Hongyun Zhao

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

Cancer-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. DOI: http://dx.doi.org/10.7554/eLife.10250.001

Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer

Glutamine 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 glutamines 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.

Exo-MFA – A 13C metabolic flux analysis framework to dissect tumor microenvironment-secreted exosome contributions towards cancer cell metabolism

Dissecting the pleiotropic roles of tumor micro-environment (TME) on cancer progression has been brought to the foreground of research on cancer pathology. Extracellular vesicles such as exosomes, transport proteins, lipids, and nucleic acids, to mediate intercellular communication between TME components and have emerged as candidates for anti-cancer therapy. We previously reported that cancer-associated fibroblast (CAF) derived exosomes (CDEs) contain metabolites in their cargo that are utilized by cancer cells for central carbon metabolism and promote cancer growth. However, the metabolic fluxes involved in donor cells towards packaging of metabolites in extracellular vesicles and exosome-mediated metabolite flux upregulation in recipient cells are still not known. Here, we have developed a novel empirical and computational technique, exosome-mediated metabolic flux analysis (Exo-MFA) to quantify flow of cargo from source cells to recipient cells via vesicular transport. Our algorithm, which is based on 13C metabolic flux analysis, successfully predicts packaging fluxes to metabolite cargo in CAFs, dynamic changes in rate of exosome internalization by cancer cells, and flux of cargo release over time. We find that cancer cells internalize exosomes rapidly leading to depletion of extracellular exosomes within 24h. However, metabolite cargo significantly alters intracellular metabolism over the course of 24h by regulating glycolysis pathway fluxes via lactate supply. Furthermore, it can supply up to 35% of the TCA cycle fluxes by providing TCA intermediates and glutamine. Our algorithm will help gain insight into (i) metabolic interactions in multicellular systems (ii) biogenesis of extracellular vesicles and their differential packaging of cargo under changing environments, and (iii) regulation of cancer cell metabolism by its microenvironment.

Abstract 4323: Metabolic reprogramming in acute myeloid leukemia cells by mesenchymal stromal cell-derived exosomes induces chemoresistance

Mesenchymal stromal cells play an important role in acute myeloid leukemia (AML) development. Altered cellular metabolism supports AML cells9 survival in multiple aspects, such as drug resistance. Here, we demonstrate the role of MSC-derived exosomes in metabolic regulation of AML cells, and put forward a combinatorial strategy to sensitize AML cells to chemodrugs. Exosomes secreted by MSCs can reprogram the metabolic machinery following their internalization by AML cells. Through 13C tracing experiments and flux analysis, we elucidate that MSC-derived exosomes enhance oxidative phosphorylation and glutamine9s entry into TCA cycle, which replenish the pool of carbon sources in mitochondria. Further, our work shows that inhibiting the interactions between MSCs and AML cells by targeting the metabolic regulation exerted by MSC-derived exosomes sensitizes AML cells to chemodrugs. Taken together, our work reveals a novel role of the TME in regulating the metabolic adaptation in AML cells and uncovers the improved strategy for AML therapy. Citation Format: Hongyun Zhao, Abhinav Achreja, Ziwen Zhu, Ahmed N. Rawi, Marina Konopleva, Michael Andreeff, Deepak Nagrath. Metabolic reprogramming in acute myeloid leukemia cells by mesenchymal stromal cell-derived exosomes induces chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4323. doi:10.1158/1538-7445.AM2017-4323

Abstract 3372: Constraints-based metabolic flux analysis approach links tumor stage to metabolic adaptations and survival in cancer cells

The Warburg effect has been observed in many cancers and their high glycolytic capacity has signified their dependence on glucose. More recently, glutamine has emerged not only as an important nutrient for many cancers, but also necessary for their elevated energetic requirements. Due to these high energetic demands, certain cancer cells become addicted to glutamine to maintain viability. We postulate that distinct metabolic reconfigurations of certain cancers define their dependence or independence on glutamine for survival while maintaining their proliferative propensity and redox status. An intricate picture of the metabolic profiles is to be drawn from estimating intracellular fluxes, by combining stable isotope tracer measurements and experimental metabolomics data from different cancer cell lines, which have been observed to be glutamine dependent and independent. To this extent, we describe an approach that utilizes a redox-balanced model incorporating the electron transport chain and comprehensive amino-acid metabolic reactions to elucidate the importance of oxidative phosphorylation, often overlooked in classical approaches. Diving deeper into the foray of metabolic reprogramming, we perform in silico experiments using a constraint-based multi-objective modeling approach. This methodology elucidates the switching of metabolic pathways in glutamine-dependent and -independent cancers under nutrient-available and nutrient-deprived conditions. Our approach assumes that cancer cells operate at optimal levels, maintaining multiple objectives under certain environmental conditions. Constraining the proliferative phenotype from a maximal to minimal levels of the cells under different nutrient conditions emulates the observed behavior of glutamine-dependent cells under deprivation conditions and contrasts their metabolic reprogramming against that of glutamine-independent cells. We corroborated our simulations with experimentally derived metabolic fluxes and found that glutamine anabolism over catabolism dictates adaptations and survival in invasive cancers. Our results will lead to identification of potential targets for inducing nutrient-sensitivity and enhance current therapeutic approaches. Citation Format: Abhinav Achreja, Lifeng Yang, Hongyun Zhao, Juan Marini, Deepak Nagrath. Constraints-based metabolic flux analysis approach links tumor stage to metabolic adaptations and survival in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2014-3372

Abstract 5413: Role of NANOG in modulating cancer cell metabolism.

Tumors and cancer cell lines are both heterogeneous populations and contain a sub-population of undifferentiated cells known as cancer stem cells (CSCs) that are able to renew, proliferate and differentiate into tumor cells. Recent studies have shown that transcription factors (TFs) that are implicated in the pluripotency maintenance of embryonic stem cells (ESCs) are over-expressed in many tumors. Origin and progression of cancer is the result of a complex network of communication between tumor initiating cells also known as cancer stem cells (CSCs) and epithelial cells. Despite the knowledge about involvement of NANOG in modulation of cancer progression, oncogenic signaling, and tumorigenesis, it is not known about how NANOG regulates metabolic fluxes and alter cancer cell9s metabolism. Here, we show that NANOG can shift cancer cells metabolism from oxidative phosphorylation to glycolysis, to meet cancer cell9s energy needs, redox balance, proliferation, and invasion. Our findings are based on the quantification of metabolites, related to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acids and nucleotide metabolism, and comparison of these pathways fluxes under induced and uninduced NANOG expression. Our results demonstrate the metabolic targets of NANOG on cancer progression. These insights will present a unique opportunity for diagnosing and treating cancer through CSCs. Citation Format: Lifeng Yang, Hongyun Zhao, Juan C. Marini, Collene Jeter, Dean Tang, Deepak Nagrath. Role of NANOG in modulating cancer cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2013-5413