Mycoplasmas and cancer

The standard of care for patients suffering cancer often includes treatment with nucleoside analogues (NAs). NAs are internalized by cell-specific nucleobase/nucleoside transporters and, after enzymatic activation (often one or more phosphorylation steps), interfere with cellular nucleo(s)(t)ide metabolism and DNA/RNA synthesis. Therefore, their efficacy is highly dependent on the expression and activity of nucleo(s)(t)ide-metabolizing enzymes, and alterations thereof (e.g. by down/upregulated expression or mutations) may change the susceptibility to NA-based therapy and/or confer drug resistance. Apart from host cell factors, several other variables including microbial presence may determine the metabolome (i.e. metabolite concentrations) of human tissues. Studying the diversity of microorganisms that are associated with the human body has already provided new insights in several diseas es (e.g. diabetes and inflammatory bowel disease) and the metabolic exchange between tissues and their specific microbiota was found to affect the bioavailability and toxicity of certain anticancer drugs, including NAs. Several studies report a preferential colonization of tumor tissues with some mycoplasma species (mostly Mycoplasma hyorhinis). These prokaryotes are also a common source of cell culture contamination and alter the cytostatic activity of some NAs in vitro due to the expression of nucleoside-catabolizing enzymes. Mycoplasma infection may therefore bias experimental work with NAs, and their presence in the tumor microenvironment could be of significance when optimizing nucleoside-based cancer treatment

Improving the metabolic fidelity of cancer models with a physiological cell culture medium

Currently available cell culture media may not reproduce the in vivo metabolic environment of tumors. To demonstrate this, we compared the effects of a new physiological medium, Plasmax, with commercial media. We prove that the disproportionate nutrient composition of commercial media imposes metabolic artifacts on cancer cells. Their supraphysiological concentrations of pyruvate stabilize hypoxia-inducible factor 1α in normoxia, thereby inducing a pseudohypoxic transcriptional program. In addition, their arginine concentrations reverse the urea cycle reaction catalyzed by argininosuccinate lyase, an effect not observed in vivo, and prevented by Plasmax in vitro. The capacity of cancer cells to form colonies in commercial media was impaired by lipid peroxidation and ferroptosis and was rescued by selenium present in Plasmax. Last, an untargeted metabolic comparison revealed that breast cancer spheroids grown in Plasmax approximate the metabolic profile of mammary tumors better. In conclusion, a physiological medium improves the metabolic fidelity and biological relevance of in vitro cancer models

Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration

Background & Aims: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. Methods: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. Results: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/β-catenin signalling and related metabolomic disturbance. Conclusions: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. Impact and implications: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally

MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer

Background: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. Methods: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. Results: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. Conclusion: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype

Abstract 1441 : MYC expression promotes lipid metabolism and metabolic plasticity in human mammary epithelial cell

MYC is one of the most commonly mutated and highly amplified oncogenes in human breast cancer. MYC amplifications occur most frequently in triple-negative breast cancers (TNBCs). TNBCs can be divided into two molecular subtypes: basal-like and claudin-low breast cancers. These cancers tend to be extremely aggressive and are strongly associated with disease recurrence, poor prognosis and high mortality. In particular, claudin-low tumors are classified by a loss of tight junctions and cell-to-cell contacts and an enrichment for genes associated with an epithelial-to-mesenchymal transition (EMT) and mammary stem cells (also known as tumor-initiating cells). Despite the high level of disease severity, there are no targeted therapies for claudin-low TNBCs. To address this unmet need, we utilized human mammary epithelial cells (HuMECs) that express oncogenic levels of MYC and a mutant MYC (T58A) to characterize the behavioral and metabolic changes that occur during the formation of MYC-driven breast cancers. We found that MYC regulates the expression of genes associated with cell stemness, EMT, lipid metabolism, and calcium (Ca2+) signaling and that the expression of this gene signature promotes cell growth, survival, migration, and metabolic plasticity. The gene signature of MYC-expressing HuMECs highly correlates with the gene signature of claudin-low breast cancers, therefore highlighting the relevance of our HuMEC model to human claudin-low breast cancer. We found the major drivers underlying the MYC-dependent changes in cell behavior to be stimulation of Ca2+ signaling and strong activation of lipid metabolism. Ca2+ signaling is stimulated through the MYC-dependent repression of Ca2+ efflux mechanisms; elevated cytosolic Ca2+ then consequently stimulates a Ca2+/calmodulin kinase kinase 2 (CAMKK2)/AMPK signaling axis that activates fatty acid scavenging and transport, as well as β-oxidation. Enhanced lipid metabolism thereby provides the necessary biomass (fatty acids) for phospholipid biosynthesis and energy (ATP) to support the metabolically demanding processes of cell growth, proliferation, and migration. In all, our findings provide a strong rationale for targeting lipid metabolism and the Ca2+/CAMKK2/AMPK signaling axis in MYC-driven, and potentially claudin-low, breast cancers

The pathogenesis of mesothelioma is driven by a dysregulated translatome.

Funder: Department of HealthMalignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-Methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a β-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC

The effect of catabolic mycoplasma enzymes on the therapeutic efficiency of nucleoside analogues

With more than 3 million new cases and 1.7 million deaths each year, cancer is the most important cause of death in Europe after cardiovascular diseases (numbers according to the World Health Organization). Strategies to manage cancer include surgery, radiation therapy, immunotherapy, hormonal therapy, chemotherapy and others. Currently, chemotherapeutic intervention of cancer is largely based on inhibition of tumor cell proliferation by the administration of nucleoside-based drugs (nucleoside analogues; NAs). After intracellular activation (often one or more phosphorylation steps), NAs may act as antimetabolites, interfering with the tumor cell nucleo(s)(t)ide metabolism and DNA/RNA synthesis. Since many cells in adults are quiescent and therefore not in the process of replication, NAs display a certain level of selectivity towards tumor cells. The microenvironment of a tumor encompasses cancer cells and resident or infiltrating non-tumor cells. In addition, several studies have reported a close association of different prokaryotes with tumor tissue in cancer patients, which may be attributed to an increased nutrient availability at the tumor site, bacterial chemotaxis or defective defense mechanisms in cancer cells. A high preferential colonization of tumor tissue with different mycoplasma species (e.g. Mycoplasma hyorhinis), compared with healthy or non-malignant diseased tissue, has been repeatedly reported. Mycoplasmas are characterized by the lack of a cell wall and a strongly reduced genome and are considered to be the smallest self-replicating organisms. Some mycoplasmas (e.g. Mycoplasma pneumoniae and Mycoplasma genitalium) are clearly associated with disease. However, most species cause asymptomatic infections and are considered to be part of the residential flora of the healthy human body. Mycoplasma-related pathogenesis is therefore mostly observed in immunocompromised individuals. The limited number of genes in mycoplasmas is reflected in their nucleo(s)(t)ide metabolism. Most mycoplasma species share the inability to synthesize purine and pyrimidine nucleotides de novo and therefore rely on the tissue of their host for preformed nucleic acid precursors which are efficiently internalized and metabolized using several transport mechanisms and salvage enzymes, respectively. Therefore we hypothesized that a mycoplasma infection may significantly affect the availability and metabolism of nucleo(s)(t)ides and nucleo(s)(t)ide analogues in the host tumor cells. The cytostatic and antiviral activity of nucleoside-based drugs was indeed dramatically altered in mycoplasma-infected tumor cell cultures due to the expression of catabolic mycoplasma-encoded enzymes. Taking into account the preferential mycoplasma colonization of tumor tissue and the occurrence of secondary mycoplasma infections in immunocompromised individuals (e.g. AIDS patients), chemotherapy of cancer/viral infections with NAs may be suboptimal. In this work, we aimed at a careful investigation of the influence of a mycoplasma infection on the biological potential of nucleo(s)(t)ide-derived drugs. Mycoplasma-encoded nucleoside phosphorylases (NPs) were found to be crucial in the altered activity of purine- and pyrimidine-based NAs. Depending on the nature of the nucleobase that was released from the drug by NP-catalyzed phosphorolysis, an increased or a decreased biological activity was observed in mycoplasma-infected tumor cell cultures. The cytostatic activity of the drugs could be restored by the co-administration of mycoplasma-targeting antibiotics or a specific NP inhibitor. We therefore cloned the thymidine phosphorylase (TP) and purine nucleoside phosphorylase (PNP) genes annotated in the genome of M. hyorhinis and expressed them as recombinant proteins in Escherichia coli. M. hyorhinis TP was found to encode a nucleoside phosphorylase that, in contrast to human TP, does not discriminate between pyrimidine-based 2 -deoxyribosides and ribosides. The kinetic parameters of this enzyme were determined in detail and surprisingly revealed that uridine was the preferred natural substrate followed by thymidine. These results therefore indicate that, even though the mycoplasma-encoded enzyme shows high sequence similarity with both human and Escherichia coli TP, it should be re-annotated as a pyrimidine nucleoside phosphorylase (PyNP-Hyor) rather than as a TP. The expression of similar enzymes has been reported for a few other prokaryotes (e.g. Geobacillus stearthermophilus) and the parasite Giardia lamblia. PyNP-Hyor also efficiently catalyzed the phosphorolysis of therapeutic 5-halogenated pyrimidine nucleosides (e.g. floxuridine) to their less active nucleobases, explaining the decreased antiviral/cytostatic activity of such compounds in mycoplasma-infected cell cultures. Also, efficient phosphorolysis of clinically approved purine-based NAs (e.g. cladribine and fludarabine) was observed in M. hyorhinis-infected tumor cells resulting in an altered biological activity of the compounds. Depending on the released nucleobase, mycoplasma infection resulted in a decreased cytostatic activity (as observed for cladribine due to the release of the poorly cytostatic 2-chloroadenine base) or increased cytostatic activity (as observed for fludarabine due to the release of the highly cytostatic 2-fluoroadenine base). The co-administration of potent PNP inhibitors efficiently restored the activity of the drugs. The recombinantly expressed PNP of M. hyorhinis (PNP-Hyor) was kinetically characterized and found to catalyze the phosphorolysis of all natural purine nucleosides [except for xanthosine; including (2 -deoxy)adenosine], and several of their therapeutic derivatives. Since (2 -deoxy)adenosine and its analogues are generally not accepted as a substrate for human PNP, the presence of mycoplasmas at a tumor site may significantly contribute to a selective phosphorolysisof such drugs. Elimination of mycoplasmas or suppression of their catabolizing enzymes may therefore potentially increase the therapeutic index of purine-based NAs such as cladribine. Alternatively, a rational choice for drugs that are activated upon mycoplasma-mediated phosphorolysis (e.g. fludarabine) may increase therapeutic selectivity. Next, a decreased cytostatic activity of gemcitabine was observed in M. hyorhinis-infected cell cultures. Strong evidence was provided for the expression of a mycoplasma-encoded (2 -deoxy)cytidine deaminase catalyzing the deamination (and therefore inactivation) of gemcitabine in such cultures. In addition, we also observed a PyNP-related inactivation of gemcitabine: its cytostatic activity could be efficiently restored by selective PyNP inhibition and was not compromised in cell cultures infected with a PyNP-deficient mycoplasma strain. These findings were surprising to us since gemcitabine proved not to be a direct substrate for NP-catalyzed phosphorolysis. Several observations suggest that mycoplasma PyNP activity may alter the host cell pyrimidine nucleo(s)(t)ide metabolism (i.e. by decreasing intracellular dTTP and increasing dCTP levels) and therefore may indirectly affect the activity of cellular 2 -deoxycytidine kinase (dCK; the enzyme responsible for the initial activation of gemcitabine). However, it is currently unclear whether dNTP levels are sufficiently different in the mycoplasma-infected tumor cell cultures to have a significant inhibitory effect on dCK. Alternatively, the formation of a tight multi-enzyme complex of catabolic mycoplasma pyrimidine nucleoside salvage enzymes [i.e. PyNP and (2 -deoxy)cytidine deaminase] may also explain our findings. However, this possibility is still very hypothetic and no solid evidence for its occurrence has yet been provided. Due to the compromising role that mycoplasma PyNP may have in the cytostatic activity of thymidine analogues, we explored the possibility to develop a prodrug of floxuridine that is not susceptible to NP-catalyzed inactivation. In collaboration with prof. C. McGuigan (Cardiff University, Cardiff, UK), the phosphoramidate prodrug (ProTide) technology was applied to design novel derivatives of floxuridine-5 -monophosphate, the active metabolite of floxuridine. We found the naphtyl/benzyl-L-alaninyl phosphate motif (present in prodrug NUC-3073) instrumental to confer resistance to enzymatic breakdown by human and prokaryotic NPs. Therefore, and in contrast to floxuridine, NUC-3073 retained its activity in mycoplasma-infected tumor cells remarkably well. Furthermore, this drug successfully delivered the active (phosphorylated) metabolite of floxuridine (5-fluoro-2 -deoxyuridine-5 -monophosphate) into the intact tumor cells, independently of the presence of thymidine kinase (TK) activity. This compound may therefore represent a more resilient alternative for floxuridine and may also prove useful in the treatment of tumors with (acquired or inherent) TK-deficiency. NUC-3073 is planned to enter phase I clinical trials for the treatment of colorectal cancer in 2014. In a next phase of our research we aimed at the design of a universal inhibitor that concomitantly targets both pyrimidine and purine NPs. Most reported NP inhibitors are nucleoside derivatives with modifications in the nucleobase and therefore show high specific inhibitory activity against one type of NP. Instead, we aimed at the development of derivatives of (2-deoxy)ribose-1-phosphate, the common product of all NP-catalyzed reactions to find a broad-acting NP inhibitor. We found that 3,5-dichlorobenzoyl-substituted 2-deoxy-D-ribose-1-phosphate (Cf2891) inhibited a variety of both mammalian and prokaryotic pyrimidine and purine NPs. Kinetic studies revealed that this compound competes with inorganic phosphate for binding to NPs and, depending on the nature of the inhibited enzyme, a competitive or non-competitive inhibition with regard to the nucleoside binding site was observed. Cf2891 may therefore serve as a lead molecule for the development of potent broad-substrate NP inhibitors to simultaneously protect pyrimidine- and purine-based NAs from catabolism when administered in combination therapies. The study of mycoplasma-infected tumor cell cultures has allowed us to explore the relationship between mycoplasma infections and the (cytostatic/antiviral) efficiency of therapeutic NAs. Our results suggest that a mycoplasma infection at the tumor site (or a secondary mycoplasma infection in immunocompromised patients) may alter the therapeutic index of such drugs. In vivo studies in mice confirmed that the antitumor activity of gemcitabine and floxuridine is indeed compromised when treating mycoplasma-infected mammary tumors compared with uninfected control tumors. Due to experimental constraints in co-culturing prokaryotes (different from mycoplasmas) along with mammalian cells, it is currently unclear whether our results are also of relevance for other bacterial infections. We demonstrated that a rational choice of (pro)drugs or a combination therapy of NAs with (i) mycoplasma-targeting antibiotics or (ii) specific inhibitors of catabolic mycoplasma-encoded enzymes, may optimize the efficiency of chemotherapeutics in cell culture. Our findings justify that this principle should be further explored and validated in the clinical setting.status: publishe

Mycoplasmas and cancer: focus on nucleoside metabolism

The standard of care for patients suffering cancer often includes treatment with nucleoside analogues (NAs). NAs are internalized by cell-specific nucleobase/nucleoside transporters and, after enzymatic activation (often one or more phosphorylation steps), interfere with cellular nucleo(s)(t)ide metabolism and DNA/RNA synthesis. Therefore, their efficacy is highly dependent on the expression and activity of nucleo(s)(t)ide-metabolizing enzymes, and alterations thereof (e.g. by down/upregulated expression or mutations) may change the susceptibility to NA-based therapy and/or confer drug resistance. Apart from host cell factors, several other variables including microbial presence may determine the metabolome (i.e. metabolite concentrations) of human tissues. Studying the diversity of microorganisms that are associated with the human body has already provided new insights in several diseases (e.g. diabetes and inflammatory bowel disease) and the metabolic exchange between tissues and their specific microbiota was found to affect the bioavailability and toxicity of certain anticancer drugs, including NAs. Several studies report a preferential colonization of tumor tissues with some mycoplasma species (mostly Mycoplasma hyorhinis). These prokaryotes are also a common source of cell culture contamination and alter the cytostatic activity of some NAs in vitro due to the expression of nucleoside-catabolizing enzymes. Mycoplasma infection may therefore bias experimental work with NAs, and their presence in the tumor microenvironment could be of significance when optimizing nucleoside-based cancer treatment.status: publishe