Mammary Progenitor Research Articles

Is BECN1 a Target Gene of Chromosome 17q21 Alteration in Breast Cancer?

Is BECN1 a Target Gene of Chromosome 17q21 Alteration in Breast Cancer?

EIF4E threshold levels differ in governing normal and neoplastic expansion of mammary stem and luminal progenitor cells.

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer.

Krt6a-positive mammary epithelial progenitors are not at increased vulnerability to tumorigenesis initiated by ErbB2.

While most breast cancers are thought to arise from the luminal layer of the breast tissue, it remains unclear which specific cells in the luminal layer are the cells of origin of breast cancer. We have previously reported that WAP-positive luminal epithelial cells are at increased susceptibility to tumor initiation by ErbB2 compared to the bulk population, while the mammary cells with canonical Wnt signaling activity fail to evolve into tumors upon ErbB2 activation. Here, we used retrovirus to introduce ErbB2 into the Krt6a-positive mammary progenitor subset of the luminal epithelium and, for comparison, into the mammary luminal epithelium indiscriminately. Tumors developed from both groups of cells with a similar latency. These data indicate that the Krt6a-positive subset of mammary epithelial cells can be induced to form cancer by ErbB2 but it is not more susceptible to tumorigenesis initiated by ErbB2 than the bulk population of the luminal epithelium.

TOX3 is expressed in mammary ER(+) epithelial cells and regulates ER target genes in luminal breast cancer.

BackgroundA breast cancer susceptibility locus has been mapped to the gene encoding TOX3. Little is known regarding the expression pattern or biological role of TOX3 in breast cancer or in the mammary gland. Here we analyzed TOX3 expression in murine and human mammary glands and in molecular subtypes of breast cancer, and assessed its ability to alter the biology of breast cancer cells.MethodsWe used a cell sorting strategy, followed by quantitative real-time PCR, to study TOX3 gene expression in the mouse mammary gland. To study the expression of this nuclear protein in human mammary glands and breast tumors, we generated a rabbit monoclonal antibody specific for human TOX3. In vitro studies were performed on MCF7, BT474 and MDA-MB-231 cell lines to study the effects of TOX3 modulation on gene expression in the context of breast cancer cells.ResultsWe found TOX3 expression in estrogen receptor-positive mammary epithelial cells, including progenitor cells. A subset of breast tumors also highly expresses TOX3, with poor outcome associated with high expression of TOX3 in luminal B breast cancers. We also demonstrate the ability of TOX3 to alter gene expression in MCF7 luminal breast cancer cells, including cancer relevant genes TFF1 and CXCR4. Knockdown of TOX3 in a luminal B breast cancer cell line that highly expresses TOX3 is associated with slower growth. Surprisingly, TOX3 is also shown to regulate TFF1 in an estrogen-independent and tamoxifen-insensitive manner.ConclusionsThese results demonstrate that high expression of this protein likely plays a crucial role in breast cancer progression. This is in sharp contrast to previous studies that indicated breast cancer susceptibility is associated with lower expression of TOX3. Together, these results suggest two different roles for TOX3, one in the initiation of breast cancer, potentially related to expression of TOX3 in mammary epithelial cell progenitors, and another role for this nuclear protein in the progression of cancer. In addition, these results can begin to shed light on the reported association of TOX3 expression and breast cancer metastasis to the bone, and point to TOX3 as a novel regulator of estrogen receptor-mediated gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1018-2) contains supplementary material, which is available to authorized users.

BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells.

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies.

A protocol for studying embryonic mammary progenitor cells during mouse mammary primordial development in explant culture.

Embryonic explant culture is a powerful technique to observe tissue morphogenesis ex vivo, and is particularly useful for monitoring embryonic mammary gland development. It has been established that mammary cell lineage specification occurs during embryogenesis, although much remains to be elucidated with respect to how this occurs. During mammary specification, mammary progenitor cells are formed. Embryonic mammary development can proceed and be monitored in embryonic explant culture. Studies using explant culture will greatly enhance our understanding of the cellular mechanisms that regulate embryonic mammary primordial development and mammary progenitor cell specification. We present a protocol for culturing explants from mid-gestation mouse embryos so that morphogenetic processes and mammary epithelial progenitor cells can be studied during embryonic mammary development ex vivo.

The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells.

BackgroundIdentification and characterization of molecular controls that regulate mammary stem and progenitor cell homeostasis are critical to our understanding of normal mammary gland development and its pathology.ResultsWe demonstrate that conditional knockout of Sox9 in the mouse mammary gland results in impaired postnatal development. In short-term lineage tracing in the postnatal mouse mammary gland using Sox9-CreER driven reporters, Sox9 marked primarily the luminal progenitors and bipotent stem/progenitor cells within the basal mammary epithelial compartment. In contrast, long-term lineage tracing studies demonstrate that Sox9+ precursors gave rise to both luminal and myoepithelial cell lineages. Finally, fate mapping of Sox9 deleted cells demonstrates that Sox9 is essential for luminal, but not myoepithelial, lineage commitment and proliferation.ConclusionsThese studies identify Sox9 as a key regulator of mammary gland development and stem/progenitor maintenance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-014-0047-4) contains supplementary material, which is available to authorized users.

Bipotent mammary stem cells: now in amazing 3D.

For many decades, developmental biologists and cancer researchers alike have been trying to understand the relationship between the basal and luminal cell compartments in the mouse mammary epithelium. Delineating the mammary stem and progenitor cell hierarchy will provide fundamental knowledge of how cell proliferation and differentiation are orchestrated to build, maintain and regenerate a complex mammalian tissue. Moreover, it is expected to offer insight into the cells of origin for human breast cancer. A new lineage-tracing study has fuelled the discussion as to the existence of bipotent stem cells in the basal layer of the mouse mammary epithelium.

Vitamin D compounds reduce mammosphere formation and decrease expression of putative stem cell markers in breast cancer

Breast cancer stem cells (BCSCs) are a subset of tumor cells that are believed to be the cells responsible for the establishment and maintenance of tumors. Moreover, BCSCs are suggested to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). We have previously reported that a vitamin D compound, BXL0124, inhibits the progression of DCIS to IDC. In the present study we sought to determine whether this effect was mediated through an influence on BCSCs. In MCF10DCIS cells treated with vitamin D compounds (1α25(OH)2D3 or BXL0124), the breast cancer stem cell-like population, identified by the CD44+/CD24−/low and CD49f+/CD24−/low subpopulations, was reduced. To determine the effects of vitamin D compounds on cancer stem cell activity, the MCF10DCIS mammosphere cell culture system, which enriches for mammary progenitor cells and putative BCSCs, was utilized. Untreated MCF10DCIS mammospheres showed a disorganized and irregular shape. When MCF10DCIS cells were treated with 1α25(OH)2D3 or BXL0124, the mammospheres that formed exhibited a more organized, symmetrical and circular shape, similar to the appearance of spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. The mammosphere forming efficiency (MFE) was significantly decreased upon treatment with 1α25(OH)2D3 or BXL0124, indicating that these compounds have an inhibitory effect on mammosphere development. Treatment with 1α25(OH)2D3 or BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, CD49f, c-Notch1, and pNFκB. Furthermore, 1α25(OH)2D3 and BXL0124 reduced the expression of pluripotency markers, OCT4 and KLF-4 in mammospheres. This study suggests that vitamin D compounds repress the breast cancer stem cell-like population, potentially contributing to their inhibition of breast cancer.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

The Stromal Vascular Fraction of Autologous Fat Graft Induces Proliferation of Epithelial Progenitor Cells in Healthy and Cancer-Containing Breast Tissue In Vitro

BACKGROUND: Autologous fat grafting is widely used for managing contour asymmetries in breast reconstruction. Adipose tissue contains a rich population of both multipotent mesenchymal stem cells and vascular progenitor cells (collectively referred to as adipose-derived stem cells, or ASCs).1 When isolated, the cellular fraction of adipose tissue containing ASCs is referred to as stromal vascular fraction (SVF). Despite widespread clinical use, interactions between the SVF of lipoaspirate and breast parenchyma remain poorly understood. Our study investigated the effect of progenitor-rich SVF on the behavior of mammary epithelial progenitor cells derived from two sources: (i) healthy breast tissue and (ii) cancer adjacent breast tissue. METHODS: Healthy breast tissue and abdominal lipoaspirate were obtained from patients undergoing elective reduction mammoplasty and liposuction. Breast tissue from outside the tumor-free margins of mastectomy specimens (termed ‘cancer-adjacent breast tissue’) was obtained from patients undergoing primary free-flap reconstruction. Samples of SVF (from lipoaspirate) and breast parenchymal cells were then isolated using established cell digestion protocols. ASCs within the SVF were characterized using established differentiation, colony-forming unit-fibroblast (CFU-f), and cell surface marker assays.1 Finally, SVF cells were co-cultured with breast parenchymal cells from (i) healthy and (ii) cancer-adjacent sources were co-cultured using a three-dimensional matrix called Matrigel. Control cultures consisted of breast cells from (i) healthy and (ii) cancer-adjacent tissue in the absence of SVF. After 14 days, the total cell numbers and mammary epithelial progenitor cell populations from each culture group were quantified using colony forming cell (CFC) assays. RESULTS: Differentiation, CFU-f, and surface marker assays demonstrated the presence of ASCs in SVF samples. Co-cultures of cancer-adjacent breast parenchymal cells with SVF showed a 9-fold expansion of mammary epithelial progenitor cells (control = 3-fold) compared to a 5.5-fold expansion in co-cultures of healthy breast parenchymal cells (control = 2-fold) with SVF based on CFC assays. CONCLUSIONS: SVF is capable of increasing the proliferation of mammary epithelial progenitor cells in both healthy and cancer-adjacent breast tissue. As a result, this study demonstrates the potential for interaction between the SVF within autologous fat graft and progenitor cells contained within breast parenchymal tissue.

Anatomical localization of progenitor cells in human breast tissue reveals enrichment of uncommitted cells within immature lobules.

IntroductionLineage tracing studies in mice have revealed the localization and existence of lineage-restricted mammary epithelial progenitor cells that functionally contribute to expansive growth during puberty and differentiation during pregnancy. However, extensive anatomical differences between mouse and human mammary tissues preclude the direct translation of rodent findings to the human breast. Therefore, here we characterize the mammary progenitor cell hierarchy and identify the anatomic location of progenitor cells within human breast tissues.MethodsMammary epithelial cells (MECs) were isolated from disease-free reduction mammoplasty tissues and assayed for stem/progenitor activity in vitro and in vivo. MECs were sorted and evaluated for growth on collagen and expression of lineages markers. Breast lobules were microdissected and individually characterized based on lineage markers and steroid receptor expression to identify the anatomic location of progenitor cells. Spanning-tree progression analysis of density-normalized events (SPADE) was used to identify the cellular hierarchy of MECs within lobules from high-dimensional cytometry data.ResultsIntegrating multiple assays for progenitor activity, we identified the presence of luminal alveolar and basal ductal progenitors. Further, we show that Type I lobules of the human breast were the least mature, demonstrating an unrestricted pattern of expression of luminal and basal lineage markers. Consistent with this, SPADE analysis revealed that immature lobules were enriched for basal progenitor cells, while mature lobules consisted of increased hierarchal complexity of cells within the luminal lineages.ConclusionsThese results reveal underlying differences in the human breast epithelial hierarchy and suggest that with increasing glandular maturity, the epithelial hierarchy also becomes more complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0453-3) contains supplementary material, which is available to authorized users.

Abstract PL04-02: The breast epithelial hierarchy and its implications for tumor heterogeneity

Abstract Breast cancer is a highly heterogeneous disease at both the molecular and pathological levels. To understand this heterogeneity and ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial hierarchy. Discrete populations of mouse and human mammary epithelial cells can be isolated on the basis of cell surface marker expression. To date, subpopulations that are highly enriched for mammary stem (MaSC), luminal progenitor and mature luminal cells have been identified. Notably, these are functionally analogous across human and mouse and share highly conserved transcriptomes. Lineage tracing is an important strategy for assessing the stem cell hierarchy as it allows stem and progenitor cell fate to be studied in situ in the context of development, tissue maintenance and disease. We have combined lineage tracing with a novel three-dimensional (3D) imaging strategy to explore the relative contributions of stem and progenitor cells to post-natal mammary gland development and tissue homeostasis. Cell lineage tracing studies also provide the current gold standard for identifying ‘cells of origin’ in cancer. Towards this end, we are utilizing newly generated transgenic strains harboring lineage-specific gene regulatory regions to direct the expression of specific mammary oncogenic lesions to discrete epithelial cell types. In terms of human breast cancer, the interrogation of molecular profiles has provided insight into potential ‘cells of origin’ in preneoplastic tissue for basal-like cancers arising in BRCA1 mutation carriers. Finally, the identification of deregulated genes/pathways in these crucial target cell populations may enable earlier detection of malignancies and lead to preventative therapies for individuals at high risk of developing breast cancer. Citation Format: Jane E. Visvader, Anne Rios, Naiyang Fu, Emma Nolan, Geoffrey Lindeman. The breast epithelial hierarchy and its implications for tumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr PL04-02. doi:10.1158/1538-7445.AM2014-PL04-02

Abstract LB-62: Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin

Abstract Basal-like breast cancers (BLBC) are heterogenous, poorly differentiated and present with limited therapeutic opportunities and a poor prognosis. We have investigated the role of the HLH transcription factor ID4 in breast stem cells and basal breast cancer aetiology. Using an Id4GFP knock-in mouse and single cell gene expression studies we show that ID4 defines a subset of Cd24+Cd29hi basal mammary epithelial cells enriched for stem cell gene expression and mammary repopulating capacity. Homozygous deletion of Id4 followed by mammary transplantation reveals a cell-autonomous requirement for Id4 in mammary ductal morphogenesis in vivo. shRNA-mediated depletion of Id4 in the Comma-D mammary stem cell line in vitro shows that Id4 is required for epithelial cell proliferation. Conversely, over-expression of Id4 maintains the stem cell pool by restricting luminal differentiation of Comma-D cells. This effect of Id4 is mediated via suppression of BrCa1 expression and the Notch-Elf5 axis. In clinical breast cancer specimens, ID4 expression is absent in luminal tumours but very high in approximately half of all BLBCs, a proportion of which possess ID4 gene amplification. These tumours are characterized by unique transcriptomes with many features of mammary stem cells. Furthermore, ID4-positive BLBCs have a very poor prognosis across multiple independent tissue cohorts. This study identifies ID4 as a master regulator of mammary stem cell homeostasis and suggests that a subset of aggressive BLBCs derive from a primitive mammary stem or progenitor cell with basal characteristics. Citation Format: Simon Junankar, Laura Baker, Kyuson Yun, Alexander Swarbrick. Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-62. doi:10.1158/1538-7445.AM2014-LB-62

Abstract SY12-04: Regulation of normal and cancer stem cell self renewal and senescence by USP16

Abstract Down syndrome (DS) is a complex clinical syndrome associated with multiple pathological conditions including deficits in motor skills and learning. DS has also been associated with early onset and higher incidence of aging-related phenomena such as Alzheimer's disease. Recently, it has been proposed that the aging process is correlated with an impaired or exhausted ability of stem cells to self-renew. we show that in Ts65Dn mice, trisomic for 132 genes homologous to HSA21, triplication of Usp16 reduces self-renewal of hematopoietic stem cells and expansion of mammary epithelial cells, neural progenitors, and fibroblasts. Moreover, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from H2AK119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal USP16 allele or by shRNAs, largely rescues all these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and post-natal neural progenitors while downregulation of USP16 partially rescues the proliferation defects of DS fibroblasts. Taken together, these results suggest that USP16 plays an important role in antagonizing the self-renewal and/or senescence pathways in Down syndrome and could serve as an attractive target to ameliorate some of the associated pathologies. Down syndrome patients also have a decreased incidence of solid tumors including breast cancer. The potential role of USP16 in human breast cancer will be discussed. Citation Format: Michael F. Clarke. Regulation of normal and cancer stem cell self renewal and senescence by USP16. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY12-04. doi:10.1158/1538-7445.AM2014-SY12-04

Abstract 3918: Characterizing growth features, allograft generation and transcriptomes of cultured conditionally reprogrammed cells (CRC) prepared from primary triple negative cancer from Brca1-mutant mice

Abstract Background: Efficient acquisition of primary cancer cells for genetic and pharmacological testing is a means towards individualized cancer treatment. Genetically engineered mouse models of triple negative mammary cancer were used to compare culture conditions conditionally reprogrammed cell culture CRC with a rho kinase inhibitor (Y-27632) and irradiated J2 feeder cells, EpiCult™, DMEM for establishment efficiency, differentiation, fibroblast contamination, and allograft generation. Goals: 1) Evaluate mammary cancer progenitor cells by allograft and secondary culture 2) Compare RNA-seq transcriptomes in cells and allografts with different Brca1 gene dosages (100%, 50%, 0%) Methods: Triple negative invasive adenocarcinomas from 4 mouse genotypes, all carrying a p53-null allele, were analyzed: A) 1 mutant Brca1 allele; B) 2 mutant Brca1 alleles; C) 1 mutant Brca1 allele and 1 human aromatase transgene; D) human aromatase transgene. Cancers were divided and growth assessed in CRC, EpiCult™ and DMEM cultures. Illumina (V2scriptseq) RNA-seq libraries were prepared from cell pellets and allografts. One million cells injected into mammary gland fat pads of nude mice generated allografts divided for histology/DNA analyses, secondary culture and RNAseq. Transcript abundance and differentially expressed genes were determined after assembling transcriptomes with Cufflinks. Results: CRC was the most efficient methodology based on rapidity and percentage cancer specimens successfully cultured and paucity of fibroblasts. Brca1 gene dosage made no difference in culture efficiency. Differentiation markers were expressed at significantly higher levels in CRC whereas markers of epithelial mesenchymal transition were expressed higher in EpiCult™. Allografts were derived from CRC and EpiCult™ but not DMEM. Genetic testing confirmed allografts were derived from original endogenous cancer cells. Some individual allografts derived from the same culture exhibited different growth curves and histology, suggesting tumor cell heterogeneity within cultures. Overall, palpable EpiCult™ allografts appeared significantly earlier than CRC allografts (p<0.05 Chi Square). RNAseq analyses distinguished gene expression patterns dependent upon culture condition as distinct from those determined by underlying genetics. Summary: CRC technology was an efficient means of generating primary cancer cell cultures, maintained epithelial cell differentiation better than other methods, had no fibroblast contamination, and preserved cancer progenitor cells. Citation Format: Ahmad M. Alamri, Svenja Groeneveld, Keunsoo Kang, Sarah Dabydeen, Weisheng Wang, Lothar Hennighausen, Bhaskar Kallakury, Xuefeng Liu, Priscilla A. Furth. Characterizing growth features, allograft generation and transcriptomes of cultured conditionally reprogrammed cells (CRC) prepared from primary triple negative cancer from Brca1-mutant mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3918. doi:10.1158/1538-7445.AM2014-3918

Abstract 1236: A gemini vitamin D analogue, BXL0124, represses mammosphere formation and decreases expression of stem cell markers in MCF10DCIS breast cancer cells

Abstract It has been suggested that cancer stem cells (CSCs), which represent a subset of tumor cells, are responsible for the possible origination and maintenance of tumors. Moreover, CSCs are believed to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). The involvement of CSCs in the progression of DCIS to IDC has yet to be elucidated. In order to investigate the role of CSCs in DCIS progression to IDC, we utilized the MCF10DCIS.com cell line in the mammosphere cell culture system, which enriches for mammary progenitor cells and mammary CSCs. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), inhibits the progression of DCIS to IDC. Therefore, we have determined whether BXL0124 plays a role in regulating the CSC or progenitor cell populations, resulting in reduced tumorigenicity. MCF10DCIS.com cells were plated in ultra-low attachment plates and treated with BXL0124 or 1α25(OH)2D3 over the course of 1, 3 and 5 days. Starting at day 2, MCF10DCIS.com cells began to form mammospheres of 100 µm in size. MCF10DCIS.com mammospheres showed enrichment in the CD44+/CD24- and the CD49f+/CD24- populations compared to monolayer cell culture. The phenotype of MCF10DCIS.com mammospheres showed a disorganized, undefined and irregular shape. However, when the MCF10DCIS.com mammospheres were treated with BXL0124 or 1α25(OH)2D3, mammospheres form a more organized, symmetrical and circular shape, which is similar to the phenotype of the spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. In addition, the mammosphere forming efficiency (MFE) was decreased upon BXL0124 and 1α25(OH)2D3 treatment, showing that there is an inhibitory effect on mammosphere development. Treatment with BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, Notch1 and JAG1. Furthermore, BXL0124 demonstrated the reduced expression of MMPs, such as MMP2, 9, 14, 15, and 16, in mammospheres, suggesting that BXL0124 reduces the tumorigenicity and invasive potential of MCF10DCIS.com cells by targeting the stem cell-like population. In conclusion, the Gemini vitamin D analog BXL0124 represses the CSC population, potentially contributing to the inhibition of the progression of DCIS to IDC. (This work was supported in part by the National Institutes of Health National Cancer Institute R01-CA127645 and the National Institute of Environmental Health Sciences Grant ES005022). Citation Format: Joseph Wahler, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. A gemini vitamin D analogue, BXL0124, represses mammosphere formation and decreases expression of stem cell markers in MCF10DCIS breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2014-1236

The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation

The transcription factor STAT5 mediates prolactin signaling and controls functional development of mammary tissue during pregnancy. This study has identified the miR-193b locus, also encoding miRNAs 365-1 and 6365, as a STAT5 target in mammary epithelium. While the locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells. Inactivation of the miR-193b locus in mice resulted in elevated mammary stem/progenitor cell activity as judged by limiting dilution transplantation experiments of primary mammary epithelial cells. Colonies formed by mutant cells were larger and contained more Ki-67 positive cells. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Cav3 and elevated levels of Elf5. Normal colony development was partially obtained upon ectopically expressing Cav3 or upon siRNA-mediated reduction of Elf5 in miR-193b-null primary mammary epithelial cells. This study reveals a previously unknown link between the mammary-defining transcription factor STAT5 and a microRNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells.

CD151 represses mammary gland development by maintaining the niches of progenitor cells

Tetraspanin CD151 interacts with laminin-binding integrins (i.e., α3β1, α6β1 and α6β4) and other cell surface molecules to control diverse cellular and physiological processes, ranging from cell adhesion, migration and survival to tissue architecture and homeostasis. Here, we report a novel role of CD151 in maintaining the branching morphogenesis and activity of progenitor cells during the pubertal development of mammary glands. In contrast to the disruption of laminin-binding integrins, CD151 removal in mice enhanced the tertiary branching in mammary glands by 2.4-fold and the number of terminal end buds (TEBs) by 30%, while having minimal influence on either primary or secondary ductal branching. Consistent with these morphological changes are the skewed distribution of basal/myoepithelial cells and a 3.2-fold increase in proliferating Ki67-positive cells. These novel observations suggest that CD151 impacts the branching morphogenesis of mammary glands by upregulating the activities of bipotent progenitor cells. Indeed, our subsequent analyses indicate that upon CD151 removal the proportion of CD24HiCD49fLow progenitor cells in the mammary gland increased by 34%, and their proliferating and differentiating activities were significantly upregulated. Importantly, fibronectin, a pro-branching extracellular matrix (ECM) protein deposited underlying mammary epithelial or progenitor cells, increased by >7.2-fold. Moreover, there was a concomitant increase in the expression and nuclear distribution of Slug, a transcription factor implicated in the maintenance of mammary progenitor cell activities. Taken together, our studies demonstrate that integrin-associated CD151 represses mammary branching morphogenesis by controlling progenitor cell activities, ECM integrity and transcription program.

Mammary stem cells: expansion and animal productivity.

Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.

The Dog as a Natural Animal Model for Study of the Mammary Myoepithelial Basal Cell Lineage and its Role in Mammary Carcinogenesis

Basal-like tumours constitute 2-18% of all human breast cancers (HBCs). These tumours have a basal myoepithelial phenotype and it has been hypothesized that they originate from either myoepithelial cells or mammary progenitor cells. They are heterogeneous in morphology, clinical presentation, outcome and response to therapy. Canine mammary carcinomas (CMCs) have epidemiological and biological similarities to HBCs, are frequently biphasic and are composed of two distinct neoplastic populations (epithelial and myoepithelial). The present study evaluates the potential of CMCs as a natural model for basal-like HBCs. Single and double immunohistochemistry was performed on serial sections of 10 normal canine mammary glands and 65 CMCs to evaluate expression of cytokeratin (CK) 8/18, CK5, CK14, α-smooth muscle actin (SMA), calponin (CALP), p63 and vimentin (VIM). The tumours were also evaluated for Ki67 and human epidermal growth factor receptor (HER)-2 expression. A hierarchical model of cell differentiation was established, similar to that for the human breast. We hypothesized that progenitor cells (CK5(+), CK14(+), p63(+) and VIM(+)) differentiate into terminally-differentiated luminal glandular (CK8/18(+)) and myoepithelial (CALP(+), SMA(+) and VIM(+)) cells via intermediary luminal glandular cells (CK5(+), CK14(+) and CK8/CK18(+)) and intermediary myoepithelial cells (CK5(+), CK14(+), p63(+), SMA(+), CALP(+) and VIM(+)). Neoplastic myoepithelial cells in canine complex carcinomas had labelling similar to that of terminally-differentiated myoepithelial cells, while those of carcinomas-and-malignant myoepitheliomas with a more aggressive biological behaviour (i.e. higher frequency of vascular/lymph node invasion and visceral metastases and higher risk of tumour-related death) were comparable with intermediary myoepithelial cells and had significantly higher Ki67 expression. The majority of CMCs examined were negative for expression of HER-2. The biphasic appearance of CMCs with involvement of the myoepithelial component in different stages of cell differentiation may help to define the role of myoepithelial cells in the mammary carcinogenetic process and the heterogeneous nature of basal-like HBCs.