Function of RasGRP3 in the formation and progression of human breast cancer

Zsuzsanna Nagy, I. Kovács, Miklós Török, Dezso Tóth, G. Vereb, Krisztina Buzás, I. Juhász, Peter M. Blumberg, T. Bíró, G. Czifra

Research output: Contribution to journalArticle

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Abstract

Introduction: 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.

Original languageEnglish
Article number96
JournalMolecular Cancer
Volume13
Issue number1
DOIs
Publication statusPublished - Apr 29 2014

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Breast Neoplasms
Insulin-Like Growth Factor I
ras Proteins
Neoplasms
ras Guanine Nucleotide Exchange Factors
Proteins
Western Blotting
Cell Proliferation
Cell Line
Severe Combined Immunodeficiency
Polymerase Chain Reaction
Estrogen Receptor alpha
Annexin A5
MCF-7 Cells
Gene Silencing
Tamoxifen
Epidermal Growth Factor
Heterografts
Cell Survival
Adenocarcinoma

Keywords

  • Chemotherapeutic resistance
  • EGF
  • Human breast cancer
  • IGF-I
  • Ras activator
  • RasGRP3
  • Signaling pathways
  • Tamoxifen
  • Trastuzumab
  • Tumorigenesis

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Medicine
  • Oncology
  • Medicine(all)

Cite this

Function of RasGRP3 in the formation and progression of human breast cancer. / Nagy, Zsuzsanna; Kovács, I.; Török, Miklós; Tóth, Dezso; Vereb, G.; Buzás, Krisztina; Juhász, I.; Blumberg, Peter M.; Bíró, T.; Czifra, G.

In: Molecular Cancer, Vol. 13, No. 1, 96, 29.04.2014.

Research output: Contribution to journalArticle

Nagy, Zsuzsanna ; Kovács, I. ; Török, Miklós ; Tóth, Dezso ; Vereb, G. ; Buzás, Krisztina ; Juhász, I. ; Blumberg, Peter M. ; Bíró, T. ; Czifra, G. / Function of RasGRP3 in the formation and progression of human breast cancer. In: Molecular Cancer. 2014 ; Vol. 13, No. 1.
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AU - Vereb, G.

AU - Buzás, Krisztina

AU - Juhász, I.

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N2 - Introduction: 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.

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