Differential effects of Mek1 and Mek2 inhibition on cellular proliferation of malignant and non-malignant human breast cells.

B127 Stimulation or inhibition of the Raf-Mek-Erk kinase cascade signaling pathway via cell surface receptors results in downstream effects on genes controlling cell cycle progression, motility and apoptosis. Because misregulation of each of these processes is a critical step in cancer progression, there has been much interest in using Mek inhibitors as anti-cancer therapeutics. These drugs target both the Mek1 and Mek2 kinases, which are assumed to act similarly on the signaling pathway. However, there is reason to believe that Mek1 and Mek2 have differential effects on proliferation, therefore complicating drug target strategies. Ussar and Voss (JBC, 279: 43861-9, 2004) showed that Mek1 causes proliferation, whereas Mek2 induces growth arrest at G1. Additionally, Scholl et al. (Cancer Research, 64:6035-40, 2004) over expressed Mek1 or Mek2 and found that only Mek1 increased proliferation. These data are consistent with our preliminarily data in cultured human mammary epithelial cells (HMECs) which indicate that cells constitutively expressing short hairpin RNAs (shRNAs) against Mek2 proliferate more rapidly than controls.
 We are determining the individual contributions of Mek1 and Mek2 to proliferation and Mek inhibitor drug sensitivity in malignant and non-malignant human breast epithelial cell lines. To date, four cell lines (MCF7, MCF10A, 184A1 and 184v-TERT) have been successfully transformed with lentiviruses expressing shRNAs against Mek1, Mek2, or both Mek1 and 2. The resulting knockdowns were robust and specific, as determined by immunoblotting. Surprisingly, these lines grew well despite decreased Mek expression, even in the case of the double knockdown. In addition, the shMek2 lines grew significantly faster than controls, regardless of cell lineage. Interestingly, immunoblots measuring total Erk and phosphorylated Erk (P-Erk) levels, indicated that P-Erk levels remain elevated in certain cell lineages despite the knockdown of Mek, suggesting that other kinases maintained phoshorylated Erk in these lines. Mek inhibitor (either U0126 or CI-1040) sensitivity assays indicated that the shMek2 lines are also more resistant than control lines to Mek inhibitors. In an effort to understand compensatory changes, we are currently profiling RNA and protein expression patterns in randomly cycling and synchronized HMEC lines stably transduced with shRNAs targeting Mek1 and /or Mek2.