Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

The tail of yeast myosin II is localized to the division site by two distinct molecular pathways and sufficient for promoting actomyosin ring assembly, furrow ingression, and guidance in ECM remodeling

Novel Non-Repulsive Outcomes From Ephrin-B Reverse Signaling

In this dissertation, I present a detailed characterization of the physiological roles for reverse signaling through B-subclass ephrins during embryonic development. The stereotypical outcome of Eph-ephrin signaling has long been established as one of cell-cell repulsion, elicited through a localized breakdown of the actin cytoskeleton. In contrast to this dogma, I have found that ephrin-B reverse signaling is instead necessary for mediating cell-cell adhesion events during several critical midline closure events. I demonstrate that mice with germline mutations specifically disrupting the ability of ephrin-B2 to conduct cell autonomous signals present with defects in urorectal septation of the hindgut, tubularization of the urethra, tracheoesophageal septation of the foregut, closure of the palatal shelves, and closure of the embryonic eyelid, defining roles for ephrin-B2 reverse signaling in each of these developmental events. Further, I show that mice with germline mutations either deleting EphB2 or similarly impairing the ability of EphB2 to conduct cell autonomous forward signals indicate that EphB2 acts non-cell autonomously to activate ephrin-B1 reverse signaling in closure of the ventral body wall. The developmental malformations in these mutant animals are each hallmarked by the failure of lateral mesenchymal folds to properly adhere, typically at the midline, which is difficult to reconcile with the canonical outcome of cell-repulsion from Eph-ephrin signaling. Consistent with a role in eliciting cell-cell adhesion at these septation events, I show that EphB and ephrin-B molecules are expressed in the epithelia where adhesion will take place. Moreover, my data specifically localizes ephrin-B reverse signaling to these adherent epithelia. Finally, an in vitro palatal shelf fusion assay used to determine the role of Eph-ephrin signaling in these developmental events indicates clear roles for ephrin-B2 reverse signaling in cell-cell adhesion. Taken together, my data leads me to propose that ephrin-B reverse signaling is not only capable of cell repulsion, but is also able to elicit cell-cell adhesion responses, which are employed in a bevy of adhesion-based septation events during embryonic development. Finally, my analysis of reverse signaling-deficient ephrin-B2 mice also indicates an additional novel, non-repulsive role for ephrin-B reverse signaling in mediating ionic homeostasis within the inner ear. I show that these ephrin-B2 mutant mice present with a circling or "waltzing" phenotype due to severe defects within the vestibular apparatus. My data shows that the disruption of bidirectional signaling between EphB2 and ephrin-B2 result in malformed structures within the vestibular apparatus and abnormal endolymph fluid. Further, I show that EphB2 and ephrin-B2 are expressed on non-motile, secretory epithelia within the inner ear, suggesting that these molecules play important roles in maintaining the proper volume and ionic makeup of the endolymph fluid running through the vestibular apparatus. My data leads me to propose that ephrin-B2 reverse signaling is therefore not only capable of regulating the cytoskeleton to produce either cell repulsion or cell adhesion outcomes, but that these reverse signals can mediate cellular responses independent of cytoskeletal dynamics, such as ionic homeostasis

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

Abstract 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</jats:p

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

Abstract 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.</jats:p

Ephrin-B reverse signaling controls septation events at the embryonic midline through separate tyrosine phosphorylation-independent signaling avenues

AbstractWe report that the disruption of bidirectional signaling between ephrin-B2 and EphB receptors impairs morphogenetic cell–cell septation and closure events during development of the embryonic midline. A novel role for reverse signaling is identified in tracheoesophageal foregut septation, as animals lacking the cytoplasmic domain of ephrin-B2 present with laryngotracheoesophageal cleft (LTEC), while both EphB2/EphB3 forward signaling and ephrin-B2 reverse signaling are shown to be required for midline fusion of the palate. In a third midline event, EphB2/EphB3 are shown to mediate ventral abdominal wall closure by acting principally as ligands to stimulate ephrin-B reverse signaling. Analysis of new ephrin-B26YFΔV and ephrin-B2ΔV mutants that specifically ablate ephrin-B2 tyrosine phosphorylation- and/or PDZ domain-mediated signaling indicates there are at least two distinct phosphorylation-independent components of reverse signaling. These involve both PDZ domain interactions and a non-canonical SH2/PDZ-independent form of reverse signaling that may utilize associations with claudin family tetraspan molecules, as EphB2 and activated ephrin-B2 molecules are specifically co-localized with claudins in epithelia at the point of septation. Finally, the developmental phenotypes described here mirror common human midline birth defects found with the VACTERL association, suggesting a molecular link to bidirectional signaling through B-subclass Ephs and ephrins

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

Abstract 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.</jats:p

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

Abstract 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</jats:p

Abstract B136: Sox10 expression labels in vitro mammary stem cell activity

Abstract Molecular pathways that guide cell proliferation and programming events during embryonic development to generate specific organs may be aberrantly re-activated in the cancer state of those organs to drive tumor progression. Recently, our laboratory has successfully demonstrated the applicability of this tenet to studies of breast cancer. We have developed an enrichment protocol for fetal mammary stem cells (fMaSCs) from embryonic mammary rudiments, and verified that particular subtypes of breast cancers, including Basal-like triple-negative breast cancers for which targeted therapies do not exist, show strong correlation with expression signatures found in fMaSCs. This suggests that elucidation of the biology behind fMaSC function will yield important insight into better treatment of these aggressive breast cancers. To identify mechanisms that govern fMaSC function, we have performed transcriptome analyses on cell populations enriched for fMaSCs and their surrounding stroma. From this data, we have identified candidate signaling and transcription networks that we are currently validating. Here we describe ongoing work with one candidate to emerge from our analyses, Sox10. Sox10 is specifically expressed in the enriched fMaSC population, and is of particular interest given the well-documented roles for other Sox family transcription factors in: 1) regulating cell fate decisions in embryonic and adult tissue, and 2) mediating the progression of some types of cancer. We show that Sox10 expression specifically correlates with MaSC function in fetal and adult mammary. We further show that Sox10 expression can be utilized to identify the cells maintaining MaSC stem cell properties in culture. Importantly, incorporation of Sox10 into our existing fMaSC purification protocol results in a twofold improvement in fMaSC enrichment. Our discoveries with Sox10 thus represent a molecular focal point around which to build signaling and transcriptional networks underlying fMaSC state and function. They further demonstrate the promise of our approach in identifying novel targets for the development of new therapies for poorly treated cancers. Citation Format: Christopher Dravis, Claire Johns, Benjamin T. Spike, Michelle Southard Smith, Geoffrey M. Wahl. Sox10 expression labels in vitro mammary stem cell activity. [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 B136.</jats:p