postdoc associate, The Rockefeller University
Stem cell plasticity drives cancer and wound repair
Human tissues harbor resident adult stem cells that, upon environmental insults, undergo tightly regulated self-renewal, lineage commitment, and fate conversion to repair damage and restore homeostasis. Although it’s long been postulated tumor is a wound that never heals, it is unclear what are the determinants of homeostasis restoration versus point-of-no-return toward malignancy. I aim to tackle this important question by developing novel tools to analyze in vivo stress response and conducting functional screens to interrogate genes that dictate stem cell behaviors at the bifurcation point between skin wound and hyperplasic disorders. Over the past decades, we start to understand some of the molecular mechanisms involved in wound repair. We have also acquired substantial insights into oncogenic and tumor suppressive pathways in cancer. However, despite the long posited hypothesis that cancer is a wound that never heals, it is still unclear whether wounds and cancer functionally rely on the same stress components and if so, at which point they diverge. These are the primary focus of my current and future proposed research.
Abstract: MicroRNAs are established as crucial modulators of skeletal myogenesis, but our knowledge about their identity and targets remains limited. In this study, we have identified microRNA-146b (miR-146b) as a novel regulator of skeletal myoblast differentiation. Following up on a previous microRNA profiling study, we establish that the expression of miR-146b is up-regulated during myoblast differentiation in vitro and muscle regeneration in vivo. Inhibition of miR-146b led to reduced myoblast differentiation, whereas overexpression of miR-146b enhanced differentiation. Computational prediction combined with gene expression information has revealed candidates for miR-146b targets in muscles. Among them, the expression of Smad4, Notch1, and Hmga2 are significantly suppressed by miR-146b overexpression in myocytes. In addition, expression levels of Smad4, Notch1 and Hmga2 are decreased during myoblast differentiation and muscle regeneration, inversely correlating to the levels of miR-146b. Importantly, inhibition of endogenous miR-146b prevents the down-regulation of Smad4, Notch1 and Hmga2 during differentiation. Furthermore, miR-146b directly targets the microRNA response elements (MREs) in the 3'UTR of those genes as assessed by reporter assays. Reporters with the seed regions of MREs mutated are insensitive to miR-146b, further confirming the specificity of targeting. In conclusion, miR-146b is a positive regulator of myogenic differentiation, possibly acting through multiple targets.
Pub.: 24 Jun '14, Pinned: 28 Jun '17
Abstract: Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases.
Pub.: 12 Jul '13, Pinned: 28 Jun '17
Abstract: Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.
Pub.: 02 Oct '09, Pinned: 28 Jun '17
Abstract: Mammalian target of rapamycin (mTOR) has emerged as a key regulator of skeletal muscle development by governing distinct stages of myogenesis, but the molecular pathways downstream of mTOR are not fully understood. In this study, we report that expression of the muscle-specific micro-RNA (miRNA) miR-1 is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of histone deacetylase 4 (HDAC4) results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the long-sought mTOR-regulated fusion factor. In summary, our findings unravel for the first time a link between mTOR and miRNA biogenesis and identify an mTOR-miR-1-HDAC4-follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo.
Pub.: 23 Jun '10, Pinned: 28 Jun '17
Abstract: It has been widely proposed that signaling by mammalian target of rapamycin (mTOR) is both necessary and sufficient for the induction of skeletal muscle hypertrophy. Evidence for this hypothesis is largely based on studies that used stimuli that activate mTOR via a phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)-dependent mechanism. However, the stimulation of signaling by PI3K/PKB also can activate several mTOR-independent growth-promoting events; thus, it is not clear whether signaling by mTOR is permissive, or sufficient, for the induction of hypertrophy. Furthermore, the presumed role of mTOR in hypertrophy is derived from studies that used rapamycin to inhibit mTOR; yet, there is very little direct evidence that mTOR is the rapamycin-sensitive element that confers the hypertrophic response. In this study, we determined that, in skeletal muscle, overexpression of Rheb stimulates a PI3K/PKB-independent activation of mTOR signaling, and this is sufficient for the induction of a rapamycin-sensitive hypertrophic response. Transgenic mice with muscle specific expression of various mTOR mutants also were used to demonstrate that mTOR is the rapamycin-sensitive element that conferred the hypertrophic response and that the kinase activity of mTOR is necessary for this event. Combined, these results provide direct genetic evidence that a PI3K/PKB-independent activation of mTOR signaling is sufficient to induce hypertrophy. In summary, overexpression of Rheb activates mTOR signaling via a PI3K/PKB-independent mechanism and is sufficient to induce skeletal muscle hypertrophy. The hypertrophic effects of Rheb are driven through a rapamycin-sensitive (RS) mechanism, mTOR is the RS element that confers the hypertrophy, and the kinase activity of mTOR is necessary for this event.
Pub.: 30 Jul '10, Pinned: 28 Jun '17
Abstract: MicroRNAs (miRNAs) have emerged as key regulators of skeletal myogenesis, but our knowledge of the identity of the myogenic miRNAs and their targets remains limited. In this study, we report the identification and characterization of a novel myogenic miRNA, miR-125b. We find that the levels of miR-125b decline during myogenesis and that miR-125b negatively modulates myoblast differentiation in culture and muscle regeneration in mice. Our results identify IGF-II (insulin-like growth factor 2), a critical regulator of skeletal myogenesis, as a direct and major target of miR-125b in both myocytes and regenerating muscles, revealing for the first time an miRNA mechanism controlling IGF-II expression. In addition, we provide evidence suggesting that miR-125b biogenesis is negatively controlled by kinase-independent mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo as a part of a dual mechanism by which mTOR regulates the production of IGF-II, a master switch governing the initiation of skeletal myogenesis.
Pub.: 05 Jan '11, Pinned: 28 Jun '17
Abstract: MicroRNAs (miRNAs) have emerged as critical regulators of numerous biological processes by modulating gene expression at the post-transcriptional level. It has become increasingly clear that almost all aspects of skeletal muscle development involve regulation by miRNAs. Many of these miRNAs have distinct expression profiles in skeletal muscles, under the regulation by the myogenic program. In the last few years the field has seen a rapid expansion of our knowledge of myogenic miRNAs that target a wide range of muscle genes to coordinately control the myogenic process. In this review we provide an up-to-date list of reported myogenic miRNAs and survey their expression patterns, regulation of biogenesis, and gene targets in skeletal muscles. Emerging themes of miRNA regulation in the context of skeletal myogenesis will also be discussed.
Pub.: 29 Jan '11, Pinned: 28 Jun '17
Abstract: The mammalian target of rapamycin (mTOR) is essential for skeletal myogenesis through controlling distinct cellular pathways. The importance of the canonical mTOR complex 1 signaling components, including raptor, S6K1, and Rheb, had been suggested in muscle maintenance, growth, and metabolism. However, the role of those components in myogenic differentiation is not entirely clear. In this study we have investigated the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. We find that although mTOR knockdown severely impairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb, enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpression of raptor or Rheb inhibits C2C12 differentiation. On the other hand, neither knockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiation elicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1 protein levels, and decreased Ser-307 (human Ser-312) phosphorylation on IRS1. Finally, IRS1 knockdown eliminated the enhancement in differentiation elicited by raptor or Rheb knockdown, suggesting that IRS1 is a critical mediator of the myogenic functions of raptor and Rheb. In conclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation by suppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTOR signaling in biological regulations and implicate the existence of novel mTOR complexes and/or signaling mechanism in skeletal myogenesis.
Pub.: 20 Aug '11, Pinned: 28 Jun '17
Abstract: Mammalian (or mechanistic) target of rapamycin (mTOR) regulates a wide range of cellular and developmental processes by coordinating signaling responses to mitogens, nutrients, and various stresses. Over the last decade, mTOR has emerged as a master regulator of skeletal myogenesis, controlling multiple stages of the myofiber formation process. In this minireview, we present an emerging view of the signaling network underlying mTOR regulation of myogenesis, which contrasts with the well established mechanisms in the regulation of cell and muscle growth. Current questions for future studies are also highlighted.
Pub.: 02 Nov '12, Pinned: 28 Jun '17
Abstract: Various cues initiate multiple signaling pathways to regulate the highly coordinated process of skeletal myogenesis. Myoblast differentiation comprises a series of ordered events starting with cell cycle withdrawal and ending with myocyte fusion, with each step probably controlled by multiple extracellular signals and intracellular signaling pathways. Here we report the identification of Fms-like tyrokine kinase 3 ligand (Flt3L) signaling as a novel regulator of skeletal myogenesis. Flt3L is a multifunctional cytokine in immune cells, but its involvement in skeletal muscle formation has not been reported. We found that Flt3L is expressed in C2C12 myoblasts, with levels increasing throughout differentiation. Knockdown of Flt3L, or its receptor Flt3, suppresses myoblast differentiation, which is rescued by recombinant Flt3L or Flt3, respectively. Differentiation is not rescued, however, by recombinant ligand when the receptor is knocked down, or vice versa, suggesting that Flt3L and Flt3 function together. Flt3L knockdown also inhibits differentiation in mouse primary myoblasts. Both Flt3L and Flt3 are highly expressed in nascent myofibers during muscle regeneration in vivo, and Flt3L siRNA impairs muscle regeneration, validating the physiological significance of Flt3L function in myogenesis. We have identified a cellular mechanism for the myogenic function of Flt3L, as we show that Flt3L promotes cell cycle exit that is necessary for myogenic differentiation. Furthermore, we identify Erk as a relevant target of Flt3L signaling during myogenesis, and demonstrate that Flt3L suppresses Erk signaling through p120RasGAP. In summary, our work reveals an unexpected role for an immunoregulatory cytokine in skeletal myogenesis and a new myogenic pathway.
Pub.: 25 May '13, Pinned: 28 Jun '17
Abstract: Previously, we identified miR-125b as a key regulator of the undifferentiated state of hair follicle stem cells. Here, we show that in both mice and humans, miR-125b is abundantly expressed, particularly at early stages of malignant progression to squamous cell carcinoma (SCC), the second most prevalent cancer worldwide. Moreover, when elevated in normal murine epidermis, miR-125b promotes tumor initiation and contributes to malignant progression. We further show that miR-125b can confer "oncomiR addiction" in early stage malignant progenitors by delaying their differentiation and favoring an SCC cancer stem cell (CSC)-like transcriptional program. To understand how, we systematically identified and validated miR125b targets that are specifically associated with tumors that are dependent on miR-125b. Through molecular and genetic analysis of these targets, we uncovered new insights underlying miR-125b's oncogenic function. Specifically, we show that, on the one hand, mir-125b directly represses stress-responsive MAP kinase genes and associated signaling. On the other hand, it indirectly prolongs activated (phosphorylated) EGFR signaling by repressing Vps4b (vacuolar protein-sorting 4 homolog B), encoding a protein implicated in negatively regulating the endosomal sorting complexes that are necessary for the recycling of active EGFR. Together, these findings illuminate miR-125b as an important microRNA regulator that is shared between normal skin progenitors and their early malignant counterparts.
Pub.: 19 Nov '14, Pinned: 28 Jun '17
Abstract: MicroRNAs play diverse roles in both normal and malignant stem cells. Focusing on miRs and/or miR(∗)s abundant in squamous cell carcinoma (SCC) stem cells, we engineer an efficient, strand-specific expression library, and apply functional genomics screening in mice to identify which of 169 cancer-associated miRs are key drivers in malignant progression. Not previously linked functionally to cancer, miR-21(∗) was the second top hit, surfacing in >12% of tumours. miR-21(∗) also correlates with poor prognosis in human SCCs and enhances tumour progression in xenografts. On deleting the miR-21 gene and rescuing each strand separately, we document the dual, but independent, oncogenicity of miR-21 and miR-21(∗). A cohort of predicted miR-21(∗) targets inversely correlate with miR-21(∗) in SCCs. Of particular interest is Phactr4, which we show is a miR-21(∗) target in SCCs, acting through the Rb/E2F cell cycle axis. Through in vivo physiological miR screens, our findings add an interesting twist to an increasingly important oncomiR locus.
Pub.: 01 Dec '15, Pinned: 28 Jun '17
Abstract: Adult tissue stem cells (SCs) reside in niches, which, through intercellular contacts and signaling, influence SC behavior. Once activated, SCs typically give rise to short-lived transit-amplifying cells (TACs), which then progress to differentiate into their lineages. Here, using single-cell RNA-seq, we unearth unexpected heterogeneity among SCs and TACs of hair follicles. We trace the roots of this heterogeneity to micro-niches along epithelial-mesenchymal interfaces, where progenitors display molecular signatures reflective of spatially distinct local signals and intercellular interactions. Using lineage tracing, temporal single-cell analyses, and chromatin landscaping, we show that SC plasticity becomes restricted in a sequentially and spatially choreographed program, culminating in seven spatially arranged unilineage progenitors within TACs of mature follicles. By compartmentalizing SCs into micro-niches, tissues gain precise control over morphogenesis and regeneration: some progenitors specify lineages immediately, whereas others retain potency, preserving self-renewing features established early while progressively restricting lineages as they experience dynamic changes in microenvironment.
Pub.: 18 Apr '17, Pinned: 28 Jun '17
Abstract: Tissue stem cells contribute to tissue regeneration and wound repair through cellular programs that can be hijacked by cancer cells. Here, we investigate such a phenomenon in skin, where during homeostasis, stem cells of the epidermis and hair follicle fuel their respective tissues. We find that breakdown of stem cell lineage confinement-granting privileges associated with both fates-is not only hallmark but also functional in cancer development. We show that lineage plasticity is critical in wound repair, where it operates transiently to redirect fates. Investigating mechanism, we discover that irrespective of cellular origin, lineage infidelity occurs in wounding when stress-responsive enhancers become activated and override homeostatic enhancers that govern lineage specificity. In cancer, stress-responsive transcription factor levels rise, causing lineage commanders to reach excess. When lineage and stress factors collaborate, they activate oncogenic enhancers that distinguish cancers from wounds.
Pub.: 25 Apr '17, Pinned: 28 Jun '17
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