Tumor Metabolism and the Microenvironment | EK14

Jan 25, 2021 ‐ Jan 28, 2021



Sessions

Crispr-engineered nanobodies a potential oncological target against extracellular laminin

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

"Crispr-engineered nanobodies a potential oncological target against extracellular laminin" Miss Akanksha Singh CSIR-National Environmental Engineering Research Institute, Nagpur. Abstract: Nanobodies due to low efficiency though effective were when targeted by CRISPR Cas 9 technology conjugated Cas 9 proteins on the surface of nanobody previously tagged with green fluorescent proteins. Laminin is a crucial extracellular proteinaous component in tumoral microenvironment. LN-332 being a multifaceted protein plays an important role in survival and proliferation of cancers thus accelerating the nutrient supply. Nanobodies can engineered specifically against the several types of cancer like colon, oral, prostrate and thyroid. CRISPR- nanostructures can hamper the transcriptome and post-transcriptome of LN-332 component as the Cas 9 proteins loaded on the nanobodies by recognition of rare markers on the surface of LN-332 with the fused fluorescent protein. Nanobodies composed of VHH domain were fused with sg-RNA Cas 9 were exposed to Ln-332 obtained from mice infected with cancer and after 24-48 hours, mice indicated diminished levels of Ln-332 protein. The review juxtaposed therapeutic effect possessed by nanobodies that was accelerated after fusion with Cas 9 proteins might inhibit the activity of LN 332 protein. Keywords: Nanobodies, CRISPR Cas 9, Laminin, LN-332, Cas 9, VHH domain References: 1. Xie, Y.J., Dougan, M., Jailkhani, N., Ingram, J., Fang, T., Kummer, L., Momin, N., Pishesha, N., Rickelt, S., Hynes, R.O. and Ploegh, H., 2019. Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice. Proceedings of the National Academy of Sciences, 116(16), pp.7624-7631. 2. Finicle, B.T., Jayashankar, V. & Edinger, A.L. Nutrient scavenging in cancer. Nat Rev Cancer 18, 619–633 (2018). https://doi.org/10.1038/s41568-018-0048-x 3. Qiu, X., Tan, H., Fu, D., Zhu, Y. and Zhang, J., 2018. Laminin is over expressed in breast cancer and facilitate cancer cell metastasis. Journal of Cancer Research and Therapeutics, 14(12), p.1170. 4. Ye, Y., Zhang, X., Xie, F., Xu, B., Xie, P., Yang, T., Shi, Q., Zhang, C.Y., Zhang, Y., Chen, J. and Jiang, X., 2020. An engineered exosome for delivering sgRNA: Cas9 ribonucleoprotein complex and genome editing in recipient cells. Biomaterials Science, 8(10), pp.2966-2976. 5. Maltseva, D.V. and Rodin, S.A., 2018. Laminins in metastatic cancer. Molecular Biology, 52(3), pp.350-371. 6. Tripathi, M., Nandana, S., Yamashita, H., Ganesan, R., Kirchhofer, D. and Quaranta, V., 2008. Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression. Journal of biological chemistry, 283(45), pp.30576-30584.

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A Novel Bioengineered Model Of Osteosarcoma Recapitulates Critical Tumor Phenotypes Within A Native Bone Microenvironment

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

A Novel Bioengineered Model Of Osteosarcoma Recapitulates Critical Tumor Phenotypes Within A Native Bone Microenvironment Alan Chramiec1, Alessandro Marturano-Kruik1,2, Luke Hao1, Keith Yeager1, Max Summers1, Miranda Wang1, Roberta Lock1, and Gordana Vunjak-Novakovic1,3 1Department of Biomedical Engineering, Columbia University, USA; 2 Department of Chemistry, Materials and Chemical Engineering “G Natta”, Politecnico de Milano, Italy; 3 Department of Medicine, Columbia University, New York, NY, USA Traditional in-vitro models of solid bone tumors used in basic and preclinical research are unable to faithfully recapitulate human physiology. Specifically, monolayer cultures of osteosarcoma fail to recapitulate various features of the 3D tumor phenotype, and they lack the native bone milieu, where tumor growth, metastasis, and response to therapy are critically dependent on cancer cell interactions with the bone matrix, supporting cells, and secreted regulatory factors. Based on our previous success in developing a biomimetic tissue model of Ewing sarcoma, we extended our approach towards bioengineering a novel human 3D tumor model of osteosarcoma within a bone microenvironment that would overcome some of these limitations. Briefly, osteosarcoma cell lines were used to generate tumor-like aggregates which were subsequently introduced into and cultured within engineered bone tissue scaffolds. Our bioengineered osteosarcoma model was able to recapitulate several aspects of native tumors and their microenvironment missing in monolayer cultures. Significantly higher expression of cancer-related genes (as seen in patients), re-activation of the hypoxic and glycolytic tumor phenotype, re-activation of hypoxia-mediated expression of vascular endothelial growth factor VEGF-α (a critical component of tumor angiogenesis), induction of vasculogenic mimicry, more physiologically relevant growth rates, and the adjacent presence of functional osteoblasts within a demineralized native bone scaffold were all achieved. Additionally, we were able to capture a both a degree of intra-tumor heterogeneity, with the presence of a necrotic core and variability in proliferation across the tumor aggregates, as well as inter-tumor heterogeneity, with notable differences observed between our models generated from metastatic and non-metastatic osteosarcoma cell lines. Finally, the osteosarcoma cell lines were transduced with a GFP-luciferase reporter, allowing us to visualize the engineered tumors in situ within the bone scaffolds, and to evaluate tumor cell responses to various canonical and experimental drug treatments. Notably, unlike monolayers, our bioengineered osteosarcoma model could be cultured stably for several weeks. This allowed us to apply a clinical treatment regimen cycle of linsitinib, an experimental drug in an ongoing phase II clinical trial for Ewing sarcoma, in also being effective at preventing the growth of non-metastatic osteosarcoma bioengineered tumors. Overall, we were able to successfully generate a new, more native-like 3D bioengineered model of osteosarcoma with significant advantages over traditional in-vitro culture that has the potential to explore novel questions regarding tumor biology, the tumor microenvironment, and translational applications.

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Exploiting a metabolic vulnerability in mTORC1-driven tumors

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Exploiting a metabolic vulnerability in mTORC1-driven tumors Alexander J. Valvezan (1), Molly C. McNamara (2), Spencer K. Miller (2), Margaret E. Torrence (2), John M. Asara (3), Elizabeth P. Henske (4), and Brendan D. Manning (2); (1) Center for Advanced Biotechnology and Medicine, Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, USA; (2) Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA; (3) Division of Signal Transduction, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA; (4) Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA. Mammalian Target of Rapamycin Complex 1 (mTORC1) is a master regulator of anabolic cell growth that is activated in the majority of human cancers across nearly all lineages. We find that mTORC1 activation creates dependence on nucleotide synthesis pathways, unveiling a targetable metabolic vulnerability. Tumor cells can acquire nucleotides in 2 ways: de novo synthesis, and/or uptake of nucleosides and nucleobases from the tumor microenvironment, which are then converted to nucleotides through salvage pathways. Inhibitors of inosine 5’-monophosphate dehydrogenase (IMPDH) block the rate-limiting step in de novo guanylate nucleotide synthesis, as well as guanylate salvage from hypoxanthine, the most abundant circulating purine nucleoside. Clinically approved IMPDH inhibitors selectively kill cells with uncontrolled mTORC1 activation and demonstrate robust anti-tumor efficacy at therapeutically relevant doses in mouse models of the mTORC1-driven genetic tumor syndrome Tuberous Sclerosis Complex (TSC). These effects stem from DNA replication stress and DNA damage caused by mTORC1-driven ribosomal RNA synthesis, which greatly increases cellular demand for nucleotides and thus renders nucleotide pools limiting. When compared directly to the mTORC1 inhibitor rapamycin, IMPDH inhibitor treatment provides a more durable anti-tumor response associated with tumor cell death. Comparison of all clinically approved IMPDH inhibitors in multiple cell and tumor models of TSC reveals mizoribine, an IMPDH inhibitor used as an immunosuppressant in Asia, as the best pre-clinical candidate for potential repurposing for the treatment of mTORC1-driven tumors.

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PYCR1 activity is essential for the maintenance of viable hypoxic regions in cancers

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

PYCR1 activity is essential for the maintenance of viable hypoxic regions in cancers Rebecca L Westbrook1, Esther Bridges2, Cristina Escribano Gonzalez1, Abeer Shaban3, Nathalie Escande-Beillard4, Katherine L Eales1, Lisa Vettore1, Paul Walker1, Federica Cuozzo1, Colin Nixon5, David J Hodson1, Bruno Reversade4,6, Adrian Harris2 and Daniel A Tennant1. 1Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT. UK 2Hypoxia and Angiogenesis Group, Cancer Research UK Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, UK 3University Hospital Birmingham NHS Foundation Trust and Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT. UK 4Institute of Medical Biology, Human Genetics and Embryology Laboratory; A*STAR, Singapore 138648, Singapore 5Beatson Institute for Cancer Research, University of Glasgow, Switchback Road, Glasgow. G61 1BD. UK 6Institute of Molecular and Cellular Biology, A*STAR, Singapore 138673, Singapore The demands of highly proliferative cancer cell populations alongside an inadequate blood supply lead to low oxygen and nutrient conditions in the tumour microenvironment. Hypoxic cells adapt their metabolic network, often increasing reliance on pathways that are independent of oxygen tension to maintain viability. However, to continue the efficient synthesis of anabolic precursors in these conditions, it is advantageous to continue use of oxidative TCA cycle metabolism, which is closely coupled to electron transport chain activity, and the reduction of molecular oxygen. We have previously shown that when redox homeostasis is perturbed by oncogenic mutations in isocitrate dehydrogenase 1, pyrroline 5-carboxylate reductase 1 (PYCR1) activity was increased to oxidise mitochondrial NADH, effectively uncoupling oxidative TCA cycle activity from respiration. We therefore hypothesised that this may also be true in hypoxic conditions. We show here that hypoxia elicits a PYCR1-dependent increase in proline synthesis and excretion, which is required for efficient growth of cells in 3D culture and in xenograft tumours. PYCR1 deficiency in hypoxia results in deficient oxidative TCA cycle and reduced growth, which cannot be recovered by exogenous proline supplementation. Finally, loss of PYCR1 in 3D spheroids and xenograft tumours increases intratumoural hypoxia through enforced respiration, which leads to loss of proliferative drive, increased cell death and necrosis. Our data therefore suggest that PYCR1 is an essential component of the cellular response to hypoxia, functionally uncoupling the mitochondrial electron transport chain from the TCA cycle to permit the efficient use of the limited supply of oxygen throughout the cell.

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Combined in vivo 13C-metabolomics and proteomics approach to optimise immunotherapy response in malignant melanoma

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Combined in vivo 13C-metabolomics and proteomics approach to optimise immunotherapy response in malignant melanoma Bernardus Evers1,2, Albert Gerding1,2, Jiske Tiersma3, Karen van Eunen1,2, Justina C. Wolters4, Dirk-Jan Reijngoud1,2, Mathilde Jalving3, and Barbara M. Bakker1,2 1) Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signalling, University of Groningen, University Medical Center Groningen, The Netherlands. 2) Systems Biology Centre for Energy Metabolism and Ageing, University of Groningen, University Medical Center Groningen, The Netherlands. 3) Department of Medical Oncology, University of Groningen, University Medical Center Groningen, The Netherlands 4) Department of Pharmacy, Analytical Biochemistry, University of Groningen, The Netherlands Metabolic reprogramming is a common feature during tumourigenesis that allows tumours to adapt to nutrient-poor microenvironments, thereby maintaining cell viability and produce biomass for cell proliferation. Increased aerobic lactate fermentation, known as the "Warburg effect" is a well-studied metabolic alteration in melanoma, as well as other cancer types, that renders the tumour microenvironment hypoglycaemic and acidic. Preclinical in vitro and in vivo data show that this phenomenon has immunosuppressive effects and may as well attenuate patient response to immunotherapy. Interestingly, this metabolic alteration distinguishes a tumour and its corresponding microenvironment from healthy tissue and make their metabolic processes susceptible to drug targeting. We hypothesise that drugs normalising tumour metabolism may revert metabolic-induced immunosuppression and increase patient response to immunotherapy. In this study, we use a combined proteomics and 13C-metabolomics approach to investigate the effect of dichloroacetate (DCA) on normalizing tumour metabolism in vivo. DCA reroutes the pyruvate produced in glycolysis to be oxidized in the mitochondria, thereby reducing the flux to lactic acid and neutralising the tumour microenvironment. A seven-day, phase 2 clinical trial of DCA has been planned in 36 patients with malignant melanoma prior to immunotherapy. Pre- and post-DCA treatment biopsies will be taken after intravenous [U-13C]glucose infusion in isotopic steady-state. We present the development of GC-EI-MS(MS) methods for quantitative analysis of 13C-label incorporation in glycolytic, TCA cycle and pentose phosphate pathway intermediates. In addition we have developed targeted proteomics for the absolute quantification of glycolytic and mitochondrial metabolic enzymes using in-house designed 13C-labelled peptide standards based on QConCat technology. These methods will be applied to the respective paired biopsies. Combing the patient-specific response to immunotherapy and DCA with subsequent analysis and computational modelling will enable detailed characterisation of metabolic activity and give insight into metabolic regulation upon treatment in vivoin melanoma patients. Thus, we expect to provide an unprecedented insight into melanoma tumour metabolism and proof-of-concept that targeting metabolism improves immunotherapy response in malignant melanoma patients.

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Targeting Leucine and Valine Catabolism to Disrupt Prostate Cancer Progression and Resistance

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Targeting Leucine and Valine Catabolism to Disrupt Prostate Cancer Progression and Resistance Charles Bidgood1, Lisa Philp1, Anja Rockstroh1, Melanie Lehman1,2, Colleen Nelson1, Martin Sadowski3, Jennifer Gunter1 1. Australian Prostate Cancer Research Centre – Queensland, Institute of Health and Biomedical Innovation, school of Biomedical Sciences, Faculty of Health, Translational Research Institute, 37 Kent St, Brisbane, 4102, Qld, Australia. 2. Vancouver Prostate Centre, Vancouver General Hospital, 2660 Oak St, Vancouver, BC V6H 3Z6, Canada. 3. Universität Bern, Institut für Pathologie, Raum L420, Murtenstrasse 31, CH-3008 Bern, Switzerland In advanced prostate cancer (PCa), the therapeutic efficacy of androgen targeted therapies (ATTs) such as enzalutamide is limited by adaptive reprogramming to androgen receptor (AR) inhibition resulting in therapy resistance. These resistance pathways include metabolic remodelling which facilitates tumour survival. As a result, there is an ever-growing need for novel strategies which can disrupt these metabolic adaptations and in turn, inhibit the progression of advanced PCa. We have analysed the transcriptome of androgen-sensitive PCa cell lines and PDX models to identify the metabolic pathways that predictably change in response to ATTs. This project aims to target the catabolism of two branched-chain amino acids: leucine and valine, which have been shown to be largely upregulated in both patient metastatic castrate resistant prostate cancer (mCRPC) biopsies and enzalutamide-treated prostate cancer cells (LNCaPs). Through modification of the extracellular amino acid environment, utilisation of RNAi technologies to suppress catabolism and strategic application of known metabolic inhibitors, we have identified key targets of leucine and valine catabolism affecting PCa cell growth, proliferation, and survival. These include two enzymes, HIBCH and MCCC2 which are specific for valine and leucine metabolites, respectively. Additionally, we have implemented a multiparametric quantitative single-cell imaging (mqSCI) approach which shows that disruption of these pathways induces fundamental changes to mitochondrial dynamics, lipid synthesis and storage, redox potential, and metabolic viability to a range of androgen-sensitive and insensitive PCa cell lines. These findings suggest the catabolism of leucine and valine are critical in the adaptive metabolic rewiring by PCa cells in response to ATTs and targeting these enzymes may serve as an effective therapeutic strategy in order to combat the progression of advanced PCa and therapeutic resistance to ATTs.

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Ascorbate availability has an impact on the hypoxic response in breast cancer cells

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Ascorbate availability has an impact on the hypoxic response in breast cancer cells Author: Citra Praditi(1), Gabi Dachs(2), Stephanie Bozonet(1), Margreet Vissers(1) 1. Centre for Free Radical Research, Dept of Pathology and Biomedical Science, University of Otago Christchurch 2. MacKenzie Cancer Research Group, Dept of Pathology and Biomedical Science, University of Otago Christchurch Background: Ascorbate (vitamin C) is an essential cofactor for the 2-oxoglutarate dependent dioxygenase (2-OGDD) class of enzymes. The HIF (hypoxia-inducible factor)-hydroxylases belong to the 2-OGDD family. Increased ascorbate availability has been shown to up-regulate the activity of the HIF hydroxylases and down-regulate the hypoxic response, which could be beneficial to patients with cancers that use the hypoxic response pathway to support cancer progression. We have investigated this relationship in breast cancer cells and aim to determine whether ascorbate availability could affect the hypoxic response in this cancer. Methods: Human breast cancer cell lines MDA-MB231 and MCF-7 were exposed to ascorbate through media supplementation and uptake monitored. Following exposure to hypoxic conditions induced by the hypoxia-mimetic agent CoCl2 or decreased oxygen tension, cell lysates were collected and analysed by western blotting to measure HIF-1α protein stabilisation and expression of the downstream marker BNIP3 protein. Results: Both breast cancer cell lines expressed the sodium-dependent vitamin C transporter SVCT2 and readily accumulate ascorbate from the medium. Ascorbate pre-treatment was able to inhibit HIF-1a stabilisation and BNIP3 expression under CoCl2 treatment in MDA-MB231 and MCF-7. However, the response to ascorbate varied under hypoxia. In MDA-MB231 cell line at 1% O2 and MCF-7 cells at 0.1% O2, ascorbate did not inhibit HIF-1α protein stabilisation, but BNIP3 protein expression was decreased in the ascorbate-treated cells. Discussion: Our results show that increased ascorbate availability is able to decrease HIF-1a protein stabilisation and expression of its target protein BNIP3 in human breast cancer cell lines. Modulation of the hypoxic response by ascorbate likely reflects capacity for ascorbate to maintain the activity of the HIF-hydroxylases (prolyl hydroxylases 1-3 (PHDs) and factor inhibiting HIF (FIH). The efficiency of this regulation may be moderated under hypoxic conditions. Conclusion: BNIP3, and other proteins expressed as a result of HIF-1 activation, contribute to cancer cell survival under stress. The activation of HIF-1 is marked in breast cancer and is closely associated with patient survival. Our results indicate that uptake of ascorbate by breast cancer cells may be a factor influencing breast cancer outcomes.

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Antioxidant protein TR1 is critical for normal melanocyte function and protect melanocytes from UVB-induced DNA damage in vivo

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Antioxidant protein TR1 is critical for normal melanocyte function and protect melanocytes from UVB-induced DNA damage in vivo Evan L. Carpenter1, Gitali Ganguli-Indra1,2, Pamela Cassidy2,3, Gary Merrill4, Arup K. Indra1,2,3,4,5 1Department of Pharmaceutical Sciences, OSU-OHSU College of Pharmacy, Corvallis, OR; 2Knight Cancer Institute, OHSU, Portland, OR; 3Department of Dermatology, Oregon Health & Science University (OHSU), Portland, OR; 4Dept. of Biochemistry & Biophysics, OSU, Corvallis, OR; 5Linus Pauling Institute, OSU, Corvallis, OR, USA Melanoma is the deadliest skin cancer and the leading cause of death due to skin disease. While melanoma is treatable during early progression, once metastasis and spread has occurred the rate of survival rapidly declines. Therefore, a better understanding of the multitude of factors that lead to disease progression and metastasis is needed for more effective treatment. Oxidative stress is a major contributing factor in cancer progression and expression of antioxidant protein thioredoxin reductase 1 (TR1) is significantly elevated during melanoma progression indicating a potential role in melanoma-genesis. TR1 is a constituent of the thioredoxin antioxidant system, which plays an important role in the cellular antioxidant stress response. Here we seek to better understand the in vivo role of TR1 in melanocyte physiology and in ultraviolet (UV) induced melanomagenesis. To that end, we have generated a knockout mouse model in which Txnrd1 encoding TR1 has been selectively ablated in melanocytes via Cre recombinase driven by the melanocyte specific tyrosinase promoter to generate Tr1mel-/- mice. The mutant mice exhibit a depigmentation phenotype that includes reduced melanin content in the extremities (i.e. ears, paws, and tail) in addition to variable amelanotic ventral fur spotting. The amelanotic ventral spots lack both melanin and melanocytes, suggesting defects during the development of the melanocyte lineage in the absence of TR1. We also subjected the Tr1mel-/- mice to UVB irradiation and have observed a significant reduction in melanocyte density, elevation of DNA damage, and reduction in proliferation early on after UVB exposure. Altogether, above data suggest that TR1 is necessary for normal melanocyte function and melanin synthesis during development and likely plays a role in preventing UV-induced melanomagenesis.

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PCK2 balances TCA cycle activity, limits mitochondrial respiration and improves the redox balance in starved lung cancer cells

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

PCK2 balances TCA cycle activity, limits mitochondrial respiration and improves the redox balance in starved lung cancer cells Gabriele Grasmann1 , Mélanie Planque2,3, Corina T. Madreiter-Sokolowski4,5, Andelko Hrzenjak1,6, Wolfgang F. Graier4,7, Sarah-Maria Fendt2,3, Horst Olschewski1,6, Katharina Leithner1,7* 1 Division of Pulmonology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 2 Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer Biology, VIB, Leuven, Belgium 3 Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), Leuven, Belgium 4 Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria 5 Energy Metabolism Laboratory, Institute of Translational Medicine, Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland 6 Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria 7 BioTechMed-Graz, Graz, Austria Cancer cells frequently lack nutrients like glucose, due to insufficient vascular networks, requiring metabolic adaptations. Gluconeogenesis, in big parts the reverse pathway of glycolysis, has been recently described as survival strategy. Phosphoenolpyruvate carboxykinase (PEPCK) is the key gluconeogenic enzyme, catalyzing the conversion of oxaloacetate, a TCA cycle intermediate, to phosphoenolpyruvate. The mitochondrial isoform of PEPCK, PCK2 is expressed in different tumor types, including lung cancer. It has pro-survival and pro-proliferative effects in vitro and in vivo and mediates the biosynthesis of gluconeogenic/glycolytic intermediates in glucose deprived cancer cells. However, it remains unknown, whether PCK2 regulates TCA cycle activity and mitochondrial respiration in cancer cells under nutrient starvation. Here we show that PCK2 silencing increased the abundance and interconversion of TCA cycle intermediates in two different lung cancer cell lines in a PCK2 re-expression reversible manner. The TCA cycle is connected to mitochondrial respiration via its production of reducing equivalents. Glucose and serum starvation led to enhanced mitochondrial respiration compared to non-starvation conditions. Under starvation conditions mitochondrial respiration was additionally augmented by PCK2 silencing and glutathione oxidation was increased. Moreover, enhancing the TCA cycle activity by PCK2 inhibition severely reduced colony formation, an effect that was antagonized by external addition of antioxidants. As a conclusion, the cataplerotic activity of PCK2 limits oxygen consumption and contributes to maintaining a reduced glutathione pool upon starvation. The study sheds light on adaptive responses in cancer cells to nutrient deprivation and identifies gluconeogenesis as starvation-induced pathway that limits respiration-induced oxidative stress.

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Fasting sensitizes hepatocellular carcinoma to sorafenib by curtailing metabolic flexibility

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Fasting sensitizes hepatocellular carcinoma to sorafenib by curtailing metabolic flexibility Jelena Krstic 1, Isabel Reinisch 1, Katharina Schindlmaier 1, Christoph Nössing 1,2, Maria R. Depaoli 3, Jeta Ramadani-Muja 3, Meritxell Huch 4, Roland Malli 3,5 and Andreas Prokesch 1,5 1 Gottfried Schatz Research Center for Cell Signaling, Metabolism & Aging, Division of Cell Biology, Histology and Embryology, Medical University of Graz, 8010 Graz, Austria 2 Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK. 3 Gottfried Schatz Research Center for Cell Signaling, Metabolism & Aging, Division of Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Austria 4 Max Planck Institute of Molecular Cell Biology and Genetics, 01307, Dresden, Germany 5 BioTechMed-Graz, 8010 Graz, Austria Metabolic flexibility is an important mediator of therapy resistance in cancer. However, while cancer cells rewire their metabolic pathways to adapt to nutritional microenvironment, they also reveal metabolic vulnerabilities which can be therapeutically exploited. Hepatocellular carcinoma (HCC) is one of the deadliest cancers with very few molecular therapeutic opportunities. Sorafenib, the most widely used clinical drug for advanced HCC provides limited extension in median overall survival of three months due to primary or acquired resistance. We explored whether nutrient deprivation can be used as an adjuvant therapy to sorafenib in the treatment of HCC. We show that fasting can sensitize resistant HCC to sorafenib in HCC-derived cells, xenografts, and in patient-derived organoids. We found that sorafenib acts as potent inhibitor of mitochondrial respiration, causing resistant HCC cells to switch to glycolysis for survival. Synergistically, reduced nutrients prevent this Warburg shift and lead to sensitisation to sorafenib in vitro and in vivo. Functional experiments show that glucose is the limiting nutrient crucial for curtailing this metabolic flexibility. We further show that p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting, as p53 knock out cells and xenografts remain resistant under starvation. p53 is also necessary for the sorafenib-enhancing effect of intermittent fasting in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for advanced-stage, and possibly even early-stage, HCC.

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