA-mediated inhibition of Gpn3 expression markedly decreased cell proliferation in all MCF-10A sublines. A fraction of the largest RNA polymerase II subunit Rpb1 was retained in the cytoplasm, but most Rpb1 remained nuclear after suppressing Gpn3 in all cell lines studied. Long-term proliferation experiments in cells with suppressed Gpn3 expression resulted in the eventual loss of all isogenic cell lines but MCF-10CA1d.cl1. In MCF-10CA1d.cl1 cells, Gpn3 knockdown reduced the proliferation of breast cancer stem cells as evaluated by mammosphere assays. After the identification that Gpn3 plays a key role in cell proliferation in mammary epithelial cells independent of the degree of transformation, we also analyzed the importance of Gpn3 in other human breast cancer cell lines from different subtypes. Gpn3 was also required for cell proliferation and nuclear translocation of RNA polymerase II in such cellular models. Altogether, our results show that Gpn3 is essential for breast cancer cell proliferation regardless of the transformation level, indicating that Gpn3 could be considered a molecular target for the development of new antiproliferative therapies. Importantly, our analysis of public data revealed that Gpn3 overexpression was associated with a significant decrease in overall survival in patients with estrogen receptor-positive and Human epidermal growth factor receptor 2 (HER2+) breast cancer, supporting our proposal that targeting Gpn3 could potentially benefit patients with breast cancer.