Abstract S5-7: Estrogen Receptor Alpha Negative (ER-) Breast Cancer Stem Cells in ER+ Cell Lines and Primary Cancers Are Enriched by Tamoxifen Therapy
Authors: O'Brien CS , Howell SJ , Gandhi A , Gee J , Lykkesfeldt A , Nicholson RI , Clarke RB. 
Published: April 2011

Introduction: Resistance to endocrine therapy in ER+ breast cancers is associated with tumour relapse and poor prognosis. Breast cancer stem cells (BCSCs), characterised by the CD44hi/CD24lo/ESA+ phenotype and mammosphere initiating capacity (MIC), show treatment resistance to chemo-/radiotherapy. We hypothesised that BCSCs may also be resistant to endocrine therapies due to absent ER expression in vitro and in vivo.

Methods: Three ER+ cell lines (MCF7, T47D and BT474) and six treatment naïve ER+ early breast cancers (5 IDC, 1 ILC) were used for in vitro and in vivo assays of BCSC activity.

Putative CSC number was assessed by the non-adherent mammosphere (MS) assay, and by Fluorescence Activated Cell Sorting (FACS) for the markers CD44+/CD24-/low/epithelial specific antigen(ESA)+ in the presence and absence of tamoxifen (TAM), and also by limiting dilution implantation of cells into NOD/SCID IL2gammaR-/- (NSG) mice. Furthermore the effect of acquired tamoxifen resistance on BCSC activity was investigated in two independent Tamoxifen-resistant (TAM-R) MCF-7 variants, from Cardiff (TAM-RCARD) and Copenhagen (TAM-RCOP) and five (ER+) endocrine resistant metastatic cancers (3 pleural effusion, 2 ascites). ER alpha expression was assessed before and after BCSC enrichment in cell lines, primary and metastatic cancers by immunohistochemistry.

Results: Putative BCSCs, (CD44+/CD24-/low/ESA or MIC phenotype) were highly enriched for ER- cells (eg. MCF7 cell line; CD44+/CD24-/low/ESA 73.2 ± 4.6% vs. 6.1 ± 1.4% p= 0.0007 or MIC 45.7 ± 1.7 vs. 6.1 ± 1.4 P&lt;0.001). 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. Similarly, the enriched BCSC population of primary and metastatic ER+ breast cancers were enriched for ER-cells (eg. BB7 metastatic cancer 91.6 ± 4.8% vs. 47.4 ± 9.5% p = 0.02). ER+ cell lines, treatment naïve primary samples and treatment resistant metastatic samples all demonstrated BCSC activity, which correlated with tumour grade and stage. BCSC activity was significantly increased in MCF7 models of acquired TAM resistance compared to parental TAM sensitive controls (mammosphere assay TAM-RCARD 4% vs. 2.5% p=0.03, and TAM-RCOP 8% vs. 3% p=0.007). A similar trend was observed in endocrine resistant (End-R) patient samples compared to treatment naïve (TN) controls (End-R 0.46 vs. TN 0.10 p = 0.1). Tumour take rates in NSG mice were significantly increased in endocrine resistant cell lines and metastatic samples. Importantly, TAM pre-treatment increased mammosphere forming efficiency in MCF7, T47D and BT474, ER+ primary and ER+ metastatic samples, suggesting BCSCs are relatively resistant to TAM compared to more differentiated cells.

Conclusions: The putative BCSC populations in ER+ cell lines and breast cancers are enriched for ER-cells and this finding is further exaggerated in acquired endocrine resistance. TAM treatment and acquired tamoxifen resistance enriches for BCSC activity in in vitro and in vivo models, presumably by preferentially inhibiting more differentiated cells. These findings establish a novel mechanism of TAM resistance in ER+ breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S5-7.