Browsing by Subject "Cell Proliferation"
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Item Open Access A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments.(Nature cell biology, 2024-05) Chidley, Christopher; Darnell, Alicia M; Gaudio, Benjamin L; Lien, Evan C; Barbeau, Anna M; Vander Heiden, Matthew G; Sorger, Peter KBlocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.Item Open Access A dual role for ErbB2 signaling in cardiac trabeculation.(Development, 2010-11) Liu, J; Bressan, M; Hassel, D; Huisken, J; Staudt, D; Kikuchi, K; Poss, KD; Mikawa, T; Stainier, DYCardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.Item Open Access A miR-34a-Numb Feedforward Loop Triggered by Inflammation Regulates Asymmetric Stem Cell Division in Intestine and Colon Cancer.(Cell Stem Cell, 2016-02-04) Bu, Pengcheng; Wang, Lihua; Chen, Kai-Yuan; Srinivasan, Tara; Murthy, Preetish Kadur Lakshminarasimha; Tung, Kuei-Ling; Varanko, Anastasia Kristine; Chen, Huanhuan Joyce; Ai, Yiwei; King, Sarah; Lipkin, Steven M; Shen, XilingEmerging evidence suggests that microRNAs can initiate asymmetric division, but whether microRNA and protein cell fate determinants coordinate with each other remains unclear. Here, we show that miR-34a directly suppresses Numb in early-stage colon cancer stem cells (CCSCs), forming an incoherent feedforward loop (IFFL) targeting Notch to separate stem and non-stem cell fates robustly. Perturbation of the IFFL leads to a new intermediate cell population with plastic and ambiguous identity. Lgr5+ mouse intestinal/colon stem cells (ISCs) predominantly undergo symmetric division but turn on asymmetric division to curb the number of ISCs when proinflammatory response causes excessive proliferation. Deletion of miR-34a inhibits asymmetric division and exacerbates Lgr5+ ISC proliferation under such stress. Collectively, our data indicate that microRNA and protein cell fate determinants coordinate to enhance robustness of cell fate decision, and they provide a safeguard mechanism against stem cell proliferation induced by inflammation or oncogenic mutation.Item Open Access A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.(2012) Yong, Sheila T.Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis, a form of programmed cell death, has been extensively studied. However, since the developmental defects observed in Bat3‐null mouse embryos cannot be explained solely by defects in apoptosis, I investigated whether BAT3 is also involved in regulating cell‐cycle progression. Using a stable‐inducible Bat3‐knockdown cellular system, I demonstrated that reduced BAT3 protein level causes a delay in both the G1/S transition and G2/M progression. Concurrent with these changes in cell‐cycle progression, I observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21. p21 is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Additionally, I observed that the p21 turnover rate was also reduced in Bat3‐knockdown cells released from G2/M synchronization. My findings indicate that in Bat3‐knockdown cells, p21 continues to be synthesized during cell‐cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent cell‐cycle delay. Finally, I showed that BAT3 co‐localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. My study reveals a novel, non‐apoptoticrole for BAT3 in cell‐cycle regulation. By maintaining low p21 protein level during G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 into S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation, an event that promotes G2/M progression. BAT3 modulates these pro‐ and anti‐proliferative roles of p21 at least in part by regulating the translocation of p21 between the cytoplasm and nucleus of the cells to ensure proper functioning and regulation of p21 in the appropriate intracellular compartments during different cell‐cycle phases.Item Restricted A switch in the control of growth of the wing imaginal disks of Manduca sexta.(PLoS One, 2010-05-19) Tobler, Alexandra; Nijhout, H FrederikBACKGROUND: Insulin and ecdysone are the key extrinsic regulators of growth for the wing imaginal disks of insects. In vitro tissue culture studies have shown that these two growth regulators act synergistically: either factor alone stimulates only limited growth, but together they stimulate disks to grow at a rate identical to that observed in situ. It is generally thought that insulin signaling links growth to nutrition, and that starvation stops growth because it inhibits insulin secretion. At the end of larval life feeding stops but the disks continue to grow, so at that time disk growth has become uncoupled from nutrition. We sought to determine at exactly what point in development this uncoupling occurs. METHODOLOGY: Growth and cell proliferation in the wing imaginal disks and hemolymph carbohydrate concentrations were measured at various stages in the last larval instar under experimental conditions of starvation, ligation, rescue, and hormone treatment. PRINCIPAL FINDINGS: Here we show that in the last larval instar of M. sexta, the uncoupling of nutrition and growth occurs as the larva passes the critical weight. Before this time, starvation causes a decline in hemolymph glucose and trehalose and a cessation of wing imaginal disks growth, which can be rescued by injections of trehalose. After the critical weight the trehalose response to starvation disappears, and the expression of insulin becomes decoupled from nutrition. After the critical weight the wing disks loose their sensitivity to repression by juvenile hormone, and factors from the abdomen, but not the brain, are required to drive continued growth. CONCLUSIONS: During the last larval instar imaginal disk growth becomes decoupled from somatic growth at the time that the endocrine events of metamorphosis are initiated. These regulatory changes ensure that disk growth continues uninterrupted when the nutritive and endocrine signals undergo the drastic changes associated with metamorphosis.Item Open Access ACE-inhibition increases podocyte number in experimental glomerular disease independent of proliferation.(Journal of the renin-angiotensin-aldosterone system : JRAAS, 2015-06) Zhang, Jiong; Yanez, David; Floege, Anna; Lichtnekert, Julia; Krofft, Ronald D; Liu, Zhi-Hong; Pippin, Jeffrey W; Shankland, Stuart JObjective
The objective of this article is to test the effects of angiotensin-converting enzyme (ACE)-inhibition on glomerular epithelial cell number in an inducible experimental model of focal segmental glomerulosclerosis (FSGS).Background
Although ACE-inhibition has been shown to limit podocyte loss by enhancing survival, little is known about its effect on podocyte number following an abrupt decline in disease.Methods
Experimental FSGS was induced with cytotoxic antipodocyte antibody. Following induction, groups were randomized to receive the ACE-inhibitor enalapril, the smooth muscle relaxant hydralazine (blood pressure control) or drinking water. Blood pressure, kidney function and histology were measured seven and 14 days following disease induction.Results
Both glomerulosclerosis and urinary albumin-to-creatinine ratio were less in the ACE-inhibition arm at day 14. At day 7 of disease, mean podocyte numbers were 26% and 29% lower in the enalapril and hydralazine arms, respectively, compared to normal mice in which no antibody was injected. At day 14, the mean podocyte number was only 18% lower in the enalapril arm, but was 39% lower in the hydralazine arm compared to normal mice. Podocyte proliferation did not occur at any time in any group. Compared to water- or hydralazine-treated mice with FSGS, the enalapril arm had a higher mean number of glomerular parietal epithelial cells that co-expressed the podocyte proteins WT-1 and synaptopodin, as well as phospho-ERK.Conclusion
The results show following an abrupt decline in podocyte number, the initiation of ACE-inhibition but not hydralazine, was accompanied by higher podocyte number in the absence of proliferation. This was accompanied by a higher number of parietal epithelial cells that co-express podocyte proteins. Increasing podocyte number appears to be accompanied by reduced glomerulosclerosis.Item Open Access Age-related effects on thymic output and homeostatic T cell expansion following depletional induction in renal transplant recipients.(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2021-09) Xu, He; Lee, Hui-Jie; Schmitz, Robin; Shaw, Brian I; Li, Shu; Kirk, Allan DThymic output and homeostatic mature cell proliferation both influence T cell repopulation following depletional induction, though the relative contribution of each and their association with recipient age have not been well studied. We investigated the repopulating T cell kinetics in kidney transplant recipients who underwent alemtuzumab induction followed by belatacept/rapamycin-based immunosuppression over 36-month posttransplantation. We focused specifically on the correlation between repopulating T cell subsets and the age of patients. Substantial homeostatic Ki67-expressing T cell proliferation was seen posttransplantation. A repertoire enriched for naïve T (TNaïve ) cells emerged posttransplantation. Analysis by generalized estimating equation linear models revealed a strong negative linear association between reconstituting TNaïve cells and advancing age. A relationship between age and persistence of effector memory cells was shown. We assessed thymic output and found an increase in the frequency of recent thymic emigrants (RTEs, CD4+ CD31+ ) at 12-month posttransplantation. Patients under 30 years of age showed significantly higher levels of CD4+ CD31+ cells than patients over 55 years of age pre- and posttransplantation. IL-7 and autologous mature dendritic cells (mDCs) induced CD57- cell proliferation. In contrast, mDCs, but not IL-7, induced CD57+ cell proliferation. This study establishes the relationship between age and thymic output during T cell homeostatic repopulation after alemtuzumab induction. Trial Registration: ClinicalTrials.gov - NCT00565773.Item Open Access An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer.(Nature genetics, 2018-02) Chen, Ming; Zhang, Jiangwen; Sampieri, Katia; Clohessy, John G; Mendez, Lourdes; Gonzalez-Billalabeitia, Enrique; Liu, Xue-Song; Lee, Yu-Ru; Fung, Jacqueline; Katon, Jesse M; Menon, Archita Venugopal; Webster, Kaitlyn A; Ng, Christopher; Palumbieri, Maria Dilia; Diolombi, Moussa S; Breitkopf, Susanne B; Teruya-Feldstein, Julie; Signoretti, Sabina; Bronson, Roderick T; Asara, John M; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Pandolfi, Pier PaoloLipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.Item Open Access Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation.(Journal of virology, 2012-10) Price, Alexander M; Tourigny, Jason P; Forte, Eleonora; Salinas, Raul E; Dave, Sandeep S; Luftig, Micah AEpstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.Item Open Access Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.(Development, 2010-09) Hutson, MR; Zeng, XL; Kim, AJ; Antoon, E; Harward, S; Kirby, MLDuring heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.Item Open Access Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.(Circ Res, 2008-07-03) Kim, Jihee; Zhang, Lisheng; Peppel, Karsten; Wu, Jiao-Hui; Zidar, David A; Brian, Leigh; DeWire, Scott M; Exum, Sabrina T; Lefkowitz, Robert J; Freedman, Neil JAtherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.Item Restricted c-Myc is required for maintenance of glioma cancer stem cells.(PLoS One, 2008) Wang, J; Wang, H; Li, Z; Wu, Q; Lathia, JD; McLendon, RE; Hjelmeland, AB; Rich, JNBACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.Item Open Access C1q/Tumor Necrosis Factor-Related Protein-9 Regulates the Fate of Implanted Mesenchymal Stem Cells and Mobilizes Their Protective Effects Against Ischemic Heart Injury via Multiple Novel Signaling Pathways.(Circulation, 2017-11) Yan, Wenjun; Guo, Yongzhen; Tao, Ling; Lau, Wayne Bond; Gan, Lu; Yan, Zheyi; Guo, Rui; Gao, Erhe; Wong, G William; Koch, Walter L; Wang, Yajing; Ma, Xin-LiangBackground
Cell therapy remains the most promising approach against ischemic heart injury. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair after myocardial infarction. CTRP9 (C1q/tumor necrosis factor-related protein-9) is a novel prosurvival cardiokine with significantly downregulated expression after myocardial infarction. Here we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting implanted stem cell survival and cardioprotection.Methods
Mice were subjected to myocardial infarction and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Survival, cardiac remodeling and function, cardiomyocytes apoptosis, and ADSCs engraftment were evaluated. Whether CTRP9 directly regulates ADSCs function was determined in vitro. Discovery-drive approaches followed by cause-effect analysis were used to uncover the molecular mechanisms of CTRP9.Results
Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 (P<0.05 or P<0.01 versus CTRP9 alone), suggesting a synergistic effect. Administration of CTRP9 at a dose recovering physiological CTRP9 levels significantly prolonged ADSCs retention/survival after implantation. Conversely, the number of engrafted ADSCs was significantly reduced in the CTRP9 knockout heart. In vitro study demonstrated that CTRP9 promoted ADSCs proliferation and migration, and it protected ADSCs against hydrogen peroxide-induced cellular death. CTRP9 enhances ADSCs proliferation/migration by extracellular regulated protein kinases (ERK)1/2-matrix metallopeptidase 9 signaling and promotes antiapoptotic/cell survival via ERK-nuclear factor erythroid-derived 2-like 2/antioxidative protein expression. N-cadherin was identified as a novel CTRP9 receptor mediating ADSCs signaling. Blockade of either N-cadherin or ERK1/2 completely abolished the previously noted CTRP9 effects. Although CTRP9 failed to promote ADSCs cardiogenic differentiation, CTRP9 promotes superoxide dismutase 3 expression and secretion from ADSCs, protecting cardiomyocytes against oxidative stress-induced cell death.Conclusions
We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms. These include binding with N-cadherin, activation of ERK-matrix metallopeptidase 9 and ERK-nuclear factor erythroid-derived 2-like 2 signaling, and upregulation/secretion of antioxidative proteins. These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy.Item Open Access Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression.(Nature cell biology, 2022-02) Huang, De; Wang, Yan; Thompson, J Will; Yin, Tao; Alexander, Peter B; Qin, Diyuan; Mudgal, Poorva; Wu, Haiyang; Liang, Yaosi; Tan, Lianmei; Pan, Christopher; Yuan, Lifeng; Wan, Ying; Li, Qi-Jing; Wang, Xiao-FanMany cancers have an unusual dependence on glutamine. However, most previous studies have focused on the contribution of glutamine to metabolic building blocks and the energy supply. Here, we report that cancer cells with aberrant expression of glutamate decarboxylase 1 (GAD1) rewire glutamine metabolism for the synthesis of γ-aminobutyric acid (GABA)-a prominent neurotransmitter-in non-nervous tissues. An analysis of clinical samples reveals that increased GABA levels predict poor prognosis. Mechanistically, we identify a cancer-intrinsic pathway through which GABA activates the GABAB receptor to inhibit GSK-3β activity, leading to enhanced β-catenin signalling. This GABA-mediated β-catenin activation both stimulates tumour cell proliferation and suppresses CD8+ T cell intratumoural infiltration, such that targeting GAD1 or GABABR in mouse models overcomes resistance to anti-PD-1 immune checkpoint blockade therapy. Our findings uncover a signalling role for tumour-derived GABA beyond its classic function as a neurotransmitter that can be targeted pharmacologically to reverse immunosuppression.Item Open Access CD142 Identifies Neoplastic Desmoid Tumor Cells, Uncovering Interactions Between Neoplastic and Stromal Cells That Drive Proliferation.(Cancer research communications, 2023-04) Al-Jazrawe, Mushriq; Xu, Steven; Poon, Raymond; Wei, Qingxia; Przybyl, Joanna; Varma, Sushama; van de Rijn, Matt; Alman, Benjamin AThe interaction between neoplastic and stromal cells within a tumor mass plays an important role in cancer biology. However, it is challenging to distinguish between tumor and stromal cells in mesenchymal tumors because lineage-specific cell surface markers typically used in other cancers do not distinguish between the different cell subpopulations. Desmoid tumors consist of mesenchymal fibroblast-like cells driven by mutations stabilizing beta-catenin. Here we aimed to identify surface markers that can distinguish mutant cells from stromal cells to study tumor-stroma interactions. We analyzed colonies derived from single cells from human desmoid tumors using a high-throughput surface antigen screen, to characterize the mutant and nonmutant cells. We found that CD142 is highly expressed by the mutant cell populations and correlates with beta-catenin activity. CD142-based cell sorting isolated the mutant population from heterogeneous samples, including one where no mutation was previously detected by traditional Sanger sequencing. We then studied the secretome of mutant and nonmutant fibroblastic cells. PTX3 is one stroma-derived secreted factor that increases mutant cell proliferation via STAT6 activation. These data demonstrate a sensitive method to quantify and distinguish neoplastic from stromal cells in mesenchymal tumors. It identifies proteins secreted by nonmutant cells that regulate mutant cell proliferation that could be therapeutically.Significance
Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors.Item Open Access Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.(Stem cells (Dayton, Ohio), 2016-09) Chinnadurai, Raghavan; Copland, Ian B; Garcia, Marco A; Petersen, Christopher T; Lewis, Christopher N; Waller, Edmund K; Kirk, Allan D; Galipeau, JacquesWe have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442.Item Open Access Cryptococcal cell morphology affects host cell interactions and pathogenicity.(PLoS Pathog, 2010-06-17) Okagaki, Laura H; Strain, Anna K; Nielsen, Judith N; Charlier, Caroline; Baltes, Nicholas J; Chrétien, Fabrice; Heitman, Joseph; Dromer, Françoise; Nielsen, KirstenCryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.Item Open Access CYLD inhibits melanoma growth and progression through suppression of the JNK/AP-1 and β1-integrin signaling pathways.(J Invest Dermatol, 2013-01) Ke, Hengning; Augustine, Christina K; Gandham, Vineela D; Jin, Jane Y; Tyler, Douglas S; Akiyama, Steven K; Hall, Russell P; Zhang, Jennifer YThe molecular mechanisms mediating cylindromatosis (CYLD) tumor suppressor function appear to be manifold. Here, we demonstrate that, in contrast to the increased levels of phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CYLD was decreased in a majority of the melanoma cell lines and tissues examined. Exogenous expression of CYLD but not its catalytically deficient mutant markedly inhibited melanoma cell proliferation and migration in vitro and subcutaneous tumor growth in vivo. In addition, the melanoma cells expressing exogenous CYLD were unable to form pulmonary tumor nodules following tail-vein injection. At the molecular level, CYLD decreased β1-integrin and inhibited pJNK induction by tumor necrosis factor-α or cell attachment to collagen IV. Moreover, CYLD induced an array of other molecular changes associated with modulation of the "malignant" phenotype, including a decreased expression of cyclin D1, N-cadherin, and nuclear Bcl3, and an increased expression of p53 and E-cadherin. Most interestingly, coexpression of the constitutively active MKK7 or c-Jun mutants with CYLD prevented the above molecular changes, and fully restored melanoma growth and metastatic potential in vivo. Our findings demonstrate that the JNK/activator protein 1 signaling pathway underlies the melanoma growth and metastasis that are associated with CYLD loss of function. Thus, restoration of CYLD and inhibition of JNK and β1-integrin function represent potential therapeutic strategies for treatment of malignant melanoma.Item Open Access Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.(Int J Obes (Lond), 2013-08) Wu, C-L; Diekman, BO; Jain, D; Guilak, FINTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.Item Open Access Drebrin regulates angiotensin II-induced aortic remodelling.(Cardiovascular research, 2018-11) Zhang, Lisheng; Wu, Jiao-Hui; Huang, Tai-Qin; Nepliouev, Igor; Brian, Leigh; Zhang, Zhushan; Wertman, Virginia; Rudemiller, Nathan P; McMahon, Timothy J; Shenoy, Sudha K; Miller, Francis J; Crowley, Steven D; Freedman, Neil J; Stiber, Jonathan AAims
The actin-binding protein Drebrin is up-regulated in response to arterial injury and reduces smooth muscle cell (SMC) migration and proliferation through its interaction with the actin cytoskeleton. We, therefore, tested the hypothesis that SMC Drebrin inhibits angiotensin II-induced remodelling of the proximal aorta.Methods and results
Angiotensin II was administered via osmotic minipumps at 1000 ng/kg/min continuously for 28 days in SM22-Cre+/Dbnflox/flox (SMC-Dbn-/-) and control mice. Blood pressure responses to angiotensin II were assessed by telemetry. After angiotensin II infusion, we assessed remodelling in the proximal ascending aorta by echocardiography and planimetry of histological cross sections. Although the degree of hypertension was equivalent in SMC-Dbn-/- and control mice, SMC-Dbn-/- mice nonetheless exhibited 60% more proximal aortic medial thickening and two-fold more outward aortic remodelling than control mice in response to angiotensin II. Proximal aortas demonstrated greater cellular proliferation and matrix deposition in SMC-Dbn-/- mice than in control mice, as evidenced by a higher prevalence of proliferating cell nuclear antigen-positive nuclei and higher levels of collagen I. Compared with control mouse aortas, SMC-Dbn-/- aortas demonstrated greater angiotensin II-induced NADPH oxidase activation and inflammation, evidenced by higher levels of Ser-536-phosphorylated NFκB p65 subunits and higher levels of vascular cell adhesion molecule-1, matrix metalloproteinase-9, and adventitial macrophages.Conclusions
We conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.