D. S. Hirsch, Y. Shen, W. J. Wu, DMA, OBP/OPS, CDER, FDA
Background: Epidermal Growth Factor Receptor (EGFR) overexpression correlates with high proliferative index, invasive capacity and poor prognosis in breast cancer. Cdc42, a member of the Ras superfamily of small GTPases, is a GTP binding protein that regulates cell proliferation and migration. Stable expression of active Cdc42 in NIH3T3 fibroblasts causes increases in EGFR protein levels and cellular transformation. Here, we used two EGFR overexpressing breast cancer cell lines, MDA-MB-231 and BT20, as models to test the hypothesis that reduction of Cdc42 protein levels would lower EGFR protein levels resulting in inhibition of processes critical to breast cancer progression.
Methods: A series of breast cancer cell lines were screened for Cdc42 protein levels by western blot. Activated Cdc42 protein levels were determined using GST-p21 binding domain assay, an affinity chromatography assay that specifically detects activated Cdc42 protein levels. Cdc42 protein levels were reduced by transfecting cells with Cdc42-targeted siRNA; non-targeting siRNA was used as a negative control. Migration was studied using boyden chamber and wound healing assays.
Results: Higher levels of activated Cdc42 were found in EGFR overexpressing MDA-MB-231 and BT20 cells as compared to MCF7 which has low EGFR protein levels. Cdc42-specific siRNA reduced Cdc42 protein levels by greater than 50%. siRNA-mediated reduction of Cdc42 protein levels lowered EGFR protein levels, leading to a marked inhibition of cell proliferation and migration.
Conclusions: Cdc42 is a critical positive regulator of EGFR protein levels, cell proliferation and cell migration. Disruption of Cdc42 signaling can reduce cell proliferation and migration in MDA-MB-231 and BT20 cells. Targeted introduction of Cdc42-specific siRNA to cancer cells may offer a novel therapeutic approach to inhibit these processes. Future studies are aimed at determining the suitability of Cdc42 as a therapeutic target.