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Metabolic rewiring is essential to enable cancer onset and progression. One important metabolic pathway that is often hijacked by cancer cells is the one-carbon cycle, in which the third carbon of serine is oxidized to formate. We have previously shown that formate production in cancer cells often exceeds the anabolic demand, resulting in formate overflow. Furthermore, we observed that high extracellular formate promotes thein vitroinvasiveness of glioblastoma (GBM) cells. However, additional data supporting thisin vitroobservation and mechanistic details remained elusive so far.In the present study, we now demonstrate that inhibition of formate overflow results in a decreased invasiveness of GBM cellsex vivoandin vivo. Additionally, we observed that exposure to exogeneous formate can induce a transiently stable pro-invasive phenotype that results in increased metastasis formationin vivo. All in all, these results suggest that a local formate increase within the tumor microenvironment may be one factor that can promote cancer cell motility and dissemination.Mechanistically, we uncover a previously undescribed interplay where formate acts as a trigger to alter fatty acid metabolism and matrix metalloproteinase (MMP) activity which in turn impacts cancer cell invasiveness. We thus highlight the role of formate as a pro-invasive metabolite. Gaining a deeper understanding of formate overflow and how it promotes invasion in cancer, may open new therapeutic opportunities to prevent cancer cell dissmination.

Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients with minimal treatment options available. Canonical drugs target mainly the proliferative capacity of cancer cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. Using a panel of anti-cancer drugs, we uncovered that antifolates, despite their strong growth arrest function, do not at all reduce the cancer cells motility potential, indicating that nucleotide synthesis is dispensable for cell motility. Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle is still highly active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate overflow are increased, which suggests that mitochondria provide a protective environment that allows serine catabolism to support cellular motility during nucleotide synthesis inhibition. Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent serine de novo synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one-carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic breast cancer model. In summary, we identified mitochondrial serine catabolism as a targetable, growth-independent metabolic vulnerability to limit metastatic progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/445928v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@cdc194org.highwire.dtl.DTLVardef@5ff6a1org.highwire.dtl.DTLVardef@38dcd6org.highwire.dtl.DTLVardef@12a9d1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients with minimal treatment options available. Canonical drugs target mainly the proliferative capacity of cancer cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions supporting resistance and metastatic dissemination are still poorly understood.In the present study, we revealed that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism allowing sustained serine catabolism and formate overflow when cytosolic 1C metabolism is impaired. Interestingly, antifolate dependent growth-arrest did not correlate with decreased migration capacity. Therefore, using the antifolate Methotrexate as a tool compound allowed us to disentangle proliferation and migration to profile the metabolic phenotype of migrating (growth-arrested) cells. Supported by an increased NAD/NADH ratio, we observed increased serine de novo synthesis and increased serine catabolism to formate.Consequently, inhibition of serine de novo synthesis using the competitive PHGDH-inhibitor BI-4916 or direct inhibition of mitochondrial 1C metabolism reduced cancer cell migration. Using an orthotopic breast cancer model, we show that sole inhibition of mitochondrial serine catabolism does not affect primary 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.Our results improve our understanding of 1C metabolism and of metabolic determinants that support the process of cancer cell migration and metastasis.

Serine catabolism via the folate cycle provides formate that is essential for nucleotide synthesis in proliferating cells. In addition to this canonical function to support biomass production in anabolic cells, we have recently demonstrated in vitro and in vivo that formate production in cancer cells is often in excess of the anabolic demand. This excess formate production is characterized by formate overflow and thus, net formate excretion into the tumor microenvironment. Interestingly, we observe increased rates of formate overflow upon different chemical perturbations that induce growth arrest. Thus, stressed cancer cells that encounter growth restriction such as upon chemotherapy, are often characterized by increased formate release rates. We demonstrated that such high formate levels in the extracellular space promote invasion of glioblastoma cells. Using ex vivo brain slice cultures and an orthotopic brain tumor model, we demonstrate that silencing MTHFD1L, the essential enzyme to enable formate overflow, results in decreased invasiveness of the tumor. Embarking from this observation, we investigated the underlying mechanism and now provide evidence that the formate-dependent increase of cell motility is mediated by an activation of Ca2+ signaling. Activation of Ca2+ signaling triggers integrin and matrix metallopeptidase (MMP) responses enabling the invasion process. Targeting either the Ca2+ response or MMP release can suppress the formate dependent increase in invasion. Finally, we tested the effect of formate also in context of breast cancer where we were able to recapitulate our observation of increased invasiveness and, in this case, formate also promoted the metastatic potential. We conclude that excreted formate might serve as a cellular stress signal that represents a promotive trigger to support tumor escape mechanisms.

Background/Aims: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. Methods: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. Results: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. Conclusion: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

Background/Aims: Similar to apoptosis of nucleated cells, red blood cells (RBC) can undergo suicidal cell death - called eryptosis. It is characterized by cell shrinkage and phosphatidylserine translocation. Eryptosis is triggered by an increase of intracellular calcium concentration due to activation of nonselective cation channels. The cation channels and consequently eryptosis are inhibited by erythropoietin. Eryptotic RBC are engulfed by macrophages and thus rapidly cleared from circulating blood. In this study, we explored whether storage of RBC influences the rate of eryptosis. Methods: Flow cytometry was employed to quantify phosphatidylserine exposing erythrocytes from annexin V binding and cytosolic Ca2+ activity from Fluo-3 fluorescence. Clearance of stored murine RBC was tested by injection of carboxyfluorescein succinimidyl ester (CFSE)-labelled erythrocytes. Results: Storage for 42 days significantly increased the percentage of phosphatidylserine exposing and haemolytic erythrocytes, an effect blunted by removal of extracellular calcium. Phosphatidylserine exposure could be inhibited by addition of erythropoietin. Upon transfusion, the clearance of murine CFSE-labelled RBC from circulating blood was significantly higher following storage for 10 days when compared to 2 days of storage. Conclusion: Storage of RBC triggers eryptosis by Ca2+ and erythropoietin sensitive mechanisms.

CD8(+) T cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T cell function which are still incompletely understood. Here we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B cell activating transcription factor (BATF) are necessary for sustained CD8(+) T cell effector function. Although Irf4( / ) CD8(+) T cells were initially capable of proliferation IRF4 deficiency resulted in limited CD8(+) T cell responses after infection with the lymphocytic choriomeningitis virus. Consequently Irf4( / ) mice established chronic infections but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T cell function limited immunopathology and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T cell immunity.Cell Death and Differentiation advance online publication 14 February 2014; doi:10.1038/cdd.2014.19.

Cluster of differentiation (CD)8(+) T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8(+) T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1(-/-)) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.

This work presents the results of a comparative study in vivo of photosensitizer based on hydrophilic derivative of chlorin e6 upon systemic administration in commercially available aqueous solution and liposomal form. It is demonstrated that newly developed liposomal form provides more selective accumulation of photosensitizer in tumor tissue and efficiency of antitumor action, as well as achieving an enhanced storage stability in comparison to the original form.

B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses., Antibody responses are regulated by selective survival of B cells with proper antigen specificity. Here the authors show that CEACAM1 is critical for B-cell survival during homeostasis and antiviral responses.

STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types. Human mutations in STAT3 cause primary immunodeficiency resulting in impaired control of a variety of infections, including reactivation of latent viruses. In this study, we investigate how T-cell functions of STAT3 contribute to responses to viral infection by inducing chronic lymphocytic choriomeningitis virus (LCMV) infection in mice lacking STAT3 specifically in T cells. Although mice with conditional disruption of STAT3 in T cells were able to mount early responses to viral infection similar to control animals, including expansion of effector T cells, we found generation of T-follicular helper (Tfh) cells to be impaired. As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum. These effects were associated with reduced control of viral replication and prolonged infection. Our results demonstrate the importance of STAT3 in T cells for the generation of functional long-term humoral immunity to viral infections.

Haponin (HLDF-alike protein) was previously identified from the human promyelocytic leukemia HL-60 cell line. For the functional study of this protein, we obtained recombinant haponin with an N-terminal hexahistidine tag using a baculovirus expression system. Antibodies against 6xHis-haponin were produced, and the expression of endogenous haponin was demonstrated in mammalian cell lines of different origin. Using affinity chromatography and immunoprecipitation methods, we have shown that in CHO-K1 cells haponin interacts with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is one of the vital glycolytic enzymes with a diverse set of noncanonical functions.