Experimental and spontaneous metastasis assays can result in divergence in clonal architecture.

Intratumoural heterogeneity is associated with poor outcomes in breast cancer. To understand how malignant clones survive and grow in metastatic niches, in vivo models using cell lines and patient-derived xenografts (PDX) have become the gold standard. Injections of cancer cells in orthotopic sites (spontaneous metastasis assays) or into the vasculature (experimental metastasis assays) have been used interchangeably to study the metastatic cascade from early events or post-intravasation, respectively. However, less is known about how these different routes of injection impact heterogeneity. Herein we directly compared the clonality of spontaneous and experimental metastatic assays using the human cell line MDA-MB-231 and a PDX model. Genetic barcoding was used to study the fitness of the subclones in primary and metastatic sites. Using spontaneous assays, we found that intraductal injections resulted in less diverse tumours compared to other routes of injections. Using experimental metastasis assays via tail vein injection of barcoded MDA-MB-231 cells, we also observed an asymmetry in metastatic heterogeneity between lung and liver that was not observed using spontaneous metastasis assays. These results demonstrate that these assays can result in divergent clonal outputs in terms of metastatic heterogeneity and provide a better understanding of the biases inherent to each technique.

Activation of the viral sensor oligoadenylate synthetase 2 (Oas2) prevents pregnancy-driven mammary cancer metastases.

BACKGROUND: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice. METHODS: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry. RESULTS: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present. CONCLUSIONS: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.

TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation.

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

Criteria-based curation of a therapy-focused compendium to support treatment recommendations in precision oncology.

While several resources exist that interpret therapeutic significance of genomic alterations in cancer, many regional real-world issues limit access to drugs. There is a need for a pragmatic, evidence-based, context-adapted tool to guide clinical management based on molecular biomarkers. To this end, we have structured a compendium of approved and experimental therapies with associated biomarkers following a survey of drug regulatory databases, existing knowledge bases, and published literature. Each biomarker-disease-therapy triplet was categorised using a tiering system reflective of key therapeutic considerations: approved and reimbursed therapies with respect to a jurisdiction (Tier 1), evidence of efficacy or approval in another jurisdiction (Tier 2), evidence of antitumour activity (Tier 3), and plausible biological rationale (Tier 4). Two resistance categories were defined: lack of efficacy (Tier R1) or antitumor activity (Tier R2). Based on this framework, we curated a digital resource focused on drugs relevant in the Australian healthcare system (TOPOGRAPH: Therapy Oriented Precision Oncology Guidelines for Recommending Anticancer Pharmaceuticals). As of November 2020, TOPOGRAPH comprised 2810 biomarker-disease-therapy triplets in 989 expert-appraised entries, including 373 therapies, 199 biomarkers, and 106 cancer types. In the 345 therapies catalogued, 84 (24%) and 65 (19%) were designated Tiers 1 and 2, respectively, while 271 (79%) therapies were supported by preclinical studies, early clinical trials, retrospective studies, or case series (Tiers 3 and 4). A companion algorithm was also developed to support rational, context-appropriate treatment selection informed by molecular biomarkers. This framework can be readily adapted to build similar resources in other jurisdictions to support therapeutic decision-making.

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype.

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Emergence of Enzalutamide Resistance in Prostate Cancer is Associated with BCL-2 and IKKB Dependencies.

PURPOSE: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. EXPERIMENTAL DESIGN: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. RESULTS: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. CONCLUSIONS: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.

MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

The Proliferative and Apoptotic Landscape of Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is an aggressive molecular subtype that represents up to 15% of breast cancers. It occurs in younger patients, and typically shows rapid development of locoregional and distant metastasis, resulting in a relatively high mortality rate. Its defining features are that it is positive for basal cytokeratins and, epidermal growth factor receptor and/or c-Kit. Problematically, it is typically negative for the estrogen receptor and human epidermal growth factor receptor 2 (HER2), which means that it is unsuitable for either hormone therapy or targeted HER2 therapy. As a result, there are few therapeutic options for BLBC, and a major priority is to define molecular subgroups of BLBC that could be targeted therapeutically. In this review, we focus on the highly proliferative and anti-apoptotic phenotype of BLBC with the goal of defining potential therapeutic avenues, which could take advantage of these aspects of tumor development.

Single-Cell Transcriptomics in Cancer Immunobiology: The Future of Precision Oncology.

Cancer is a heterogeneous and complex disease. Tumors are formed by cancer cells and a myriad of non-cancerous cell types that together with the extracellular matrix form the tumor microenvironment. These cancer-associated cells and components contribute to shape the progression of cancer and are deeply involved in patient outcome. The immune system is an essential part of the tumor microenvironment, and induction of cancer immunotolerance is a necessary step involved in tumor formation and growth. Immune mechanisms are intimately associated with cancer progression, invasion, and metastasis; as well as to tumor dormancy and modulation of sensitivity to drug therapy. Transcriptome analyses have been extensively used to understand the heterogeneity of tumors, classifying tumors into molecular subtypes and establishing signatures that predict response to therapy and patient outcomes. However, the classification of the tumor cell diversity and specially the identification of rare populations has been limited in these transcriptomic analyses of bulk tumor cell populations. Massively-parallel single-cell RNAseq analysis has emerged as a powerful method to unravel heterogeneity and to study rare cell populations in cancer, through unsupervised sampling and modeling of transcriptional states in single cells. In this context, the study of the role of the immune system in cancer would benefit from single cell approaches, as it will enable the characterization and/or discovery of the cell types and pathways involved in cancer immunotolerance otherwise missed in bulk transcriptomic information. Thus, the analysis of gene expression patterns at single cell resolution holds the potential to provide key information to develop precise and personalized cancer treatment including immunotherapy. This review is focused on the latest single-cell RNAseq methodologies able to agnostically study thousands of tumor cells as well as targeted single-cell RNAseq to study rare populations within tumors. In particular, we will discuss methods to study the immune system in cancer. We will also discuss the current challenges to the study of cancer at the single cell level and the potential solutions to the current approaches.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy.

Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

Myeloid cell leukemia 1 (MCL-1), an unexpected modulator of protein kinase signaling during invasion.

Myeloid cell leukemia-1 (MCL-1), closely related to B-cell lymphoma 2 (BCL-2), has a well-established role in cell survival and has emerged as an important target for cancer therapeutics. We have demonstrated that inhibiting MCL-1 is efficacious in suppressing tumour progression in pre-clinical models of breast cancer and revealed that in addition to its role in cell survival, MCL-1 modulated cellular invasion. Utilizing a MCL-1-specific genetic antagonist, we found two possible mechanisms; firstly MCL-1 directly binds to and alters the phosphorylation of the cytoskeletal remodeling protein, Cofilin, a protein important for cytoskeletal remodeling during invasion, and secondly MCL-1 modulates the levels SRC family kinases (SFKs) and their targets. These data provide evidence that MCL-1 activities are not limited to endpoints of extracellular and intracellular signaling culminating in cell survival as previously thought, but can directly modulate the output of SRC family kinases signaling during cellular invasion. Here we review the pleotropic roles of MCL-1 and discuss the implications of this newly discovered effect on protein kinase signaling for the development of cancer therapeutics.

Andy's Algorithms: new automated digital image analysis pipelines for FIJI.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Andy's Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. Andy's algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy's Algorithms are available at GitHub, publicly accessed at https://github.com/andlaw1841/Andy-s-Algorithm .

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.

Grid therapy using high definition multileaf collimators: realizing benefits of the bystander effect.

BACKGROUND: In microbeam radiotherapy (MRT), parallel arrays of high-intensity synchrotron x-ray beams achieve normal tissue sparing without compromising tumor control. Grid-therapy using clinical linacs has spatial modulation on a larger scale and achieves promising results for palliative treatments of bulky tumors. The availability of high definition multileaf collimators (HDMLCs) with 2.5 mm leaves provides an opportunity for grid-therapy to more closely approach MRT. However, challenges to the wider implementation of grid-therapy remain because spatial modulation of the target volume runs counter to current radiotherapy practice and mechanisms for the beneficial effects of MRT are not fully understood. Without more knowledge of cell dose responses, a quantitative basis for planning treatments is difficult. The aim of this study is to determine if therapeutic benefits of MRT can be achieved using a linac with HDMLCs and if so, to develop a predictive model to support treatment planning. MATERIAL AND METHODS: HD120-MLCs of a Varian Novalis TXTM were used to generate grid patterns of 2.5 and 5.0 mm spacing, which were characterized dosimetrically using GafchromicTM EBT3 film. Clonogenic survival of normal (HUVEC) and cancer (NCI-H460, HCC-1954) cell lines following irradiation under the grid and open fields using a 6 MV photon beam were compared in-vitro for the same average dose. RESULTS AND CONCLUSIONS: Relative to an open field, survival of normal cells in a 2.5 mm striped field was the same, while the survival of both cancer cell lines was significantly lower. A mathematical model was developed to incorporate dose gradients of the spatial modulation into the standard linear quadratic model. Our new bystander extended LQ model assumes spatial gradients drive the diffusion of soluble factors that influence survival through bystander effects, successfully predicting the experimental results that show an increased therapeutic ratio. Our results challenge conventional radiotherapy practice and propose that additional gain can be realized by prescribing spatially modulated treatments to harness the bystander effect.

MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib.

BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

ELF5 isoform expression is tissue-specific and significantly altered in cancer.

BACKGROUND: E74-like factor 5 (ELF5) is an epithelial-specific member of the E26 transforming sequence (ETS) transcription factor family and a critical regulator of cell fate in the placenta, pulmonary bronchi, and milk-producing alveoli of the mammary gland. ELF5 also plays key roles in malignancy, particularly in basal-like and endocrine-resistant forms of breast cancer. Almost all genes undergo alternative transcription or splicing, which increases the diversity of protein structure and function. Although ELF5 has multiple isoforms, this has not been considered in previous studies of ELF5 function. METHODS: RNA-sequencing data for 6757 samples from The Cancer Genome Atlas were analyzed to characterize ELF5 isoform expression in multiple normal tissues and cancers. Extensive in vitro analysis of ELF5 isoforms, including a 116-gene quantitative polymerase chain reaction panel, was performed in breast cancer cell lines. RESULTS: ELF5 isoform expression was found to be tissue-specific due to alternative promoter use but altered in multiple cancer types. The normal breast expressed one main isoform, while in breast cancer there were subtype-specific alterations in expression. Expression of other ETS factors was also significantly altered in breast cancer, with the basal-like subtype demonstrating a distinct ETS expression profile. In vitro inducible expression of the full-length isoforms 1 and 2, as well as isoform 3 (lacking the Pointed domain) had similar phenotypic and transcriptional effects. CONCLUSIONS: Alternative promoter use, conferring differential regulatory responses, is the main mechanism governing ELF5 action rather than differential transcriptional activity of the isoforms. This understanding of expression and function at the isoform level is a vital first step in realizing the potential of transcription factors such as ELF5 as prognostic markers or therapeutic targets in cancer.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

Acquired convergence of hormone signaling in breast cancer: ER and PR transition from functionally distinct in normal breast to predictors of metastatic disease.

Cumulative exposure to estrogen (E) and progesterone (P) over the menstrual cycle significantly influences the risk of developing breast cancer. Despite the dogma that PR in the breast merely serves as a marker of an active estrogen receptor (ER), and as an inhibitor of the proliferative actions of E, it is now clear that in the breast P increases proliferation independently of E action. We show here that the progesterone receptor (PR) and ER are expressed in different epithelial populations, and target non-overlapping pathways in the normal human breast. In breast cancer, PR becomes highly correlated with ER, and this convergence is associated with signaling pathways predictive of disease metastasis. These data challenge the established paradigm that ER and PR function co-operatively in normal breast, and have significant implications not only for our understanding of normal breast biology, but also for diagnosis, prognosis and/or treatment options in breast cancer patients.

The mammary cellular hierarchy and breast cancer.

Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer.