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The cytokine interleukin-6 (IL6) and its downstream effector STAT3 constitute a key oncogenic pathway, which has been thought to be functionally connected to estrogen receptor α (ER) in breast cancer. We demonstrate that IL6/STAT3 signaling drives metastasis in ER+ breast cancer independent of ER. STAT3 hijacks a subset of ER enhancers to drive a distinct transcriptional program. Although these enhancers are shared by both STAT3 and ER, IL6/STAT3 activity is refractory to standard ER-targeted therapies. Instead, inhibition of STAT3 activity using the JAK inhibitor ruxolitinib decreases breast cancer invasion in vivo. Therefore, IL6/STAT3 and ER oncogenic pathways are functionally decoupled, highlighting the potential of IL6/STAT3-targeted therapies in ER+ breast cancer.

Background BCSC are considered to be involved in the recurrence of breast cancer and its resistance to the systemic therapies. Their detection and targeting remain challenging. Patients and methods: The study was conducted in vitro, in xenografted immunecompromised mice and samples of 38 patients with early stage BC having biopsies and blood samples before and after anthraycycline + taxane-based neoadjuvant chemotherapy (NAC). All patients gave written informed consent before inclusion. 1) MCF7 cells grown as mammospheres (MS) for BCSC enrichment were treated with 5FU or paclitaxel (Pac) to select chemo-resistant BCSC. miRNA microarray was performed to identify specific miRNAs for chemo-resistant BCSC. The results were compared to miRNAs found in immortalized non-tumorigenic MCF10A cells to exclude miRNAs related to normal stem cells. 2) The correlation between the most highly expressed miRNA and the BCSC was confirmed by RT-qPCR in ALDH+ and ALDH- cells sorted from MCF7 and MDA-MB-231 cells by flow cytometry. 3) The impact of the miRNA on MS and colony development was assessed by up- and down-regulating its expression. MCF7 cells transfected with ectopic expression of miRNA, anti-miRNA or miRNA control were grown in MS before being injected in mice using a mouse INtraDuctal xenograft Model (MIND). In vivo tumor growth was assessed by luciferase imaging, then measured and quantified with human GAPDH ex vivo at 6 weeks. 4) The miRNA was quantified by RT-qPCR in tumor samples and sera of the patients before and after treatment, and its levels were correlated with pathological complete response and patients' outcomes. Results: 379 miRNAs out of 2006 were altered in chemo-resistant versus untreated MCF7 MS. Thirteen were specific for 5FU and 5 for Pac. Three were common for both drugs. Of these, miR-363-3p was overexpressed specifically in BCSC-enriched chemo-resistant MCF7 cells and all the other tested BC cell lines, but not in non-tumorigenic MCF10A cells. Compared to adherent MCF7 cells, miR-363-3p was 12-, 60-, and 10-folds more expressed in BCSC-enriched MS treated with 5FU, Pac, or without treatment, respectively. miR-363-3p was 20- and 100-folds higher in ALDH+ compared to ALDH- in MCF7 and MDA-MB-231 cells. Anti-miR-363-3p reduced MS size and decreased their number 50%. A significant decrease of the number of colonies was also observed in soft agar. Consistently, miR-363-3p downregulation decreased tumor growth and metastasis by MCF7 cells transplanted in mice. In patients' sera with lower baseline level (n=15), miR-363-3p appeared decreased upon NAC. Patients with high miR-363-3p serum levels (n=22) had more risk to maintain higher level after chemotherapy. Triple-negative and HER2+ BC were more frequent in this second group. No significant difference was observed in term of pCR between the 2 groups. However 3 patients relapsed with distant metastases and all were in the second group with high baseline level and no decrease after NAC. Conclusions: miR363-3p appeared to be a mediator of chemo-resistant BCSC. Its measurement in the serum of BC patients may predict resistance to neo-/adjuvant chemotherapy and higher risk of distant recurrence. Further investigations are warranted to confirm its role as biomarker and potential therapeutic target against BCSC. Citation Format: Renaud S, Fiche M, Stravodimou A, Scabia V, Dormoy VM, Galmiche Rindisbacher M, Brisken C, Mermod N, Zaman K. miR363-3p mediates maintenance and resistance of breast cancer stem cells (BCSC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-11.

Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome‐wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBPα. Using ChIP‐seq, we identify around 1,000 sites where C/EBPα binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBPα maintains an open chromatin conformation that facilitates loading of ligand‐activated PR. Prior to hormone exposure, C/EBPα favors promoter–enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBPα disrupts enhancer–promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBPα fulfills a previously unknown function as a potential growth modulator in hormone‐dependent breast cancer., The experimental work was supported by grants from the Departament d'Innovació Universitat i Empresa (DIUiE), and the Spanish Ministry of Economy and Competitiveness (SAF2016‐75006P), “Centro de Excelencia Severo Ochoa 2013‐2017”, SEV‐2012‐0208 and ERC Synergy Grant “4DGenome” nr: 609989.

Estrogen receptor α positive (ER+) or “luminal” breast cancers were notoriously difficult to establish as patient-derived xenografts (PDXs). We and others recently demonstrated that the microenvironment is critical for ER+ tumor cells; by grafting them into milk ducts >90% take rates are achieved and many features of the human disease are recapitulated. This intra-ductal (ID) approach holds promise for personalized medicine, yet human and murine stroma are organized differently and this and other species specificities may limit the value of this model. Here, we analyzed 21 ER+ ID-PDXs histopathologically. We find that ID-PDXs vary in extent and define four histopathological patterns: flat, lobular,in situ, and invasive, which occur in pure and combined forms. The ID-PDXs replicate earlier stages of tumor development than their clinical counterparts. Micrometastases are already detected when lesions appearin situ. Tumor extent, histopathological patterns, and metastatic load correlate with biological properties of their tumors of origin. Our findings add evidence to the validity of the intraductal model forin vivostudies of ER+ breast cancer and raise the intriguing possibility that tumor cell dissemination may occur earlier than currently thought.Conflict of interest statement:The authors declare no conflict of interest.

Estrogen receptor α‐positive (ER‐positive) or ‘luminal’ breast cancers were notoriously difficult to establish as patient‐derived xenografts (PDXs). We and others recently demonstrated that the microenvironment is critical for ER‐positive tumor cells; when grafted as single cells into milk ducts of NOD Scid gamma females, >90% of ER‐positive tumors can be established as xenografts and recapitulate many features of the human disease in vivo. This intraductal approach holds promise for personalized medicine, yet human and murine stroma are organized differently and this and other species specificities may limit the value of this model. Here, we analyzed 21 ER‐positive intraductal PDXs histopathologically. We found that intraductal PDXs vary in extent and define four histopathological patterns: flat, lobular, in situ and invasive, which occur in pure and combined forms. The intraductal PDXs replicate earlier stages of tumor development than their clinical counterparts. Micrometastases are already detected when lesions appear in situ. Tumor extent, histopathological patterns and micrometastatic load correlate with biological properties of their tumors of origin. Our findings add evidence to the validity of the intraductal model for in vivo studies of ER‐positive breast cancer and raise the intriguing possibility that tumor cell dissemination may occur earlier than currently thought. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational pre-clinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and “Triple-negative” (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward “credentialing” of PDX models as surrogates to represent individual patients for use in pre-clinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research.

Summary Seventy-five percent of breast cancers are estrogen receptor α positive (ER + ). Research on these tumors is hampered by lack of adequate in vivo models; cell line xenografts require non-physiological hormone supplements, and patient-derived xenografts (PDXs) are hard to establish. We show that the traditional grafting of ER + tumor cells into mammary fat pads induces TGFβ/SLUG signaling and basal differentiation when they require low SLUG levels to grow in vivo. Grafting into the milk ducts suppresses SLUG; ER + tumor cells develop, like their clinical counterparts, in the presence of physiological hormone levels. Intraductal ER + PDXs are retransplantable, predictive, and appear genomically stable. The model provides opportunities for translational research and the study of physiologically relevant hormone action in breast carcinogenesis.