CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells

INTRODUCTION: Breast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells. METHODS: Mouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation. RESULTS: Three populations of CD24 expressing cells were identified: CD24(Negative), CD24(Low )and CD24(High). Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24(Low )cells, whilst CD24(High )cells have limited repopulation capacity. CONCLUSION: Differential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24(Low )population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential

CCAAT/enhancer binding proteins in normal mammary development and breast cancer

CCAAT/enhancer binding proteins (C/EBPs) are a family of leucine zipper, transcription factors that bind to DNA as homodimers and heterodimers. They regulate cellular proliferation, differentiation and apoptosis in the mammary gland. Multiple protein isoforms, including truncated, dominant negatives, are generated by translation of the C/EBPβ transcript or via proteolytic cleavage of the full-length C/EBPβ protein. Gene deletion of individual C/EBP family members has demonstrated an essential role for C/EBPβ in normal mammary development, while transgenic and overexpression studies provide evidence that the dominant-negative C/EBPβ-liver-enriched inhibitory protein isoform induces proliferation in mammary epithelial cells. Mounting evidence suggests that alterations in the ratio of the C/EBPβ-liver-enriched inhibitory protein isoform and the C/EBPβ-liver-enriched activating protein isoform may play a role in the development of breast cancer. This review will consequently focus on C/EBP actions in normal mammary development and on the emerging data that supports a role in breast cancer

Comprehensive characterization of 536 patient-derived xenograft models prioritizes candidatesfor targeted treatment

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors

Stem cells in normal breast development and breast cancer

The main focus of this review is the role of mammary stem cells in normal breast development and carcinogenesis. We have developed a new in vitro culture system that permits, for the first time, the propagation of mammary stem and progenitor cells in an undifferentiated state, which should facilitate the elucidation of pathways that regulate normal mammary stem-cell self-renewal and differentiation. Furthermore, we propose a model in which transformation of stem cells, or early progenitor cells, results in carcinogenesis. A key event in this process is the deregulation of normal self-renewal in these cells. Transformed mammary stem or progenitor cells undergo aberrant differentiation processes that result in generation of the phenotypic heterogeneity found in human and rodent breast cancers. This phenotypic diversity is driven by a small subset of mammary tumour stem cells. We will discuss the important implications of this mammary tumour stem-cell model.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72182/1/j.1365-2184.36.s.1.6.x.pd

Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor-Positive Breast Cancer.

UNLABELLED: Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated β2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of β2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer. SIGNIFICANCE: Adding SMAC mimetics to endocrine therapy enhances tumor regression in a cell autonomous manner while increasing tumor immunogenicity, indicating that this combination could be an effective treatment for HR+ patients with breast cancer

Keratin 6 is not essential for mammary gland development

INTRODUCTION: Keratin 6 (K6) has previously been identified as a marker of early mammary gland development and has also been proposed to be a marker of mammary gland progenitor cells. However, the function of K6 in the mammary gland was not known, so we examined the expression pattern of the protein during both embryonic and postnatal mammary development, as well as the mammary gland phenotype of mice that were null for both K6a and K6b isoforms. METHOD: Immunostaining was performed to determine the expression pattern of K6a throughout mammary gland development, from the embryonic mammary bud to lactation. Double immunofluorescence was used to co-localize K6 with known markers of mammary gland development. Wild-type and K6ab-null mammary tissues were transplanted into the cleared fat pads of nude mice and the outgrowths were analyzed for morphology by whole-mount staining and for markers of mammary epithelium by immunostaining. Finally, progesterone receptor (PR) and bromodeoxyuridine co-localization was quantified by double immunofluorescence in wild-type and K6ab-null mammary outgrowths. RESULTS: Here we report that K6 is expressed earlier than described previously, by embryonic day 16.5. K6a is the predominant isoform expressed in the mammary gland, localized in the body cells and luminal epithelial cells but not in the cap cells or myoepithelial cells. Co-localization studies showed that most K6a-positive cells express steroid receptors but do not proliferate. When both the K6a and K6b genes are deleted, mammary gland development appears normal, with similar expression of most molecular markers examined in both the pubertal gland and the mature gland. Loss of K6a and K6b, however, leads to an increase in the number of steroid-receptor-positive cells, and increased co-localization of steroid receptor expression and proliferation was observed. CONCLUSION: Although K6a was not essential for mammary gland development, loss of both K6a and K6b resulted in an increase in PR-positive mammary epithelial cells and decreased proliferation after exposure to steroid hormones. There was also increased co-localization of PR and bromodeoxyuridine, suggesting alterations in patterning events important for normal lobuloalveolar development

Predicting cancer involvement of genes from heterogeneous data

<p>Abstract</p> <p>Background</p> <p>Systematic approaches for identifying proteins involved in different types of cancer are needed. Experimental techniques such as microarrays are being used to characterize cancer, but validating their results can be a laborious task. Computational approaches are used to prioritize between genes putatively involved in cancer, usually based on further analyzing experimental data.</p> <p>Results</p> <p>We implemented a systematic method using the PIANA software that predicts cancer involvement of genes by integrating heterogeneous datasets. Specifically, we produced lists of genes likely to be involved in cancer by relying on: (i) protein-protein interactions; (ii) differential expression data; and (iii) structural and functional properties of cancer genes. The integrative approach that combines multiple sources of data obtained positive predictive values ranging from 23% (on a list of 811 genes) to 73% (on a list of 22 genes), outperforming the use of any of the data sources alone. We analyze a list of 20 cancer gene predictions, finding that most of them have been recently linked to cancer in literature.</p> <p>Conclusion</p> <p>Our approach to identifying and prioritizing candidate cancer genes can be used to produce lists of genes likely to be involved in cancer. Our results suggest that differential expression studies yielding high numbers of candidate cancer genes can be filtered using protein interaction networks. </p