From ERα66 to ERα36, a new marker for breast tumor therapeutic response ?

Breast cancer is the main cause of cancer-induced morbidity and mortality in women. Breast tumors are usually classified according to their ERa66 status. Such a classification led to the use of endocrine therapeutic agents against ER-positive tumors [ER+]. Nevertheless, numerous therapeutic failures are observed due to unclear resistance mechanism. ERa66 was considered as the unique functional estrogen receptor in hormone sensitive breast tumor until the recent identification of membrane bound new estrogen receptors: the G protein coupled estrogen receptor (GPER) and the 36kDa ERa splice variant (ERa36). Surprisingly, ERa36 stimulates cell proliferation in response to tamoxifen treatment and could therefore be involved in the acquired resistance to this compound. Moreover, a high ERa36 expression level correlates with a short term survival for ERa66-negative patients as well as enhanced tumorigenesis and metastatic potential of triple-negative cells in vitro. The aim of our project was to improve the classification of so-called "ER-positive" breast tumors by taking into account the key role of ERa36 and GPER in the control of non genomic estrogen response and metastatic potential. We set up a retrospective study, performed on hundreds of breast tumor samples, in order to better define the field of acceptance of [ER+] versus [ER-] classification and to help the clinicians choosing the best therapeutic compounds. We addressed two main questions: 1- Are ERa36 and GPER predictive markers of therapeutic response in ER-positive tamoxifen-treated patients [ER+,TAM+]? 2- Is a high ERa36 or/and GPER expression level of poor prognosis because these receptors stimulate tumor progression and metastatic potential? ERa36, GPER and metastatic marker expressions were measured by real-time PCR in almost 100 [ER+,TAM] as well as 60 triple-negatives [ER-,TAM-] tumor samples. Then, we performed statistical analyses between gene expression levels and clinical parameters (grade, survival, treatment). Modeling of the potential relationship between the genes tested using nonlinear correlation analyses, Bayesian inference and transfer entropy computation led to the characterization of complex network connecting non genomic estrogen signaling and metastatic process. Hence, ERa36 expression is strongly related to GPER, Snail1, Vim and MMP9A in [ER+, TAM+] samples. This suggests that, after tamoxifen treatment, ERa36 may stimulate the metastatic potential of [ER+] tumor in vivo. Such a model will be first tested in vitro in hormone-dependent or triple negative cell lines. Taken together, the results from this project should lead to (i) a better understanding of the breast tumor hormone sensitive status, (ii) a validation of new predictive markers of response in order to improve therapeutic orientation, (iii) the potential discovery of new therapeutic targets in triple-negative tumors.