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Estrogen and progesterone receptor (ER, PR) signaling control breast development and impinge on breast carcinogenesis. ER is an established driver of ER + disease but the role of the PR, itself an ER target gene, is debated. We assess the issue in clinically relevant settings by a genetic approach and inject ER + breast cancer cell lines and patient-derived tumor cells to the milk ducts of immunocompromised mice. Such ER + xenografts were exposed to physiologically relevant levels of 17-β-estradiol (E2) and progesterone (P4). We find that independently both premenopausal E2 and P4 levels increase tumor growth and combined treatment enhances metastatic spread. The proliferative responses are patient-specific with MYC and androgen receptor (AR) signatures determining P4 response. PR is required for tumor growth in patient samples and sufficient to drive tumor growth and metastasis in ER signaling ablated tumor cells. Our findings suggest that endocrine therapy may need to be personalized, and that abrogating PR expression can be a therapeutic option. The role of progesterone receptor (PR) and its interplay with estrogen receptor (ER) in breast cancer is controversial. Here, the authors demonstrate that PR can have an ER-independent role in breast cancer growth and metastasis and that its effects are dependent on MYC and androgen receptor signatures.

More than 70% of human breast cancers (BCs) are estrogen receptor α-positive (ER + ). A clinical challenge of ER + BC is that they can recur decades after initial treatments. Mechanisms governing latent disease remain elusive due to lack of adequate in vivo models. We compare intraductal xenografts of ER + and triple-negative (TN) BC cells and demonstrate that disseminated TNBC cells proliferate similarly as TNBC cells at the primary site whereas disseminated ER + BC cells proliferate slower, they decrease CDH1 and increase ZEB1 , 2 expressions, and exhibit characteristics of epithelial-mesenchymal plasticity (EMP) and dormancy. Forced E-cadherin expression overcomes ER + BC dormancy. Cytokine signalings are enriched in more active versus inactive disseminated tumour cells, suggesting microenvironmental triggers for awakening. We conclude that intraductal xenografts model ER + BC dormancy and reveal that EMP is essential for the generation of a dormant cell state and that targeting exit from EMP has therapeutic potential. The study of tumour dormancy is limited by suitable in vivo models. Here the authors show that mammary intraductal breast cancer (BC) xenografts model estrogen receptor α-positive (ER+) BC dormancy and rapid metastatic progression characteristic of triple-negative (TN) BC. The dormant disseminated ER+ BC cells display characteristics of epithelial-mesenchymal plasticity and forced expression of E-cadherin allows them to overcome dormancy.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC‐derived breast cancer cell line, designated CTC‐ITB‐01, established from a patient with metastatic estrogen receptor‐positive (ER + ) breast cancer, resistant to endocrine therapy. CTC‐ITB‐01 remained ER + in culture, and copy number alteration (CNA) profiling showed high concordance between CTC‐ITB‐01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA‐sequencing data indicate that CTC‐ITB‐01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER + breast cancer. Downstream ER signaling was constitutively active in CTC‐ITB‐01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC‐ITB‐01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology. Synopsis Blood‐born dissemination and subsequent outgrowth of tumor cells ‐ a process called metastasis ‐ is the leading cause of cancer‐related death. Cell lines derived from circulating tumor cells (CTCs) in blood of cancer patients provide excellent models to study the largely unknown biology of CTCs. The cell line established from CTCs of an estrogen receptor (ER)‐positive breast cancer patient mirrored the in situ CTCs and provided therefore a realistic model to investigate CTC biology. Xenograft experiments demonstrated a pattern of metastasis similar to ER‐positive breast cancer patients involving bone, liver and lung as secondary organs. Growth pattern and protein analyses revealed subtle signs of epithelial‐mesenchymal transition (EMT) with CTCs falling on the more epithelial end of the EMT spectrum. CTCs carried mutations in druggable genes relevant to cancer therapy (e.g., PIK3CA mutations). In vitro drug screening experiments indicated a cytostatic activity of the CDK4/6 inhibitor Palbociclib on CTCs. Graphical Abstract Blood‐born dissemination and subsequent outgrowth of tumor cells ‐ a process called metastasis ‐ is the leading cause of cancer‐related death. Cell lines derived from circulating tumor cells (CTCs) in blood of cancer patients provide excellent models to study the largely unknown biology of CTCs.

Oestrogen receptor α (ERα) is a transcription factor with ligand-independent and ligand-dependent activation functions (AF)-1 and -2. Oestrogens control postnatal mammary gland development acting on a subset of mammary epithelial cells (MECs), termed sensor cells, which are ERα-positive by immunohistochemistry (IHC) and secrete paracrine factors, which stimulate ERα-negative responder cells. Here we show that deletion of AF-1 or AF-2 blocks pubertal ductal growth and subsequent development because both are required for expression of essential paracrine mediators. Thirty percent of the luminal cells are ERα-negative by IHC but express Esr1 transcripts. This low level ERα expression through AF-2 is essential for cell expansion during puberty and growth-inhibitory during pregnancy. Cell-intrinsic ERα is not required for cell proliferation nor for secretory differentiation but controls transcript levels of cell motility and cell adhesion genes and a stem cell and epithelial mesenchymal transition (EMT) signature identifying ERα as a key regulator of mammary epithelial cell plasticity. Oestrogen receptors α (ERα) are expressed in a subset of mammary epithelial cells. Here, the authors identify cells with low-ERα protein levels and show that distinct cell populations have distinct requirements for the AF1 and AF2 domains of the ERα, and ERα acts in a biphasic manner dependent on developmental stage.

Invasive lobular carcinoma (ILC) is the most frequent special histological subtype of breast cancer, typically characterized by loss of E‐cadherin. It has clinical features distinct from other estrogen receptor‐positive (ER + ) breast cancers but the molecular mechanisms underlying its characteristic biology are poorly understood because we lack experimental models to study them. Here, we recapitulate the human disease, including its metastatic pattern, by grafting ILC‐derived breast cancer cell lines, SUM‐44 PE and MDA‐MB‐134‐VI cells, into the mouse milk ducts. Using patient‐derived intraductal xenografts from lobular and non‐lobular ER + HER2 − tumors to compare global gene expression, we identify extracellular matrix modulation as a lobular carcinoma cell‐intrinsic trait. Analysis of TCGA patient datasets shows matrisome signature is enriched in lobular carcinomas with overexpression of elastin, collagens, and the collagen modifying enzyme LOXL1 . Treatment with the pan LOX inhibitor BAPN and silencing of LOXL1 expression decrease tumor growth, invasion, and metastasis by disrupting ECM structure resulting in decreased ER signaling. We conclude that LOXL1 inhibition is a promising therapeutic strategy for ILC. Synopsis Intraductal xenografts of invasive lobular carcinoma (ILC) cells faithfully model this breast cancer subtype, and reveal tumor cell intrinsic ECM remodeling as a critical feature of disease progression that can be exploited therapeutically by targeting LOXL1. In vivo ILC models from MDA‐MB134 and SUM44 cell lines are developed and characterized. Additional ILC PDX models are established through the intraductal xenografting approach. Global gene expression profiling reveals ECM remodeling as a key tumor cell intrinsic ILC feature. ILC patient tumors show enhanced ECM production and LOXL1 expression. Inhibition of LOXL1 slows tumor progression in preclinical ILC models. Graphical Abstract Intraductal xenografts of invasive lobular carcinoma (ILC) cells faithfully model this breast cancer subtype, and reveal tumor cell intrinsic ECM remodeling as a critical feature of disease progression that can be exploited therapeutically by targeting LOXL1.

The energetic demands of proliferating cells during tumorigenesis require close coordination between the cell cycle and metabolism. While CDK4 is known for its role in cell proliferation, its metabolic function in cancer, particularly in triple-negative breast cancer (TNBC), remains unclear. Our study, using genetic and pharmacological approaches, reveals that CDK4 inactivation only modestly impacts TNBC cell proliferation and tumor formation. Notably, CDK4 depletion or long-term CDK4/6 inhibition confers resistance to apoptosis in TNBC cells. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERCs) formation, promoting mitochondrial fission and ER-mitochondrial calcium signaling, which are crucial for TNBC metabolic flexibility. Phosphoproteomic analysis identified CDK4’s role in regulating PKA activity at MERCs. In this work, we highlight CDK4’s role in mitochondrial apoptosis inhibition and suggest that targeting MERCs-associated metabolic shifts could enhance TNBC therapy. CDK4/6 inhibitors (CDK4/6i) have improved cancer patient outcomes but shown limited benefits for those with triple-negative breast cancer (TNBC). Here, the authors report that CDK4/6 inhibition prevents CDK4 enhanced mitochondria-endoplasmic reticulum interactions, inhibiting mitochondrial apoptosis and driving resistance to CDK4/6i in TNBC models.

Hormonal contraception exposes women to synthetic progesterone receptor (PR) agonists, progestins, and transiently increases breast cancer risk. How progesterone and progestins affect the breast epithelium is poorly understood because we lack adequate models to study this. We hypothesized that individual progestins differentially affect breast epithelial cell proliferation and hence breast cancer risk. Using mouse mammary tissue ex vivo, we show that testosterone‐related progestins induce the PR target and mediator of PR signaling‐induced cell proliferation receptor activator of NF‐κB ligand (Rankl), whereas progestins with anti‐androgenic properties in reporter assays do not. We develop intraductal xenografts of human breast epithelial cells from 36 women, show they remain hormone‐responsive and that progesterone and the androgenic progestins, desogestrel, gestodene, and levonorgestrel, promote proliferation but the anti‐androgenic, chlormadinone, and cyproterone acetate, do not. Prolonged exposure to androgenic progestins elicits hyperproliferation with cytologic changes. Androgen receptor inhibition interferes with PR agonist‐ and levonorgestrel‐induced RANKL expression and reduces levonorgestrel‐driven cell proliferation. Thus, different progestins have distinct biological activities in the breast epithelium to be considered for more informed choices in hormonal contraception. Synopsis Hormonal contraception exposes women to different progestins in conjunction or without estrogen. The androgenic properties of progestins determine their biological activity in the breast epithelium and reveal an unexpected role for AR activity in breast epithelial cell proliferation. Androgenic progestins induce expression of Rankl, an important mediator of PR signaling‐induced cell proliferation in the mammary epithelium, whereas anti‐androgenic progestins fail to do so. AR activity is required for the induction of Rankl transcripts. Human breast epithelial cells engraft and proliferate in mouse milk ducts maintaining nuclear hormone receptor expression and hormone responsiveness. Androgenic but not anti‐androgenic progestins promote cell proliferation in xenografted human breast epithelia. Prolonged exposure to androgenic progestins causes hyperproliferation and cytological changes associated with early premalignant lesions in xenografted human breast epithelia. Graphical Abstract Hormonal contraception exposes women to different progestins in conjunction or without estrogen. The androgenic properties of progestins determine their biological activity in the breast epithelium and reveal an unexpected role for AR activity in breast epithelial cell proliferation.

Invasive lobular carcinoma (ILC) accounts for up to 15% of all breast cancer (BC) cases and responds well to endocrine treatment when estrogen receptor α-positive (ER+) yet differs in many biological aspects from other ER+ BC subtypes. Up to 30% of patients with ILC will develop late-onset metastatic disease up to ten years after initial tumor diagnosis and may experience failure of systemic therapy. Unfortunately, preclinical models to study ILC progression and predict the efficacy of novel therapeutics are scarce. Here, we review the current advances in ILC modeling, including cell lines and organotypic models, genetically engineered mouse models, and patient-derived xenografts. We also underscore four critical challenges that can be addressed using ILC models: drug resistance, lobular tumor microenvironment, tumor dormancy, and metastasis. Finally, we highlight the advantages of shared experimental ILC resources and provide essential considerations from the perspective of the European Lobular Breast Cancer Consortium (ELBCC), which is devoted to better understanding and translating the molecular cues that underpin ILC to clinical diagnosis and intervention. This review will guide investigators who are considering the implementation of ILC models in their research programs.

Background Estrogen receptor α (ERα) signaling is a defining and driving event in most breast cancers; ERα is detected in malignant epithelial cells of 75% of all breast cancers (classified as ER-positive breast cancer) and, in these cases, ERα targeting is the main therapeutic strategy. However, the biological determinants of ERα heterogeneity and the mechanisms underlying therapeutic resistance are still elusive, hampered by the challenges in developing experimental models recapitulative of intra-tumoral heterogeneity and in which ERα signaling is sustained. Ex vivo cultures of human breast cancer tissue have been proposed to retain the original tissue architecture, epithelial and stromal cell components and ERα. However, loss of cellularity, viability and ERα expression are well-known culture-related phenomena. Methods BC samples were collected and brought to the laboratory. Then they were minced, enzymatically digested, entrapped in alginate and cultured for 1 month. The histological architecture, cellular composition and cell proliferation of tissue microstructures were assessed by immunohistochemistry. Cell viability was assessed by measurement of cell metabolic activity and histological evaluation. The presence of ERα was accessed by immunohistochemistry and RT-qPCR and its functionality evaluated by challenge with 17-β-estradiol and fulvestrant. Results We describe a strategy based on entrapment of breast cancer tissue microstructures in alginate capsules and their long-term culture under agitation, successfully applied to tissue obtained from 63 breast cancer patients. After 1 month in culture, the architectural features of the encapsulated tissue microstructures were similar to the original patient tumors: epithelial, stromal and endothelial compartments were maintained, with an average of 97% of cell viability compared to day 0. In ERα-positive cases, fibers of collagen, the main extracellular matrix component in vivo, were preserved. ERα expression was at least partially retained at gene and protein levels and response to ERα stimulation and inhibition was observed at the level of downstream targets, demonstrating active ER signaling. Conclusions The proposed model system is a new methodology to study ex vivo breast cancer biology, in particular ERα signaling. It is suitable for interrogating the long-term effects of anti-endocrine drugs in a set-up that closely resembles the original tumor microenvironment, with potential application in pre- and co-clinical assays of ERα-positive breast cancer.

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 preclinical 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 preclinical 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.