RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells

Abstract

Inflammatory breast cancer (IBC) is a distinct and aggressive form of locally advanced breast cancer. IBC is highly angiogenic, invasive, and metastatic at its inception. Previously, we identified specific genetic alterations of IBC that contribute to this highly invasive phenotype. RhoC GTPase was overexpressed in 90% of archival IBC tumor samples, but not in stage-matched, non-IBC tumors. To study the role of RhoC GTPase in contributing to an IBC-like phenotype, we generated stable transfectants of human mammary epithelial cells overexpressing the RhoC gene, and studied the effect of RhoC GTPase overexpression on the modulation of angiogenesis in IBC. Levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) were significantly higher in the conditioned media of the HME-RhoC transfectants than in the untransfected HME and HME-beta-galactosidase control media, similar to the SUM149 IBC cell line. Inhibition of RhoC function by introduction of C3 exotransferase decreased production of angiogenic factors by the HME-RhoC transfectants and the SUM149 IBC cell line, but did not affect the control cells. These data support the conclusion that overexpression of RhoC GTPase is specifically and directly implicated in the control of the production of angiogenic factors by IBC cells.

Figure 1

Comparison of levels of angiogenic factors by HME-RhoC transfectants, SUM149 IBC cell line and the HME-β-gal control cell line as determined by ELISA. The HME-RhoC cells produced significantly higher levels of angiogenic factors compared with the HME-β-gal control cell line, nearly recapitulating the levels produced by the SUM149 IBC cell line. Significant differences (p<0.001) between the control cells and the HME-RhoC cells are denoted by an asterisk (*) with standard deviations within 10% of the reported values.

Figure 2

Results of a rat aortic ring assay for functional angiogenic factors produced by untransfected HME (panel A), HME-β-gal control transfectants (panel B), HME-RhoC transfectants (panel C) and the SUM149 IBC cell line (panel D). Segments of rat aorta were embedded in Matrigel and cultured in the corresponding cell-conditioned media for 4 days and then observed for microvessel outgrowth. Conditioned media from the control cell lines (panels A and B) did not induce microvessel outgrowth. However, conditioned medium from the HME-RhoC transfectants (panel C) produced similar levels of new vessel growth as the SUM149 IBC cell line (panel D).

Figure 3

Panel A demonstrates the effect on production of angiogenic factors by inhibition of RhoC GTPase with C3 exotransferase. Similar levels of inhibition were accomplished by expressing a C3 exotransferase construct or introducing the active protein directly into the cells. Significant differences (p<0.05) between the untreated and C3 exotransferase treated cells are denoted by an asterisk (*) with standard deviations within 10% of the reported values. Panel B demonstrates the results of an in vitro ADP-ribosylation study to determine the in vivo efficiency of C3 exotransferase inhibition of Rho activity. The assay was performed as outlined in the Materials and Methods section. The potential ADP-ribosylated sites in both the HME-RhoC and SUM149 cell lines were significantly reduced after C3 treatment, thus indicating efficient in vivo inhibition of RhoC GTPase.

Figure 4

Rhodamine-labeled C3 exotransferase was introduced into cells using a lipid mediated transfer method (see Materials and Methods section). The presence and efficiency of C3 protein transfer was determined by visualizing cells under a fluorescent microscope.