Luís G. Gonçalves

Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

Immunometabolic Pathways in BCG-Induced Trained Immunity

Summary The protective effects of the tuberculosis vaccine Bacillus Calmette-Guerin (BCG) on unrelated infections are thought to be mediated by long-term metabolic changes and chromatin remodeling through histone modifications in innate immune cells such as monocytes, a process termed trained immunity. Here, we show that BCG induction of trained immunity in monocytes is accompanied by a strong increase in glycolysis and, to a lesser extent, glutamine metabolism, both in an in-vitro model and after vaccination of mice and humans. Pharmacological and genetic modulation of rate-limiting glycolysis enzymes inhibits trained immunity, changes that are reflected by the effects on the histone marks (H3K4me3 and H3K9me3) underlying BCG-induced trained immunity. These data demonstrate that a shift of the glucose metabolism toward glycolysis is crucial for the induction of the histone modifications and functional changes underlying BCG-induced trained immunity. The identification of these pathways may be a first step toward vaccines that combine immunological and metabolic stimulation.

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

Butyrate-rich Colonic Microenvironment Is a Relevant Selection Factor for Metabolically Adapted Tumor Cells

The short chain fatty acid (SCFA) buyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.

Occurrence of 1-Glyceryl-1-myo-Inosityl Phosphate in Hyperthermophiles

ABSTRACT The accumulation of compatible solutes was studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of α- and β-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated by A. pyrophilus are negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while α- and β-glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo- and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected in Archaeoglobus fulgidus (L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only in Archaeoglobus species), di-myo-inositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.

Evolution of the biosynthesis of di‐myo‐inositol phosphate, a marker of adaptation to hot marine environments

The synthesis of di-myo-inositol phosphate (DIP), a common compatible solute in hyperthermophiles, involves the consecutive actions of inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol phosphate phosphate synthase (DIPPS). In most cases, both activities are present in a single gene product, but separate genes are also found in a few organisms. Genes for IPCT and DIPPS were found in the genomes of 33 organisms, all with thermophilic/hyperthermophilic lifestyles. Phylogeny of IPCT/DIPPS revealed an incongruent topology with 16S RNA phylogeny, thus suggesting horizontal gene transfer. The phylogenetic tree of the DIPPS domain was rooted by using phosphatidylinositol phosphate synthase sequences as out-group. The root locates at the separation of genomes with fused and split genes. We propose that the gene encoding DIPPS was recruited from the biosynthesis of phosphatidylinositol. The last DIP-synthesizing ancestor harboured separated genes for IPCT and DIPPS and this architecture was maintained in a crenarchaeal lineage, and transferred by horizontal gene transfer to hyperthermophilic marine Thermotoga species. It is plausible that the driving force for the assembly of those two genes in the early ancestor is related to the acquired advantage of DIP producers to cope with high temperature. This work corroborates the view that Archaea were the first hyperthermophilic organisms.

Mannosylglycerate: structural analysis of biosynthesis and evolutionary history

Halophilic and halotolerant microorganisms adapted to thrive in hot environments accumulate compatible solutes that usually have a negative charge either associated with a carboxylic group or a phosphodiester unit. Mannosylglycerate (MG) has been detected in several members of (hyper)thermophilic bacteria and archaea, in which it responds primarily to osmotic stress. The outstanding ability of MG to stabilize protein structure in vitro as well as in vivo has been convincingly demonstrated. These findings led to an increasingly supported link between MG and microbial adaptation to high temperature. However, the accumulation of MG in many red algae has been known for a long time, and the peculiar distribution of MG in such distant lineages was intriguing. Knowledge on the biosynthetic machinery together with the rapid expansion of genome databases allowed for structural and phylogenetic analyses and provided insight into the distribution of MG. The two pathways for MG synthesis have distinct evolutionary histories and physiological roles: in red algae MG is synthesised exclusively via the single-step pathway and most probably is unrelated with stress protection. In contrast, the two-step pathway is strongly associated with osmoadaptation in (hyper)thermophilic prokaryotes. The phylogenetic analysis of the two-step pathway also reveals a second cluster composed of fungi and mesophilic bacteria, but MG has not been demonstrated in members of this cluster; we propose that the synthase is part of a more complex pathway directed at the synthesis of yet unknown molecules containing the mannosyl-glyceryl unit.

HNF1β drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC).

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1β, experiments were also performed in an OSC cell line, which does not express HNF1β. Metabolic profiles, GSH quantification, HNF1β, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1β knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1β regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1β as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

STAT3:FOXM1 and MCT1 drive uterine cervix carcinoma fitness to a lactate-rich microenvironment

Uterine cervix cancer is the second most common malignancy in women worldwide with human papillomavirus (HPV) as the etiologic factor. The two main histological variants, squamous cell carcinomas (SCC) and adenocarcinomas (AC), resemble the cell morphology of exocervix and endocervix, respectively. Cancer metabolism is a cancer hallmark conditioned by the microenvironment. As uterine cervix homeostasis is dependent on lactate, we hypothesized lactate plays a role in uterine cervix cancer progression. Using in vitro (SiHa-SCC and HeLa-AC) and BALB-c/SCID models, we demonstrated that lactate metabolism is linked to histological types, with SCC predominantly consuming and AC producing lactate. MCT1 is a key factor, allowing lactate consumption and being regulated in vitro by lactate through the FOXM1:STAT3 pathway. In vivo models showed that SCC (SiHa) expresses MCT1 and is dependent on lactate to grow, whereas AC (HeLa) expresses MCT1 and MCT4, with higher growth capacities. Immunohistochemical analysis of tissue microarrays (TMA) from human cervical tumors showed that MCT1 expression associates with the SCC type and metastatic behavior of AC, whereas MCT4 expression concomitantly increases from in situ SCC to invasive SCC and is significantly associated with the AC type. Consistently, FOXM1 expression is statistically associated with MCT1 positivity in SCC, whereas the expression of FOXO3a, a FOXM1 functional antagonist, is linked to MCT1 negativity in AC. Our study reinforces the role of the microenvironment in the metabolic adaptation of cancer cells, showing that cells that retain metabolic features of their normal counterparts are positively selected by the organ’s microenvironment and will survive. In particular, MCT1 was shown to be a key element in uterine cervix cancer development; however, further studies are needed to validate MCT1 as a suitable therapeutic target in uterine cervix cancer.

Monocarboxylate transporter 1 (MCT1), a tool to stratify acute myeloid leukemia (AML) patients and a vehicle to kill cancer cells

Dysregulation of glucose/lactate dynamics plays a role in cancer progression, and MCTs are key elements in metabolic remodeling. VEGF is a relevant growth factor in the maintenance of bone marrow microenvironment and it is also important in hematological diseases. Our aim was to investigate the role of VEGF in the metabolic adaptation of Acute myeloid leukemia (AML) cells by evaluating the metabolic profiles and cell features according to the AML lineage and testing lactate as a metabolic coin. Our in vitro results showed that AML promyelocytic (HL60) and monocytic (THP1) (but not erythroid- HEL) lineages are well adapted to VEGF and lactate rich environment. Their metabolic adaptation relies on high rates of glycolysis to generate intermediates for PPP to support cell proliferation, and on the consumption of glycolysis-generated lactate to supply biomass and energy production. VEGF orchestrates this metabolic network by regulating MCT1 expression. Bromopyruvic acid (BPA) was proven to be an effective cytotoxic in AML, possibly transported by MCT1. Our study reinforces that targeting metabolism can be a good strategy to fight cancer. MCT1 expression at the time of diagnosis can assist on the identification of AML patients that will benefit from BPA therapy. Additionally, MCT1 can be used in targeted delivery of conventional cytotoxic drugs.