Promotes Stem Cell Maintenance Research Articles

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis.

A deeper understanding of the metastatic process is required for the development of new therapies that improve patient survival. Metastatic tumor cell growth and survival in distant organs is facilitated by the formation of a pre-metastatic niche composed of hematopoietic cells, stromal cells, and extracellular matrix (ECM). Perivascular cells, including vascular smooth muscle cells (vSMCs) and pericytes, are involved in new vessel formation and in promoting stem cell maintenance and proliferation. Given the well-described plasticity of perivascular cells, we hypothesize that perivascular cells similarly regulate tumor cell fate at metastatic sites. Using perivascular cell-specific and pericyte-specific lineage-tracing models, we trace the fate of perivascular cells in the pre-metastatic and metastatic microenvironments. We show that perivascular cells lose the expression of traditional vSMC/pericyte markers in response to tumor-secreted factors and exhibit increased proliferation, migration, and ECM synthesis. Increased expression of the pluripotency gene Klf4 in these phenotypically-switched perivascular cells promotes a less differentiated state characterized by enhanced ECM production that establishes a pro-metastatic fibronectin-rich environment. Genetic inactivation of Klf4 in perivascular cells decreases pre-metastatic niche formation and metastasis. Our data reveal a previously unidentified role for perivascular cells in pre-metastatic niche formation and uncover novel strategies for limiting metastasis.

Redox Homeostasis Plays Important Roles in the Maintenance of the Drosophila Testis Germline Stem Cells.

SummaryOxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. We utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in Keap1/Nrf2 activity decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells.

Elucidating the full repertoire of molecular mechanisms that promote stem cell maintenance requires sophisticated techniques for identifying and characterizing gene function in stem cells in their native environment. Ovarian germline stem cells in the fruit fly, Drosophila melanogaster, are an ideal model to study the complex molecular mechanisms driving stem cell function in vivo. A variety of new genetic tools make RNAi a useful complement to traditional genetic mutants for the investigation of the molecular mechanisms guiding ovarian germline stem cell function. Here, we provide a detailed guide for using targeted RNAi knockdown for the discovery of gene function in ovarian germline stem cells and their progeny.

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning

The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer.

BackgroundNotch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC).Patients and MethodsWe analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays.ResultsWe show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1, when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P=0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P=0.029).ConclusionWe anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer.

Parent stem cells can serve as niches for their daughter cells.

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance, while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation. Recently, stem cells have been shown to regulate themselves using an autocrine mechanism. The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells. Since then, an increasing body of work has focused on defining stem cell niches. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium, basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly, this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation, lineage tracing and signalling pathway modulation, we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals, the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus, a parent stem/progenitor cell can serve as a functional daughter cell niche.

The hypoxia-inducible epigenetic regulators Jmjd1a and G9a provide a mechanistic link between angiogenesis and tumor growth.

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.

Coupling of Hedgehog and Hippo pathways promotes stem cell maintenance by stimulating proliferation

It is essential to define the mechanisms by which external signals regulate adult stem cell numbers, stem cell maintenance, and stem cell proliferation to guide regenerative stem cell therapies and to understand better how cancers originate in stem cells. In this paper, we show that Hedgehog (Hh) signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yorkie (Yki), the transcriptional coactivator of the Hippo pathway, by inducing yki transcription. Moreover, both Hh signaling and Yki positively regulate the rate of FSC proliferation, both are essential for FSC maintenance, and both promote increased FSC longevity and FSC duplication when in excess. We also found that responses to activated Yki depend on Cyclin E induction while responses to excess Hh signaling depend on Yki induction, and excess Yki can compensate for defective Hh signaling. These causal connections provide the most rigorous evidence to date that a niche signal can promote stem cell maintenance principally by stimulating stem cell proliferation.

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3' processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification.

Understanding the role of the microenvironment during definitive hemopoietic development

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Trachea-Derived Dpp Controls Adult Midgut Homeostasis in Drosophila

Homeostasis in adult tissues is maintained by resident stem cells and their progeny. Little is known about the regulation of tissue homeostasis by organ-organ interaction. Here we demonstrate that trachea-derived Decapentaplegic (Dpp), the main bone morphogenetic protein ligand in Drosophila, is essential for adult midgut homeostasis. We show that Dpp signaling is primarily activated in enterocytes (ECs). Depletion of Dpp signaling in ECs results in excess amounts of intestinal stem-cell-like cells and their progeny. Importantly, we find that Dpp is expressed specifically in tracheal cells that reach the intestinal cells through the visceral muscles. Depletion of dpp expression in tracheal cells phenocopies the Dpp loss-of-function defects in ECs. Our data demonstrate that the Drosophila trachea not only exchanges air for bodily needs but also produces a Dpp morphogen essential for neighboring tissue homeostasis. This work will provide important insights into the mechanisms of tissue homeostasis control by interorgan communication.

Prdm3 and Prdm16 are H3K9me1 Methyltransferases Required for Mammalian Heterochromatin Integrity

Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.

Transcriptional Analysis of Histone Deacetylase Family Members Reveal Similarities Between Differentiating and Aging Spermatogonial Stem Cells

The differentiation of adult stem cells involves extensive chromatin remodeling, mediated in part by the gene products of histone deacetylase (HDAC) family members. While the transcriptional downregulation of HDACs can impede stem cell self-renewal in certain contexts, it may also promote stem cell maintenance under other circumstances. In self-renewing, differentiating, and aging spermatogonial stem cells (SSCs), the gene expression dynamics of HDACs have not yet been characterized. To gain further insight with these studies, we analyzed the transcriptional profiles of six HDAC family members, previously identified to be the most highly expressed in self-renewing SSCs, during stem cell differentiation and aging. Here we discovered that in both differentiating and aging SSCs the expression of Sirt4 increases, while the expression of Hdac2, Hdac6, and Sirt1 decreases. When SSCs are exposed to the lifespan-enhancing drug rapamycin in vivo, the resultant HDAC gene expression patterns are opposite of those seen in the differentiating and aging SSCs, with increased Hdac2, Hdac6, and Sirt1 and decreased Hdac8, Hdac9, and Sirt4. Our findings suggest that HDACs important for stem cell maintenance and oxidative capacity are downregulated as adult stem cells differentiate or age. These results provide important insights into the epigenetic regulation of stem cell differentiation and aging in mammals.

302 Dynamic Regulation of Intestinal Crypt Base Columnar Stem Cells by Notch Signaling

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

303 High Altitude Journeys and Flights are Associated With the Increased Risk of Flares in IBD Patients

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

Abstract SY28-02: Interactions between cancer stem cells and their niche govern metastatic colonization

Abstract Metastatic growth in distant organs is the major cause of cancer mortality. Formation of metastasis is a multi-step process with several rate-limiting steps. While dissemination of tumour cells appears to be an early and frequent event, successful initiation of metastatic growth, a process termed “metastatic colonization,” is rather inefficient for many cancer types and accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic for many tumour entities. The inefficiency of the metastatic process suggests two possibilities: a differential potential of the disseminated cancer cells to grow at the secondary sites or an inadequate support by distant tissues. We aimed at exploring limiting factors that determine metastatic success using the MMTVPyMT mouse breast cancer model which spontaneously metastasizes to the lungs. Considering that the disseminated cancer cells might have a differential metastatic potential, we investigated whether the recently identified cancer stem cells (CSCs, also termed tumour initiating cells), might be also relevant for the metastatic process. We found that a small population of cancer stem cells (CSCs) is critical for metastatic colonization. These CSCs represent a stable population of tumour cells which selectively survive and proliferate upon metastatic seeding and thereby drive the initial expansion of cancer cells at the secondary site. In contrast, nonCSCs fail to grow, are rapidly lost and do not de-differentiate into CSCs. Yet, the numbers of CSCs appear to be too high to fully explain the inefficiency of metastatic nodule formation, indicating that additional factors restrict successful metastatic colonization. Stem cells are suspected to rely on signals from their stromal environment such as localized growth factors that can affect stem cell maintenance and proliferation. In this study, we find stromal niche signals to be crucial for this process. We identify the extracellular matrix (ECM) component POSTN to be expressed by fibroblasts in the normal tissue exclusively in response to tumour cell infiltration. Infiltrating tumour cells induce stromal POSTN expression in the secondary target organ via TGFb3 secretion. Importantly the induction of POSTN at the target site is essential for the metastatic process. Indeed, generation of a POSTN null mouse revealed that the metastatic process is heavily compromised in the absence of POSTN. Similarly, cancer cells unable to induce POSTN expression at the target site due to blocking of TGFb3 signaling show poor metastatic activity. We further show that this ECM protein is required to allow maintenance of cancer stem cells: the cells driving the metastatic process. To understand how POSTN connects to the mechanisms that control stem cell maintenance we characterize its binding partners. We found that POSTN is able to bind Wnt ligands and therefore increase Wnt signaling. In vivo we observed Wnt signaling activity selectively in the CSCs population and this activity is abrogated in absence of POSTN. Finally, the metastatic activity of tumour cells with constitutively active Wnt signaling is independent of POSTN presence. Thus, POSTN acts as a niche component that can promote stem cell maintenance and metastatic colonization by augmenting Wnt signaling. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de-novo niche formation may represent a novel treatment strategy against metastatic disease. 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 SY28-02. doi:1538-7445.AM2012-SY28-02

In Vivo Clonal Analysis Reveals Self-Renewing and Multipotent Adult Neural Stem Cell Characteristics

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing invivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into invivo properties of adult neural stem cells.

Abstract SY21-01: Networks of proto-oncogenes and tumor suppressors regulate stem cell self-renewal

Abstract Advances in several areas over the past year have illuminated the critical roles played by networks of proto-oncogenes and tumor suppressors in the regulation of stem cell maintenance. To better understand the mechanisms that regulate stem cell identity and function we sought to identify genes that are preferentially expressed by stem cells and critical for their function in multiple tissues. Prdm16 is a transcription factor that regulates leukemogenesis and brown fate development, but which was not known to be required for stem cell function. We discovered that Prdm16 is preferentially expressed by stem cells throughout the nervous and hematopoietic systems and required for their maintenance [1]. Prdm16 deficiency led to changes in reactive oxygen species (ROS) levels, increased cell death, altered cell cycle distribution, and stem cell depletion in the hematopoietic and nervous systems. In neural stem/progenitor cells, Prdm16 bound the Hgf promoter and in the absence of Prdm16 Hgf expression declined. Addition of recombinant HGF to culture partially rescued the increase in ROS levels and the depletion of Prdm16 deficient neural stem cells. Administration of the antioxidant, N-acetyl-cysteine, to Prdm16 deficient mice partially rescued defects in neural stem/progenitor cell function and neural development. The physiological function of the Prdm16 proto-oncogene is to promote stem cell maintenance in multiple tissues, partly by modulating oxidative stress. The Pten tumor suppressor is also critically required for hematopoietic stem cell (HSC) maintenance, by virtue of its role in the negative regulation of PI 3-kinase pathway signaling. Pten deficiency depletes HSCs but expands leukemia-initiating cells and the mTOR inhibitor, rapamycin, blocks these effects [2]. Understanding the opposite effects of mTOR activation on HSCs versus leukemia-initiating cells could improve antileukemia therapies. We found that the depletion of Pten-deficient HSCs was not caused by oxidative stress and could not be blocked by N-acetyl-cysteine. Instead, Pten deletion induced, and rapamycin attenuated, the expression of p16Ink4a and p53 in HSCs, and p19Arf and p53 in other hematopoietic cells [3]. p53 suppressed leukemogenesis and promoted HSC depletion after Pten deletion. p16Ink4a also promoted HSC depletion but had a limited role suppressing leukemogenesis. p19Arf strongly suppressed leukemogenesis but did not deplete HSCs. Secondary mutations attenuated this tumor suppressor response in some leukemias that arose after Pten deletion. mTOR activation therefore depletes HSCs by a tumor suppressor response that is attenuated by secondary mutations in leukemogenic clones. Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumor suppression. We studied the Lkb1 tumor suppressor, and its substrate AMPK, kinases that coordinate metabolism with cell growth [4]. Lkb1 deletion caused increased HSC division, rapid HSC depletion, and pancytopenia. HSCs depended more acutely on Lkb1 for cell cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP. AMPK-deficient HSCs showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell cycle regulation between HSCs and some other hematopoietic progenitors.

MicroRNA - a contributor to age-associated neural stem cell dysfunction?

In the adult mammalian brain, new neurons are continuously generated from neural stem cells in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus [1]. Increasing evidence for the functional importance of adult-generated neurons in these regions for learning, complex behavior, and mood regulation, as well as a potential link of impaired adult neurogenesis to cognitive deficits in ageing and neurodegeneration have sparked great interest in the regulatory mechanisms underlying the coordinated generation of new functional neurons. Neurogenesis includes self-renewal and fate specification of neural stem cells, migration and maturation of young neurons, and functional integration of new neurons into the neural circuitry [1]. A large body of work has demonstrated that neurogenesis is regulated in a complex manner by the dynamic interplay of cell-extrinsic signals derived from the neurogenic “niche” and cell-intrinsic transcriptional and epigenetic regulators. More recently, microRNAs (miRNAs) have emerged as potent modulators of adult neurogenesis. miRNA 132 has been linked to the maturation and functional integration of newly generated hippocampal neurons and – in this context – is potentially regulated by hippocampal network activity and CREB-signalling [2]. Several miRNAs have also been implied in controlling the balance of stem cell maintenance and differentiation - a crucial checkpoint for sustaining adult neurogenesis throughout adulthood. In this context, miRNA-137 [3] and miRNA-184 [4] have been found to promote stem cell maintenance at the expense of differentiation, while miRNA-9 [5], miRNA lethal-7b (let-7b) [6, 7], and miRNA-124 [8] tip the balance from stem cell maintenance towards differentiation through the negative regulation of cyclin D1 and the stem cell maintenance factors TLX, Hmga2, and Sox9. Intriguingly, the latter factors are transcriptional regulators and thus are likely to control the expression of larger sets of genes, which may explain the profound effects of those miRNAs in stem cell maintenance and differentiation. In the February issue of Aging Brett and colleagues link the microRNA cluster miR-106b~25 to neural stem cell expansion and neuronal differentiation [9]. In this work, they demonstrate that miR-106b~25 promotes proliferation in primary neural stem cell cultures. Moreover they report that overexpression of miR-106b~25 enhances neural stem cell differentiation toward the neuronal lineage. Although the in vitro modulatory effects of miRNA-106b~25 on neural stem cell behaviour are relatively modest, the authors made several notable observations that imply this miRNA cluster as a critical regulator of adult neurogenesis and warrant further investigation of its function and regulation: Firstly, in silico predictions suggest that miRNA-25 may be involved in the modulation of transforming growth factor β (TGFβ)/bone morphogenic protein (BMP) and insulin/IGF signalling, i.e., signalling pathways that control neural stem cell quiescence, proliferation, and fate as well as age-related stem cell dysfunction in other organs. Secondly, the authors identify a functional FoxO3 binding site near the promoter for miR-106b~25, which modulates the activity of the miRNA cluster. Recent work identified FoxO transcription factors as crucial regulators of stem cell maintenance in the hematopoietic and the adult central nervous system, whose loss results in premature depletion of the stem cell pool. Notably, FoxO transcription factor-dependent pathways control ageing and longevity in C.elegans and Drosophila melanogaster and certain FoxO3 gene variants are associated with increased lifespan in people [10]. Hence, the miRNA-106b~25 cluster may emerge as an important modulator of ageing in neural stem cells and the neurogenic niches. The in vivo function of miRNA-106b~25 in neural stem cell behaviour and neurogenesis has to be determined and it will be particularly interesting to determine the impact of ageing on the activity of the miRNA-106b~25 cluster in neural stem cells and their progeny. Moreover, it will be important to understand if and how the miRNA-106b~25 cluster is controlled by signals derived from the neurogenic environment, given the evidence that major shifts in pathway activities contribute to stem cell dysfunction during ageing. In the long run, determining functional targets of miRNA-106b~25 in the adult neurogenic lineage may reveal novel pathways in the control of neurogenesis, which may be harnessed for treatment of age-associated cognitive deficits.