Am J Cancer Res 2013;3(2):173-195
Original Article
Dissecting GRB7-mediated signals for proliferation and migration in HER2
overexpressing breast tumor cells: GTP-ase rules
De Pradip, Mark Bouzyk, Nandini Dey, Brian Leyland-Jones
Edith Sanford Breast Cancer Research, Sanford Research/USD, Sioux Falls, SD; AKESOgen, Inc., Atlanta, GA, USA
Received March 14, 2013; Accepted March 29, 2013; Epub April 3, 2013; Published April 13, 2013
Abstract: Amplification of human Her2 and its aberrant signaling in 20-30% of early breast cancer patients is responsible for
highly aggressive tumors with poor outcome. Grb7 is reported to be co-amplified with Her2. We report a concurrent high
expression of mRNA (from FFPE tumor samples; mRNA correlation, Pearson r2= 0.806), and high levels of GRB7 protein
(immunoblot) in HER2+ breast cancer cell lines. We demonstrated the signaling mechanism of HER2 and downstream
effectors that contributes to proliferation and migration. Using HER2+ and trastuzumab-resistant breast cancer cell lines, we
identified the interaction between GRB7 and HER2 in the control of HER2+ cell proliferation. Our co-IP data show that GRB7
recruits SHC into the HER2-GRB7 signaling complex. This complex formation leads to activation of RAS-GTP. We also observed
that following integrin engagement, GRB7 is phosphorylated at tyrosine in a p-FAK (Y397) dependent manner. This FAK-GRB7
complex leads to downstream activation of RAC1-GTP (responsible for migration) probably through the recruitment of VAV2. Our
CO-IP data demonstrate that GRB7 directly binds with VAV2 following fibronectin engagement in HER2+ cells. To address
whether GRB7 could serve as a pathway specific therapeutic target, we used siRNA to suppress GRB7 expression. Knockdown
of GRB7 expression in the HER2+ breast cancer cell lines decreases RAS activation, cell proliferation, 2D and 3D colony
formation and also blocked integrin-mediated RAC1 activation along with integrin-directed cell migration. These findings
dissected the HER2-mediated signaling cascade into (1) HER2+ cell proliferation (HER2-GRB7-SHC-RAS) and (2) HER2+ cell
migration (alpha5 beta1/alpha4 beta1-FAK-GRB7-VAV2-RAC1). Our data clearly demonstrate that a coupling of GRB7 with HER2
is required for the proliferative and migratory signals in HER2+ breast tumor cells. (AJCR0000188)
Keywords: Her2/Neu, GRB7, adapter protein, RAS, RAC, proliferation and migration
Address correspondence to: Dr. Brian Leyland-Jones, Edith Sanford Breast Cancer Research, Sanford Research/USD, 2301 E
60th Street N, Sioux Falls, SD, 57104, USA. Phone: 605-312-6007; Fax: 605-312-6071; E-mail:
Brian.leyland-jones@sanfordhealth.org
Original Article
Dissecting GRB7-mediated signals for proliferation and migration in HER2
overexpressing breast tumor cells: GTP-ase rules
De Pradip, Mark Bouzyk, Nandini Dey, Brian Leyland-Jones
Edith Sanford Breast Cancer Research, Sanford Research/USD, Sioux Falls, SD; AKESOgen, Inc., Atlanta, GA, USA
Received March 14, 2013; Accepted March 29, 2013; Epub April 3, 2013; Published April 13, 2013
Abstract: Amplification of human Her2 and its aberrant signaling in 20-30% of early breast cancer patients is responsible for
highly aggressive tumors with poor outcome. Grb7 is reported to be co-amplified with Her2. We report a concurrent high
expression of mRNA (from FFPE tumor samples; mRNA correlation, Pearson r2= 0.806), and high levels of GRB7 protein
(immunoblot) in HER2+ breast cancer cell lines. We demonstrated the signaling mechanism of HER2 and downstream
effectors that contributes to proliferation and migration. Using HER2+ and trastuzumab-resistant breast cancer cell lines, we
identified the interaction between GRB7 and HER2 in the control of HER2+ cell proliferation. Our co-IP data show that GRB7
recruits SHC into the HER2-GRB7 signaling complex. This complex formation leads to activation of RAS-GTP. We also observed
that following integrin engagement, GRB7 is phosphorylated at tyrosine in a p-FAK (Y397) dependent manner. This FAK-GRB7
complex leads to downstream activation of RAC1-GTP (responsible for migration) probably through the recruitment of VAV2. Our
CO-IP data demonstrate that GRB7 directly binds with VAV2 following fibronectin engagement in HER2+ cells. To address
whether GRB7 could serve as a pathway specific therapeutic target, we used siRNA to suppress GRB7 expression. Knockdown
of GRB7 expression in the HER2+ breast cancer cell lines decreases RAS activation, cell proliferation, 2D and 3D colony
formation and also blocked integrin-mediated RAC1 activation along with integrin-directed cell migration. These findings
dissected the HER2-mediated signaling cascade into (1) HER2+ cell proliferation (HER2-GRB7-SHC-RAS) and (2) HER2+ cell
migration (alpha5 beta1/alpha4 beta1-FAK-GRB7-VAV2-RAC1). Our data clearly demonstrate that a coupling of GRB7 with HER2
is required for the proliferative and migratory signals in HER2+ breast tumor cells. (AJCR0000188)
Keywords: Her2/Neu, GRB7, adapter protein, RAS, RAC, proliferation and migration
Address correspondence to: Dr. Brian Leyland-Jones, Edith Sanford Breast Cancer Research, Sanford Research/USD, 2301 E
60th Street N, Sioux Falls, SD, 57104, USA. Phone: 605-312-6007; Fax: 605-312-6071; E-mail:
Brian.leyland-jones@sanfordhealth.org
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American Journal of Cancer Research
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