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

The innate and adaptive infiltrating immune systems as targets for breast cancer immunotherapy.

A cancer cell-centric view has long dominated the field of cancer biology. Research efforts have focussed on aberrant cancer cell signalling pathways and on changes to cancer cell DNA. Mounting evidence demonstrates that many cancer-associated cell types within the tumour stroma co-evolve and support tumour growth and development, greatly modifying cancer cell behaviour, facilitating invasion and metastasis and controlling dormancy and sensitivity to drug therapy. Thus, these stromal cells represent potential targets for cancer therapy. Among these cell types, immune cells have emerged as a promising target for therapy. The adaptive and the innate immune system play an important role in normal mammary development and breast cancer. The number of infiltrating adaptive immune system cells with tumour-rejecting capacity, primarily, T lymphocytes, is lower in breast cancer compared with other cancer types, but infiltration occurs in a large proportion of cases. There is strong evidence demonstrating the importance of the immunosuppressive role of the innate immune system during breast cancer progression. A consideration of components of both the innate and the adaptive immune system is essential for the design and development of immunotherapies in breast cancer. In this review, we focus on the importance of immunosuppressive myeloid-derived suppressor cells (MDSCs) as potential targets for breast cancer therapy

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

Y4 receptor knockout rescues fertility in ob/ob mice

Hypothalamic neuropeptide Y (NPY) has been implicated in the regulation of energy balance and reproduction, and chronically elevated NPY levels in the hypothalamus are associated with obesity and reduced reproductive function. However, it is not known which one of the five cloned Y receptors mediates these effects. Here we show that crossing the Y4 receptor knockout mouse (Y4−/−) onto the ob/ob background restores the reduced plasma testosterone levels of ob/ob mice as well as the reduced testis and seminal vesicle size and morphology to control values. Fertility in the sterile ob/ob mice was greatly improved by Y4 receptor deletion, with 100% of male and 50% of female Y4−/−,ob/ob double knockout mice producing live offspring. Development of the mammary ducts and lobuloalveoli was significantly enhanced in pregnant Y4−/− and Y4−/−,ob/ob females. Consistent with the improved fertility and enhanced mammary gland development, gonadotropin releasing hormone (GnRH) expression was significantly increased in Y4−/− and Y4−/−,ob/ob animals. Y4−/− mice displayed lower body weight and reduced white adipose tissue mass accompanied by increased plasma levels of pancreatic polypeptide (PP). However, Y4 deficiency had no beneficial effects to reduce body weight or excessive adiposity of ob/ob mice. These data suggest that central Y4 receptor signaling specifically inhibits reproductive function under conditions of elevated central NPY-ergic tonus

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

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

Gata-3 and mammary cell fate

Genomic regulatory networks specify how cellular gene expression responds to external temporal and spatial stimuli, ensuring that correct cell fate decisions are made and the appropriate cell phenotypes are adopted. In mammary epithelial cells, the hierarchy of stem and progenitor cells and the genetically specified program of transcriptional activity are beginning to be elucidated and integrated. A novel role for Gata-3 in specifying and maintaining mammary cell fate has recently been identified. These reports offer an understanding of how mammary cells assume and maintain a variety of cell behaviours and functions, and how a mammary cell may potentially subvert these constraints during carcinogenesis

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

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