Christopher Dravis

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells

To discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.

Stem Cells and the Developing Mammary Gland

The mammary gland undergoes dynamic changes throughout life. In the mouse, these begin with initial morphogenesis of the gland in the mid-gestation embryo followed by hormonally regulated changes during puberty and later in adulthood. The adult mammary gland contains a hierarchy of cell types with varying potentials for self-maintenance and differentiation. These include cells able to produce complete, functional mammary glands in vivo and that contain daughter cells with the same remarkable regenerative potential, as well as cells with more limited clonogenic activity in vitro. Here we review how applying in vitro and in vivo methods for quantifying these cells in adult mammary tissue to fetal mammary cells has enabled the first cells fulfilling the functional criteria of transplantable, isolated mammary stem cells to be identified a few days before birth. Thereafter, the number of these cells increases rapidly. Populations containing these fetal stem cells display growth and gene expression programs that differ from their adult counterparts but share signatures characteristic of certain types of breast cancer. Such observations reinforce growing evidence of important differences between tissue-specific fetal and adult cells with stem cell properties and emphasize the merits of investigating their molecular basis.

Lgr5 is a marker for fetal mammary stem cells, but is not essential for stem cell activity or tumorigenesis

The search for the bipotent mammary stem cells that drive mammary development requires markers to enable their prospective isolation. There is general agreement that bipotent mouse mammary stem cells are abundant in late fetal development, but their existence in the adult is vigorously debated. Among markers useful for mammary stem cell identification, the Wnt co-receptor Lgr5 has been suggested by some to be both “necessary and sufficient” for bipotency and transplantation of adult mammary stem cell activity, though other studies disagree. Importantly, the relevance of Lgr5 to the bipotency of fetal mammary stem cells has not been studied. We show here that expression of a fluorescent protein driven by the endogenous Lgr5 promoter enables significant fetal mammary stem cell enrichment. We used lineage tracing to demonstrate embryonic cells expressing Lgr5 are bipotent, while their adult counterparts are myoepithelial restricted. Importantly, our data conclusively demonstrate that Lgr5 is dispensable for both fetal and adult mammary stem cell activity and for the development of mammary tumors.Stem cells: protein not needed for stem cell activity in mammary glandA protein considered necessary for mammary stem cell activity turns out to be dispensable. What’s more, it’s not required for tumor development either. Geoffrey Wahl, Christy Trejo, and colleagues from the Salk Institute for Biological Studies in La Jolla, CA, USA, investigated whether the expression of a protein called Lgr5 denotes mammary stem cells and whether it’s truly needed for the cells to be capable of giving rise to two cell lineages—a subject of active debate among researchers. Wahl’s team showed that mouse fetal mammary stem cells expressing Lgr5 had this dual-lineage capacity; their counterparts in the adult mouse did not. Yet, even though Lgr5 expression marks a population of fetal mammary stem cells, the protein is not required for stem cell activity or for tumors to form in a mouse model of breast cancer.

Single-Cell Transcriptomes Distinguish Stem Cell State Changes and Lineage Specification Programs in Early Mammary Gland Development

SUMMARY The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.

Epigenetic and Transcriptomic Profiling of Mammary Gland Development and Tumor Models Disclose Regulators of Cell State Plasticity

Cell state reprogramming during tumor progression complicates accurate diagnosis, compromises therapeutic effectiveness, and fuels metastatic dissemination. We used chromatin accessibility assays and transcriptional profiling during mammary development as an agnostic approach to identify factors that mediate cancer cell state interconversions. We show that fetal and adult basal cells share epigenetic features consistent with multi-lineage differentiation potential. We find that DNA-binding motifs for SOX transcription factors are enriched in chromatin that is accessible in stem/progenitor cells and inaccessible in differentiated cells. In both mouse and human tumors, SOX10 expression correlates with stem/progenitor identity, dedifferentiation, and invasive characteristics. Strikingly, we demonstrate that SOX10 binds to genes that regulate neural crest cell identity, and that SOX10-positive tumor cells exhibit neural crest cell features.

Abstract IA10: Sox10 regulates stem/progenitor and mesenchymal cell states in mammary epithelial cells

To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor, Sox10. Here we report that Sox10 is specifically expressed in mammary cells that exhibit the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we find that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, and that Sox10+ tumor cells utilize FGF signaling for growth and invasive behaviors, which suggest that Sox10 may reprise these same functions during tumorigenesis. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 regulates stem/progenitor and mesenchymal cell states in mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA10.

Abstract 914: Sox10 regulates stem- and mesenchymal-like features in mammary cells

To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor, Sox10. Here we report that Sox10 is specifically expressed in mammary cells that exhibit the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we report that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, which suggest that Sox10 may reprise these same functions during tumorigenesis. Indeed, we find that in an autochthonous mouse model of basal-like breast cancer, Sox10 is expressed at high levels and that these Sox10+ tumor cells exhibit characteristics that are similar to mammary cells in which Sox10 is ectopically expressed. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 regulates stem- and mesenchymal-like features in mammary cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 914.

Abstract 2326: Sox10 promotes both stem-like and EMT properties in mammary cells

Molecular pathways that guide cell proliferation and programming events during embryonic development may be aberrantly re-activated in the cancer state to drive tumor progression. To identify mechanisms that drive progression of aggressive breast cancers, we identified molecules that are present in the fetal mammary stem cells (fMaSCs) that drive early development of the mammary gland. These analyses revealed that Sox10 is one of the most highly expressed transcription factors within fMaSCs, and prompted us to investigate the function of Sox10 in the mammary gland and in subtypes of cancer originating from it. Our studies described here indicate that Sox10 is specifically expressed in mammary cells that exhibit higher levels of stem or progenitor cell functions using in vitro or in vivo assays. This includes fetal and adult mammary cells in vivo, as well as mammary cells cultured in vitro as organoids, and for the first time, male fetal mammary stem cells. Sox10 appears to contribute to these stem/progenitor behaviors, as the genetic deletion of Sox10 limits stem/progenitor activities, while the overexpression of Sox10 significantly expands clonogenic behavior. Intriguingly, ectopic Sox10 expression is also able to elicit an EMT-like response in mammary organoids. Sox10 thus represents a single molecule capable of directly mediating both stem-like and EMT-like properties in mammary cells. Furthermore, by modulating Sox10 levels in 3D mammary organoids, we are able to induce cells into sequential motile and stem/progenitor states. This provides a striking in vitro surrogate for metastatic behavior. Consistent with these findings, we find that Sox10 is highly expressed in basal-like and claudin-low subtypes of breast cancer, which are the most stem-like and EMT-like manifestations of the disease, respectively. Finally, we show that Sox10 expression is regulated through a feedback loop involving FGF signaling, and that inhibition of FGF signaling can block EMT-like cell behaviors mediated by Sox10. These findings have important implications in how stem-like and metastatic properties may be specified in mammary cells. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 promotes both stem-like and EMT properties in mammary cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2015-2326

Abstract 1960: Sox10 expression labels mammary stem cell activity in fetal, adult, cultured, and male mammary tissues and is attenuated by FGF signaling inhibition

Molecular pathways that guide cell proliferation and programming events during embryonic development may be aberrantly re-activated in the cancer state to drive tumor progression. We have demonstrated the applicability of this tenet to studies of breast cancer. We have isolated fetal mammary stem cells (fMaSCs) from embryonic mammary rudiments, and verified that particular subtypes of breast cancers, including Basal-like triple-negative breast cancers, show strong correlation with expression signatures found in fMaSCs. This suggests that elucidation of the biology behind fMaSC function will yield insight into better treatment of these aggressive breast cancers. Through transcriptome analysis of fMaSCs, the transcription factor Sox10 was identified as a potential mediator of MaSC-activity. By analyzing a Sox10-Venus reporter line, we have discovered that Sox10 labels fMaSCs in the embryo, and improves recovery of fMaSC-enriched populations threefold over previous markers. Intriguingly, Sox10 also permits the first identification and recovery of male fMaSCs, which also demonstrate stem cell behaviors in vitro and in vivo. In the adult mammary gland, Sox10 only labels a subset of mammary cells, yet these cells possess all in vitro colony-forming potential compared to Sox10-negative cells. Of interest, both Sox10+ and Sox10-negative adult mammary cells demonstrate in vivo stem cell behaviors, suggesting the presence of unique multipotent MaSC populations. We find that Sox10 is also highly enriched in Basal-like breast cancers. Per previous studies with Sox9 and Sox10 in pancreatic and skin cancer models, Sox10 may play critical roles in the progression of Basal-like breast cancer. Molecular pathways that regulate Sox10 expression may therefore be useful therapeutic targets for treating Basal-like breast cancers. We find that perturbation of FGF signaling significantly attenuates Sox10 expression in organoids grown in vitro that are derived from either fMaSCs or luminal progenitors. As Sox10+ cells in mammary organoids specifically possess and label retained MaSC properties that are not seen in Sox10-negative cells, this may indicate a mechanism to attenuate MaSC specification or function in mammary tissue. Our discoveries with Sox10 thus represent a molecular focal point around which to build signaling and transcriptional networks underlying fMaSC state and function. To capitalize on this, we have performed RNA-sequencing on fetal and adult mammary populations using Sox10 as a marker. These analyses combine significantly purer cell fractions with a sequencing tool capable of most accurately profiling transcription events. This is expected to provide highest quality transcriptome data describing signaling events in these mammary tissues, which will identify potential therapeutic targets for different subtypes of breast cancer. Citation Format: Christopher Dravis, Geoff Wahl. Sox10 expression labels mammary stem cell activity in fetal, adult, cultured, and male mammary tissues and is attenuated by FGF signaling inhibition. [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 1960. doi:10.1158/1538-7445.AM2014-1960

Abstract A136: Deconstructing stemness from the fetal mammary perspective

Somatic stem cells govern the development, maintenance and regeneration of tissues, and their dysregulation is associated with diverse pathologies including cancer. Given the significance of these cells both biologically and therapeutically, it is critical to elucidate the signaling mechanisms that dictate their behavior. Our recent work on the fetal mammary gland has dissected complementary roles for the stem cell compartment and stromal cues in fetal mammary stem cell function. We also identified significant similarities between the gene expression profiles of some of the most aggressive and untreatable forms of human breast cancer and those of the mouse fetal stem cell enriched mammary epithelial and stromal fractions. These similarities suggest that stem cell regulatory mechanisms operative during organogenesis, including those specified by the stromal niche, may serve as novel therapeutic targets in these cancers. We also discovered significant gene expression heterogeneity within the fetal stem cell compartment when analyzed at single cell resolution. While such heterogeneity likely reflects the natural diversity of stem and non-stem cell states comprising the system, it confounds the interpretation of expression profiles from mixed populations and the delineation of stem cell specific and niche specific molecular components. Our current objective is to utilize genome wide single cell RNA-Sequencing to hasten the identification of functionally relevant, stem cell specific regulators and markers in the context of this multi-cellular/multi-signal system. Among other candidates, we have identified the Cripto/GRP78 signaling axis as a critical regulator of mammary stem cell activity, and have developed novel molecular genetic tools to temporally control the activity of candidate stem cell regulators in vivo in normal and neoplastic contexts. Integration of these tools promises to deliver a new molecular understanding of stem cell biology in the mammary gland and other systems and of stem cell-like cancer cells. Citation Format: Benjamin T. Spike, Danielle D. Engle, Jennifer C. Lin, Jonathan A. Kelber, Justin La, Samantha K. Cheung, Evan Booker, Rose Rodewald, Christopher Dravis, Peter C. Gray, Geoffrey M. Wahl. Deconstructing stemness from the fetal mammary perspective. [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 A136.