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Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build‐up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3‐ubiquitin ligase complex for Nrf2. Here, we report that the Bax‐binding protein MOAP‐1 regulates p62‐Keap1‐Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP‐1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP‐1 interacts with the PB1‐ZZ domains of p62 and interferes with its self‐oligomerization and liquid–liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP‐1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP‐1‐deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)‐induced hepatocarcinogenesis model. Together, our data define MOAP‐1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies., Stress‐induced p62/SQSTM1 bodies sequester the E3 ubiquitin ligase Keap1 from Nrf2, which is free to mediate transactivation of its target genes. MOAP‐1 recruitment to p62 bodies promotes their disassembly and reduces Nrf2 activation.

The mammary gland is a highly dynamic organ that undergoes profound changes within its epithelium during puberty and the reproductive cycle. These changes are fueled by dedicated stem and progenitor cells. Both short- and long-lived lineage-restricted progenitors have been identified in adult tissue as well as a small pool of multipotent mammary stem cells (MaSCs), reflecting intrinsic complexity within the epithelial hierarchy. While unipotent progenitor cells predominantly execute day-to-day homeostasis and postnatal morphogenesis during puberty and pregnancy, multipotent MaSCs have been implicated in coordinating alveologenesis and long-term ductal maintenance. Nonetheless, the multipotency of stem cells in the adult remains controversial. The advent of large-scale single-cell molecular profiling has revealed striking changes in the gene expression landscape through ontogeny and the presence of transient intermediate populations. An increasing number of lineage cell-fate determination factors and potential niche regulators have now been mapped along the hierarchy, with many implicated in breast carcinogenesis. The emerging diversity among stem and progenitor populations of the mammary epithelium is likely to underpin the heterogeneity that characterizes breast cancer.

Emerging evidence suggests that hepatocytes are primarily maintained by self-renewal during normal liver homeostasis, as well as in response to a wide variety of hepatic injuries. However, how hepatocytes in distinct anatomic locations within the liver lobule are replenished under homeostasis and injury-induced regeneration remains elusive. Using a newly developed bacterial artificial chromosome (BAC)-transgenic mouse model, we demonstrate that Lgr5 expression in the liver is restricted to a unique subset of hepatocytes most adjacent to the central veins. Genetic lineage tracing revealed that pericentral Lgr5(+) hepatocytes have a long lifespan and mainly contribute to their own lineage maintenance during postnatal liver development and homeostasis. Remarkably, these hepatocytes also fuel the regeneration of their own lineage during the massive and rapid regeneration process following two-thirds partial hepatectomy. Moreover, Lgr5(+) hepatocytes are found to be the main cellular origin of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and are highly susceptible to neoplastic transformation triggered by activation of Erbb pathway. Our findings establish an unexpected self-maintaining mode for a defined subset of hepatocytes during liver homeostasis and regeneration, and identify Lgr5(+) pericentral hepatocytes as major cells of origin in HCC development.

Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5+ basal epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5+Tspan8hi) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5+Tspan8hi cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.

Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1-deficient glands were highly enriched for quiescent Tspan8hi MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development.

Expansion and remodelling of the mammary epithelium requires a tight balance between cellular proliferation, differentiation and death. To explore cell survival versus cell death decisions in this organ, we deleted the pro-survival gene Mcl-1 in the mammary epithelium. Mcl-1 was found to be essential at multiple developmental stages including morphogenesis in puberty and alveologenesis in pregnancy. Moreover, Mcl-1-deficient basal cells were virtually devoid of repopulating activity, suggesting that this gene is required for stem cell function. Profound upregulation of the Mcl-1 protein was evident in alveolar cells at the switch to lactation, and Mcl-1 deficiency impaired lactation. Interestingly, EGF was identified as one of the most highly upregulated genes on lactogenesis and inhibition of EGF or mTOR signalling markedly impaired lactation, with concomitant decreases in Mcl-1 and phosphorylated ribosomal protein S6. These data demonstrate that Mcl-1 is essential for mammopoiesis and identify EGF as a critical trigger of Mcl-1 translation to ensure survival of milk-producing alveolar cells.

The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells., The mammary epithelium comprises two cell lineages but the heterogeneity amongst these during development is unclear. Here, the authors report single-cell RNA sequencing of the mouse mammary epithelium at four developmental stages, revealing diversity in both compartments and a transcriptional shift with puberty onset.

T-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.

Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors.

Lineage tracing analysis allows mammary epithelial cells to be tracked in their natural environment, thereby revealing cell fate and proliferation choices in the intact tissue. This technique is particularly informative for studying how stem cells build and maintain the mammary epithelium during development and pregnancy. Here we describe two experimental systems based on Cre/loxP technology (Cre(ERT2)/loxP and rtTA/tetO-Cre/loxP), which allow the inducible, permanent labeling of mammary epithelial cells following the administration of either tamoxifen or doxycycline.

SummaryThe mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland.

Lineage tracing is increasingly being utilised to probe different cell types that exist within the mammary gland. Whilst this technique is powerful for tracking cells in vivo and dissecting the roles of different cellular subsets in development, homeostasis and oncogenesis, there are important caveats associated with lineage tracing strategies. Here we highlight key parameters of particular relevance for the mammary gland. These include tissue preparation for whole-mount imaging, whereby the inclusion of enzymatic digestion can drastically alter tissue architecture and cell morphology, and therefore should be avoided. Other factors include the scoring of clones in three dimensions versus two dimensions, the timing of induction, and the marked variability in labelling efficiency that is evident not only between different mouse models harbouring a similar gene promoter but also within a given strain and even within a single mammary gland. Thus, it becomes crucial to visualise extensive areas of ductal tissue and to consider the intricacies of the methodology for lineage tracing studies on normal mammary development and on potential ‘cells of origin’ of cancer.

Lineage tracing analysis allows mammary epithelial cells to be tracked in their natural environment, thereby revealing cell fate and proliferation choices in the intact tissue. This technique is particularly informative for studying how stem cells build and maintain the mammary epithelium during development and pregnancy. Here we describe two experimental systems based on Cre/loxP technology (Cre

To understand relationships between the mammary epithelia, ‘cells of origin’ and cancer stem cells in breast tumors, it is necessary to dissect the normal mammary epithelial hierarchy. We have isolated discrete populations of mouse and human mammary epithelial cells on the basis of cell-surface markers and identified cell subsets that are highly enriched for mammary stem (MaSC), luminal progenitor and mature luminal cells. Analysis of different mouse models of mammary tumorigenesis using syngeneic transplantation assays and interrogation of molecular profiles has revealed potential ‘cells of origin’ in preneoplastic tissue and the presence of a definitive cancer stem cell subset in different mouse mammary tumors. Cell lineage tracing studies in the mouse provide the current gold standard for identifying cells of origin. Towards this end, we have constructed a series of transgenic mice harboring lineage-specific gene regulatory regions (180 kB) that can be used to direct the expression of specific mammary oncogenes to discrete epithelial cell types. This will allow us to address the contribution of ‘cells of origin’ and the nature of the oncogene itself to mouse mammary tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY10-04. doi:1538-7445.AM2012-SY10-04

Breast cancer is a highly heterogeneous disease at both the molecular and pathologic levels. To understand this heterogeneity and the cells of origin of breast cancer, it is important to dissect the normal mammary epithelial hierarchy. Both transplantation and lineage tracing studies, a strategy for tracking stem and progenitor cell fate in situ, have been implemented to explore the contribution of stem and progenitor cells to postnatal mammary gland development and tissue homeostasis. The combination of lineage tracing with a novel three-dimensional imaging strategy, which enables visualization of large regions of intact tissue at cellular resolution, has provided insights into the normal differentiation hierarchy as well as potential cells of origin of breast cancer. In a further layer of investigation, we have applied single cell RNA-seq analysis to both mouse and human mammary tissue. This type of analysis has revealed unexpected heterogeneity among epithelial cells that reside in the normal mammary gland. In the context of human tissue, the identification of cells of origin of cancer and deregulated genes/pathways in crucial target cell populations may enable earlier detection of malignancies and lead to preventive therapies for individuals at high risk of developing breast cancer. Towards this end, we recently showed that the RANK receptor marks a small subset of luminal progenitors and this subset is expanded in precancerous tissue from BRCA1 mutation carriers. In preclinical Brca1-deficient mouse models, inhibition of the RANK ligand (RANKL) prevented or delayed tumorigenesis. These data implicate blockade of the RANK-RANKL signaling axis as a promising breast cancer prevention strategy. Citation Format: Nai Yang Fu, Emma Nolan, Francois Vaillant, Rachel Joyce, Geoffrey Lindeman, Jane Visvader. Mapping stem and progenitor cells to decipher the origins of breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA02.

T cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function. Age-related thymic involution is recognised as a potentially fatal brake on immune recovery following haematopoietic stem/progenitor cell transplantation and cancer treatments. To uncover new strategies to restore thymic function, we determined the molecular mechanisms that control life and death decisions in TEC. We recently published that while TEC-specific deletion of pro-survival proteins BCL-2 and BCL-XL did not cause gross abnormalities, MCL-1 deficiency impacted on TEC as early as E15.5, resulting in early thymic atrophy, T cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was continually required for the survival of medullary TEC, including autoimmune regulator (AIRE) expressing TECs and the maintenance of overall thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via the MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. However, these findings do not imply that the intrinsic pathway of apoptosis necessarily controls TEC death during homeostasis. Therefore, we removed essential effectors of this pathway, BAX and BAK, specifically in TECs and found that there was a specific increase in a medullary TEC subset leading to an overall increase in TEC numbers both in young and old mice. This signalling axis governing TEC survival, death and thymic function represent a compelling strategy for protecting the thymus during blood cancer treatment to prevent immunodeficiency, without counterproductive survival of haematopoietic lineages.

Inhibition of apoptotic response of host cells during an early phase of infection is a strategy used by many enteroinvasive bacterial pathogens to enhance their survival. Here, we report the identification of a soluble form of the pilus protein FimA from the culture supernatants of E. coli K1, Salmonella, and Shigella that can potently inhibit Bax-mediated release of cytochrome c from isolated mitochondria. Similar to the infected cells, HCT116 cells stably expressing FimA display a delay in the integration of Bax into outer mitochondrial membrane induced by apoptotic stimuli. FimA targets to mitochondria through binding to VDAC1, which is a prerequisite step for E. coli K1 to render the short-term blockade of apoptotic death in the host cells. Interestingly, FimA strengthens the VDAC1-hexokinase interaction and prevents dissociation of hexokinase from VDAC1 triggered by apoptotic stimuli. Together, these data thus reveal a paradigm of antiapoptosis mechanism undertaken by the enteroinvasive bacteria.

Bax, a multi-domain pro-apoptotic Bcl-2 family member, is a key regulator for the release of apoptogenic factors from mitochondria. MOAP-1, which was first isolated from a screen for Bax-associating proteins, interacts with Bax upon apoptotic induction. MOAP-1 is a short-lived protein that is constitutively degraded by the ubiquitin-proteasome system. Apoptotic stimuli upregulate MOAP-1 rapidly through inhibition of its poly-ubiquitination process. However, cellular factors that regulate the stability of MOAP-1 have not yet been identified. In this study, we report the identification of TRIM39 as a MOAP-1-binding protein. TRIM39 belongs to a family of proteins characterized by a Tripartite Motif (TRIM), consisting of RING domain, B-box and coiled-coil domain. Several TRIM family members are known to demonstrate E3 ubiquitin ligase activity. Surprisingly, TRIM39 significantly extends the half-life of MOAP-1 by inhibiting its poly-ubiquitination process. In agreement with its effect on enhancing MOAP-1 stability, TRIM39 sensitizes cells to etoposide-induced apoptosis. Conversely, knockdown of TRIM39 reduces the sensitivity of cells to etoposide-stimulated apoptosis. Furthermore, TRIM39 elevates the level of MOAP-1 in mitochondria and promotes cytochrome c release from isolated mitochondria stimulated by recombinant Bax. Together, these data suggest that TRIM39 can promote apoptosis signalling through stabilization of MOAP-1.

Although mRNAs of multiple isoforms of Bax, which encodes a central regulator of apoptosis signaling, have been reported, only Baxalpha protein has been well documented and studied. Baxalpha exists in latent form and is activated upon apoptosis induction through conformational changes. Here we demonstrate that Baxbeta protein is ubiquitously present among human cells, but its activity is restricted through stringent regulation by proteasomal degradation. In contrast to Baxalpha, native Baxbeta spontaneously integrates into mitochondrial membrane and is highly potent in inducing cytochrome c release from mitochondria. Remarkably, Baxbeta protein is upregulated by apoptotic stimuli via inhibition of its ubiquitination process, and stable expression of Baxbeta in HCT116-Bax(-/-) cells restores their sensitivity to multiple stimuli. Baxbeta associates with and promotes Baxalpha activation. Moreover, selective knockdown of Baxbeta desensitizes HCT116-Bax(+/-) cells to Bax-dependent apoptosis signaling. These observations underscore the plasticity of human Bax in serving its role as a "gatekeeper" for apoptosis.

SummaryFas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

The multidomain proapoptotic protein Bax of the Bcl-2 family is a central regulator for controlling the release of apoptogenic factors from mitochondria. Recent evidence suggests that the Bax-associating protein MOAP-1 may act as an effector for promoting Bax function in mitochondria. Here, we report that MOAP-1 protein is rapidly up-regulated by multiple apoptotic stimuli in mammalian cells. MOAP-1 is a short-lived protein ( t 1/2 ≈ 25 min) that is constitutively degraded by the ubiquitin-proteasome system. Induction of MOAP-1 by apoptotic stimuli ensues through inhibition of its polyubiquitination process. Elevation of MOAP-1 levels sensitizes cells to apoptotic stimuli and promotes recombinant Bax-mediated cytochrome c release from isolated mitochondria. Mitochondria depleted of short-lived proteins by cycloheximide (CHX) become resistant to Bax-mediated cytochrome c release. Remarkably, incubation of these mitochondria with in vitro -translated MOAP-1 effectively restores the cytochrome c releasing effect of recombinant Bax. We propose that apoptotic stimuli can facilitate the proapoptotic function of Bax in mitochondria through stabilization of MOAP-1.

The mammary gland represents a unique tissue to study organogenesis as it predominantly develops in the post-natal animal and undergoes dramatic morphogenetic changes during puberty and the reproductive cycle. The physiological function of the mammary gland is to produce milk to sustain the newborn. Here we view the lactating gland through three-dimensional confocal imaging of intact tissue. We observed that the majority of secretory alveolar cells are binucleated. These cells first arise in very late pregnancy due to failure of cytokinesis and are larger than mononucleated cells. Augmented expression of Aurora kinase-A and Polo-like kinase-1 at the lactogenic switch likely mediates the formation of binucleated cells. Our findings demonstrate an important physiological role for polyploid mammary epithelial cells in lactation, and based on their presence in five different species, suggest that binucleated cells evolved to maximize milk production and promote the survival of offspring across all mammalian species., The main function of the mammary gland is to produce milk to sustain offspring. Here, the authors show that secretory alveolar cells in the lactating gland in several species are binucleated, which increases milk production, and that binucleation is regulated by Aurora kinase-A and Polo-like kinase-1.

Apoptotic stimuli induce conformational changes in Bax and trigger its translocation from cytosol to mitochondria. Upon assembling into the mitochondrial membrane, Bax initiates a death program through a series of events, culminating in the release of apoptogenic factors such as cytochrome c . Although it is known that Bax is one of the key factors for integrating multiple death signals, the mechanism by which Bax functions in mitochondria remains controversial. We have previously identified modulator of apoptosis-1 (MAP-1) as a Bax-associating protein, but its functional relationship with Bax in contributing to apoptosis regulation remains to be established. In this study, we show that MAP-1 is a critical mitochondrial effector of Bax. MAP-1 is a mitochondria-enriched protein that associates with Bax only upon apoptotic induction, which coincides with the release of cytochrome c from mitochondria. Small interfering RNAs that diminish MAP-1 levels in mammalian cell lines confer selective inhibition of Bax-mediated apoptosis. Mammalian cells with stable expression of MAP-1 small interfering RNAs are resistant to multiple apoptotic stimuli in triggering apoptotic death as well as in inducing conformation change and translocation of Bax. Similar to Bax-deficient cells, MAP-1-deficient cells exhibit aggressive anchorage-independent growth. Remarkably, recombinant Bax- or tBid-mediated release of cytochrome c from isolated mitochondria is significantly compromised in the MAP-1 knockdown cells. We propose that MAP-1 is a direct mitochondrial target of Bax.

The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins.

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. Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5+ versus Lgr5– basal epithelial cells, we identify a novel marker that led to the fractionation of three distinct mammary stem cell (MaSC) subsets in the adult gland. These exist in a largely quiescent state but differ in their repopulating ability, spatial localisation, and their molecular signatures. Interestingly, the dormant MaSC subset localises to the proximal region of the gland throughout life. These cells appear to originate from the embryonic mammary primordia before switching to a quiescent state post-natally but can be recruited into the cell cycle in response to hormones. Single cell gene expression analyses have also revealed unexpected complexity within the basal and luminal compartments. Moreover, analyses at different stages of development have provided insights into the earliest ‘lineage priming’ events. Citation Format: Nai Yang Fu, Anne Rios, Bhupinder Pal, Charity Law, Paul Jamieson, Francois Vaillant, Gordon K. Smyth, Matthew E. Ritchie, Geoffrey J. Lindeman, Jane E. Visvader. Unmasking heterogeneity within the adult mammary stem cell compartment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5024. doi:10.1158/1538-7445.AM2017-5024

The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.

The mammary epithelium undergoes enormous morphogenetic changes during the lifespan of a mammal. The recent elucidation of an epithelial differentiation hierarchy in the mouse mammary gland through classical transplantation and clonogenic assays has pointed to the existence of multipotent mammary stem cells (MaSCs) and at least two distinct luminal progenitor types. Moreover, an analogous functional hierarchy has been defined in human breast tissue. The existence of slow cycling stem cells, both long- and short-term repopulating cells, and a unique fetal MaSC population, imply a complex stem cell compartment within the mammary gland. The recent discovery of unipotent stem-like cells from lineage tracing studies has added a further layer of complexity to the emerging differentiation hierarchy. Although the precise relationships between stem and progenitor cells have yet to be resolved, the epithelial hierarchy has provided an important framework for elucidating the roles of molecular regulators of mammary gland ontogeny and understanding potential cells of origin in breast cancer.

The mammary epithelium undergoes profound morphogenetic changes during development. Architecturally, it comprises two primary lineages, the inner luminal and outer myoepithelial cell layers. Two opposing concepts on the nature of mammary stem cells (MaSCs) in the postnatal gland have emerged. One model, based on classical transplantation assays, postulates that bipotent MaSCs have a key role in coordinating ductal epithelial expansion and maintenance in the adult gland, whereas the second model proposes that only unipotent MaSCs identified by lineage tracing contribute to these processes. Through clonal cell-fate mapping studies using a stochastic multicolour cre reporter combined with a new three-dimensional imaging strategy, we provide evidence for the existence of bipotent MaSCs as well as distinct long-lived progenitor cells. The cellular dynamics at different developmental stages support a model in which both stem and progenitor cells drive morphogenesis during puberty, whereas bipotent MaSCs coordinate ductal homeostasis and remodelling of the mouse adult gland.

RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-alpha stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-X L , a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-X L and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-X L was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-X L . Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-X L are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-X L also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-X L at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments.

Recent data on gene deletion analyses revealed that cells derived from animals that are deficient in both Bax and Bak (two key members of the “multidomain” proapoptotic Bcl-2 subfamily), but not cells lacking one of the two genes, are completely resistant to apoptotic death triggered by diverse stimuli. These data suggest that engagement of a “multidomain” proapoptotic member, BAX or BAK, is essential for apoptotic signaling. To gain further understanding of the molecular functions of the “multidomain” proapoptotic molecules, we used yeast two hybrid screen to identify protein partners of BAX. A proapoptotic protein, termed MAP-1 (Modulator of Apoptosis), was identified as a BAX-interacting protein from a human brain cDNA library. MAP-1 is a member of a growing family of proteins that were initially identified as onconeural antigens. MAP-1 contains a BH3-like motif and mediates caspase-dependent death in mammalian cells when overexpressed. Mutagenesis analyses revealed that the BH3-like domain of MAP-1 is required for association with BAX and for mediating apoptosis. Interestingly, in contrast to all other previously identified BAX-associating proteins that require only one of the three BH domains of BAX for binding, all the three BH (BH1, BH2 and BH3) domains of BAX are necessary for binding to MAP-1. Taken together, these data suggest that MAP-1 may mediate its proapoptotic function by engaging its BH3-like domain in binding the Bc1-XL-like hydrophobic pocket of BAX.

Effects of mitochondrial permeability transition pore-specific inhibitor cyclosporine A (CsA) and highly expressed Bcl-2 on the apoptosis of HL-60 cells induced by EGTA were studied. Detection of apoptotic peak by flow cytometry, fluorescent microscope observation of chromatin condensation with double staining of PI and Hoechst33342 and DNA ladder analysis all demonstrated that CsA obviously enhanced the apoptosis of HL-60 cells induced by EGTA, while highly expressed Bcl-2 completely blocked it. It is revealed by mitochondrial membrane potential (deltapsi(m)) fluorescent probes rhodamine 123 and CMXRos that the deltapsi(m) decreased in the apoptosis of HL-60 cells induced by EGTA. CsA enhanced the decrease of deltapsi(m), but highly expressed Bcl-2 increased deltapsi(m) of HL-60 cells about 2-fold and completely blocked the decrease of deltapsi(m).

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

To understand relationships between breast tissue 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 markers and subsets that are highly enriched for mammary stem (MaSC), luminal progenitor and mature luminal cells have been identified. These are functionally analogous across species and have highly conserved transcriptomes. Lineage tracing is an important approach 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. Indeed, recent studies in the mammary gland have described unipotent stem cells that appear to control the different stages of development. Here we have combined lineage tracing with a novel three-dimensional (3D) imaging strategy to explore the relative contributions of stem and progenitor cells in the post-natal mammary gland. Newly generated transgenic strains harboring lineage-specific gene regulatory regions are also being used to direct the expression of specific mammary oncogenes to discrete epithelial cell types to gain insight into cells of origin of breast cancer. Citation Format: Anne Rios, Naiyang Fu, Bhupinder Pal, Geoffrey Lindeman, Jane Visvader. Mammary epithelial subtypes and their implications for breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA12.

Transcription factors are essential for controlling cell fate decisions and differentiation during embryonic and adult development. Although transcription factors have been implicated in regulating development of the mammary gland, most of these have not been defined at a cellular level. Recent studies in the laboratory have identified the transcriptional regulator Inhibitor of Differentiation 4 (Id4) as a highly expressed gene in the mammary stem cell (MaSC)-enriched population of both mouse and human mammary glands. This population also contains progenitors and mature myoepithelial cells. Germ-line deletion of Id4 in mice has previously revealed a role for this gene in neuronal differentiation1, and more recently, as a regulator of mammary gland development through its suppression of p38MAPK2. Id4 has been identified as differentially regulated in breast cancers and plays a role in the negative regulation of BRCA13,4. We have developed a conditional knockout mouse model to investigate the function of Id4 specifically in the mammary epithelium. Id4 is expressed in myoepithelial cells of the terminal end buds and ducts, with expression reducing after puberty. Consistent with previous findings2, Id4-deficient mammary glands displayed a prominent reduction in ductal elongation and branching. We found that Id4-deficient mammary epithelial cells also have a diminished capacity to maintain the balance of luminal to myoepithelial cells, and that the MaSC/basal subset had significantly reduced repopulating capacity when assayed by transplantation. Understanding the mechanism and role of Id4 in the developing mammary gland may reveal important parallels with the role of Id4 in breast cancer. 1. Yun et al. Development, 2004. 131(21):5441-8 2. Dong et al. Development, 2011. 138(23):5247-56 3. Beger et al. PNAS, 2001. 98(1):130-5 4. Roldan et al. Cancer Biology and Therapy, 2006. 5(5):505-10 Citation Format: Sarah A. Best, NaiYang Fu, Francois Vaillant, Geoff J. Lindeman, Jane E. Visvader. Role of the transcription factor Id4 in the developing mammary gland. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A035.