Tamoxifen Resistance in Estrogen Receptor Positive (ER+) Breast Cancer Is Driven by Estrogen Receptor Negative (ER-) Cancer Stem-Like Cells.

Abstract

Abstract Introduction: Intrinsic and acquired resistance to endocrine therapy significantly reduces survival rates of women with ER+ breast cancer. In the normal breast, multipotent stem cells are ER-. We hypothesised that tumourigenic breast cancer stem-like cells (CSCs) are ER- and therefore function independently of estrogen, representing a novel mechanism of resistance to endocrine therapy.Methods: Three ER+ cell lines, MCF-7, T47D and BT474 were assessed for ER expression by immunocytochemistry (ICC) in monolayer and after CSC enrichment. Putative CSC number was assessed by the non-adherent mammosphere (MS) assay, in the presence and absence of tamoxifen, and by Fluorescence Activated Cell Sorting (FACS) for the markers CD44+/CD24-/low/epithelial specific antigen(ESA)+. The effect of acquired Tamoxifen resistance on the CSC population of two independent Tamoxifen-resistant (TAM-R) MCF-7 variants, from Cardiff (TAM-RCARD) and Copenhagen (TAM-RCOP) was compared to parental, tamoxifen sensitive (TAM-S) controls. In the MS assay both the number and size of colonies were assessed.Results: A small sub-population of ER_ cells were demonstrated in MCF7 (6.1 ± 1.4%), T47D (6.8± 0.5) and BT474 (3.7±1.3) cells by ICC. However, the majority of putative breast CSCs (CD44+/CD24-/low/ESA-) in MCF7 cells were ER_ (73.2 ± 4.6 % p= 0.0007). There was a significant increase in the proportion of ER- cells in TAM-R cell lines (TAM-RCARD 11.6 ± 1.4% p=0.01 and TAM-RCOP 22 ± 4.8% p=0.02 respectively) compared to the parental TAM-S controls. Both TAM-R cell lines demonstrated increased mammosphere forming efficiency (MFE) compared to TAM-S cells (TAM-RCARD 4% vs. 2.5% p=0.03, and TAM-RCOP 8% vs. 3% p=0.007). The putative CSC population (CD44+/CD24-/low/ESA+) was enriched more than 2.5 fold in TAM-R compared to TAM-S parental controls. Compared with controls, the absolute MFE was not affected by Tamoxifen treatment, although mammospheres formed were significantly smaller.Conclusions: The majority of the putative CSC populations in ER+ breast cancer do not express ER. There is an increase in the proportion of putative ER- CSCs in TAM-R MCF7 cell lines. The reduction in MS size suggests that Tamoxifen targets transit amplifying cells but does not reduce the absolute number of CSC in ER+ breast cancers. These findings demonstrate a novel mechanism of endocrine resistance in ER+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5125.