Investigation of RasGRP3 expression and function in human breast cancers and breast-derived ductal adenocarcinoma cell lines

RasGRP3 is a member of the Ras guanine nucleotide releasing protein (RasGRP) family of the Ras-specific guanine nucleotide exchange factors. These proteins play important role in the regulation of the activity of Ras signaling pathway which constitutive activation is demonstrated in many cancer types. The RasGRP3 proteins have potential oncogenic effect, the amplification of the gen of the protein is observed in many malignant cancer types, for example in Burkitt's lymphoma and pre-B-cell leukemia. Our recent investigations have highlighted that RasGRP3 plays a role in tumorigenesis, hence it exerts effect on the proliferation, migration, survival and tumorigenecity of prostate adenocarcinoma-derived and melanoma cells. In light of this potential oncogenic effect we have examined the change of expression and potencial function of RasGRP3 in one of the most malignant cancer type, the breast-derived ductal adenocarcinoma. The RasGRP3 and phosphoRasGRP3 expressions were examined in human ductal adenocarcinoma-derived samples from different grades and in 1 primary ductal adenocarcinoma-derived cell line called BT-474 and 5 different metastatic: JIMT-1, MCF7, SK-BR-3, MDA-MB-453 és T-47D cell lines both in mRNA (Q-PCR) and protein levels (Western blot; immunhisto- and cytochemistry). To explore the biological function of the protein RasGRP3 knockdown cultures were created on MCF7 and T-47D cell lines using retroviral transfection. To examine the role of RasGRP3 in the viability of cells annexin-V/PI staining analyzed by flow citometry was performed. To clarify the protein's function in cell proliferation and in the developement of resistance to the most common clinical chemotherapeutic drugs Tamoxifen and Herceptin CyQuant assay were performed. In addition to observe the RasGRP3 function in tumor formation and maintance, the SCID mouse model was used. According to our results the expression of RasGRP3 and the active phosphoRasGRP3 were elevated both at the mRNA and protein levels on the human ductal adenocarcinoma samples. This expression was increased in the tumor samples compared to the normal. RasGRP3 was found typically in the cytoplasm of the cells, while the phosphoRasGRP3 showed strong nuclear reaction. The RasGRP3 expression of the BT-474 cell line was significantly lower then in the metastatics. The downregulation of RasGRP3 did not induce significant apoptosis or necrosis, but inhibited cell proliferation and sensitizied the T-47D cells to killing by Tamoxifen and Herceptin. In vivo tumor growth in mouse xenografts of both cell lines was decreased. Our results suggest the RasGRP3 may have an importante role in the regulation of cell growth, chemotherapeutic resistance, and tumor formation in breast cancer.