The Influence of Metabolism on Drug Response in Cancer

Resistance to therapeutic agents, either intrinsic or acquired, is currently a major problem in the treatment of cancers and occurs in virtually every type of anti-cancer therapy. Therefore, understanding how resistance can be prevented, targeted and predicted becomes increasingly important to improve cancer therapy. In the last decade, it has become apparent that alterations in cellular metabolism are a hallmark of cancer cells and that a rewired metabolism is essential for rapid tumor growth and proliferation. Recently, metabolic alterations have been shown to play a role in the sensitivity of cancer cells to widely-used first-line chemotherapeutics. This suggests that metabolic pathways are important mediators of resistance toward anticancer agents. In this review, we highlight the metabolic alterations associated with resistance toward different anticancer agents and discuss how metabolism may be exploited to overcome drug resistance to classical chemotherapy

Retinyl Ester Analysis by Orbitrap Mass Spectrometry

Retinoids are light-sensitive molecules that are normally detected by UV absorption techniques. Here we describe the identification and quantification of retinyl ester species by high-resolution mass spectrometry. Retinyl esters are extracted by the method of Bligh and Dyer and subsequently separated by HPLC in runs of 40 min. The retinyl esters are identified and quantified by mass spectrometry analysis. This procedure enables the highly sensitive detection and characterization of retinyl esters in biological samples such as hepatic stellate cells

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R&gt;C) in lung tumors specifically. Most R&gt;C events did not coincide with genetically encoded R&gt;C mutations but were likely products of tRNA misalignments. The expression of R&gt;C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R&gt;C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.</p

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R&gt;C) in lung tumors specifically. Most R&gt;C events did not coincide with genetically encoded R&gt;C mutations but were likely products of tRNA misalignments. The expression of R&gt;C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R&gt;C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.</p

Metabolic changes underlying drug resistance in the multiple myeloma tumor microenvironment

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow (BM). MM remains an incurable disease, with the majority of patients experiencing multiple relapses from different drugs. The MM tumor microenvironment (TME) and in particular bone-marrow stromal cells (BMSCs) play a crucial role in the development of drug resistance. Metabolic reprogramming is emerging as a hallmark of cancer that can potentially be exploited for cancer treatment. Recent studies show that metabolism is further adjusted in MM cells during the development of drug resistance. However, little is known about the role of BMSCs in inducing metabolic changes that are associated with drug resistance. In this Perspective, we summarize current knowledge concerning the metabolic reprogramming of MM, with a focus on those changes associated with drug resistance to the proteasome inhibitor Bortezomib (BTZ). In addition, we present proof-of-concept fluxomics (glucose isotope-tracing) and Seahorse data to show that co-culture of MM cells with BMSCs skews the metabolic phenotype of MM cells towards a drug-resistant phenotype, with increased oxidative phosphorylation (OXPHOS), serine synthesis pathway (SSP), TCA cycle and glutathione (GSH) synthesis. Given the crucial role of BMSCs in conveying drug resistance, insights into the metabolic interaction between MM and BMSCs may ultimately aid in the identification of novel metabolic targets that can be exploited for therapy

TNFR2 Costimulation Differentially Impacts Regulatory and Conventional CD4+ T-Cell Metabolism

CD4+ conventional T cells (Tconvs) mediate adaptive immune responses, whereas regulatory T cells (Tregs) suppress those responses to safeguard the body from autoimmunity and inflammatory diseases. The opposing activities of Tconvs and Tregs depend on the stage of the immune response and their environment, with an orchestrating role for cytokine- and costimulatory receptors. Nutrient availability also impacts T-cell functionality via metabolic and biosynthetic processes that are largely unexplored. Many data argue that costimulation by Tumor Necrosis Factor Receptor 2 (TNFR2) favors support of Treg over Tconv responses and therefore TNFR2 is a key clinical target. Here, we review the pertinent literature on this topic and highlight the newly identified role of TNFR2 as a metabolic regulator for thymus-derived (t)Tregs. We present novel transcriptomic and metabolomic data that show the differential impact of TNFR2 on Tconv and tTreg gene expression and reveal distinct metabolic impact on both cell types

Metabolic changes underlying drug resistance in the multiple myeloma tumor microenvironment

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow (BM). MM remains an incurable disease, with the majority of patients experiencing multiple relapses from different drugs. The MM tumor microenvironment (TME) and in particular bone-marrow stromal cells (BMSCs) play a crucial role in the development of drug resistance. Metabolic reprogramming is emerging as a hallmark of cancer that can potentially be exploited for cancer treatment. Recent studies show that metabolism is further adjusted in MM cells during the development of drug resistance. However, little is known about the role of BMSCs in inducing metabolic changes that are associated with drug resistance. In this Perspective, we summarize current knowledge concerning the metabolic reprogramming of MM, with a focus on those changes associated with drug resistance to the proteasome inhibitor Bortezomib (BTZ). In addition, we present proof-of-concept fluxomics (glucose isotope-tracing) and Seahorse data to show that co-culture of MM cells with BMSCs skews the metabolic phenotype of MM cells towards a drug-resistant phenotype, with increased oxidative phosphorylation (OXPHOS), serine synthesis pathway (SSP), TCA cycle and glutathione (GSH) synthesis. Given the crucial role of BMSCs in conveying drug resistance, insights into the metabolic interaction between MM and BMSCs may ultimately aid in the identification of novel metabolic targets that can be exploited for therapy

Specific labeling of newly synthesized lipopolysaccharide via metabolic incorporation of azido-galactose

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy

Extracellular vesicles from seminal plasma interact with T cells in vitro and drive their differentiation into regulatory T-cells

Seminal plasma induces immune tolerance towards paternal allogenic antigens within the female reproductive tract and during foetal development. Recent evidence suggests a role for extracellular vesicles in seminal plasma (spEVs). We isolated spEVs from seminal plasma that was donated by vasectomized men, thereby excluding any contributions from the testis or epididymis. Previous analysis demonstrated that such isolated spEVs originate mainly from the prostate. Here we observed that when isolated fluorescently labelled spEVs were mixed with peripheral blood mononuclear cells, they were endocytosed predominantly by monocytes, and to a lesser extent also by T-cells. In a mixed lymphocyte reaction, T-cell proliferation was inhibited by spEVs. A direct effect of spEVs on T-cells was demonstrated when isolated T cells were activated by anti-CD3/CD28 coated beads. Again, spEVs interfered with T cell proliferation, as well as with the expression of CD25 and the release of IFN-γ, TNF, and IL-2. Moreover, spEVs stimulated the expression of Foxp3 and IL-10 by CD4+CD25+CD127- T cells, indicating differentiation into regulatory T-cells (Tregs). Prior treatment of spEVs with proteinase K revoked their effects on T-cells, indicating a requirement for surface-exposed spEV proteins. The adenosine A2A receptor-specific antagonist CPI-444 also reduced effects of spEVs on T-cells, consistent with the notion that the development of Tregs and their immune suppressive functions are under the influence of adenosine-A2A receptor signalling. We found that adenosine is highly enriched in spEVs and propose that spEVs are targeted to and endocytosed by T-cells, after which they may release their adenosine content into the lumen of endosomes, thus allowing endosome-localized A2A receptor signalling in spEVs targeted T-cells. Collectively, these data support the idea that spEVs can prime T cells directly for differentiation into Tregs

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions