ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

Direct Synthesis of 3-Aryl Substituted Isocoumarins and Phthalides through Palladium Acetate Catalyzed C(sp2 )-H Activation in Ionic Liquids.

A novel Pd-catalysed oxidative coupling between benzoic acids and vinylarenes or acrylates to furnish isocoumarins and phthalides is reported. The reaction proceeds smoothly in molten tetrabutylammonium acetate via a selective C-H bond activation, with very low percentage of ligand-free palladium acetate as the catalyst, under atmospheric pressure of oxygen. Sub-stoichiometric amount of copper acetate is also required as a reoxidant for the palladium.

Synthesis and Investigation of Novel CHCA-Derived Matrices for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Lipids.

A significant area of study and upgrading for increasing sensitivity and general performances of matrix-assisted laser-desorption ionization (MALDI) mass spectrometry (MS) is related to matrix design. Several efforts have been made to address the challenge of low-mass-region interference-free for metabolomics analysis and specifically for lipidomics. To this aim, rationally designed matrices as 4-chloro-α-cyanocinnamic acid (ClCCA) were introduced and reported to provide enhanced analytical performances. We have taken this rational design one step further by developing and optimizing new MALDI matrices with a range of modifications on the CHCA core, involving different functionalities and substituents. Of particular interest was the understanding of the electron-withdrawing (e.g., nitro-) or donating (e.g., methoxy-) effects along with the extent of conjugation on the ionization efficiency. In the present work, ten matrices were designed on a reasonable basis, synthesized, and characterized by NMR and UV spectroscopies and laser desorption ionization. With the assistance of these putative MALDI matrices, samples containing phospholipids (PL), and neutral di-/tri-acylglycerols (DAG, TAG) were investigated using milk, fish, blood, and human plasma extracts. In comparison with CHCA and ClCCA, four of them, viz. [(2E,4E)-2-cyano-5-(4-methoxyphenyl)penta-2,4-dienoic acid] (1), [(2E,4E)-2-cyano-5-(4-nitrophenyl)penta-2,4-dienoic acid] (2), [(E)-2-cyano-3-(6-methoxynaphthalen-2-yl)acrylic acid] (6) and [(E)-2-cyano-3-(naphthalen-2-yl)acrylic acid] (7) displayed good to even excellent performances as MALDI matrices in terms of ionization capability, interference-free spectra, S/N ratio, and reproducibility. Especially compound 7 (cyano naphthyl acrylic acid, CNAA) was the election matrix for PL analysis and matrix 2 (cyano nitrophenyl dienoic acid, CNDA) for neutral lipids such as DAG and TAG in positive ion mode.

A Fatty Acid Oxidation-dependent Metabolic Shift Regulates the Adaptation of BRAF-mutated Melanoma to MAPK Inhibitors.

PURPOSE: Treatment of BRAFV600E -mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi. EXPERIMENTAL DESIGN: By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre- and on-treatment tumor specimens. RESULTS: Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARα-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E -mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo. CONCLUSIONS: Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E -mutant melanoma.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Decreased transcriptional activity of calcium-sensing receptor gene promoter 1 is associated with calcium nephrolithiasis.

BACKGROUND: CaSR gene is a candidate for calcium nephrolithiasis. Single-nucleotide polymorphisms (SNPs) encompassing its regulatory region were associated with calcium nephrolithiasis. AIMS: We tested SNPs in the CaSR gene regulatory region associated with calcium nephrolithiasis and their effects in kidney. SUBJECTS AND METHODS: One hundred sixty-seven idiopathic calcium stone formers and 214 healthy controls were genotyped for four CaSR gene SNPs identified by bioinformatics analysis as modifying transcription factor binding sites. Strontium excretion after an oral load was tested in 55 stone formers. Transcriptional activity induced by variant alleles at CaSR gene promoters was compared by luciferase reporter gene assay in HEK-293 and HKC-8 cells. CaSR and claudin-14 mRNA levels were measured by real-time PCR in 107 normal kidney medulla samples and compared in patients with different CaSR genotype. RESULTS: Only rs6776158 (A>G), located in the promoter 1, was associated with nephrolithiasis. Its minor G allele was more frequent in stone formers than controls (37.8% vs 26.4%, P = .001). A reduced strontium excretion was observed in GG homozygous stone formers. Luciferase fluorescent activity was lower in cells transfected with the promoter 1 including G allele at rs6776158 than cells transfected with the A allele. CaSR mRNA levels were lower in kidney medulla samples from homozygous carriers for the G allele at rs6776158 than carriers for the A allele. Claudin-14 mRNA levels were also lower in GG homozygous subjects. CONCLUSIONS: Minor allele at rs6776158 may predispose to calcium stones by decreasing transcriptional activity of the CaSR gene promoter 1 and CaSR expression in kidney tubules.

The melatonin receptor 1A (MTNR1A) gene is associated with recurrent and idiopathic calcium nephrolithiasis.

BACKGROUND: Experimental evidence indicate that melatonin regulates some renal tubular functions via specific melatonin receptors (MTNRs) located in the kidney of several avian and mammalian species, including humans. We hypothesized that single nucleotide polymorphisms (SNPs) in the melatonin receptor 1A gene (MTNR1A) might influence the risk of calcium nephrolithiasis. METHODS: We performed a systematic analysis of the MTNR1A gene in 246 recurrent calcium stone formers (136 men, 110 women; mean age 40.2 ± 12.0 years; body mass index 25.8 ± 4.5 kg/m2) and 269 healthy controls comparable for age and gender without a history of nephrolithiasis. RESULTS: Two SNPs in Intron 1 of MTNR1A were significantly associated with calcium nephrolithiasis: rs13140012 (P = 0.0004) and rs6553010 (P = 0.009). The haplotypes resulting from the two SNPs were also differently distributed between stone formers and controls, the haplotype A-T being more represented among stone formers (P = 0.00001) and the haplotype T-C being more common in healthy controls (P = 0.00001). Preliminary functional studies showed that the SNP rs13140012 could modify the binding sites for transcription factors. CONCLUSION: The results of this case-control study indicate a strong association between allelic variants of MTNR1A and recurrent calcium nephrolithiasis.

Calcium-sensing receptor and calcium kidney stones.

Calcium nephrolithiasis may be considered as a complex disease having multiple pathogenetic mechanisms and characterized by various clinical manifestations. Both genetic and environmental factors may increase susceptibility to calcium stones; therefore, it is crucial to characterize the patient phenotype to distinguish homogeneous groups of stone formers. Family and twin studies have shown that the stone transmission pattern is not mendelian, but complex and polygenic. In these studies, heritability of calcium stones was calculated around 50%Calcium-sensing receptor (CaSR) is mostly expressed in the parathyroid glands and in renal tubules. It regulates the PTH secretion according to the serum calcium concentration. In the kidney, it modulates electrolyte and water excretion regulating the function of different tubular segments. In particular, CaSR reduces passive and active calcium reabsorption in distal tubules, increases phosphate reabsorption in proximal tubules and stimulates proton and water excretion in collecting ducts. Therefore, it is a candidate gene for calcium nephrolithiasis.In a case-control study we found an association between the normocitraturic stone formers and two SNPs of CaSR, located near the promoters region (rs7652589 and rs1501899). This result was replicated in patients with primary hyperparathyroidism, comparing patients with or without kidney stones. Bioinformatic analysis suggested that the minor alleles at these polymorphisms were able to modify the binding sites of specific transcription factors and, consequently, CaSR expression.Our studies suggest that CaSR is one of the candidate genes explaining individual predisposition to calcium nephrolithiasis. Stone formation may be favored by an altered CaSR expression in kidney medulla involving the normal balance among calcium, phosphate, protons and water excretion.

[Hyperparathyroidism as a cardiovascular risk factor in chronic kidney disease: an update from a biological-cellular perspective]. / L'iperparatiroidismo come fattore di rischio cardiovascolare nell'insufficienza renale: un update dal punto di vista biologico-cellulare.

Cardiovascular complications are the main cause of death in patients with chronic kidney disease (CKD). Among these complications, calcific arteriosclerosis and myocardial hypertrophy are the main predictors of cardiovascular morbidity and mortality. Epidemiological studies have shown their association with hyperparathyroidism, which has therefore been included among the non-traditional cardiovascular risk factors. Studies in laboratory animals have shown that PTH administration may induce calcific arteriosclerosis and myocardial hypertrophy. The former develops independently of hyperphosphatemia, but its mechanisms remain unknown. The latter is characterized by increased thickness of the myocardial fibers and especially the fibrous interstitium; its development is influenced by protein kinase C activation and the subsequent increase in cytosolic calcium as well as activation of intracellular signaling pathways inducing protein synthesis and proliferation. Different from these findings, in other studies PTH infusion was able to produce vasodilatation and to favor myocardial cell contraction and regeneration. These effects depend on protein kinase A activation. PTH may produce different and sometimes contradictory functional effects in the arteries and myocardium that are probably related to different experimental or clinical conditions. In patients with CKD and hyperparathyroidism, PTH may be considered a uremic toxin exerting its effects mainly by increasing cellular calcium. Thus, hyperparathyroidism is confirmed to be a target for the conservative therapy of CKD.

Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor.

Previous studies have demonstrated a gain-of-function of the calcium-sensing receptor (CASR) gene R990G polymorphism. In this study, activation of the R990G CASR stably transfected in HEK-293 (HEK-990G) cells compared with that of the common variant (HEK-wild-type (WT)) by increasing concentrations of CaCl(2) or calcimimetic R-568 caused significantly higher intracellular free calcium concentration ([Ca(2+)](i)) and lower Ca-EC(50). Moreover, the [Ca(2+)](i) oscillation percentage was higher with a larger sinusoidal pattern in HEK-990G. R-568 induced a shift of the oscillatory events from 4 to 2  mmol/l extracellular calcium concentration in HEK-990G cells and increased the sinusoidal oscillation percentage in comparison with HEK-WT. Preincubation with thapsigargin or phospholipase C inhibitors completely prevented oscillations in both cell lines, consistent with the involvement of the inositol trisphosphate pathway, while protein kinase C inhibitor prevented oscillations in HEK-WT cells only. Finally, CaCl(2) and R-568 caused a significant increase in p44/42 extracellular signaling-regulated kinase phosphorylation, with the mean Ca-EC(50) values being significantly lower in HEK-990G. Our findings demonstrated that the 990G allele is associated with high sensitivity to R-568, which provided new evidence for differences in CASR signaling.

Common variants in the regulative regions of GRIA1 and GRIA3 receptor genes are associated with migraine susceptibility.

BACKGROUND: Glutamate is the principal excitatory neurotransmitter in the central nervous system which acts by the activation of either ionotropic (AMPA, NMDA and kainate receptors) or G-protein coupled metabotropic receptors. Glutamate is widely accepted to play a major role in the path physiology of migraine as implicated by data from animal and human studies. Genes involved in synthesis, metabolism and regulation of both glutamate and its receptors could be, therefore, considered as potential candidates for causing/predisposing to migraine when mutated. METHODS: The association of polymorphic variants of GRIA1-GRIA4 genes which encode for the four subunits (GluR1-GluR4) of the alpha-amino-3- hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor for glutamate was tested in migraineurs with and without aura (MA and MO) and healthy controls. RESULTS: Two variants in the regulative regions of GRIA1 (rs2195450) and GRIA3 (rs3761555) genes resulted strongly associated with MA (P = 0.00002 and P = 0.0001, respectively), but not associated with MO, suggesting their role in cortical spreading depression. Whereas the rs548294 variant in GRIA1 gene showed association primarily with MO phenotype, supporting the hypothesis that MA and MO phenotypes could be genetically related. These variants modify binding sites for transcription factors altering the expression of GRIA1 and GRIA3 genes in different conditions. CONCLUSIONS: This study represents the first genetic evidence of a link between glutamate receptors and migraine.