RNA sequencing-based single sample predictors of molecular subtype and risk of recurrence for clinical assessment of early-stage breast cancer.

Multigene assays for molecular subtypes and biomarkers can aid management of early invasive breast cancer. Using RNA-sequencing we aimed to develop single-sample predictor (SSP) models for clinical markers, subtypes, and risk of recurrence (ROR). A cohort of 7743 patients was divided into training and test set. We trained SSPs for subtypes and ROR assigned by nearest-centroid (NC) methods and SSPs for biomarkers from histopathology. Classifications were compared with Prosigna in two external cohorts (ABiM, n = 100 and OSLO2-EMIT0, n = 103). Prognostic value was assessed using distant recurrence-free interval. Agreement between SSP and NC for PAM50 (five subtypes) was high (85%, Kappa = 0.78) for Subtype (four subtypes) very high (90%, Kappa = 0.84) and for ROR risk category high (84%, Kappa = 0.75, weighted Kappa = 0.90). Prognostic value was assessed as equivalent and clinically relevant. Agreement with histopathology was very high or high for receptor status, while moderate for Ki67 status and poor for Nottingham histological grade. SSP and Prosigna concordance was high for subtype (OSLO-EMIT0 83%, Kappa = 0.73 and ABiM 80%, Kappa = 0.72) and moderate and high for ROR risk category (68 and 84%, Kappa = 0.50 and 0.70, weighted Kappa = 0.70 and 0.78). Pooled concordance for emulated treatment recommendation dichotomized for chemotherapy was high (85%, Kappa = 0.66). Retrospective evaluation suggested that SSP application could change chemotherapy recommendations for up to 17% of postmenopausal ER+/HER2-/N0 patients with balanced escalation and de-escalation. Results suggest that NC and SSP models are interchangeable on a group-level and nearly so on a patient level and that SSP models can be derived to closely match clinical tests.

Estrogen receptor positive breast cancers have patient specific hormone sensitivities and rely on progesterone receptor.

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-ß-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.

Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1.

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.

Intraductal patient-derived xenografts of estrogen receptor α-positive breast cancer recapitulate the histopathological spectrum and metastatic potential of human lesions.

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.

Oestrogen receptor α AF-1 and AF-2 domains have cell population-specific functions in the mammary epithelium.

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.

A Preclinical Model for ERα-Positive Breast Cancer Points to the Epithelial Microenvironment as Determinant of Luminal Phenotype and Hormone Response.

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 TGFß/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.

Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk.

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.

Progesterone/RANKL is a major regulatory axis in the human breast.

Estrogens and progesterones are major drivers of breast development but also promote carcinogenesis in this organ. Yet, their respective roles and the mechanisms underlying their action in the human breast are unclear. Receptor activator of nuclear factor κB ligand (RANKL) has been identified as a pivotal paracrine mediator of progesterone function in mouse mammary gland development and mammary carcinogenesis. Whether the factor has the same role in humans is of clinical interest because an inhibitor for RANKL, denosumab, is already used for the treatment of bone disease and might benefit breast cancer patients. We show that progesterone receptor (PR) signaling failed to induce RANKL in PR(+) breast cancer cell lines and in dissociated, cultured breast epithelial cells. In clinical specimens from healthy donors and intact breast tissue microstructures, hormone response was maintained and RANKL expression was under progesterone control, which increased RNA stability. RANKL was sufficient to trigger cell proliferation and was required for progesterone-induced proliferation. The findings were validated in vivo where RANKL protein expression in the breast epithelium correlated with serum progesterone levels and the protein was expressed in a subset of luminal cells that express PR. Thus, important hormonal control mechanisms are conserved across species, making RANKL a potential target in breast cancer treatment and prevention.

Perinatal exposure to bisphenol a increases adult mammary gland progesterone response and cell number.

Bisphenol A [BPA, 2,2,-bis (hydroxyphenyl) propane] is one of the highest-volume chemicals produced worldwide. It is detected in body fluids of more than 90% of the human population. Originally synthesized as an estrogenic compound, it is currently utilized to manufacture food and beverage containers resulting in uptake with food and drinks. There is concern that exposure to low doses of BPA, defined as less than or equal to 5 mg/kg body weight /d, may have developmental effects on various hormone-responsive organs including the mammary gland. Here, we asked whether perinatal exposure to a range of low doses of BPA is sufficient to alter mammary gland hormone response later on in life, with a possible impact on breast cancer risk. To mimic human exposure, we added BPA to the drinking water of C57/Bl6 breeding pairs. Analysis of the mammary glands of their daughters at puberty showed that estrogen-dependent transcriptional events were perturbed and the number of terminal end buds, estrogen-induced proliferative structures, was altered in a dose-dependent fashion. Importantly, adult females showed an increase in mammary epithelial cell numbers comparable to that seen in females exposed to diethylbestrol, a compound exposure to which was previously linked to increased breast cancer risk. Molecularly, the mRNAs encoding Wnt-4 and receptor activator of nuclear factor κB ligand, two key mediators of hormone function implicated in control of mammary stem cell proliferation and carcinogenesis, showed increased induction by progesterone in the mammary tissue of exposed mice. Thus, perinatal exposure to environmentally relevant doses of BPA alters long-term hormone response that may increase the propensity to develop breast cancer.

Epithelial-mesenchymal transition-derived cells exhibit multilineage differentiation potential similar to mesenchymal stem cells.

The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-beta in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44(+), CD24(-), and CD45(-). Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-beta), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.

Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland.

The mouse mammary gland develops postnatally under the control of female reproductive hormones. Estrogens and progesterone trigger morphogenesis by poorly understood mechanisms acting on a subset of mammary epithelial cells (MECs) that express their cognate receptors, estrogen receptor alpha (ERalpha) and progesterone receptor (PR). Here, we show that in the adult female, progesterone drives proliferation of MECs in two waves. The first, small wave, encompasses PR(+) cells and requires cyclin D1, the second, large wave, comprises mostly PR(-) cells and relies on the tumor necrosis factor (TNF) family member, receptor activator of NF-kappaB-ligand (RANKL). RANKL elicits proliferation by a paracrine mechanism. Ablation of RANKL in the mammary epithelium blocks progesterone-induced morphogenesis, and ectopic expression of RANKL in MECs completely rescues the PR(-/-) phenotype. Systemic administration of RANKL triggers proliferation in the absence of PR signaling, and injection of a RANK signaling inhibitor interferes with progesterone-induced proliferation. Thus, progesterone elicits proliferation by a cell-intrinsic and a, more important, paracrine mechanism.

The epithelial-mesenchymal transition generates cells with properties of stem cells.

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Prolactin signaling and Stat5: going their own separate ways?

Miyoshi et al. compared the role of the prolactin receptor (PrlR) and its downstream mediator, the signal transducer and activator of transcription 5 (Stat5), in mammary epithelial cells in vivo by studying PrlR-/- and Stat5ab-/- mouse mammary epithelial transplants during pregnancy. At first glance, the two mutant epithelia appear to have similar defects in the differentiation of the alveolar epithelium. However, a closer examination by Miyoshi et al. revealed defects in the epithelial architecture of the smallest ducts of Stat5ab-/- transplants not apparent in the PrlR-/- transplants, suggesting that Stat5 is more than a simple mediator of PrlR action.