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Breast cancer presents as ERα positive, or negative. ERα- tumors have a poor prognosis and are resistant to anti-hormone therapy, and frequently show overexpression, amplification, and/or hyperactivation of growth factor signaling, especially the erbB family. We have developed cell line models for elevated growth factor signaling in the MCF-7 breast cancer cells, and have shown that hyperactivation of the p42/44 MAPK pathway results in a reversible loss of ERα expression. Here, we have further explored the mechanisms involved in this ERα downregulation due to hyperactive MAPK signaling. Our results show that MAPK activity affects ERα at multiple levels. First, elevated MAPK activity represses ERα transcription - run-on assays demonstrate a decreased rate of transcription, and ERα promoter activity is lowered. Inhibition of MAPK activity restores ER promoter activity, and the MAPK sensitive region maps to a short length of the ER promoter. Secondly, while treatment of cells with hyperactive MAPK with a MEK inhibitor results in an increase in ERα mRNA levels, withdrawal of the MEK inhibitor leads to a very rapid loss of ERα mRNA indicating decreased mRNA stability. We have also found that ERα protein levels are significantly affected by MAPK activity - elevation in MAPK activity through co-expression of constitutively active Raf results in a rapid (within 1-2 hours) loss of wild-type ERα protein in MCF-7 cells. Interestingly, a mutant ERα construct (S118A) which cannot be phosphorylated by MAPK is unaffected by this increase in MAPK signaling, suggesting that Ser-118 phosphorylation of ERα targets it for degradation. We also find that inhibition of the ubiquitin-proteasome pathway restores ERα expression in our model cell lines to levels similar to that seen with inhibition of MAPK activation, suggesting that hyperactive MAPK targets ERα for ubiquitination and subsequent degradation. This is further reinforced by the finding that an ERα mutant that is resistant to degradation via the ubiquitin-proteasome pathway is stabilized in our cell lines relative to the wild-type receptor. Thus, we find that downregulation of ERα by hyperactive MAPK occurs at the transcriptional, post-transcriptional and post-translational levels. While determining which mechanism is the prevalent one will be important for developing a therapeutic strategy for restoring ERα in ERα- tumors, our data so far suggests that post-translational targeting of ERα for degradation is the primary mechanism involved in ERα downregulation by hyperactive MAPK.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]