Purpose Anti-endocrine therapy remains the most effective treatment for ER+ breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAFs), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and anti-endocrine resistance. Experimental Design Tissues from ER+ breast cancer patients were analyzed for the presence of CD146 positive (CD146pos) and CD146 negative (CD146neg) fibroblasts. ER dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells co-cultured with CD146pos or CD146neg fibroblasts. RNAseq was used to develop a high confidence gene signature that predicts for disease recurrence in tamoxifen treated patients with ER+ breast cancer. Results We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells is sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer, and should be considered a target for drug development.

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Bypassing estrogen receptor (ER) signaling during development of endocrine resistance remains the most common cause of disease progression and mortality in breast cancer patients. To date, the majority of molecular research on ER action in breast cancer has occurred in cell line models derived from late stage disease. Here we describe patient-derived ER + luminal breast tumor models for the study of intratumoral hormone and receptor action. Human breast tumor samples obtained from patients post surgery were immediately transplanted into NOD/SCID or NOD/SCID/ILIIrg−/− mice under estrogen supplementation. Five transplantable patient-derived ER + breast cancer xenografts were established, derived from both primary and metastatic cases. These were assessed for estrogen dependency, steroid receptor expression, cancer stem cell content, and endocrine therapy response. Gene expression patterns were determined in select tumors ±estrogen and ±endocrine therapy. Xenografts morphologically resembled the patient tumors of origin, and expressed similar levels of ER (5–99 %), and progesterone and androgen receptors, over multiple passages. Four of the tumor xenografts were estrogen dependent, and tamoxifen or estrogen withdrawal (EWD) treatment abrogated estrogen-dependent growth and/or tumor morphology. Analysis of the ER transcriptome in select tumors revealed notable differences in ER mechanism of action, and downstream activated signaling networks, in addition to identifying a small set of common estrogen-regulated genes. Treatment of a na¨ıve tumor with tamoxifen or EWD showed similar phenotypic responses, but relatively few similarities in estrogen-dependent transcription, and affected signaling pathways. Several core estrogen centric genes were shared with traditional cell line models. However, novel tumor-specific estrogen-regulated potential target genes, such as cancer/testis antigen 45, were uncovered. These results evoke the importance of mapping both conserved and tumor-unique ER programs in breast cancers. Furthermore, they underscore the importance of primary xenografts for improved understanding of ER+ breast cancer heterogeneity and development of personalized therapies.

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER) and progesterone receptor (PR) positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5), and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44+ cell population. Downregulation of miR-29 members potentiates the expansion of CK5+ and CD44+ cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone responsive breast cancers.