Normal Mammary Stem Cells Research Articles

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macro-Bound Enhancers’, that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Abstract 1531: CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity

Abstract CD146 (Mcam; Muc18; s-endo; and gicerin) is an integral membrane glycoprotein originally identified in a human malignant melanoma, but mis-expressed in a wider variety of malignancies including breast cancer, where it has been implicated in controlling tumor growth and dissemination. The mechanisms by which CD146 conveys extracellular cues into malignant cellular outputs is not well understood, but its high-level expression in early development and normal mouse mammary stem cells, as well as expression in highly plastic adult pericytes (formerly known as mesenchymal stem cells) suggested to us that it may control cell state plasticity. We tested the role of CD146 in a plastic, stem cell-like mouse mammary tumor cell line derived from the MMTV-PyMT mouse (PY230). We identified domains in the CD146 cytoplasmic tail essential for controlling substrate utilization by upstream kinases in Akt, Mapk, Stat3, and integrin-related signaling pathways that alter cell state plasticity and tumorigenicity. This work suggests that MCAM plays a key role in controlling lineage plasticity in normal and neoplastic mammary cells with relevance for understanding tumor cell plasticity, progression and evasion of targeted cancer therapies. Citation Format: Ozlen Balcioglu, Brooke L. Gates, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1531.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies.

Simple SummaryA limitation to successful therapeutic outcomes for breast and other cancer patients is the ability of small subsets of tumor cells to resist the apoptotic cell death provoked by currently employed therapeutic agents. These therapy-resistant cancer stem cell populations can then seed recurrent tumors and metastatic lesions, compromising the efficacy of the treatment regimen. The aim of our study was to assess the hypothesis that cationic amphiphilic drugs (CADs), which induce tumor cell death via the unrelated programmed necrotic mechanism, exhibit efficacy toward cancer stem cell populations that are resistant to currently employed therapeutics. We found that the therapy-resistant stem-like subpopulation of cells from a variety of breast cancer models are as sensitive to CADs as the bulk population. Our observations imply that the incorporation of cationic amphiphilic anticancer agents into existing therapeutic regimens could ultimately improve breast cancer patient outcomes by minimizing tumor recurrence and metastatic outgrowth.The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Abstract 2667: RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process

Abstract Despite an enhanced interest, the origin of cancer stem cells (CSCs) remains equivocal. A strong body of evidence suggests that, in some adult carcinomas, CSC may originate from normal mature differentiated cells through a dedifferentiation process and the acquisition of stem-like properties. At the crux of this concept is the epithelial-mesenchymal transition (EMT), an embryonic transdifferentiation process that can be abnormally reactivated over the course of tumor development as a result of oncogenic or microenvironmental cues. High expression of EMT genes and stemness features are hallmarks of claudin-low (CL) breast cancers, a rare subtype of breast malignancies, generally considered as the most primitive breast cancers. We have recently reported that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups (CL1, CL2 and CL3), that emerge from unique evolutionary processes. Genetic and epigenetic analyses support the hypothesis that CL1 tumors generate from the transformation of a normal mammary stem cells, whereas CL2 and CL3 arise from the reactivation of an EMT process over the course of tumor progression. Interestingly, CL tumors share the frequent activation of the RAS-MAPK pathway, suggesting a potent role for the RAS pathway in the development of CL tumors. Based on these findings, we wondered whether RAS pathway activation could induce the reactivation of EMT-TFs allowing the emergence of CSCs displaying CL2 and CL3 characteristics. By expressing an oncogenic form of RAS in differentiated epithelial cells (HME), we observed a phenotypic switch towards a CD24-/low/CD44+ phenotype, associated with CSC properties, and suggesting an acquisition of cellular plasticity after RAS activation. By using CRISPR/Cas9 knock-down experiments we have shown i) the emergence of CD24-/low/CD44+ cells, ii) the acquisition of transformation and stemness features, and iii) that the cellular plasticity acquired after RAS activation in differentiated HME strongly rely on ZEB1. Single-cell RNA sequencing analyses after RAS activation in HME-RAS cells suggested two different outcomes: senescence or EMT. Treatment with a senolytic drug resulted in reduction of RAS-induced senescent cells associated with a decrease in the emergence of CD24-/low/CD44+ cells and ZEB1 expression. Finally, a co-culture reporter system and a depletive approach with cytokine-neutralizing antibodies highlighted a paracrine effect of IL-6 and IL-1α, produced by senescent RAS-expressing cells on their neighboring differentiated cells, leading to the acquisition of ZEB1-dependent cellular plasticity. Hence, we unveiled a new paracrine process, in which cytokines secreted by cells undergoing oncogene-induced senescence drive ZEB1 expression, thus promoting cellular plasticity correlated with the emergence of CSCs. Citation Format: ALAIN PUISIEUX, Hadrien De Blander, Laurie Tonon, Frédérique Fauvet, Roxane Pommier, Christelle Lamblot, Rahma Benhassoun, Benjamin Gibert, Maria Ouzounova, Anne-Pierre Morel. RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2667.

Abstract 6024: An integrin αvβ3+ stem-like tumor cell subset promotes breast cancer progression via c-Jun/AXL signaling

Abstract Breast cancer cells bearing similarities to normal mammary stem cells play a key role in tumor progression. Here, we characterize a unique stem-like tumor cell population that expresses the integrin αvβ3 and is associated with aggressive patient disease. We now show that αvβ3+ cells that also express EpCAMLow represent a functionally and transcriptomically distinct breast cancer cell type. Rare αvβ3+ cells enrich for stemness properties such as anchorage-independent colony formation, self-renewal, differentiation and tumor initiation at limiting dilution. Unbiased whole transcriptome analysis by RNA-Seq identified several genes specifically upregulated in αvβ3+ cells that are also enriched in normal adult mammary stem cells. Phospho-array profiling of major signaling pathways revealed increased activation of the proto-oncogene c-Jun in αvβ3+ stem-like cells. Inhibition of JNK/c-Jun signaling further identified the receptor tyrosine kinase AXL as a critical downstream target gene necessary for tumorsphere formation. Our findings reveal a c-Jun/AXL signaling pathway required for stemness properties in αvβ3+ tumor cells important for breast cancer progression. Citation Format: Qi Sun, Yufen Wang, Adam Officer, Olivier Harismendy, Jay S. Desgrosellier. An integrin αvβ3+ stem-like tumor cell subset promotes breast cancer progression via c-Jun/AXL signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6024.

Comprehensive characterization of claudin-low breast tumors reflects the impact of the cell-of-origin on cancer evolution

Claudin-low breast cancers are aggressive tumors defined by the low expression of key components of cellular junctions, associated with mesenchymal and stemness features. Although they are generally considered as the most primitive breast malignancies, their histogenesis remains elusive. Here we show that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups that emerge from unique evolutionary processes. Genetic, gene methylation and gene expression analyses reveal that two of the subgroups relate, respectively, to luminal breast cancers and basal-like breast cancers through the activation of an EMT process over the course of tumor progression. The third subgroup is closely related to normal human mammary stem cells. This unique subgroup of breast cancers shows a paucity of genomic aberrations and a low frequency of TP53 mutations, supporting the emerging notion that the intrinsic properties of the cell-of-origin constitute a major determinant of the genetic history of tumorigenesis.

Hybrid Stem Cell States: Insights Into the Relationship Between Mammary Development and Breast Cancer Using Single-Cell Transcriptomics.

Similarities between stem cells and cancer cells have implicated mammary stem cells in breast carcinogenesis. Recent evidence suggests that normal breast stem cells exist in multiple phenotypic states: epithelial, mesenchymal, and hybrid epithelial/mesenchymal (E/M). Hybrid E/M cells in particular have been implicated in breast cancer metastasis and poor prognosis. Mounting evidence also suggests that stem cell phenotypes change throughout the life course, for example, through embryonic development and pregnancy. The goal of this study was to use single cell RNA-sequencing to quantify cell state distributions of the normal mammary (NM) gland throughout developmental stages and when perturbed into a stem-like state in vitro using conditional reprogramming (CR). Using machine learning based dataset alignment, we integrate multiple mammary gland single cell RNA-seq datasets from human and mouse, along with bulk RNA-seq data from breast tumors in the Cancer Genome Atlas (TCGA), to interrogate hybrid stem cell states in the normal mammary gland and cancer. CR of human mammary cells induces an expanded stem cell state, characterized by increased expression of embryonic stem cell associated genes. Alignment to a mouse single-cell transcriptome atlas spanning mammary gland development from in utero to adulthood revealed that NM cells align to adult mouse cells and CR cells align across the pseudotime trajectory with a stem-like population aligning to the embryonic mouse cells. Three hybrid populations emerge after CR that are rare in NM: KRT18+/KRT14+ (hybrid luminal/basal), EPCAM+/VIM+ (hybrid E/M), and a quadruple positive population, expressing all four markers. Pseudotime analysis and alignment to the mouse developmental trajectory revealed that E/M hybrids are the most developmentally immature. Analyses of single cell mouse mammary RNA-seq throughout pregnancy show that during gestation, there is an enrichment of hybrid E/M cells, suggesting that these cells play an important role in mammary morphogenesis during lactation. Finally, pseudotime analysis and alignment of TCGA breast cancer expression data revealed that breast cancer subtypes express distinct developmental signatures, with basal tumors representing the most “developmentally immature” phenotype. These results highlight phenotypic plasticity of normal mammary stem cells and provide insight into the relationship between hybrid cell populations, stemness, and cancer.

Two Sides of the Same Coin: The Role of Developmental pathways and pluripotency factors in normal mammary stem cells and breast cancer metastasis.

Breast cancer initiation and progression are often observed as the result of dysregulation of normal developmental processes and pathways. Studies focused on normal mammary stem/progenitor cell activity have led to an understanding of how breast cancer cells acquire stemness-associated properties including tumor initiation, survival and multi-lineage differentiation into heterogeneous tumors that become difficult to target therapeutically. Importantly, more recent investigations have provided valuable insight into how key developmental regulators can impact multiple phases of metastasis, where they are repurposed to not only promote metastatic phenotypes such as migration, invasion and EMT at the primary site, but also to regulate the survival, initiation and maintenance of metastatic lesions at secondary organs. Herein, we discuss findings that have led to a better understanding of how embryonic and pluripotency factors contribute not only to normal mammary development, but also to metastatic progression. We further examine the therapeutic potential of targeting these developmental pathways, and discuss how a better understanding of compensatory mechanisms, crosstalk between pathways, and novel experimental models could provide critical insight into how we might exploit embryonic and pluripotency regulators to inhibit tumor progression and metastasis.

Abstract P3-03-10: Targeting Wnt/β-catenin pathway by antidepressant imipramine for triple negative breast cancer treatment

Abstract Aberrant activation of Wnt signaling has been implicated in various human cancers including breast cancer. Several intra-cellular signal transduction pathways are induced by Wnt, notably by Wnt/β-catenin dependent pathway. In breast cancer, triple negative breast cancer (TNBC) subtypes have increased Wnt/β-catenin signaling associated with high grade tumors, metastasis, poor prognosis and tumor regression. Importantly, significant upregulation of Wnt/β-catenin pathway and overexpression of Wnt transcriptional targets have emerged in breast cancer stem cells (BCSCs) than normal mammary epithelial stem cells. Blockade of Wnt/β-catenin signaling has been reported to suppress breast cancer growth, invasion, migration and induced apoptosis and “stemness”. WNT pathway inhibition preferentially reduces stem cell like activity in patient-derived metastatic breast cancer compared to normal cells. Collectively, these studies suggest that Wnt/β-catenin pathway could be the potential target in inhibiting TNBC growth, metastatic progression and “stemness”. We recently identified that imipramine, an FDA approved antidepressant used to treat chronic depression, inhibits TNBC growth and progression. Imipramine treatment significantly reduced cell viability, migration and invasion of breast cancer cells in general and TNBC cells in particular. Our preliminary findings demonstrated that systemic delivery of imipramine suppressed TNBC growth in orthotopic mouse xenograft model. Our studies further show that imipramine inhibits several Wnt/β-catenin catenin pathway proteins including WNT1, FZD1, β-catenin, cyclin D1 and c-myc, which are reported to support breast cancer “stemness” and metastasis. Importantly, our preliminary studies revealed that imipramine inhibits TNBC stem cells as evidenced by reduced CD44+ population. Our results suggest the therapeutic efficacy of imipramine as a single/or neo-adjuvant agent for treating TNBC patients. Citation Format: Subapriya Rajamanickam, Santosh Timilsina, Ismoil Jatoi, Virginia Kaklamani, Ratna K Vadlamudi, Manjeet K Rao. Targeting Wnt/β-catenin pathway by antidepressant imipramine for triple negative breast cancer treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-10.

Abstract SY17-02: Solid tumor cancer stem cells: From bench to bedside

Abstract Stem cells are essential for maintenance of multiple tissues including the intestine, skin, brain and breast. Remarkably, cancers invariably arise in tissues that contain stem cells whose function is to maintain and repair tissues for the life of the animal. Self renewal, the mechanisms by which stem cells regenerate themselves, is the fundamental property by which stem cells maintain themselves. Our laboratory investigates the fundamental molecular mechanisms of self renewal. Our studies have found that stem cells can be identified by the expression of genes required for self renewal AND by the lack of expression of genes that suppress self renewal. The specific components of self renewal pathways in normal and malignant colon and mammary gland stem cells will be discussed. Stem cell pathways contribute to minimal residual disease after cancer therapy will be discussed. Finally, the application of stem cell biology to cancer therapy will be described. Approximately 4-7% of stage 2 colon cancers consist of homogeneous populations of undiffentiated CDX2- cancer cells. Stage 2 colon cancer patients are generally not thought to benefit from adjuvant chemotherapy. However, stage 2 colon cancer patients with CDX 2- cancer do appear to benefit. Citation Format: Michael F. Clarke. Solid tumor cancer stem cells: From bench to bedside [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY17-02.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

Plasticity and Potency of Mammary Stem Cell Subsets During Mammary Gland Development

It is now widely believed that mammary epithelial cell plasticity, an important physiological process during the stages of mammary gland development, is exploited by the malignant cells for their successful disease progression. Normal mammary epithelial cells are heterogeneous and organized in hierarchical fashion, in which the mammary stem cells (MaSC) lie at the apex with regenerative capacity as well as plasticity. Despite the fact that the majority of studies supported the existence of multipotent MaSCs giving rise to both basal and luminal lineages, others proposed lineage restricted unipotent MaSCs. Consistent with the notion, the latest research has suggested that although normal MaSC subsets mainly stay in a quiescent state, they differ in their reconstituting ability, spatial localization, and molecular and epigenetic signatures in response to physiological stimuli within the respective microenvironment during the stages of mammary gland development. In this review, we will focus on current research on the biology of normal mammary stem cells with an emphasis on properties of cellular plasticity, self-renewal and quiescence, as well as the role of the microenvironment in regulating these processes. This will include a discussion of normal breast stem cell heterogeneity, stem cell markers, and lineage tracing studies.

Identification and clinical validation of a multigene assay that interrogates the biology of cancer stem cells and predicts metastasis in breast cancer: A retrospective consecutive study.

BackgroundBreast cancers show variations in the number and biological aggressiveness of cancer stem cells that correlate with their clinico-prognostic and molecular heterogeneity. Thus, prognostic stratification of breast cancers based on cancer stem cells might help guide patient management.MethodsWe derived a 20-gene stem cell signature from the transcriptional profile of normal mammary stem cells, capable of identifying breast cancers with a homogeneous profile and poor prognosis in in silico analyses. The clinical value of this signature was assessed in a prospective-retrospective cohort of 2, 453 breast cancer patients. Models for predicting individual risk of metastasis were developed from expression data of the 20 genes in patients randomly assigned to a training set, using the ridge-penalized Cox regression, and tested in an independent validation set.FindingsAnalyses revealed that the 20-gene stem cell signature provided prognostic information in Triple-Negative and Luminal breast cancer patients, independently of standard clinicopathological parameters. Through functional studies in individual tumours, we correlated the risk score assigned by the signature with the proliferative and self-renewal potential of the cancer stem cell population. By retraining the 20-gene signature in Luminal patients, we derived the risk model, StemPrintER, which predicted early and late recurrence independently of standard prognostic factors.InterpretationOur findings indicate that the 20-gene stem cell signature, by its unique ability to interrogate the biology of cancer stem cells of the primary tumour, provides a reliable estimate of metastatic risk in Triple-Negative and Luminal breast cancer patients independently of standard clinicopathological parameters.

Assays for functionally defined normal and malignant mammary stem cells.

The discovery of rare, heterogeneous self-renewing stem cells with shared developmental and molecular features within epithelial components of mammary gland and breast cancers has provided a conceptual framework to understand cellular composition of these tissues and mechanisms that control their number. These normal mammary epithelial stem cells (MaSCs) and breast cancer stem cells (BCSCs) were identified and analyzed using transplant assays (namely mammary repopulating unit (MRU) assay, mammary tumor-initiating cell (TIC) assay), which reveal their latent ability to regenerate respective normal and malignant epithelial tissues with self-renewing units displaying hierarchical cellular differentiation over multiple generations in recipient mice. "Next-generation" methods using "barcoded" normal and malignant mammary cells, with the help of next-generation sequencing (NGS) technology, have revealed hidden complexity and heterogeneous growth potential of MaSCs and BCSCs. Several single markers or combinations of markers have been reported to prospectively enrich MaSCs and BCSCs. Such markers and the extent to which they enrich for MaSCs and BCSCs activity require a critical appraisal. Also, knowledge of the functional assays and their limitations and harmonious reporting of results is a prerequisite to improve our understanding of MaSCs and BCSCs. This chapter describes evolution of the concept of MaSCs and BCSCs, and specific methodologies to investigate them.

Nuclear respiratory factor 1 promotes spheroid survival and mesenchymal transition in mammary epithelial cells.

Epithelial cells aggregate into spheroids when deprived of matrix, and the proclivity for spheroid formation and survival is a hallmark of normal and tumorigenic mammary stem cells. We show here that Nuclear Respiratory Factor 1 (NRF1) is a spheroid promoter by in silico identification of this transcription factor as highly connected to top shRNA-hits deduced from re-iterative selections for shRNAs enriched in MCF10A spheroids. NRF1-promoted spheroid survival is linked to its stimulation of mitochondrial OXPHOS, cell migration, invasion, and mesenchymal transition. Conversely, NRF1 knockdown in breast cancer MDA-MB-231 cells reduced spheroids, migration, invasion, and mesenchymal marker expression. NRF1 knockdown also reduced tumor burden in mammary fat pads and lungs of orthotopic- or tail vein-transplanted mice. With the Luminal A subtype of breast cancer, higher NRF1 expression is associated with lower survival. These results show that NRF1, an activator of mitochondrial metabolism, supports mammary spheroid survival and tumor development.

MicroRNA-221 may be involved in lipid metabolism in mammary epithelial cells.

Milk lipids, important for infant growth and development, are produced and secreted by mammary gland under the regulation of steroid hormones, growth factors, and microRNAs (miRNAs). miR-221 has been identified in milk and adipocytes and it plays important roles in regulating normal mammary epithelial hierarchy and breast cancer stem cells; however, its roles in lipid metabolism in mammary epithelial cells (MECs), the cells of lipid synthesis and secretion, are as yet unknown. Through overexpression or inhibition of miR-221 expression, we found that it regulated lipid metabolism in MECs and was expressed differentially at various stages during murine mammary gland development. Inhibition of miR-221 expression increased lipid content in MECs through elevation of the lipid synthesis enzyme FASN, while overexpression of miR-221 reduced MEC lipid content. Moreover, the steroid hormones estradiol and progesterone decreased miR-221 expression with a subsequent increase in lipid formation in MECs. The expression of miR-221 was lower during lactation, which suggests that it may be involved in milk production. Therefore, miR-221 might be a useful target for influencing milk lipid production.

Abstract ES9-2: Tracking progesterone receptor actions in breast cancer progression: Jekyll and Hyde

Abstract Recent work on the actions for progesterone receptors (PRs) in breast cancer has raised the profile of PRs as players rather than simply markers of estrogen (E)-responsive luminal disease. Indeed, estrogen receptors (ERs) and PRs are intimate partners at the genome regulatory level rather than having a simple driver/passenger relationship. PRs alter breast cancer cell proliferation in complex ways and can be either growth inhibitory or stimulatory depending on the cell and hormonal context. Additionally, PRs are important drivers of normal mammary stem cell as well as cancer stem cell expansion. Along with ERs, targeting PRs as part of modernized endocrine therapies may provide a rational treatment alternative for women with ER- and PR-positive breast cancers. However, should PR agonists or antagonists be paired with anti-estrogens or estrogen-ablative therapies and when? This lecture will review historical and current literature on the complex topic of PR action in luminal breast cancer, with focus on key knowledge gaps and future steps that could speed translation of exciting research findings into useful clinical therapeutic applications. Citation Format: Lange CA. Tracking progesterone receptor actions in breast cancer progression: Jekyll and Hyde [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr ES9-2.