Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.In this study, Green, Marttila, Kiweler et al. characterize one-carbon metabolism rewiring in response to a dual MTHFD1 and MTHFD2 inhibitor. This work provides insight into one-carbon fluxes, and reveals a previously uncharacterized vulnerability in cancer cells created by folate trapping

Impact of class I HDACs and their inhibitors on renal and colorectal tumor cell fate decisions

Advanced, metastasized renal and colorectal carcinomas are difficult to treat and frequently incurable diseases. Therefore, a key focus of modern cancer research is the identification of improved chemotherapeutic treatment options. Overexpression of histone deacetylases (HDACs) is a frequent characteristic of malignant neoplasms and often correlates with disadvantageous disease progression and therapy outcome. In recent years, inhibitors of histone deacetylases (HDACi) were introduced as a new class of small-molecule anticancer therapeutics. Since HDACs deacetylate numerous histones and non-histone proteins, the inhibition of these enzymes has implications for a large number of cellular processes. As a result of the pleiotropic actions of HDACs, the precise impact of HDACi on cellular signaling pathways remains poorly understood. This work investigated the effects of HDACs and HDACi on key cellular signaling pathways in kidney and colon tumor cell lines on a global and functional scale. Having found that HDACi induce morphological alterations in renal cell carcinoma (RCC) cells that resemble the characteristics of a mesenchymal phenotype, this work investigated individual and proteomics-based analyses of epithelial-mesenchymal transition (EMT) signaling pathways. Proteomics and GO-term enrichment analyses revealed disturbed EMT signaling and an associated loss of cell cycle control. Detailed analysis of EMT transcription factor expression by qPCR, epithelial and mesenchymal marker protein expression by Western blot, migratory potential, and β-catenin localization by immunofluorescence confirmed these findings. Furthermore, HDACi significantly induced cell death that due to caspase-mediated apoptosis. These findings were confirmed in primary renal cells. RNAi-based knockdown experiments unveiled HDAC1 and HDAC2 as mechanistic targets of HDACi. In colorectal cancer (CRC) cells, the specific modification of HDAC2 by sumoylation affects p53- and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB)-dependent gene expression. The work presented demonstrates that a loss of HDAC2 sumoylation enhances CRC sensitivity towards the antimetabolite 5-fluorouracil (5-FU) as well as the DNA-crosslinker nimustine (ACNU). HDAC2 sumoylation did thereby not affect p53 activation and ATM- and ATR-dependent DNA damage signaling. In contrast, DNA damage-induced accumulation of γH2AX and γH2AX foci formation depended on HDAC2 sumoylation. Mass spectrometric analysis of global protein expression, knockdown experiments, and analyses of DNA damage repair by immunofluorescence identified the specific loss of the chromatin remodeling complex-associated DNA-helicases Brahma-related gene 1 (BRG1) and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1 (SMARCAD1) as potential cause for the observed sensitization effects. Curiously, the main cytotoxic effects of 5-FU were rather based on its RNA damage than on its DNA damage. Thus, this work reveals a multitude of novel effects of commonly used chemotherapeutics.Fortgeschrittene, metastasierende renale und kolorektale Karzinome sind schwer behandelbare und häufig unheilbare Erkrankungen. Ein Hauptaugenmerk gegenwärtiger Krebsforschung liegt daher auf der Identifikation verbesserter chemotherapeutischer Behandlungsoptionen. Die Überexpression von Histondeacetylasen (HDACs) ist ein häufiges Merkmal maligner Neoplasien und korreliert oftmals mit einem unvorteilhaften Krankheitsverlauf und Therapieausgang. Im Laufe der letzten Jahre wurden Inhibitoren von Histondeactelyasen (HDACi) als neue Klasse niedermolekularer Chemotherapeutika vorgestellt. Da HDACs eine Vielzahl an Histonen und Nicht-Histonproteinen deacetylieren, hat die Inhibierung dieser Enzyme Auswirkungen auf zahlreiche zelluläre Prozesse. Aufgrund dieses pleiotropen Einflusses von HDACs sind die konkreten Wirkungsweisen von HDACi auf zelluläre Signalwege jedoch weiterhin kaum verstanden. Diese Arbeit untersuchte den Einfluss von HDACs und HDACi auf zentrale, zelluläre Signalwege in renalen und kolorektalen Tumorzelllinien auf globaler und funktionaler Ebene. Nachdem gefunden wurde, dass HDACi in renalen Karzinomzellen (RCC) morphologische Veränderungen auslösen, die denen eines mesenchymalen Phänotypus ähneln, untersucht diese Arbeit individuelle und Proteom-basierte Analysen von Signalwegen der epithelial-mesenchymalen Transition (EMT). Diese zeigten eine Störung von EMT-Signalwegen sowie einen damit assoziierten Verlust der Zellzykluskontrolle. Detaillierte Analysen der Expression von EMT Transkriptionsfaktoren durch qPCR, der Expression epithelialer und mesenchymaler Markerproteine durch Western Blot, des Migrationspotentials und der β-Catenin-Lokalisation durch Immunfluoreszenz bestätigten diese Befunde. Weiterhin induzierten HDACi signifikant Zelltod durch Caspase-vermittelte Apoptose. Diese Ergebnisse wurden bestätigt in primären renalen Zellen. RNAi-basierte Knockdown-Experimente identifizierten HDAC1 und HDAC2 als mechanistischen Zielstrukturen der HDACi. In kolorektalen Tumorzellen (CRC) beeinflusst die Modifizierung von HDAC2 durch Sumoylierung die p53- und NF-κB-abhängige Genexpression. Die vorliegende Arbeit zeigt, dass ein Verlust der HDAC2-Sumoylierung die Sensitivität von CRC gegenüber dem Antimetaboliten 5-Fluorouracil (5-FU) sowie dem DNA-Vernetzer Nimustin (ACNU) steigert. Die HDAC2-Sumoylierung beeinflusste dabei weder die p53-Aktivierung noch die ATM- und ATR-abhängige DNA-Schadenssignalisierung. Dagegen waren die schadensinduzierte Anreicherung von γH2AX und die γH2AX-Focibildung von der HDAC2-Sumoylierung abhängig. Die massenspektrometrische Untersuchung der globalen Proteinexpression, Knockdown-Experimente und Analysen der DNA-Schadensreparatur durch Immunfluoreszenz identifizierten den Expressionsverlust der DNA-Helikasen BRG1 und SMARCAD1 als potentielle Ursache der beobachteten Sensitivierungseffekte. Eigenartigerweise basierten die zytotoxischen Effekte von 5-FU eher auf dessen RNA-Schaden als auf dessen DNA-Schaden. Diese Arbeit offenbart daher eine Vielzahl neuer Effekte häufig verwendeter Chemotherapeutika

Histone deacetylase inhibitors dysregulate DNA repair proteins and antagonize metastasis-associated processes

Purpose!#!We set out to determine whether clinically tested epigenetic drugs against class I histone deacetylases (HDACs) affect hallmarks of the metastatic process.!##!Methods!#!We treated permanent and primary renal, lung, and breast cancer cells with the class I histone deacetylase inhibitors (HDACi) entinostat (MS-275) and valproic acid (VPA), the replicative stress inducer hydroxyurea (HU), the DNA-damaging agent cis-platinum (L-OHP), and the cytokine transforming growth factor-β (TGFβ). We used proteomics, quantitative PCR, immunoblot, single cell DNA damage assays, and flow cytometry to analyze cell fate after drug exposure.!##!Results!#!We show that HDACi interfere with DNA repair protein expression and trigger DNA damage and apoptosis alone and in combination with established chemotherapeutics. Furthermore, HDACi disrupt the balance of cell adhesion protein expression and abrogate TGFβ-induced cellular plasticity of transformed cells.!##!Conclusion!#!HDACi suppress the epithelial-mesenchymal transition (EMT) and compromise the DNA integrity of cancer cells. These data encourage further testing of HDACi against tumor cells

Class I histone deacetylases regulate p53/NF-κB crosstalk in cancer cells

The transcription factors NF-kappa B and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytolcines promote signaling cascades that lead to the co-regulation of p53 and NF-kappa B. Consequently, nuclear p53/NF-kappa B signaling complexes activate NF-kappa B-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-kappa B, we addressed whether clinically relevant HDACi affect the NF-kappa B/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-kappa B-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class lib deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-kappa B. HDACi decrease the basal and stress-induced expression of p53 and block NF-kappa B-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53. (C) 2016 Elsevier Inc. All rights reserved

Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential

Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential

Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism