Abhinav Achreja

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.

Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime ‘collateral lethality’ therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.

Role of Increased n-acetylaspartate Levels in Cancer

BACKGROUND The clinical and biological effects of metabolic alterations in cancer are not fully understood. METHODS In high-grade serous ovarian cancer (HGSOC) samples (n = 101), over 170 metabolites were profiled and compared with normal ovarian tissues (n = 15). To determine NAT8L gene expression across different cancer types, we analyzed the RNA expression of cancer types using RNASeqV2 data available from the open access The Cancer Genome Atlas (TCGA) website (http://www.cbioportal.org/public-portal/). Using NAT8L siRNA, molecular techniques and histological analysis, we determined cancer cell viability, proliferation, apoptosis, and tumor growth in in vitro and in vivo (n = 6-10 mice/group) settings. Data were analyzed with the Students t test and Kaplan-Meier analysis. Statistical tests were two-sided. RESULTS Patients with high levels of tumoral NAA and its biosynthetic enzyme, aspartate N-acetyltransferase (NAT8L), had worse overall survival than patients with low levels of NAA and NAT8L. The overall survival duration of patients with higher-than-median NAA levels (3.6 years) was lower than that of patients with lower-than-median NAA levels (5.1 years, P = .03). High NAT8L gene expression in other cancers (melanoma, renal cell, breast, colon, and uterine cancers) was associated with worse overall survival. NAT8L silencing reduced cancer cell viability (HEYA8: control siRNA 90.61% ± 2.53, NAT8L siRNA 39.43% ± 3.00, P < .001; A2780: control siRNA 90.59% ± 2.53, NAT8L siRNA 7.44% ± 1.71, P < .001) and proliferation (HEYA8: control siRNA 74.83% ± 0.92, NAT8L siRNA 55.70% ± 1.54, P < .001; A2780: control siRNA 50.17% ± 4.13, NAT8L siRNA 26.52% ± 3.70, P < .001), which was rescued by addition of NAA. In orthotopic mouse models (ovarian cancer and melanoma), NAT8L silencing reduced tumor growth statistically significantly (A2780: control siRNA 0.52 g ± 0.15, NAT8L siRNA 0.08 g ± 0.17, P < .001; HEYA8: control siRNA 0.79 g ± 0.42, NAT8L siRNA 0.24 g ± 0.18, P = .008, A375-SM: control siRNA 0.55 g ± 0.22, NAT8L siRNA 0.21 g ± 0.17 g, P = .001). NAT8L silencing downregulated the anti-apoptotic pathway, which was mediated through FOXM1. CONCLUSION These findings indicate that the NAA pathway has a prominent role in promoting tumor growth and represents a valuable target for anticancer therapy.Altered energy metabolism is a hallmark of cancer (1). Proliferating cancer cells have much greater metabolic requirements than nonproliferating differentiated cells (2,3). Moreover, altered cancer metabolism elevates unique metabolic intermediates, which can promote cancer survival and progression (4,5). Furthermore, emerging evidence suggests that proliferating cancer cells exploit alternative metabolic pathways to meet their high demand for energy and to accumulate biomass (6-8).

Amplification of USP13 drives ovarian cancer metabolism

Dysregulated 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.

Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma

Kras 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.

Abstract 4904: GLUT4 exhibits a non-canonical role of regulating lung cancer metastasis

Lung cancer continues to be fatal, in part due to the inability to prevent and treat metastases. Highly metastatic cancers exhibit enhanced glucose uptake to sustain proliferation and importantly, tumor invasion. Among the SLC2A family of facilitative glucose transporters, GLUT1 is largely attributed to be responsible for increased glucose uptake of cancer cells. GLUT1 is however responsible for glucose transport across the blood-brain barrier, expressed in many normal cell types and therefore a less desirable therapeutic target. We previously reported that multiple myeloma cells rely on overexpression and constitutive plasma membrane localization of insulin-responsive glucose transporter, GLUT4. In this study we investigated a role for GLUT4 in lung cancer. To interrogate contributions of GLUT1 and GLUT4 in proliferation, invasion and migration we generated H1299 and A549 GLUT 1 or GLUT4 knockdowns. Knockdown (KD) of GLUT4 did not inhibit proliferation but suppressed migration and invasion assessed through scratch and Boyden chamber assays, respectively. On the contrary, knockdown of GLUT1 reduced proliferation of these lines. Treatment of H1299 and A549 with our newly developed GLUT4-selective inhibitors also reduced invasion, phenocopying the effects detected with GLUT4 KD. GLUT4 inhibition also reduced H1299 invasion in a spheroid invasion model. We utilized H1299 cells to isolate highly invasive less proliferative “leader cells” and less invasive but highly proliferative “follower cells”. Interestingly, examination of these two cell types exhibited a differential expression pattern of GLUT1/GLUT4. Leader cells have elevated expression of GLUT4 and decreased GLUT1. On the contrary, follower cells have high GLUT1 and low GLUT4 expression. Leader cells are more sensitive to GLUT4 inhibitors indicating they are more dependent on GLUT4 than follower cells. In addition, leader cells are more sensitive to mitochondrial complex I inhibitors compared to follower cells, suggesting they rely more on oxidative phosphorylation. A differential reliance on glycolysis/OXPHOS was further supported by evaluation of glucose uptake/oxygen consumption. Isotope tracer and bioenergetics analyses further support altered nutrient dependencies of leader and follower cells. Lastly, we found that GLUT4 is expressed in patient lung adenocarcinoma specimens including more aggressive micropapillary lung adenocarcinoma. Examination of collective invasion packs in human adenocarcinoma demonstrated patchy GLUT1 expression suggestive of a subset of more proliferative “follower” cells. These results suggest that in a lung cancer population a subset of more invasive cells are reliant on GLUT4 with reduced GLUT1 expression while more proliferative cells rely on high GLUT1 expression, making GLUT4 a promising candidate for targeting metastasis in lung cancer. Citation Format: Changyong Wei, Abhinav Achreja, Jessica Konen, Gabriel Sica, Melissa Gilbert-Ross, Deepak Nagrath, Adam Marcus, Mala Shanmugam. GLUT4 exhibits a non-canonical role of regulating lung cancer metastasis [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 4904. doi:10.1158/1538-7445.AM2017-4904

Abstract 1204: Design of phenotype-driven flux analysis approach for personalized metabolic models of cancer patients

It is well established that departure from healthy and tightly-controlled metabolism is a hallmark of cancer and hence, studying these metabolic changes can help us gain useful therapeutic insights. Several mathematical methodologies have been developed to quantify these changes to better understand metabolic adaptations in tumor cells. However, they (i) do not consider different genetic profiles and protein signatures leading to diversity among cancer patients and (ii) do not mimic biological interactions between metabolism and cancer phenotypes (such as chemo-resistance, infinite growth potential and invasive capacities). To successfully deconstruct metabolic and phenotypic links in tumorigenesis, we have developed a novel mathematical framework. First, to achieve patient-specificity we have devised a technique for reconstructing metabolic models that integrate high-throughput transcriptomic and proteomic data using a biased random-walk algorithm. The NetWalker software effectively implements this technique to score interactions between genes and proteins based on their expression levels and connectivity with each other and their metabolic targets. These scores are then used to identify contextually important sub-networks of the central carbon metabolism. Our reconstruction of the OVCAR-3 metabolic network independently identifies metabolic features in low-invasive ovarian cancers as highlighted in previously published literature and recent discoveries in our lab. These personalized models are then used to estimate phenotypically-consistent intracellular metabolic fluxes, with our novel metabolic flux analysis technique. Our approach is designed on the rationale that cancer cells regulate metabolism to maintain multiple phenotypic functions by optimally using the limited nutrients available in their environments. We quantitatively show via correlation studies and principal component analyses on NCI-60 and CCLE cell-lines’ gene-expression data that certain phenotypes are inherently competitive. This competition is manifested in metabolic functions, which is effectively modeled using our 13 C-Multiobjective Metabolic Flux Analysis ( 13 C-MOMFA) tool. It utilizes genome-scale metabolic models by integrating high-throughput omics data, along with measurements from metabolic assays and 13-Carbon ( 13 C) stable isotope-labeled tracer studies, to quantify fluxes in cancer cells. Our approach is the first to connect cancer metabolism to their malignant phenotype - a crucial step to discover the metabolic underpinnings of cancer pathology. It also captures the heterogeneity in cancer cells and patients, hence providing a useful framework for targeted therapy. Citation Format: Abhinav Achreja, Lifeng Yang, Tyler Moss, Vasudha Sehgal, Juan Marini, Prahlad T. Ram, Deepak Nagrath. Design of phenotype-driven flux analysis approach for personalized metabolic models of cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1204. doi:10.1158/1538-7445.AM2015-1204

Abstract 1208: Glutamine modulates cellular NAD+/NADH homeostasis thereby regulating cancer metastasis, drug sensitivity in cancer cells

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. Recently, we 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 cellular NAD+/NADH homeostasis, which mediates cancer metastasis and progression. On the other hand, the overexpression of NAD+ biosynthesis pathway enhances glutamine9s entry into TCA cycle for cancer metastasis, as well as chemo-drug resistance. Our findings suggest that a combined approach of targeting high-invasive OVCA cells by blocking glutamine9s entry into the TCA cycle, along with targeting NAD+ biosynthesis pathway may lead to potential therapeutic approaches for treating OVCAs. Our insights will present a unique opportunity for overcoming the drug resistance limitation in clinical trials in ovarian cancers. Citation Format: Lifeng Yang, Abhinav Achreja, Tyler Moss, Joelle Baddour, Katherine Stilles, Lisa Chiba, Sun Hye Kim, Josh Morse, Juan Marini, Anil K. Sood, Prahlad T. Ram, Deepak Nagrath. Glutamine modulates cellular NAD+/NADH homeostasis thereby regulating cancer metastasis, drug sensitivity in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1208. doi:10.1158/1538-7445.AM2015-1208

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