Function of RasGRP3 in the formation and progression of human breast cancer.
BACKGROUND
Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer.


METHODS
The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed.


RESULTS
RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells.


CONCLUSIONS
Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.
Zsuzsanna
Z
Nagy
Ilona
I
Kovács
Miklós
M
Török
Dezső
D
Tóth
György
G
Vereb
Krisztina
K
Buzás
István
I
Juhász
Peter M
PM
Blumberg
Tamás
T
Bíró
DE-MTA "Lendület" Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Medical and Health Science Center, Research Center for Molecular Medicine, Nagyerdei krt, 98, PO Box 22, Debrecen H-4032, Hungary. czifrag@gmail.com.
Gabriella
G
Czifra
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D016513:Mice, SCID; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009361:Neoplasm Invasiveness; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013629:Tamoxifen / Q000494:pharmacology; D000068878:Trastuzumab; D023041:Xenograft Model Antitumor Assays; D020663:ras Guanine Nucleotide Exchange Factors
D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D020662:Guanine Nucleotide Exchange Factors; C413212:RASGRP3 protein, human; D034741:RNA, Small Interfering; D020663:ras Guanine Nucleotide Exchange Factors; D013629:Tamoxifen; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000068878:Trastuzumab