Abstract 3174: Visualization of the activation/deactivation cycle of RhoC in inflammatory breast cancer

Abstract

Abstract A key event in the development of Inflammatory Breast Cancer, a particularly aggressive, metastatic form of breast cancer, is the over-expression of the GTPase protein RhoC. RhoC plays a role in regulating cell shape, attachment and motility and over-expression is likely associated with tumor proliferation. RhoC is activated into a GTP-bound state by the regulatory proteins GEFs (guanine nucleotide exchange factor proteins) and is deactivated to a GDP-bound state by GAPs (GTPase activating proteins). Stimulation of IBC cells with LPA (lysophophatidic acid), an activator of the cycle, showed a transient RhoC-GTP increase, peaking at 2 minutes and then diminishing until 20 minutes. It has been suggested that LPA can activate both GEF and GAP proteins, and that a delay in the GAP protein activation could explain this RhoC-GTP behavior. Because RhoC activity occurs at the plasma membrane, the translocation of GEF and GAP proteins was studied as an indication of RhoC interaction. Immunofluorescent stains for the three key proteins_RhoC, GAP, and GEF_were performed on an IBC cell line before and after stimulation with LPA and observed using confocal microscopy. Preliminary images revealed a co-localization of three proteins RhoC, p190B (a GAP protein), and PDZ (a GEF protein) in the cytosol, forming a gradient of high to low protein concentration from the nuclear membrane to the plasma membrane. Further analysis with ImageJ software showed slight differences in protein concentration at the two membranes upon stimulation with LPA. RhoC protein levels increased at both membranes, PDZ quantities increased slightly at the plasma membrane, and the p190B levels decreased at both membranes as a result of stimulation. This data suggests that after 5 minutes, LPA has a positive regulatory effect on the GEF protein and a negative one on the GAP protein at the plasma membrane. In addition, co-localization of the proteins RhoC and p190B was analyzed at different time points. The data indicates a decrease in co-localization after LPA stimulation that is slightly recovered 20 minutes after treatment. A possible explanation for these results is that 5 minutes after stimulation, RhoC undergoes translocation to the nuclear and plasma membranes, and is not followed by the GAP protein until later times. As of now, these conclusions are preliminary. These results will be verified using a new RhoC antibody, and additional co-localization analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3174.