Therapeutic IMC-C225 antibody inhibits breast cancer cell invasiveness via Vav2-dependent activation of RhoA GTPase

Abstract

Purpose: Abnormalities in the expression and signaling pathways downstream of epidermal growth factor receptor (EGFR) contribute to progression, invasion, and maintenance of the malignant phenotype in human cancers. Accordingly, biological agents, such as the EGFR-blocking antibody IMC-C225 have promising anticancer potential and are currently in various stages of clinical development. Because use of IMC-C225 is limited, at present, only for treatment of cancer with high EGFR expression, the goal of the present study was to determine the effect of IMC-C225 on the invasiveness of breast cancer cells with high and low levels of EGFR expression.

Experimental design: The effect of IMC-C225 on invasion was studied using breast cancer cell lines with high and low levels of EGFR expression.

Results: The addition of EGF led to progressive stress fiber dissolution. In contrast, cells treated with IMC-C225 showed reduced invasiveness and increased stress-fiber formation. Interestingly, IMC-C225 pretreatment was accompanied by EGFR phosphorylation, as detected using an anti-phosphorylated tyrosine antibody (PY99), which correlated with phosphorylation of Vav2 guanine nucleotide exchange factor and activation of RhoA GTPase irrespective of EGFR level, and Vav2 interacted with EGFR only in IMC-C225-treated cells. The underlying mechanism involved an enhanced interaction between beta1 integrins and EGFR upon IMC-C225 treatment.

Conclusion: Here, we defined a new mechanism for IMC-C225 that cross-links integrins with EGFR, leading to activation of RhoA and inhibition of breast cancer cell invasion irrespective of the level of EGFR in the cells, thus providing a rationale for using IMC-C225 in the metastatic setting independent of the levels of EGFR.

Figure 1. Cytoskeleton rearrangement and modulation of the signal transduction pathway by IMC-C225

(A) Representative immunofluorescence image showing distribution of phospho-EGFR (Y1086 and Y1068, green) and total pools of EGFR (blue) in MDA-MB468 cells in the presence or absence of EGF and IMC-C225. F-actin is shown in red. (B) MDA-MB468 cells treated with EGF for various time points in the presence or absence of IMC-C225 and processed for immunoblotting using specific antibodies. Western blot showing specific inhibition of EGFR and MAPK phosphorylation by the blocking antibody IMC-C225. (C) Distribution of phospho-EGFR (Y1086 and Y1068, green) in MDA-MB231 cells in the presence or absence of EGF and IMC-C225. F-actin is shown in red. Bar, 20 μm. (D) Bar plot showing inhibition of migration of MDA-MB468 and MDA-MB231 cells on treatment with IMC-C225.

Figure 2. Intracellular trafficking of EGFR

Representative immunofluorescence images showing distribution of total EGFR in response to EGF stimulation for 5 and 10 min in control (A) and in IMC-C225 pretreated (B) MDA-MB468 cells. EGFR (green), F-actin (red), and DNA (blue). Bar, 20 μm.

Figure 3. Effect of IMC-C225 on β-catenin subcellular localization and RhoA activation

MDA-MB468 cells (A) or MDA-MB231 cells (C) were serum-starved or pretreated with IMC-C225 before stimulation with EGF. Western blot showing levels of Rho GTP and total RhoA. (B) Vav2 immunoprecipitated from MDA-MB468 cells treated as mentioned above (first panel) and probed for EGFR (second panel) and phospho Vav2 (PY20, third panel). Total EGFR immunoprecipitated (last panel) and probed for phospho-EGFR using anti phosphor-tyrosine antibody (PY99, fourth panel). IgG was used as negative control. (D) Vav2 immunoprecipitated from MDA-MB231 cells treated as mentioned above and probed for EGFR (first panel). Immunoblot showing interaction of Vav2 with EGFR in IMC-C225–treated cells independent of EGF stimulation (second panel). EGFR was immunoprecipitated from MDA-MB231 cells (last panel) treated as mentioned above and probed for phospho-EGFR (PY99). Immunoblot showing increased levels of phospho-EGFR (PY99) in MDA-MB231 cells treated with either EGF or IMC-C225 compared to untreated cells (Third panel).

Figure 4. IMC-C225–mediated activation of Rac1

MDA-MB468 cells were serum-starved and pretreated with IMC-C225 before stimulation with EGF. Western blot showing levels of Rac1 GTP and total Rac1 for longer time points (A) and shorter time points (B) of EGF stimulation. (C) Immunofluorescence image showing transfection of myc tagged V14 RhoA (dominant active, DA, green) or V12 Rac (DA, green) and DNA (blue) in MDA-MB468 cells. (D) Bar plot showing effect of V14RhoA and V12Rac transfection on migration of MDA-MB468 in presence or absence of IMC-C225 pre-treatment.

Figure 5. IMC-C225–mediated activation of EGFR via integrins

Immunoblot showing effect of IMC-C225 treatment on EGFR phosphorylation at various tyrosine residues in MDA-MB468 (A) and MDA-MB231 cells (B). Total EGFR was used as an internal control. (C) EGFR was immunoprecipitated from MDA-MB231 cells after treatment with IMC-C225 or IMC-C225 and Src inhibitor PP2 and probed with phospho-EGFR antibody. Western blot showing effect of Src inhibitor PP2 on Src phosphorylation on Y215. Total Src was used as an internal control. (D) Immunoblot showing EGFR in a complex with β1 integrin only in response to IMC-C225 treatment in MDA-MB468 cells. EGFR immunoprecipitated with β1 integrin in response to IMC-C225 treatment was phosphorylated on PY99.

Figure 6

Proposed model of activation of Rho GTPase caused by IMC-C225–mediated cross linking between β1 integrins and EGFR on the cell membrane leading to inhibition of cell migration.