Browsing by Author "Nagrath, Deepak"
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Item A Novel Human Adipocyte-Derived Basement Membrane for Tissue Engineering Applications(2012-09-05) Damm, Aaron; Nagrath, Deepak; Zygourakis, Kyriacos; Gonzalez, Ramon; Sabek, OmaimaTissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.Item Amplification of USP13 drives ovarian cancer metabolism(Springer Nature, 2016) Han, Cecil; Yang, Lifeng; Choi, Hyun Ho; Baddour, Joelle; Achreja, Abhinav; Liu, Yunhua; Li, Yujing; Li, Jiada; Wan, Guohui; Huang, Cheng; Ji, Guang; Zhang, Xinna; Nagrath, Deepak; Lu, XiongbinDysregulated energetic metabolism has been recently identified as a hallmark of cancer. Although mutations in metabolic enzymes hardwire metabolism to tumourigenesis, they are relatively infrequent in ovarian cancer. More often, cancer metabolism is re-engineered by altered abundance and activity of the metabolic enzymes. Here we identify ubiquitin-specific peptidase 13 (USP13) as a master regulator that drives ovarian cancer metabolism. USP13 specifically deubiquitinates and thus upregulates ATP citrate lyase and oxoglutarate dehydrogenase, two key enzymes that determine mitochondrial respiration, glutaminolysis and fatty acid synthesis. The USP13 gene is co-amplified with PIK3CA in 29.3% of high-grade serous ovarian cancers and its overexpression is significantly associated with poor clinical outcome. Inhibiting USP13 remarkably suppresses ovarian tumour progression and sensitizes tumour cells to the treatment of PI3K/AKT inhibitor. Our results reveal an important metabolism-centric role of USP13, which may lead to potential therapeutics targeting USP13 in ovarian cancers.Item Designing synthetic biological circuits using optimality and nonequilibrium thermodynamics(2013-08-06) Nagrath, Deepak; Avila-Elchiver, Marco; Yarmush, Martin; Rice University; General Hospital Corporation; United States Patent and Trademark OfficeIn general, the invention relates to a method for designing a biological circuit. The method includes obtaining a target circuit objective for the biological circuit, determining an objective function corresponding to the target circuit objective, obtaining a number of nodes for the biological circuit, obtaining a set of possible circuit subgraphs using the number of nodes, obtaining a specific dissipation energy (SDE) for each one of the set of possible circuit subgraphs by optimizing the objective function, selecting at least one circuit subgraph from the set of possible circuit subgraphs with the lowest SDE, and designing the biological circuit using the at least one selected one circuit subgraph.Item Glutaminolysis: A Hallmark of Cancer Metabolism(2016-04-15) Yang, Lifeng; Nagrath, DeepakThe goals of these projects are to study the critical role of glutamine (Gln) in ovarian cancer growth, metastasis, drug resistance and sources of glutamine in tumor microenvironment. 1): Gln‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. 1): we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs. 2): Reactive stromal cells are an integral part of tumor microenvironment (TME) in tumors and interact with cancer cells to regulate their growth and survival. Although targeting stromal cells could be a viable therapy to regulate the communication between TME and cancer cells, identification of stromal targets which make cancer cells vulnerable has remained challenging and still elusive. Here, we identify a previously unrecognized mechanism whereby metabolism of reactive stromal cells is reprogrammed through upregulated glutamine anabolic pathway. This dysfunctional stromal metabolism confers atypical metabolic flexibility and adaptive mechanisms in stromal cells allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in TME. We demonstrate that targeting cancer associated fibroblasts (CAFs), a major component of reactive stroma that expresses high glutamine synthetase (GLUL), disrupts metabolic crosstalk between stromal and cancer cells. Our work underscores reliance of cancer cells on CAFs and presents a synthetic lethal approach to target tumor stroma and cancer cells simultaneously for desirable therapeutic outcomes.Item HSulf-1 deficiency dictates a metabolic reprograming of glycolysis and TCA cycle in ovarian cancer(Impact Journals, LLC., 2015) Mondal, Susmita; Roy, Debarshi; Camacho-Pereira, Juliana; Khurana, Ashwani; Chini, Eduardo; Yang, Lifeng; Baddour, Joelle; Stilles, Katherine; Padmabandu, Seth; Leung, Sam; Kalloger, Steve; Gilks, Blake; Lowe, Val; Dierks, Thomas; Hammond, Edward; Dredge, Keith; Nagrath, Deepak; Shridhar, VijiWarburg effect has emerged as a potential hallmark of many cancers. However, the molecular mechanisms that led to this metabolic state of aerobic glycolysis, particularly in ovarian cancer (OVCA) have not been completely elucidated. HSulf-1 predominantly functions by limiting the bioavailability of heparan binding growth factors and hence their downstream signaling. Here we report that HSulf-1, a known putative tumor suppressor, is a negative regulator of glycolysis. Silencing of HSulf-1 expression in OV202 cell line increased glucose uptake and lactate production by upregulating glycolytic genes such as Glut1, HKII, LDHA, as well as metabolites. Conversely, HSulf-1 overexpression in TOV21G cells resulted in the down regulation of glycolytic enzymes and reduced glycolytic phenotype, supporting the role of HSulf-1 loss in enhanced aerobic glycolysis. HSulf-1 deficiency mediated glycolytic enhancement also resulted in increased inhibitory phosphorylation of pyruvate dehydrogenase (PDH) thus blocking the entry of glucose flux into TCA cycle. Consistent with this, metabolomic and isotope tracer analysis showed reduced glucose flux into TCA cycle. Moreover, HSulf-1 loss is associated with lower oxygen consumption rate (OCR) and impaired mitochondrial function. Mechanistically, lack of HSulf-1 promotes c-Myc induction through HB-EGF-mediated p-ERK activation. Pharmacological inhibition of c-Myc reduced HB-EGF induced glycolytic enzymes implicating a major role of c-Myc in loss of HSulf-1 mediated altered glycolytic pathway in OVCA. Similarly, PG545 treatment, an agent that binds to heparan binding growth factors and sequesters growth factors away from their ligand also blocked HB-EGF signaling and reduced glucose uptake in vivo in HSulf-1 deficient cells.Item Human Omental-Derived Adipose Stem Cells Increase Ovarian Cancer Proliferation, Migration, and Chemoresistance(Public Library of Science, 2013) Nowicka, Aleksandra; Marini, Frank C.; Solley, Travis N.; Elizondo, Paula B.; Zhang, Yan; Sharp, Hadley J.; Broaddus, Russell; Kolonin, Mikhail; Mok, Samuel C.; Thompson, Melissa S.; Woodward, Wendy A.; Lu, Karen; Salimian, Bahar; Nagrath, Deepak; Klopp, Ann H.Objectives: Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination. Materials and Methods: We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment. Results: O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries. Conclusions: ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro.Item Kinetic and Stoichiometric Modeling of the Metabolism of Escherichia coli for the Synthesis of Biofuels and Chemicals(2013-09-16) Cintolesi Makuc, Angela; Gonzalez, Ramon; Nagrath, Deepak; Nakhleh, Luay K.This thesis presents the mathematical modeling of two new Escherichia coli platforms with economical potential for the production of biofuels and chemicals, namely glycerol fermentation and the reversal of the β-oxidation cycle. With the increase in traditional fuel prices, alternative renewable energy sources are needed, and the efficient production of biofuels becomes imperative. So far studies have focused on using glucose as feedstock for the production of ethanol and other fuels, but a recent increase in glycerol availability and its consequent decrease in price make it an attractive feedstock. Furthermore, the reversed β-oxidation cycle is a highly efficient mechanism for the synthesis of long-chain products. These two platforms have been reported experimentally in E. coli but their mathematical modeling is presented for the first time here. Because mathematical models have proved to be useful in the optimization of microbial metabolism, two complementary models were used in this study: kinetic and stoichiometric. Kinetic models can identify the control structure within a specific pathway, but they require highly detailed information, making them applicable to small sets of reactions. In contrast, stoichiometric models require only mass balance information, making them suitable for genome-scale modeling to study the effect of adding or removing reactions for the optimization of the synthesis of desired products. To study glycerol fermentation, a kinetic model was implemented, allowing prediction of the limiting enzymes of this process: glycerol dehydrogenase and di-hydroxyacetone kinase. This prediction was experimentally validated by increasing their enzymatic activities, resulting in a two-fold increase in the rate of ethanol production. Additionally, a stoichiometric genome-scale model (GEM) was modified to represent the fermentative metabolism of glycerol, identifying key metabolic pathways for glycerol fermentation (including a new glycerol dissimilation pathway). The GEM was used to identify genetic modifications that would increase the synthesis of desired products, such as succinate and butanol. Finally, glucose metabolism using the reversal β-oxidation cycle was modeled using a GEM to simulate the synthesis of a variety of medium and long chain products (including advanced biofuels). The model was used to design strategies that can lead to increase the productivity of target products.Item Mapping tumor metabolism: from personalized multiobjective metabolic flux analysis to intercellular metabolite transport via exosomes(2016-12-02) Achreja, Abhinav; Nagrath, DeepakDeparture from healthy and tightly-regulated metabolism is an emerging hallmark of cancer that facilitates tumorigenic characteristics of uncontrolled proliferation, metastasis, and resistance to chemotherapy. Our aim is to elucidate underlying mechanisms of metabolic shifts that satisfy enhanced energetic and biochemical demands of cancer cells. This is essential to develop therapies that sensitize resistant tumors to front-line treatments and alleviate side-effects or attack novel targets that are robust to acquired resistance. Study of metabolic reprogramming involves a marriage of empirical techniques that measure biophysical parameters and computational algorithms to estimate useful but unmeasurable parameters of metabolic activity, i.e. fluxes. This thesis describes novel computational algorithms designed to quantify metabolic fluxes vis a vis metabolic reprogramming in cancer cells and metabolic interaction within tumor microenvironment (TME). The first part describes reconstruction of metabolic models using transcriptomic and proteomic data from cancer cell-lines to address the need for personalized medicine. The second part discusses the implementation of currently used 13-carbon metabolic flux analysis (13C-MFA) to demonstrate how cancer-associated fibroblasts (CAFs) in TME, reprogram metabolism to utilize nutrients atypically, to synthesize glutamine for glutamine deprived cancer cells. In the third part, the 13C multiobjective MFA (13C-MOMFA) algorithm is presented that removes the restrictions of 13C-MFA to simple models and abundant empirical data. The empirical data utilized is same as conventional techniques, but with a multiobjective optimization approach to capture competing metabolic objectives facilitating various tumorigenic functions in larger “personalized” models. 13C-MOMFA is applied in ovarian cancer cell-lines subjected to glutamine catabolism inhibition, to uncover mechanisms linking invasiveness to glutamine-dependence. Finally, exosome-mediated MFA (Exo-MFA) technique is designed to elucidate tumor growth-supporting transport of metabolites from tumor stroma to nutrient-deprived cancer cells via secreted exosomes. Exo-MFA is the first to capture multicellular metabolic interaction between TME components. Results demonstrate packaging of nutrients into exosomes by CAFs, and sufficient supply of metabolites to central carbon metabolism of nutrient-deprived cancer cells. This thesis addresses two burgeoning fields of cancer biology focused on improving therapeutic outcomes – (i) precision medicine that recognizes patient-heterogeneity and complex metabolic programs that support tumorigenic phenotypes, and (ii) systems approach to understanding metabolism of whole tumors.Item Metabolic regulation of collagen gel contraction by porcine aortic valvular interstitial cells(The Royal Society, 2014) Kamel, Peter I.; Qu, Xin; Geiszler, Andrew M.; Nagrath, Deepak; Harmancey, Romain; Taegtmeyer, Heinrich; Grande-Allen, K.JaneDespite a high incidence of calcific aortic valve disease in metabolic syndrome, there is little information about the fundamental metabolism of heart valves. Cell metabolism is a first responder to chemical and mechanical stimuli, but it is unknown how such signals employed in valve tissue engineering impact valvular interstitial cell (VIC) biology and valvular disease pathogenesis. In this study porcine aortic VICs were seeded into three-dimensional collagen gels and analysed for gel contraction, lactate production and glucose consumption in response to manipulation of metabolic substrates, including glucose, galactose, pyruvate and glutamine. Cell viability was also assessed in two-dimensional culture. We found that gel contraction was sensitive to metabolic manipulation, particularly in nutrient-depleted medium. Contraction was optimal at an intermediate glucose concentration (2 g l−1) with less contraction with excess (4.5 g l−1) or reduced glucose (1 g l−1). Substitution with galactose delayed contraction and decreased lactate production. In low sugar concentrations, pyruvate depletion reduced contraction. Glutamine depletion reduced cell metabolism and viability. Our results suggest that nutrient depletion and manipulation of metabolic substrates impacts the viability, metabolism and contractile behaviour of VICs. Particularly, hyperglycaemic conditions can reduce VIC interaction with and remodelling of the extracellular matrix. These results begin to link VIC metabolism and macroscopic behaviour such as cell–matrix interaction.Item Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer(EMBO, 2014) Yang, Lifeng; Moss, Tyler; Mangala, Lingegowda S.; Marini, Juan; Zhao, Hongyun; Wahlig, Stephen; Armaiz-Pena, Guillermo; Jiang, Dahai; Achreja, Abhinav; Win, Julia; Roopaimoole, Rajesha; Rodriguez-Aguayo, Cristian; Mercado-Uribe, Imelda; Lopez-Berestein, Gabriel; Liu, Jinsong; Tsukamoto, Takashi; Sood, Anil K.; Ram, Prahlad T.; Nagrath, DeepakGlutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.Item Modeling a Reversed β-oxidation Cycle Into the Genome Scale Model of Zymomonas mobilis(2013-09-16) Dash, Satyakam; Gonzalez, Ramon; Nagrath, Deepak; Segatori, LauraThis study proposes simulations which present optimized methods for producing fatty acids, fatty alcohols and alkanes using Zymomonas mobilis bacterium by the energy efficient β-oxidation reversal pathway, an eco-friendly alternative to the present petrolItem Modeling Integrated Cellular Machinery Using Hybrid Petri-Boolean Networks(Public Library of Science, 2013) Berestovsky, Natalie; Zhou, Wanding; Nagrath, Deepak; Nakhleh, LuayThe behavior and phenotypic changes of cells are governed by a cellular circuitry that represents a set of biochemical reactions. Based on biological functions, this circuitry is divided into three types of networks, each encoding for a major biological process: signal transduction, transcription regulation, and metabolism. This division has generally enabled taming computational complexity dealing with the entire system, allowed for using modeling techniques that are specific to each of the components, and achieved separation of the different time scales at which reactions in each of the three networks occur. Nonetheless, with this division comes loss of information and power needed to elucidate certain cellular phenomena. Within the cell, these three types of networks work in tandem, and each produces signals and/or substances that are used by the others to process information and operate normally. Therefore, computational techniques for modeling integrated cellular machinery are needed. In this work, we propose an integrated hybrid model (IHM) that combines Petri nets and Boolean networks to model integrated cellular networks. Coupled with a stochastic simulation mechanism, the model simulates the dynamics of the integrated network, and can be perturbed to generate testable hypotheses. Our model is qualitative and is mostly built upon knowledge from the literature and requires fine-tuning of very few parameters. We validated our model on two systems: the transcriptional regulation of glucose metabolism in human cells, and cellular osmoregulation in S. cerevisiae. The model produced results that are in very good agreement with experimental data, and produces valid hypotheses. The abstract nature of our model and the ease of its construction makes it a very good candidate for modeling integrated networks from qualitative data. The results it produces can guide the practitioner to zoom into components and interconnections and investigate them using such more detailed mathematical models.Item Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma(Springer Nature, 2017) Ju, Huai-Qiang; Ying, Haoqiang; Tian, Tian; Ling, Jianhua; Fu, Jie; Lu, Yu; Wu, Min; Yang, Lifeng; Achreja, Abhinav; Chen, Gang; Zhuang, Zhuonan; Wang, Huamin; Nagrath, Deepak; Yao, Jun; Hung, Mien-Chie; DePinho, Ronald A.; Huang, Peng; Xu, Rui-Hua; Chiao, Paul J.; Laboratory for Systems Biology of Human DiseasesKras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC.Item Nitric Oxide: The Missing Link in Omentum-Induced Metabolic reprogramming of ovarian cancers(2015-11-24) Salimian Rizi, Bahar; Nagrath, Deepak; Zygourakis, Kyriacos; Bennett, George; Klopp, Ann HA novel metabolic regulatory mechanism of ovarian cancer by omentum adipose-derived stroma cells (O-ASCs) has been discovered. O-ASCs induce survival, migration, and chemoresistance of ovarian cancer cells. However, the underpinning mechanism behind the metabolic modulation was not understood. Here, O-ASCs are shown to promote nitric oxide (NO) homeostasis in ovarian cancers by generating the pool of arginine. Ovarian cancer cells benefit from tumor microenvironment’s elements and expand their growth. In turn, cancer cells modify the elements’ fate to further take advantage of nutrients and resources. A unique combinatory drug treatment is proposed to target O-ASCs-induced chemoresistance of ovarian cancer cells.Item Ovarian Cancer Metabolism: Effect of Anoikis Condition and Nitric Oxide on Ovarian Cancer Metabolism, and Effect of Metabolites on Ovarian Cancer Migration(2013-12-06) Caneba, Christine; Nagrath, Deepak; Mikos, Antonios G.; Zygourakis, KyriacosOvarian cancer remains the most lethal gynecological malignancy worldwide, with most of the disease detected at later stages. Elucidating pathways based on upregulation of proteins and genes involved in the development and progression of ovarian cancer is underway. However, understanding of the metabolic regulation and changes in metabolism involved in ovarian cancer is lacking, and this understanding could lead to development of therapies for ovarian cancer. In order for ovarian cancer cells to metastasize, they must be able to survive deprived of extracellular matrix attachment in the peritoneal cavity. In the first part of this thesis, the effect of cell detachment on the metabolism of highly-invasive and less-invasive ovarian cancer cells was explored by employing culture methods that induced cell detachment. Experiments were designed to collect media from the cells for metabolic analysis to gain insight into changes in the glycolytic and oxidative phosphorylation pathways. Results showed that oxidative phosphorylation was higher for highly-invasive versus less-invasive ovarian cancer cells in detachment. It was also observed that highly-invasive ovarian cancer cells consumed more pyruvate than less-invasive ovarian cancer cells, which indicated that the ovarian cancer cells had functional mitochondria. In the second part of this thesis, the role of metabolites in cancer cell migration was investigated. Results showed that pyruvate increased the cancer cell migration, indicating that mitochondria were important for the migration of ovarian cancer cells. In the last part of this thesis, the role of nitric oxide on ovarian cancer cell proliferation and metabolism was explored. Results showed that nitric oxide increased proliferation of ovarian cancer cells, and also maintained their high glycolytic rate (i.e. – the Warburg Effect). This was paired with a decrease in oxidative phosphorylation, which was due to inhibition by nitric oxide of complexes II/III and complex IV in the mitochondria. Thus, nitric oxide plays an important role in the metabolism of ovarian cancer cells. Our work is one of the first to elucidate the interactions between non-adherent/nitric oxide stress and ovarian cancer metabolism, and could pave the way for development of metabolically-based therapies that could halt progression of ovarian cancer.Item Reprogramming the proteostasis network to prevent the accumulation of alpha-synuclein aggregates(2014-03-14) Kilpatrick, Kiri; Segatori, Laura; Gonzalez, Ramon; Silberg, Jonathan J.; Nagrath, DeepakProtein misfolding and aggregation characterizes the development of a number of neurodegenerative diseases, such as Parkinson’s, Alzheimer’s and Huntington’s disease. The hallmark of Parkinson’s disease is the formation of proteinaceous inclusions, which consist primarily of α-synuclein (α-syn), a natively unstructured protein with propensity to misfold and aggregate. Cells have evolved sophisticated systems of protein quality control to prevent accumulation of non-native proteins and maintain protein homeostasis. However, the load of misfolded α-syn typically exceeds the capacity of the quality control system. Aberrant accumulation of misfolded α-syn leads to proteotoxic stress, eventually resulting in neurodegeneration. The objective of this project is to investigate chemical and genetic approaches to modulate the protein quality control system and reduce the accumulation of aberrant α-syn species. Studying α-syn aggregation in cells presents a number of challenges mainly due to the limited availability of tools to quantitatively distinguish between different α-syn conformational species within the cellular environment. To address this need, we engineered an in vitro model system based on neuroglioma cells that accumulate α-syn aggregates and developed a set of analytical tools based on the use of aggregation responsive probes to quantify α-syn aggregation in cells. To test whether modulating the protein quality control system affects the accumulation of α-syn aggregates, we investigated a series of complementary approaches aimed at i) enhancing the innate cellular chaperone machinery, which promotes folding and prevents aggregation, and ii) upregulating the autophagy pathway, which mediates clearance of aggregated proteins. We demonstrated that chemical modulation of Hsp70, a ubiquitously expressed molecular chaperone, affects the accumulation of α-syn aggregates. Particularly, the Hsp70 upregulator carbenoxolone was found to reduce α-syn aggregation and prevent α-syn-induced cytotoxicity via activation of the heat shock response. We also found that activation of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, results in enhanced autophagic clearance of α-syn aggregates. We demonstrated that cell treatment with 2-hydroxypropyl-β-cyclodextrin reduces the accumulation of aggregated α-syn specifically by upregulating TFEB-mediated autophagic clearance. These findings lay the foundation for the development of pharmacological strategies to reduce the accumulation of misfolded and aggregated α-syn for the treatment of Parkinson’s disease.Item Towards integrated computational models of cellular networks(2013-09-16) Berestovsky, Natalie; Nakhleh, Luay K.; Kavraki, Lydia E.; Nagrath, DeepakThe whole-cell behavior arises from the interplay among signaling, metabolic, and regulatory processes, which differ not only in their mechanisms, but also in the time scale of their execution. Proper modeling of the overall function of the cell requires development of a new modeling approach that accurately integrates these three types of processes, using the representation that best captures each one of them, and the interconnections between them. Traditionally, signaling networks have been modeled with ordinary differential equations (ODEs), regulation with Boolean networks, and metabolic pathways with Petri nets – these approaches are widely accepted and extensively used. Nonetheless, each of these methods, while being effective, have had limitations pointed out to them. Particularly, ODEs generally require very thorough parameterization, which is difficult to acquire, Boolean networks have been argued to be not capable of capturing complex systems dynamics, and the effectiveness of Petri nets when comparing to other, steady-state methods, have been debated. The main goal of this dissertation is to devise an integrated model that capture the whole-cell behavior and accurately combines these three components in the interplay between them. I provide a systematic study on using particle swarm optimization (PSO) as an effective approach for parameterizing ODEs. I survey different inference method for Boolean networks on the sets of complex dynamic data and demonstrate that they are, in fact, capable of capturing a variety of different systems. I review the existing use of Petri nets in modeling of biochemical system to show their effectiveness and, particularly, the ease for their integration with other methods. Finally, I propose an integrated hybrid model (IHM) that uses Petri nets to represent metabolic and signaling components, and Boolean networks to model regulation. The interconnections between these models allow to overcome the time scale differences of the processes by adding appropriate delay mechanisms. I validate IHM on two data sets. The significant advantage of IHM over other models is that it is able to capture the dynamics of all three components and can potentially identify novel and important cross-talk within the cell.Item Tumor Microenvironment Derived Exosomes Pleiotropically Modulate Cancer Cell Metabolism(2016-12-01) Zhao, Hongyun; Nagrath, DeepakExosomes are extracellular vesicles responsible for efficient cell-to-cell communications. We intended to investigate the role of tumor microenvironment (TME) derived exosomes in regulating cancer cell metabolism. We found that the isolated exosomes from cancer-associated fibroblasts (CAFs), a major cellular type in TME were uptaken by cancer cells efficiently. Viability assays proved that CAF-derived exosomes (CDEs) enhanced cancer cells viability in nutrient deprived medium to a large extent. In the followed metabolic tests, we found that CDEs downregulate mitochondrial function in both prostate and pancreatic cancer cells, which causes compensatory upregulation of glycolysis in these cells. We found that the possible mechanism of this metabolic regulation is through transfer of miRNAs by CDEs into the cancer cells. We showed that miRNAs contained in CDEs induced mitochondrial dysfunction. Considering that cancer cells in vivo reside in a nutrient-deprived microenvironment of nutrients deprivation and cancer cells’ avid growth needs huge amounts of nutrients, we proposed that CDEs were able to supply nutrients such as lipids, TCA cycle metabolites and amino acids to cancer cells. To prove this, we cultured cancer cells in nutrients deprived medium which is deprived of glutamine, pyruvate, lysine, phenylalanine, and leucine. CDEs were added to the medium to prove that CDEs can maintain cancer cells growth in nutrient deprived conditions similar to complete medium. Surprisingly, the CDEs added to the nutrient-deprived medium endowed cancer cells ability to maintain growth similar to complete medium. The direct rationale to explain it is that CDEs transferred nutrients to and were being utilized by cancer cells. The high throughput metabolomics methods showed that CDEs contained lipids, TCA cycle metabolites, and amino acids. Our results convincingly demonstrate that not only do exosomes enhance the phenomenon of “Warburg effect” in tumors, but remarkably, contain de novo “off-the-shelf” metabolites within their cargo that can contribute to the entire compendia of central carbon metabolism within cancer cells. Disruption of this CDEs-induced metabolic adaptation in cancer cells might provide a novel therapeutic avenue for exploitation.Item Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism(eLife Sciences Publications Ltd., 2016) Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C.; Tudawe, Thavisha; Seviour, Elena G.; San Lucas, F. Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N.; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T.; Maitra, Anirban; Nagrath, Deepak; Laboratory for Systems Biology of Human DiseasesCancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.