Tumor dormancy: EMT beyond invasion and metastasis.

More than two-thirds of cancer-related deaths are attributable to metastases. In some tumor types metastasis can occur up to 20 years after diagnosis and successful treatment of the primary tumor, a phenomenon termed late recurrence. Metastases arise from disseminated tumor cells (DTCs) that leave the primary tumor early on in tumor development, either as single cells or clusters, adapt to new environments, and reduce or shut down their proliferation entering a state of dormancy for prolonged periods of time. Dormancy has been difficult to track clinically and study experimentally. Recent advances in technology and disease modeling have provided new insights into the molecular mechanisms orchestrating dormancy and the switch to a proliferative state. A new role for epithelial-mesenchymal transition (EMT) in inducing plasticity and maintaining a dormant state in several cancer models has been revealed. In this review, we summarize the major findings linking EMT to dormancy control and highlight the importance of pre-clinical models and tumor/tissue context when designing studies. Understanding of the cellular and molecular mechanisms controlling dormant DTCs is pivotal in developing new therapeutic agents that prevent distant recurrence by maintaining a dormant state.

The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells.

Stem-like cells may be integral to the development and maintenance of human cancers. Direct proof is still lacking, mainly because of our poor understanding of the biological differences between normal and cancer stem cells (SCs). Using the ErbB2 transgenic model of breast cancer, we found that self-renewing divisions of cancer SCs are more frequent than their normal counterparts, unlimited and symmetric, thus contributing to increasing numbers of SCs in tumoral tissues. SCs with targeted mutation of the tumor suppressor p53 possess the same self-renewal properties as cancer SCs, and their number increases progressively in the p53 null premalignant mammary gland. Pharmacological reactivation of p53 correlates with restoration of asymmetric divisions in cancer SCs and tumor growth reduction, without significant effects on additional cancer cells. These data demonstrate that p53 regulates polarity of cell division in mammary SCs and suggest that loss of p53 favors symmetric divisions of cancer SCs, contributing to tumor growth.

C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells.

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.

Membrane expression of the estrogen receptor ERα is required for intercellular communications in the mammary epithelium.

17ß-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ERα. ERα acts as a transcription factor but also elicits rapid signaling through a fraction of ERα expressed at the membrane. Here, we have used the C451A-ERα mouse model mutated for the palmitoylation site to understand how ERα membrane signaling affects mammary gland development. Although the overall structure of physiological mammary gland development is slightly affected, both epithelial fragments and basal cells isolated from C451A-ERα mammary glands failed to grow when engrafted into cleared wild-type fat pads, even in pregnant hosts. Similarly, basal cells purified from hormone-stimulated ovariectomized C451A-ERα mice did not produce normal outgrowths. Ex vivo, C451A-ERα basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ERα basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ERα-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ERα expression in promoting intercellular communications that are essential for mammary gland development.

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.

Deep Learning Enables Individual Xenograft Cell Classification in Histological Images by Analysis of Contextual Features.

Patient-Derived Xenografts (PDXs) are the preclinical models which best recapitulate inter- and intra-patient complexity of human breast malignancies, and are also emerging as useful tools to study the normal breast epithelium. However, data analysis generated with such models is often confounded by the presence of host cells and can give rise to data misinterpretation. For instance, it is important to discriminate between xenografted and host cells in histological sections prior to performing immunostainings. We developed Single Cell Classifier (SCC), a data-driven deep learning-based computational tool that provides an innovative approach for automated cell species discrimination based on a multi-step process entailing nuclei segmentation and single cell classification. We show that human and murine cell contextual features, more than cell-intrinsic ones, can be exploited to discriminate between cell species in both normal and malignant tissues, yielding up to 96% classification accuracy. SCC will facilitate the interpretation of H&E- and DAPI-stained histological sections of xenografted human-in-mouse tissues and it is open to new in-house built models for further applications. SCC is released as an open-source plugin in ImageJ/Fiji available at the following link: https://github.com/Biomedical-Imaging-Group/SingleCellClassifier .

Two-Tier Mapper, an unbiased topology-based clustering method for enhanced global gene expression analysis.

MOTIVATION: Unbiased clustering methods are needed to analyze growing numbers of complex datasets. Currently available clustering methods often depend on parameters that are set by the user, they lack stability, and are not applicable to small datasets. To overcome these shortcomings we used topological data analysis, an emerging field of mathematics that discerns additional feature and discovers hidden insights on datasets and has a wide application range. RESULTS: We have developed a topology-based clustering method called Two-Tier Mapper (TTMap) for enhanced analysis of global gene expression datasets. First, TTMap discerns divergent features in the control group, adjusts for them, and identifies outliers. Second, the deviation of each test sample from the control group in a high-dimensional space is computed, and the test samples are clustered using a new Mapper-based topological algorithm at two levels: a global tier and local tiers. All parameters are either carefully chosen or data-driven, avoiding any user-induced bias. The method is stable, different datasets can be combined for analysis, and significant subgroups can be identified. It outperforms current clustering methods in sensitivity and stability on synthetic and biological datasets, in particular when sample sizes are small; outcome is not affected by removal of control samples, by choice of normalization, or by subselection of data. TTMap is readily applicable to complex, highly variable biological samples and holds promise for personalized medicine. AVAILABILITY AND IMPLEMENTATION: TTMap is supplied as an R package in Bioconductor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

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.

In vivo reprogramming of non-mammary cells to an epithelial cell fate is independent of amphiregulin signaling.

Amphiregulin (AREG)-/- mice demonstrate impaired mammary development and form only rudimentary ductal epithelial trees; however, AREG-/- glands are still capable of undergoing alveologenesis and lactogenesis during pregnancy. Transplantation of AREG-/- mammary epithelial cells into cleared mouse mammary fat pads results in a diminished capacity for epithelial growth (∼15%) as compared to that of wild-type mammary epithelial cells. To determine whether estrogen receptor α (ERα, also known as ESR1) and/or AREG signaling were necessary for non-mammary cell redirection, we inoculated either ERα-/- or AREG-/- mammary cells with non-mammary progenitor cells (WAP-Cre/Rosa26LacZ+ male testicular cells or GFP-positive embryonic neuronal stem cells). ERα-/- cells possessed a limited ability to grow or reprogram non-mammary cells in transplanted mammary fat pads. AREG-/- mammary cells were capable of redirecting both types of non-mammary cell populations to mammary phenotypes in regenerating mammary outgrowths. Transplantation of fragments from AREG-reprogrammed chimeric outgrowths resulted in secondary outgrowths in six out of ten fat pads, demonstrating the self-renewing capacity of the redirected non-mammary cells to contribute new progeny to chimeric outgrowths. Nestin was detected at the leading edges of developing alveoli, suggesting that its expression may be essential for lobular expansion.

Control of hair follicle cell fate by underlying mesenchyme through a CSL-Wnt5a-FoxN1 regulatory axis.

Epithelial-mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jkappa gene, the effector of "canonical" Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch-CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.

Patient-derived xenograft (PDX) models in basic and translational breast cancer research.

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.

ID4 regulates mammary gland development by suppressing p38MAPK activity.

The ID family of helix-loop-helix proteins regulates cell proliferation and differentiation in many different developmental pathways, but the functions of ID4 in mammary development are unknown. We report that mouse Id4 is expressed in cap cells, basal cells and in a subset of luminal epithelial cells, and that its targeted deletion impairs ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone. We discover that p38MAPK is activated in Id4-null mammary cells. p38MAPK is also activated following siRNA-mediated Id4 knockdown in transformed mammary cells. This p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands. Therefore, ID4 promotes mammary gland development by suppressing p38MAPK activity.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1.

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

Does cancer start in the womb? altered mammary gland development and predisposition to breast cancer due to in utero exposure to endocrine disruptors.

We are now witnessing a resurgence of theories of development and carcinogenesis in which the environment is again being accepted as a major player in phenotype determination. Perturbations in the fetal environment predispose an individual to disease that only becomes apparent in adulthood. For example, gestational exposure to diethylstilbestrol resulted in clear cell carcinoma of the vagina and breast cancer. In this review the effects of the endocrine disruptor bisphenol-A (BPA) on mammary development and tumorigenesis in rodents is used as a paradigmatic example of how altered prenatal mammary development may lead to breast cancer in humans who are also widely exposed to it through plastic goods, food and drink packaging, and thermal paper receipts. Changes in the stroma and its extracellular matrix led to altered ductal morphogenesis. Additionally, gestational and lactational exposure to BPA increased the sensitivity of rats and mice to mammotropic hormones during puberty and beyond, thus suggesting a plausible explanation for the increased incidence of breast cancer.

Is There a Special Role for Ovarian Hormones in the Pathogenesis of Lobular Carcinoma?

Lobular carcinoma represent the most common special histological subtype of breast cancer, with the majority classed as hormone receptor positive. Rates of invasive lobular carcinoma in postmenopausal women have been seen to increase globally, while other hormone receptor-positive breast cancers proportionally have not followed the same trend. This has been linked to exposure to exogenous ovarian hormones such as hormone replacement therapy. Reproductive factors resulting in increased lifetime exposure to endogenous ovarian hormones have also been linked to an increased risk of lobular breast cancer, and taken together, these data make a case for the role of ovarian hormones in the genesis and progression of the disease. In this review, we summarize current understanding of the epidemiological associations between ovarian hormones and lobular breast cancer and highlight mechanistic links that may underpin the etiology and biology.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

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.

Targeting the Progesterone Receptor in Breast Cancer: Mind the Short Form!

SUMMARY: The presurgical window of opportunity trial (WOT) MIPRA provides evidence that neoadjuvant treatment with the progesterone receptor (PR) antagonist mifepristone (RU486) may benefit patients with estrogen receptor-positive (ER+) breast cancer characterized by a high ratio of PR-A versus PR-B isoform (>1.5), suggesting that PR may be targeted in a subset of patients. See related article by Elía et al., p. 866.

Optimized Modeling of Metastatic Triple-Negative Invasive Lobular Breast Carcinoma.

Invasive lobular carcinoma (ILC) is a common breast cancer subtype that is often diagnosed at advanced stages and causes significant morbidity. Late-onset secondary tumor recurrence affects up to 30% of ILC patients, posing a therapeutic challenge if resistance to systemic therapy develops. Nonetheless, there is a lack of preclinical models for ILC, and the current models do not accurately reproduce the complete range of the disease. We created clinically relevant metastatic xenografts to address this gap by grafting the triple-negative IPH-926 cell line into mouse milk ducts. The resulting intraductal xenografts accurately recapitulate lobular carcinoma in situ (LCIS), invasive lobular carcinoma, and metastatic ILC in relevant organs. Using a panel of 15 clinical markers, we characterized the intratumoral heterogeneity of primary and metastatic lesions. Interestingly, intraductal IPH-926 xenografts express low but actionable HER2 and are not dependent on supplementation with the ovarian hormone estradiol for their growth. This model provides a valuable tool to test the efficiency of potential new ILC therapeutics, and it may help detect vulnerabilities within ILC that can be exploited for therapeutic targeting.