Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Lactate Upregulates the Expression of DNA Repair Genes, Causing Intrinsic Resistance of Cancer Cells to Cisplatin

Intrinsic or acquired drug resistance is one of the major problems compromising the success of antineoplastic treatments. Several evidences correlated some therapeutic failures with changes in cell metabolic asset and in line with these findings, hindering the glycolytic metabolism of cancer cells via lactate dehydrogenase (LDH) inhibition was found to overcome the resistance to chemotherapeutic agents. Lactate, the product of LDH reaction, was shown to be involved in epigenetic regulation of gene expression. The experiments described in this paper were aimed at highlighting a possible direct effect of lactate in modifying the response of cancer cells to a chemotherapeutic treatment. To discriminate between the effects potentially caused by glycolytic metabolism from those directly referable to lactate, we selected cancer cell lines able to grow in glucose deprived conditions and evaluated the impact of lactate on the cellular response to cisplatin-induced DNA damage. In lactate-exposed cells we observed a reduced efficacy of cisplatin, which was associated with reduced signatures of DNA damage, enhanced DNA recombination competence and increased expression of a panel of genes involved in DNA repair. The identified genes take part in mismatch and nucleotide excision repair pathways, which were found to contribute in restoring the cisplatin-induced DNA damage. The obtained results suggest that this metabolite could play a role in reducing the efficacy of antineoplastic treatments.

Differential Response of Human Amniotic Membrane Proteins in Hepatocellular Carcinoma: Analysis of the Glycolytic Metabolism

Background: The human Amniotic Membrane (hAM) has been studied as a potential therapeutic option in cancer, namely in hepatocellular carcinoma. Previously, our research group evaluated the effect of human Amniotic Membrane Protein Extracts (hAMPE) in cancer therapy, demonstrating that hAMPE inhibit the metabolic activity of human hepatocellular carcinoma cell lines: Hep3B2.1-7, HepG2 and Huh7. Therefore, the aim of this study was to evaluate the effect of hAMPE treatment in glucose metabolism of hepatocellular carcinoma. Methods and Results: Glucose uptake and lactate production was assessed by 1H-NMR, and the expression of several mediators of the glycolytic pathway was evaluated by Western blot or fluorescence. Our results showed that hAMPE treatment increased glucose consumption on Hep3B2.1-7, HepG2, and Huh7 through the increase of GLUT1 in Hep3B2.1-7 and Huh7, and GLUT3 in HepG2 cells. It was observed increased expression of 6-phosphofrutokinase (PFK-1L) in all cell lines, indicating that glucose can be converted to pyruvate. Also, it was verified that glucose seems not to be converted to lactate on HepG2 and Huh7 cells, suggesting that hAMPE treatment may contradict the Warburg effect observed in carcinogenesis. In Hep3B2.1-7, the hAMPE treatment induced an increase in expression of lactate dehydrogenase (LDH) and monocarboxylate transporter isoform 4 (MCT4). Conclusions: Overall, this work highlighted the potential usefulness of hAMPE as anticancer therapy through the modulation of the glycolytic metabolism in human hepatocellular carcinoma.

Knockdown of OLR1 weakens glycolytic metabolism to repress colon cancer cell proliferation and chemoresistance by downregulating SULT2B1 via c-MYC

Chemoresistance is one of the major problems of colon cancer treatment. In tumors, glycolytic metabolism has been identified to promote cell proliferation and chemoresistance. However, the molecular mechanisms underlying glycolytic metabolism and chemoresistance in colon cancer remains enigmatic. Hence, this research was designed to explore the mechanism underlying the OLR1/c-MYC/SULT2B1 axis in the regulation of glycolytic metabolism, to affect colon cancer cell proliferation and chemoresistance. Colon cancer tissues and LoVo cells were attained, where OLR1, c-MYC, and SULT2B1 expression was detected by immunohistochemistry, RT-qPCR, and western blot analysis. Next, ectopic expression and knockdown assays were implemented in LoVo cells. Cell proliferation was detected by MTS assay and clone formation. Extracellular acidification, glucose uptake, lactate production, ATP/ADP ratio, and GLUT1 and LDHA expression were measured to evaluate glycolytic metabolism. Then, the transfected cells were treated with chemotherapeutic agents to assess drug resistance by MTS experiments and P-gp and SMAD4 expression by RT-qPCR. A nude mouse model of colon cancer transplantation was constructed for in vivo verification. The levels of OLR1, c-MYC, and SULT2B1 were upregulated in colon cancer tissues and cells. Mechanistically, OLR1 increased c-MYC expression to upregulate SULT2B1 in colon cancer cells. Moreover, knockdown of OLR1, c-MYC, or SULT2B1 weakened glycolytic metabolism, proliferation, and chemoresistance of colon cancer cells. In vivo experiments authenticated that OLR1 knockdown repressed the tumorigenesis and chemoresistance in nude mice by downregulating c-MYC and SULT2B1. Conclusively, knockdown of OLR1 might diminish SULT2B1 expression by downregulating c-MYC, thereby restraining glycolytic metabolism to inhibit colon cancer cell proliferation and chemoresistance.

Analogous Metabolic Decoupling in Pseudomonas putida and Comamonas testosteroni Implies Energetic Bypass to Facilitate Gluconeogenic Growth

Glycolytic metabolism of sugars is extensively studied in the Proteobacteria , but gluconeogenic carbon sources (e.g., organic acids, amino acids, aromatics) that feed into the tricarboxylic acid (TCA) cycle are widely reported to produce a fast-growth phenotype, particularly in species with biotechnological relevance. Much remains unknown about the importance of glycolysis-associated pathways in the metabolism of gluconeogenic carbon substrates.

Metformin Suppresses Monocyte Inflammation and Metabolic Reprogramming by SARS-CoV-2 Spike Protein

COVID-19 disproportionately affects older adults, and a hallmark of the disease is a hyperinflammatory state that is associated with severity. Various anti-inflammatory therapeutics have shown mixed efficacy in treating COVID-19, and the mechanisms by which hyperinflammation occurs are not well understood. Previous research indicated that monocytes, a key innate immune cell, undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism – including the geroprotector drug metformin – could inhibit the inflammatory response in monocytes. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism that was associated with production of pro-inflammatory cytokines including interleukin-6 and tumor necrosis factor-alpha. This response was dependent on hypoxia-inducible factor-1alpha, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production in monocytes, and abrogated glycolytic and mitochondrial metabolism. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by treatments that interfere with glycolytic activation, including metformin. This has potential implications for the treatment of hyperinflammation during COVID-19, which disproportionately affects older adults.

Long noncoding RNA GSEC promotes neutrophil inflammatory activation by supporting PFKFB3-involved glycolytic metabolism in sepsis

Metabolic reprogramming is a hallmark of neutrophil activation in sepsis. LncRNAs play important roles in manipulating cell metabolism; however, their specific involvement in neutrophil activation in sepsis remains unclear. Here we found that 11 lncRNAs and 105 mRNAs were differentially expressed in three transcriptome datasets (GSE13904, GSE28750, and GSE64457) of gene expression in blood leukocytes and neutrophils of septic patients and healthy volunteers. After Gene Ontology biological process analysis and lncRNA–mRNA pathway network construction, we noticed that GSEC lncRNA and PFKFB3 were co-expressed and associated with enhanced glycolytic metabolism. Our clinical observations confirmed the expression patterns of GSEC lncRNA and PFKFB3 genes in neutrophils in septic patients. Performing in vitro experiments, we found that the expression of GSEC lncRNA and PFKFB3 was increased when neutrophils were treated with inflammatory stimuli. Knockdown and overexpression experiments showed that GSEC lncRNA was essential for mediating PFKFB3 mRNA expression and stability in neutrophil-like dHL-60 cells. In addition, we found that GSEC lncRNA-induced PFKFB3 expression was essential for mediating dHL-60 cell inflammatory cytokine expression. Performing mechanistic experiments, we found that glycolytic metabolism with PFKFB3 involvement supported inflammatory cytokine expression. In summary, our study uncovers a mechanism by which GSEC lncRNA promotes neutrophil inflammatory activation in sepsis by supporting glycolytic metabolism with PFKFB3.

Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 Spike Protein Subunit 1

A hallmark of COVID-19 is a hyperinflammatory state associated with severity. Monocytes undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism associated with production of pro-inflammatory cytokines. This response was dependent on hypoxia-inducible factor-1α, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production and metabolic reprogramming. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.

N6-methyladenosine reader IMP2 stabilizes the ZFAS1/OLA1 axis and activates the Warburg effect: implication in colorectal cancer

Background Accumulating evidence shows that N6-methyladenine (m6A) modulators contribute to the etiology and progression of colorectal cancer (CRC). However, the exact mechanisms of m6A reader involved in glycolytic metabolism remain vague. This article aimed to crosstalk the m6A reader with glycolytic metabolism and reveal a new mechanism for the progression of CRC. Methods The relationship between candidate lncRNA and m6A reader was analyzed by bioinformatics, ISH and IHC assays. In vivo and in vitro studies (including MTT, CFA, trans-well, apoptosis, western blot, qRT-PCR and xenograft mouse models) were utilized to explore the biological functions of these indicators. Lactate detection, ATP activity detection and ECAR assays were used to verify the biological function of the downstream target. The bioinformatics, RNA stability, RIP experiments and RNA pull-down assays were used to explore the potential molecular mechanisms. Results We identified that the crosstalk of the m6A reader IMP2 with long-noncoding RNA (lncRNA) ZFAS1 in an m6A modulation-dependent manner, subsequently augmented the recruitment of Obg-like ATPase 1 (OLA1) and adenosine triphosphate (ATP) hydrolysis and glycolysis during CRC proliferation and progression. Specifically, IMP2 and ZFAS1 are significantly overexpressed with elevated m6A levels in CRC cells and paired CRC cohorts (n = 144). These indicators could be independent biomarkers for CRC prognostic prediction. Notably, IMP2 regulated ZFAS1 expression and enhanced CRC cell proliferation, colony formation, and apoptosis inhibition; thus, it was oncogenic. Mechanistically, ZFAS1 is modified at adenosine +843 within the RGGAC/RRACH element in an m6A-dependent manner. Thus, direct interaction between the KH3–4 domain of IMP2 and ZFAS1 where IMP2 serves as a reader for m6A-modified ZFAS1 and promotes the RNA stability of ZFAS1 is critical for CRC development. More importantly, stabilized ZFAS1 recognizes the OBG-type functional domain of OLA1, which facilitated the exposure of ATP-binding sites (NVGKST, 32–37), enhanced its protein activity, and ultimately accelerated ATP hydrolysis and the Warburg effect. Conclusions Our findings reveal a new cancer-promoting mechanism, that is, the critical modulation network underlying m6A readers stabilizes lncRNAs, and they jointly promote mitochondrial energy metabolism in the pathogenesis of CRC.

Genomic Characterization of in Vitro Acquired-Resistance to Proteasome Inhibitors

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novels agents that have significantly improved clinical outcomes, MM patients invariably relapse. A better understanding of the drug resistance mechanisms and development of biomarkers remain of major interest to improve the treatment of patients. In order to further investigate the mechanisms involved in resistance to proteasome inhibitors (PI), we have derived and characterized PI-resistant human myeloma cells lines (HMCLs) from different molecular subgroups including XG2BR t(12;14), XG7BR t(4;14), XG19BR t(14;16), and XG1BR t(11;14). These cell lines were cultured continuously with escalating concentrations of Bortezomib (Btz) during 12 months and showed a significant resistance to Bortezomib compared to their parental cell lines (mean IC50: Btz-resistant HMCLs =5.5nM vs parental HMCLs=2.5nM, p<0.05). Of interest, we demonstrated that Btz-resistant HMCLs are also significantly more resistant to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (mean IC50: Btz-resistant HMCLs =6nM for Cfz and =70nM for Ixa vs parental HMCLs=3nM, for Cfz, p<0.05 ; and =21nM for Ixa, p<0.05. No significant cross-resistance was observed with other therapeutic agents including melphalan, dexamethasone and IMIDs indicating that the observed drug resistance mechanisms are specifically related to PIs. In order to understand the PIs resistance mechanisms acquired by MM cells, we used a combination of genomic approaches including whole genome sequencing, and comparative transcriptomic analysis. Among the 40 mutations identified in Btz-resistant HMCLs compared to the parental ones, a mutation residing in the Btz-binding pocket in the proteasome beta5-subunit (PSMB5) gene was identified. This mutation has already been found in other models of PIs-resistant MM cell lines and in relapsed MM patients, and is associated to PIs- resistance by reducing the PI binding capacity and impairing the chemotrypsin-like catalytic activity of the 20S proteasome. When we compared the gene expression profiling of Btz-resistant HMCLs with the parental ones, we identified a gene expression signature significantly enriched in nucleotide excision repair (NER) pathway with an increased expression of ERCC1, ERCC5, LIG1, POLD1 in the Btz-resistant HMCLs (FC >1.5). Since, protein ubiquitination is essential in regulation and coordination of various pathways of DNA damage recognition, signaling and repair, proteasome inhibitors affect DNA repair, overexpression of DNA repair pathways may participate in drug resistance mechanisms. Using gene expression profiling data, we also identified a significant downregulation of 8 solute carrier protein (SLC) intake transporters (SLC6A6, SLC16A1, SLC16A14, SLC16A10, SLC25A13, SLC5A6, SLCO3A1, SLCO4A1) together with a significant upregulation of xenobiotic receptors (RXRA, RXRB) in Btz-resistant HMCLs compared to parental HMCLs (FC> or < 1.5; p value <0.05). In addition, several genes involved in antioxidant response (NQO1), and in glutathione regulation (MGST1, MGST2, GSTO1) were also overexpressed in Btz-resistant HMCLs (FC>1.5 p value <0.05). Investigating the deregulated genes involved on energy metabolism that is often associated with resistance, we identified an upregulation of glycolytic enzymes directly involved in glycolytic metabolism (ALDOC, ENO3, HK1, PDK1, PDK3, PFKB3, PFKB4, PFKL, SLC2A1 (FC>1.5 p value <0.05) in the Btz-resistant HMCLs. Altogether our data underline a significant deregulation of genes involved in cell metabolism and drug clearance system that allow the PI resistant-MM cells to maintain metabolic homeostasis and survival in stringent redox conditions. This is in accordance with described mechanisms linking drug resistance and glycolytic metabolism in cancers cells. Metabolomic analyzes are currently ongoing for functional validation. Altogether, drug-resistant cell lines represent an attractive preclinical model to test molecules targeting these pathways in order to identify new therapeutic strategies to overcome PI resistance in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Kassambara: Diag2Tec: Consultancy. Machura: Diag2Tec: Current Employment. Chemlal: Diag2Tec: Current Employment. Vincent: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Herbaux: Abbvie: Honoraria, Research Funding; Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding. Bruyer: Diag2Tec: Current Employment. Moreaux: Diag2Tec: Consultancy.