Abstract P4-02-09: Extranuclear Estrogen Receptors in Human Breast Cancer
Authors: Márquez-Garbán DC , Chen H-w , Goodglick L , Fishbein MC , Pietras RJ. 
Published: April 2011

Estrogens are well known to promote growth and progression of human breast cancer. These effects are exerted by binding of estradiol to estrogen receptors (ER), ER-alpha and ER-beta. Immunohistochemical (IHC) detection of nuclear ER-alpha in breast tumors is a standard assessment in the clinic to plan patient management decisions. However, ERs also occur at extranuclear sites, and numerous studies now show that extranuclear

ER (exER) play important roles in regulating downstream signaling by activating kinase cascades that impact gene expression and tumor progression. Nonetheless, we still have poor understanding of the relative contributions of nuclear ER versus extranuclear ER in mediating estrogen action. Based on current IHC assays for nuclear estrogen receptors, only about half of advanced breast cancers with expression of ER and/or progesterone receptor respond to endocrine therapy, indicating a need for improved assays designed to correlate not only nuclear ER but possibly also exER with patient outcome. In this study we focused on development of innovative methods for IHC detection of exER-alpha as well as nuclear ER-alpha and correlation of expression with clinical outcome. IHC results depend greatly on the type of antibody used, the detection procedure, subcellular densities of ER and problems due to long-term formalin fixation (antigen masking). We have tested and compared different methods and antibodies that specifically detect nuclear and extranuclear forms of ER in formalin-fixed, paraffin-embedded tissues. Although some exER staining is apparent in clinical samples that test positive for nuclear ER with antibodies commonly used for standard ER assays, our findings suggest that carboxy-terminal antibodies are more sensitive in the detection of specific exER staining. Such C-terminal antibodies can also detect exER staining even in the absence of significant nuclear staining in about one third of 16 breast cancer cases analyzed from the clinic. Extranuclear ER staining is specific, but exhibits lower intensity than nuclear ER staining, and it does not occur in breast tumor cells negative for ER by Western blot or when antibodies are pre-absorbed with specific peptides. Further, subsets of human breast tumor cells selected for membrane-associated exER in vitro using flow cytometry and ligand-affinty binding methods (about 4-5% of the total tumor cell population) show enhanced growth responses to estradiol (P&lt;0.001) and increased expression of CD133, polycomb gene Bmi-1 and ALDH-1. These biomarkers are often associated with high tumor-initiating activity and poor clinical outcome. The findings suggest that tumor cell subpopulations with relatively high expression of exER may represent tumor progenitor cells with a capacity to drive tumor progression. Despite advances indicating the role of exER in regulating cancer progression, there is no current assay in the clinic to detect exER, nor to consider its contribution in managing hormonal therapy. Assay of breast tumor exER may prove important in predicting endocrine response and resistance as well as in development of new therapies to target these tumor cell subsets. [Supported by California Breast Cancer Research Program and Stiles Program funding]

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-09.