Understanding the role of RhoC GTPase in inflammatory breast

ALEJANDRA C VENTURA; ZHIFEN WU; JACQUES-A. SEPULCHRE; MEI WU; JORGE R TREDICCE; SOFIA D MERAJVER

Congreso; Breast Cancer Research Program (BCRP) Leading Innovative Networking and Knowledge Sharing (LINKS) Meeting; 2010

The most damaging change during cancer progression is the growth of metastases. Understanding genes that drive metastasis and how to correctly inhibit them is paramount to improve survival. The protein RhoC GTPase was found to be crucial in that process in different cancers, particularly in a highly aggressive form of breast cancer termed inflammatory breast cancer (IBC). RhoC is a molecular switch cycling between inactive (GDP-bound) and active (GTP-bound) states, tightly regulated by several regulatory proteins.We have developed a dual mathematical-experimental approach to understand the mechanism of RhoC deregulation in breast cancer. A major impact of this work is, for the first time, to quantitatively predict the effects of drugs targeted against RhoC in cancer. The methodologies include biochemical experiments for active and total RhoC, quantitative imaging, and mathematical modeling.We have found that the activation profile of RhoC in IBC cells (SUM149) after stimulation with lysophosphatidic acid (LPA) is similar to those of other small GTPases. Unexpectedly, however, a significant and rapid increase in RhoC protein abundance accompanies this activation profile. Several tests and alternative experiments were performed to ensure that this last observation was not an experimental artifact.The bacterial exotransferase from Clostridium botulinum (C3 transferase) irreversibly ADP ribosylates RhoA, RhoB, and RhoC proteins and inhibits their downstream signaling interactions. After a recovery period following C3 transfection, SUM149 cells were stimulated with LPA. RhoC activation was effectively suppressed by C3 as expected. Interestingly, the increase in total protein level was also blocked by C3. These data suggest that inhibition of RhoC activation by C3 concurrently suppressed the increase in RhoC protein. The pathway initiated by LPA that produces the increase in total RhoC is therefore dependent on RhoC-GTP, revealing the existence of a previously unrecognized positive feedback loop between the activation cycle and the protein synthesis-degradation balance for RhoC.RhoC protein half-life was measured after treatment with the protein synthesis inhibitor cycloheximide (CHX) in a time course experiment with SUM149 cells. The half-life of the protein was estimated around 40 minutes. Less protein degradation was observed in cells pretreated with LPA than in cells treated with CHX only. In addition, RhoC protein production rate was measured after treatment with the proteasome inhibitor MG132. Dual LPA and MG132 treatment indicates that this production rate is not modified by LPA. The CHX and MG132 experiments indicate that the increase in protein level after LPA stimulation is due to a decrease in the protein degradation rate, and the C3 experiment indicates that this mechanism is mediated by RhoC-GTP.We have developed a set of ordinary differential equations to describe RhoC-GTP and RhoC protein temporal courses. The equations take into account the mentioned positive feedback loop between the activation cycle and the control of the protein degradation rate. The contribution of this regulatory mechanism to the aggressive IBC phenotype is studied, as well as its impact in planning RhoC-directed drug strategies.This work was supported by the U.S. Army Medical Research and Materiel Command under W81XWH-06-1-0489; Burroughs Wellcome Funds; the Breast Cancer Research Foundation; and National Institutes of Health (R01 CA77612).