Restoration of ER expression and antiestrogen response in ER-negative breast tumors: Strategies for treatment and prevention.

CS09-02

Breast cancer presents as either estrogen receptor alpha (ERα) positive or negative, with ERα+ tumors responding to anti-estrogen therapy and having better prognosis. In ERα- tumors, overexpression of the epidermal growth factor receptor (EGFR) or c-erbB-2 leads to increased growth factor signaling including hyperactivation of mitogen-activated protein kinase (MAPK) compared to ERα+ breast cancer. Using cell line models that mimic this elevated growth factor signaling by EGFR and erbB-2 in MCF-7 breast cancer cells, we have previously shown that hyperactivation of downstream p42/44 MAPK results in the reversible loss of ERα expression. The primary mechanism underlying this MAPK-induced downregulation of ERα involves targeting ERα for degradation via the ubiquitin-proteasome pathway. Recently, we established a hyperactive MAPK gene profile from these cell line models. This MAPK signature accurately predicts ERα- breast cancer lending further support to our hypothesis that hyperactivation of MAPK by increased growth factor signaling plays a role in the generation of the ERα- phenotype. In addition, it indicates these cell lines are excellent, highly relevant models for the investigation of mechanisms underlying the ERα- phenotype. We have since gone on to ascertain the role of these mechanisms in established ERα- breast cancer cell lines and in ERα- tumor specimens. Importantly, we asked if inhibition of MAPK activity in established ERα- breast cancer cell lines and then in ERα- tumor specimens could restore ERα expression and further, if this re-expressed ERα could now restore an anti-estrogen response. MAPK inhibition in a series of ERα- breast cancer cell lines results in re-expressed ERα protein. In ERα- cell lines with overexpression of EGFR or erbB-2 and a dependence upon MAPK for proliferation, re-expression of ERα upon MAPK inhibition correlated with a restoration of estrogen-dependence and anti-estrogen sensitivity. However, in a cell line with hyperactivation of NFkB due to RhoC overexpression and no dependence on MAPK for proliferation, the re-expressed ERα could not restore anti-estrogen sensitivity. Interestingly, in ERα- breast cancer cell lines with a basal rather than luminal phenotype, MAPK inhibition could not restore ERα expression suggesting that more permanent repression mechanisms such as ERα promoter hypermethylation are responsible for the ERα- phenotype in these cell types. To determine if these in vitro cell line results would extrapolate to a real breast tumor situation, ERα- tumor specimens were subjected to MAPK inhibition. Of the 12 tumors examined, 6 displayed increased ERα mRNA levels after treatment with MEK inhibitor. Dissociated tumor cell cultures established from ERα- tumors confirm the ability of MAPK inhibition to restore ERα expression in ERα- tumors. Importantly, re-expression of ERα upon MAPK inhibition restored anti-estrogen sensitivity. These data suggest that we need to alter our interpretation of ERα status - rather than thinking of ERα expression as being positive or negative, we need to think of it as a dynamic event that is strongly impacted by other cellular signaling pathways. Collectively, these data support our hypothesis that there exists a population of ERα- tumors where upregulated growth factor signaling is directly involved in the generation of the ERα- phenotype by repressing ERα expression and that further, this reversible downregulation may be targeted clinically such that this repression can be reversed by inhibiting signaling through MAPK, resulting in re-expression of ERα and perhaps restoration of tamoxifen sensitivity.