Metabolic Reprogramming of Glioblastoma Cells during HCMV Infection Induces Secretome-Mediated Paracrine Effects in the Microenvironment

Glioblastoma (GBM) is an aggressive primary central nervous system neoplasia with limited therapeutic options and poor prognosis. Following reports of cytomegalovirus (HCMV) in GBM tumors, the anti-viral drug Valganciclovir was administered and found to significantly increase the longevity of GBM patients. While these findings suggest a role for HCMV in GBM, the relationship between them is not clear and remains controversial. Treatment with anti-viral drugs may prove clinically useful; however, their results do not explain the underlying mechanism between HCMV infection and GBM progression. We hypothesized that HCMV infection would metabolically reprogram GBM cells and that these changes would allow for increased tumor progression. We infected LN-18 GBM cells and employed a Seahorse Bioanalyzer to characterize cellular metabolism. Increased mitochondrial respiration and glycolytic rates were observed following infection. These changes were accompanied by elevated production of reactive oxygen species and lactate. Due to lactate’s numerous tumor-promoting effects, we examined the impact of paracrine signaling of HCMV-infected GBM cells on uninfected stromal cells. Our results indicated that, independent of viral transmission, the secretome of HCMV-infected GBM cells was able to alter the expression of key metabolic proteins and epigenetic markers. This suggests a mechanism of action where reprogramming of GBM cells alters the surrounding tumor microenvironment to be permissive to tumor progression in a manner akin to the Reverse-Warburg Effect. Overall, this suggests a potential oncomodulatory role for HCMV in the context of GBM.

Disrupting the Reverse Warburg Effect As a Therapeutic Strategy in Multiple Myeloma

Introduction: Altered cellular metabolism is a hallmark of every cancer cell. Aerobic glycolysis ("The Warburg Effect") is one of the earliest recognized metabolic abnormalities in cancer cells whereby extracellular glucose is preferentially metabolized and eventually processed to generate lactate and energy in the form of ATP before the former is released extracellularly, irrespective of oxygen availability. While extracellular lactate produced and released from cancer cells has traditionally been considered a waste metabolic by-product, recent understanding of cell metabolism suggests that it can also serve as a primary metabolic fuel for cancer cells via uptake by monocarboxylate transporters (MCTs). Our goal was to evaluate this "Reverse Warburg Effect" phenomenon in multiple myeloma (MM) cells and determine if it can be exploited for therapeutic purposes. Methods: All HMCLs, MM1S, RPMI-8226 and U266, were grown in RPMI-1640 cell culture medium containing 11 mM glucose and supplemented with 10% dialyzed fetal bovine serum (FBS) and 2 mM Glutamine. Primary MM cells were extracted using magnetic bead CD138 positive selection from MM patient bone marrow aspirates. For 13C-labeling experiments, HMCLs and primary MM cells were suspended in RPMI-1640 cell culture media containing 13C-labeled isotopes. Isotopomer analysis of glycolytic and tricarboxylic acid (TCA) cycle metabolites from HMCL and primary MM cell pellets was performed using Agilent Technologies 5975C gas chromatography-mass spectrometry. Small molecule inhibitors, AZD3965 and syrosingopine, were purchased from Selleck Chemicals and Sigma respectively. Cellular viability and proliferation were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT) and CCK-8 assays respectively. MCT-1 and MCT-4 antibodies for western blotting were utilized to evaluate their cell membrane expression on HMCLs. Results: The HMCLs, MM1S and RPMI-8226 as well as primary CD138+ cells from MM patient bone marrow were cultured in cell culture media containing physiological levels (1 mM) of U-13C-Lactate. The incorporation of extracellular 13C into the intracellular glycolytic and TCA cycle metabolite pool was observed (Fig 1) based on the expected isotopomeric patterns, demonstrating the Reverse Warburg Effect in MM cells. The relative contribution of carbon substrate by extracellular lactate compared to extracellular glucose was assessed in the following HMCLs: MM1S, RPMI-8226 and U266 cells by culturing in cell culture media containing 3-13C-Lactate and U-13C-Glucose. Extracellular lactate (yellow bar) contribution to the formation of TCA metabolites equaled that of glucose (red bar) based on the expected isotopomer patterns, suggesting the relative importance of extracellular lactate as an essential nutrient like glucose (Fig 2). Since MCT-1 and MCT-4 are key bidirectional cell membrane transporters of lactate in and out of cells, we explored the clinical significance of their gene expression level on clinical outcomes using the COMMPASS dataset provided by the Multiple Myeloma Research Foundation (MMRF). When MM patients were dichotomized at above or below the median of the expression levels of fragments per kilobase of transcript per million (FPKM), MCT-1 and MCT-4 overexpression conferred a worse progression free survival and overall survival (Fig 3). The MCT-1/MCT-4 protein expression was detectable across the various HMCLs: MM1S, U266 and RPMI-8226 (Fig 4). Inhibition of MCT-1 by specific inhibitor AZD3965 was able to reduce proliferation but not affect viability of HMCLs at 48 hours (Fig 5). However, dual inhibition of MCT-1/MCT-4 using syrosingopine was able to significantly reduce proliferation and decrease viability of HMCLs in a dose dependent fashion (Fig 6). Finally, dual inhibition of MCT-1/MCT-4 using syrosingopine reduced the utilization of extracellular lactate into the TCA cycle pool by HMCLs in media containing 3-13C-Lactate (Fig 7). Conclusion: Utilization of extracellular lactate via Reverse Warburg Effect phenomenon appears highly active in MM cells. Disrupting the utilization of extracellular lactate by dual inhibition of both MCT-1 and MCT-4 appears therapeutic. In the future, dual inhibition of MCT-1/MCT-4 in combination with other anti-MM therapies should be evaluated to determine synergistic therapeutic potential. Figure 1 Figure 1. Disclosures Kumar: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Antengene: Consultancy, Honoraria; Beigene: Consultancy; Bluebird Bio: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding.

Microeconomics of Metabolism: The Warburg Effect as Giffen Behaviour

Metabolic behaviours of proliferating cells are often explained as a consequence of rational optimization of cellular growth rate, whereas microeconomics formulates consumption behaviours as optimization problems. Here, we pushed beyond the analogy to precisely map metabolism onto the theory of consumer choice. We thereby revealed the correspondence between long-standing mysteries in both fields: the Warburg effect, a seemingly wasteful but ubiquitous strategy where cells favour aerobic glycolysis over more energetically efficient oxidative phosphorylation, and Giffen behaviour, the unexpected consumer behaviour where a good is demanded more as its price rises. We identified the minimal, universal requirements for the Warburg effect: a trade-off between oxidative phosphorylation and aerobic glycolysis and complementarity, i.e. impossibility of substitution for different metabolites. Thus, various hypotheses for the Warburg effect are integrated into an identical optimization problem with the same universal structure. Besides, the correspondence between the Warburg effect and Giffen behaviour implies that oxidative phosphorylation is counter-intuitively stimulated when its efficiency is decreased by metabolic perturbations such as drug administration or mitochondrial dysfunction; the concept of Giffen behaviour bridges the Warburg effect and the reverse Warburg effect. This highlights that the application of microeconomics to metabolism can offer new predictions and paradigms for both biology and economics.

Innovative Surgical System for Breast Cancer Based on Real-time Excising of Hypoxic Lesions Significantly Reduced the Positive Cavity Side Margin

Background: For most people, the first step in treatment is to take out the tumor (surgery), so precise and fast diagnosis of any sign of high-risk cells in surgical cavity margins is significant. The frozen pathology method is the conventional standard of intraoperative diagnosis, but the low number of slides prepared from non-fixed tissues prevents us to achieve a perfect diagnosis. Although many improvements in intraoperative margin detection were achieved, still no clinically approved intra-operative technique has been reported for the detection of surgical margins with pathologically approved classification in breast cancer.Methods: From November 2018 to April 2020, 227 patients were registered and 213 with different types of breast tumors (IDC: n=151 (70.9%), ILC: n=6 (2.8%), DCIS: n=38 (17.8%), Atypia: n=8 (3.8%), Benign tumors: n=10 (%4.7)) were randomly assigned for CDP clinical trials. Men made up 2 (1%) of the patients; 211 (99%) were women. Functionalized carbon nanotubes grown on the electrode needles lively and selectively determine the H2O2 released from cancer/atypical cells, through reverse Warburg effect and hypoxia assisted glycolysis pathways, in a quantitative electrochemical manner. The study is registered at Iran National Committee for Ethics in Biomedical Research (IR.TUMS.VCR.REC.1397.355).Result: A real-time electrotechnical system, named cancer diagnostic probe (CDP) (Patent Pub. No.: US 2018/0299401A1), has been developed and clinically approved for breast cancer surgery (National Certificate ID:14006918495) to find the presence of pre-neoplastic/neoplastic cells in-vivo. Here, Functionalized carbon nanotubes grown on the electrode needles lively and selectively determine the H2O2 released from cancer or atypical cells, through reverse Warburg effect and hypoxia assisted glycolysis pathways, in quantitative electrochemical manner. A matched clinical diagnostic categorization between the pathological results and response peaks of CDP was proposed based on pathological classifications of WHO. Conclusion: The clinical ability of CDP was verified on more than 1300 human in-vivo breast samples with sensitivity and specificity of 93%, and 97%, respectively. After passing many trials and standard examinations, the system received production and clinical use certifications as a surgeon assistant system for usage in the operating room.

A tumour suppressive relationship between mineralocorticoid and retinoic acid receptors activates a transcriptional program consistent with a reverse Warburg effect in breast cancer

Background The role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective. Methods We used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments. Results Using this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARβ. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival. Conclusion Our study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.

A Scaffold-Free 3-D Co-Culture Mimics the Major Features of the Reverse Warburg Effect In Vitro

Most tumors consume large amounts of glucose. Concepts to explain the mechanisms that mediate the achievement of this metabolic need have proposed a switch of the tumor mass to aerobic glycolysis. Depending on whether primarily tumor or stroma cells undergo such a commutation, the terms ‘Warburg effect’ or ‘reverse Warburg effect’ were coined to describe the underlying biological phenomena. However, current in vitro systems relying on 2-D culture, single cell-type spheroids, or basal-membrane extract (BME/Matrigel)-containing 3-D structures do not thoroughly reflect these processes. Here, we aimed to establish a BME/Matrigel-free 3-D microarray cancer model to recapitulate the metabolic interplay between cancer and stromal cells that allows mechanistic analyses and drug testing. Human HT-29 colon cancer and CCD-1137Sk fibroblast cells were used in mono- and co-cultures as 2-D monolayers, spheroids, and in a cell-chip format. Metabolic patterns were studied with immunofluorescence and confocal microscopy. In chip-based co-cultures, HT-29 cells showed facilitated 3-D growth and increased levels of hexokinase-2, TP53-induced glycolysis and apoptosis regulator (TIGAR), lactate dehydrogenase, and: translocase of outer mitochondrial membrane 20 (TOMM20), when compared with HT-29 mono-cultures. Fibroblasts co-cultured with HT-29 cells expressed higher levels of mono-carboxylate transporter 4, hexokinase-2, microtubule-associated proteins 1A/1B light chain 3, and ubiquitin-binding protein p62 than in fibroblast mono-cultures, in both 2-D cultures and chips. Tetramethylrhodamin-methylester (TMRM) live-cell imaging of chip co-cultures revealed a higher mitochondrial potential in cancer cells than in fibroblasts. The findings demonstrate a crosstalk between cancer cells and fibroblasts that affects cellular growth and metabolism. Chip-based 3-D co-cultures of cancer cells and fibroblasts mimicked features of the reverse Warburg effect.

Cancer Development and Damped Electromagnetic Activity

Cancer can be initiated in a cell or a fibroblast by short-circuiting of the cellular electromagnetic field by various fibers, parasitic energy consumption, virus infections, and mitochondrial defects, leading to a damped cellular electromagnetic field. Except short-circuiting (e.g., by asbestos fibers), the central process is mitochondrial dysfunction in cancer cells (the Warburg effect) or in fibroblasts associated with a cancer cell (the reverse Warburg effect), critically lowered respiration, reversed polarity of the ordered water layers around mitochondria, and damped electromagnetic activity of the affected cells. Frequency and power changes of the generated electromagnetic field result in broken communication between cells and possibly in reduced control over chemical reactions, with an increased probability of random genome mutations. An interdisciplinary framework of phenomena related to cancer development is presented, with special attention to the causes and consequences of disturbed cellular electromagnetic activity. Our framework extends the current knowledge of carcinogenesis, to clarify yet unexplained phenomena leading to genome mutation and cancer initiation.

The Reverse Warburg Effect Is Associated with Fbp2-Dependent Hif1α Regulation in Cancer Cells Stimulated by Fibroblasts

Fibroblasts are important contributors to cancer development. They create a tumor microenvironment and modulate our metabolism and treatment resistance. In the present paper, we demonstrate that healthy fibroblasts induce metabolic coupling with non-small cell lung cancer cells by down-regulating the expression of glycolytic enzymes in cancer cells and increasing the fibroblasts’ ability to release lactate and thus support cancer cells with energy-rich glucose-derived metabolites, such as lactate and pyruvate—a process known as the reverse Warburg effect. We demonstrate that these changes result from a fibroblasts-stimulated increase in the expression of fructose bisphosphatase (Fbp) in cancer cells and the consequent modulation of Hif1α function. We show that, in contrast to current beliefs, in lung cancer cells, the predominant and strong interaction with the Hif1α form of Fbp is not the liver (Fbp1) but in the muscle (Fbp2) isoform. Since Fbp2 oligomerization state and thus, its role is regulated by AMP and NAD+—crucial indicators of cellular metabolic conditions—we hypothesize that the Hif1α-dependent regulation of the metabolism in cancer is modulated through Fbp2, a sensor of the energy and redox state of a cell.