Association of marine PUFAs intakes with cardiovascular disease, all-cause mortality, and cardiovascular mortality in American adult male patients with dyslipidemia: the U.S. National Health and Nutrition Examination Survey, 2001 to 2016.

BACKGROUND: The relationship between marine polyunsaturated fatty acid (PUFA) intake and cardiovascular disease and mortality in dyslipidemic patients is unclear. Men with dyslipidemia have a higher risk of cardiovascular disease than women, and PUFA supplementation may be more beneficial in men. OBJECTIVE: The purpose of this study was to assess the relationship between different types of marine polyunsaturated fatty acids intakes and cardiovascular disease, all-cause mortality, and cardiovascular mortality in adult U.S. males with dyslipidemia. METHODS: The study ultimately included 11,848 adult men with dyslipidemia who were screened from the National Health and Nutrition Examination Survey (NHANES) between 2001 and 2016. This was linked to the 2019 National Death Index (NDI) records to establish a prospective cohort. In the study, a logistic regression model was established to assess the relationship between PUFA intake and prevalent CVD, and a Cox proportional hazards regression model was established to assess the relationship between PUFA intake and death. RESULTS: In the fully adjusted models, compared with participants in the lowest tertile, participants with the highest DPA intake were associated with a lower risk of CVD (CVD: OR = 0.71, 95%CI: 0.55, 0.91; angina: OR = 0.54, 95%CI: 0.38, 0.79; stroke: OR = 0.62, 95%CI: 0.43, 0.89), but not with three subtypes of congestive heart failure, coronary heart disease, and myocardial infarction. And the highest tertile level of DPA intake can reduce all-cause mortality (HR = 0.77, 95%CI: 0.64, 0.91) and CVD mortality (HR = 0.68, 95%CI: 0.52, 0.90). CONCLUSIONS: Cardiovascular disease risk, all-cause mortality, and CVD mortality were inversely associated with dietary DPA intake but not EPA and DHA intakes in U.S. male participants with dyslipidemia.

Apelin/APJ system protects placental trophoblasts from hypoxia-induced oxidative stress through activating PI3K/Akt signaling pathway in preeclampsia.

BACKGROUND: Preeclampsia is a placentally induced syndrome with diverse clinical presentation that currently has no cure. Oxidative stress is a potent inducer of placental dysfunction. The apelin receptor (APJ) system is a pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. This study examines the alteration of circulating apelin levels and placental APJ expression in preeclampsia and investigates whether apelin/APJ system can protect placental trophoblast from hypoxia-induced oxidative stress injury through PI3K/AKT signaling pathway. RESULTS: Our results confirmed that maternal apelin concentration was increased in women with preeclampsia, but APJ expression was reduced in the preeclamptic placentas. Apelin-13 treatment not only specifically attenuated CoCl2-induced superoxide production, but also prevented CoCl2-induced reduction of SOD activity and SOD1 expression. In addition, apelin-13 suppressed CoCl2-induced apoptosis by increasing the expression of bcl-2/bax ratio and by decreasing the expression of active caspase-3 in placental trophoblasts. Furthermore, we found that apelin-13 binding APJ activated the PI3K and AKT kinases and inhibition of PI3K kinase significantly blocked the anti-oxidative effects of apelin-13 in placental trophoblasts. CONCLUSIONS: Decrease of placental APJ expression is associated with oxidative stress-induced placental dysfunction in preeclampsia, and increased circulating apelin could be a moderately successful marker to differentiate subjects with preeclampsia from healthy pregnant women. Inhibition of superoxide production and caspase-3 cleavage, together with upregulation of SOD activity/expression and bcl-2/bax ratio, could be the potential molecular mechanisms by which apelin-13/APJ protects placental trophoblasts from oxidative stress injury.

Causal associations of histidine and 12 site-specific cancers: a bidirectional Mendelian randomization study.

An increasing number of studies indicate that cancer patients' histidine (HIS) circulating levels have changed. However, the causality between HIS and cancer is still not well established. Thus, to ascertain the causal link between HIS and cancers, we performed a bidirectional Mendelian randomization (MR) analysis. Summary-level data are derived from publicly available genome-wide association studies (GWAS). The causal effects were mainly estimated using the inverse-variance weighted method (IVW). The weighted-median (WM) method and MR-Egger regression were conducted as sensitivity analyses. In the forward-MR, we found malignant neoplasm of respiratory system and intrathoracic organs (OR: 1.020; 95% CI: 1.006-1.035; pIVW = 0.007) genetically associated with circulating HIS. And there was no significant genetic correlation between HIS and another 11 site-specific cancers using IVW method. In the reversed-MR, we did not observe the causal relationship between HIS and 12 site-specific cancers. Our findings help clarify that HIS, as a biomarker for malignant neoplasms of respiratory system and intrathoracic organs, is causal rather than a secondary biomarker of the cancerous progression. The mechanism between histidine and cancer progression deserves further investigation.

Analysis of serum fatty acid, amino acid, and organic acid profiles in gestational hypertension and gestational diabetes mellitus via targeted metabolomics.

This study aimed to characterize metabolite differences and correlations between hypertensive disorders of pregnancy (HP) and gestational diabetes mellitus (GDM) using univariate, multivariate analyses, RF, and pathway analyses in a cross-sectional study. Dietary surveys were collected and targeted metabolomics was applied to measure levels of serum fatty acids, amino acids, and organic acids in 90 pregnant women at 24-28 weeks gestation at the First Affiliated Hospital of Harbin Medical University. Principal components analysis (PCA) and partial least squares-discriminatory analysis (PLS-DA) models were established to distinguish HP, GDM, and healthy, pregnant control individuals. Univariate and multivariate statistical analyses and Random Forest (RF) were used to identify and map co-metabolites to corresponding pathways in the disease states. Finally, risk factors for the disease were assessed by receiver operating characteristics (ROC) analysis. Dietary survey results showed that HP and GDM patients consumed a high-energy diet and the latter also consumed a high-carbohydrate and high-fat diet. Univariate analysis of clinical indices revealed HP and GDM patients had glycolipid disorders, with the former possessing more severe organ dysfunction. Subsequently, co-areas with significant differences identified by basic discriminant analyses and RF revealed lower levels of pyroglutamic acid and higher levels of 2-hydroxybutyric acid and glutamic acid in the GDM group. The number of metabolites increased in the HP group as compared to the healthy pregnant control group, including pyroglutamic acid, γ-aminobutyric acid (GABA), glutamic acid, oleic acid (C18:1), and palmitic acid (C16:0). ROC curves indicated that area under curve (AUC) for pyroglutamic acid in the GDM group was 0.962 (95% CI, 0.920-1.000), and the AUC of joint indicators, including pyroglutamic acid and GABA, in the HP group was 0.972 (95% CI, 0.938-1.000). Collectively, these results show that both GDM and HP patients at mid-gestation possessed dysregulated glucose and lipid metabolism, which may trigger oxidative stress via glutathione metabolism and biosynthesis of unsaturated fatty acids.

The metabolic adaptation of the adult offspring after maternal high-dosed folic acid supplementation based on the proteomics and metabolomics in rats.

The onset of complex diseases at a later stage of life has been linked with maternal folic acid (FA) ingestion. However, little is known regarding the underlying molecular fingerprints of the offspring. We integrated proteomics-metabolomics profiles and analyzed the influence of maternal FA supplementation on the metabolism of adult offspring rats. Twenty pregnant female rats were randomly assigned to a FA supplementation (FolS group, 10 mg/kg FA) or control group (2 mg/kg FA respectively). Such an omics approach revealed that the dopaminergic synapse pathway, tricarboxylic acid cycle and neural development-related metabolites such as glutamic acid and γ-aminobutyric acid were significantly up-regulated in the FolS group, whereas pyruvic acid, oxalic acid and adipic acid were reduced. Maternal FA supplementation can cause alterations of metabolites and protein in the offspring rats.

Mangiferin Improved Palmitate-Induced-Insulin Resistance by Promoting Free Fatty Acid Metabolism in HepG2 and C2C12 Cells via PPARα: Mangiferin Improved Insulin Resistance.

Elevated free fatty acid (FFA) is a key risk factor for insulin resistance (IR). Our previous studies found that mangiferin could decrease serum FFA levels in obese rats induced by a high-fat diet. Our research was to determine the effects and mechanism of mangiferin on improving IR by regulating FFA metabolism in HepG2 and C2C12 cells. The model was used to quantify PA-induced lipid accumulation in the two cell lines treated with various concentrations of mangiferin simultaneously for 24 h. We found that mangiferin significantly increased insulin-stimulated glucose uptake, via phosphorylation of protein kinase B (P-AKT), glucose transporter 2 (GLUT2), and glucose transporter 4 (GLUT4) protein expressions, and markedly decreased glucose content, respectively, in HepG2 and C2C12 cells induced by PA. Mangiferin significantly increased FFA uptake and decreased intracellular FFA and triglyceride (TG) accumulations. The activity of the peroxisome proliferator-activated receptor α (PPARα) protein and its downstream proteins involved in fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT1) and the fatty acid ß-oxidation rate corresponding to FFA metabolism were also markedly increased by mangiferin in HepG2 and C2C12 cells. Furthermore, the effects were reversed by siRNA-mediated knockdown of PPARα. Mangiferin ameliorated IR by increasing the consumption of glucose and promoting the FFA oxidation via the PPARα pathway in HepG2 and C2C12 cells.

Recurrent Tumor Cell-Intrinsic and -Extrinsic Alterations during MAPKi-Induced Melanoma Regression and Early Adaptation.

Treatment of advanced BRAFV600-mutant melanoma using a BRAF inhibitor or its combination with a MEK inhibitor typically elicits partial responses. We compared the transcriptomes of patient-derived tumors regressing on MAPK inhibitor (MAPKi) therapy against MAPKi-induced temporal transcriptomic states in human melanoma cell lines or murine melanoma in immune-competent mice. Despite heterogeneous dynamics of clinical tumor regression, residual tumors displayed highly recurrent transcriptomic alterations and enriched processes, which were also observed in MAPKi-selected cell lines (implying tumor cell-intrinsic reprogramming) or in bulk mouse tumors (and the CD45-negative or CD45-positive fractions, implying tumor cell-intrinsic or stromal/immune alterations, respectively). Tumor cell-intrinsic reprogramming attenuated MAPK dependency, while enhancing mesenchymal, angiogenic, and IFN-inflammatory features and growth/survival dependence on multi-RTKs and PD-L2. In the immune compartment, PD-L2 upregulation in CD11c+ immunocytes drove the loss of T-cell inflammation and promoted BRAFi resistance. Thus, residual melanoma early on MAPKi therapy already displays potentially exploitable adaptive transcriptomic, epigenomic, immune-regulomic alterations.Significance: Incomplete MAPKi-induced melanoma regression results in transcriptome/methylome-wide reprogramming and MAPK-redundant escape. Although regressing/residual melanoma is highly T cell-inflamed, stromal adaptations, many of which are tumor cell-driven, could suppress/eliminate intratumoral T cells, reversing tumor regression. This catalog of recurrent alterations helps identify adaptations such as PD-L2 operative tumor cell intrinsically and/or extrinsically early on therapy. Cancer Discov; 7(11); 1248-65. ©2017 AACR.See related commentary by Haq, p. 1216This article is highlighted in the In This Issue feature, p. 1201.

JUN dependency in distinct early and late BRAF inhibition adaptation states of melanoma.

A prominent mechanism of acquired resistance to BRAF inhibitors in BRAF (V600) -mutant melanoma is associated with the upregulation of receptor tyrosine kinases. Evidences suggested that this resistance mechanism is part of a more complex cellular adaptation process. Using an integrative strategy, we found this mechanism to invoke extensive transcriptomic, (phospho-) proteomic and phenotypic alterations that accompany a cellular transition to a de-differentiated, mesenchymal and invasive state. Even short-term BRAF-inhibitor exposure leads to an early adaptive, differentiation state change-characterized by a slow-cycling, persistent state. The early persistent state is distinct from the late proliferative, resistant state. However, both differentiation states share common signaling alterations including JUN upregulation. Motivated by the similarities, we found that co-targeting of BRAF and JUN is synergistic in killing fully resistant cells; and when used up-front, co-targeting substantially impairs the formation of the persistent subpopulation. We confirmed that JUN upregulation is a common response to BRAF inhibitor treatment in clinically treated patient tumors. Our findings demonstrate that events shared between early- and late-adaptation states provide candidate up-front co-treatment targets.

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.

BACKGROUND: Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).

Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma.

PD-1 immune checkpoint blockade provides significant clinical benefits for melanoma patients. We analyzed the somatic mutanomes and transcriptomes of pretreatment melanoma biopsies to identify factors that may influence innate sensitivity or resistance to anti-PD-1 therapy. We find that overall high mutational loads associate with improved survival, and tumors from responding patients are enriched for mutations in the DNA repair gene BRCA2. Innately resistant tumors display a transcriptional signature (referred to as the IPRES, or innate anti-PD-1 resistance), indicating concurrent up-expression of genes involved in the regulation of mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, and wound healing. Notably, mitogen-activated protein kinase (MAPK)-targeted therapy (MAPK inhibitor) induces similar signatures in melanoma, suggesting that a non-genomic form of MAPK inhibitor resistance mediates cross-resistance to anti-PD-1 therapy. Validation of the IPRES in other independent tumor cohorts defines a transcriptomic subset across distinct types of advanced cancer. These findings suggest that attenuating the biological processes that underlie IPRES may improve anti-PD-1 response in melanoma and other cancer types.

Acquired BRAF inhibitor resistance: A multicenter meta-analysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms.

BACKGROUND: Acquired resistance to BRAF inhibitors (BRAFi) is a near-universal phenomenon caused by numerous genetic and non-genetic alterations. In this study, we evaluated the spectrum, onset, pattern of progression, and subsequent clinical outcomes associated with specific mechanisms of resistance. METHODS: We compiled clinical and genetic data from 100 patients with 132 tissue samples obtained at progression on BRAFi therapy from 3 large, previously published studies of BRAFi resistance. These samples were subjected to whole-exome sequencing and/or polymerase chain reaction-based genetic testing. RESULTS: Among 132 samples, putative resistance mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAF(V600E/K) amplifications (13%), MEK1/2 mutations (7%), and non-mitogen-activated protein kinase pathway alterations (11%). Marked heterogeneity was observed within tumors and patients; 18 of 19 patients (95%) with more than one progression biopsy had distinct/unknown drivers of resistance between samples. NRAS mutations were associated with vemurafenib use (p = 0.045) and intracranial metastases (p = 0.036), and MEK1/2 mutations correlated with hepatic progression (p = 0.011). Progression-free survival and overall survival were similar across resistance mechanisms. The median survival after disease progression was 6.9 months, and responses to subsequent BRAF and MEK inhibition were uncommon (2 of 15; 13%). Post-progression outcomes did not correlate with specific acquired BRAFi-resistance mechanisms. CONCLUSIONS: This is the first study to systematically characterise the clinical implications of particular acquired BRAFi-resistance mechanisms in patients with BRAF-mutant melanoma largest study to compile the landscape of resistance. Despite marked heterogeneity of resistance mechanisms within patients, NRAS mutations correlated with vemurafenib use and intracranial disease involvement.

Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.

Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.

Therapy-induced tumour secretomes promote resistance and tumour progression.

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

Tunable-combinatorial mechanisms of acquired resistance limit the efficacy of BRAF/MEK cotargeting but result in melanoma drug addiction.

Combined BRAF- and MEK-targeted therapy improves upon BRAF inhibitor (BRAFi) therapy but is still beset by acquired resistance. We show that melanomas acquire resistance to combined BRAF and MEK inhibition by augmenting or combining mechanisms of single-agent BRAFi resistance. These double-drug resistance-associated genetic configurations significantly altered molecular interactions underlying MAPK pathway reactivation. (V600E)BRAF, expressed at supraphysiological levels because of (V600E)BRAF ultra-amplification, dimerized with and activated CRAF. In addition, MEK mutants enhanced interaction with overexpressed (V600E)BRAF via a regulatory interface at R662 of (V600E)BRAF. Importantly, melanoma cell lines selected for resistance to BRAFi+MEKi, but not those to BRAFi alone, displayed robust drug addiction, providing a potentially exploitable therapeutic opportunity.

Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma.

Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas.

Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors.

RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2-18 months. Here we demonstrate that the mixed lineage kinases (MLK1-4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1-4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors.

A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition.

BRAF inhibitor (BRAFi) therapy leads to remarkable anti melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants, causing late, acquired resistance. We show here that BRAFi (or BRAFi + MEKi) therapy in patients frequently led to rebound phosphorylated AKT (p-AKT) levels in their melanomas early on-treatment. In cell lines, BRAFi treatment led to rebound levels of receptor tyrosine kinases (RTK; including PDGFRß), phosphatidyl (3,4,5)-triphosphate (PIP3), pleckstrin homology domain recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by the introduction of a mutant AKT1 (Q79K) known to confer acquired BRAFi resistance. Functionally, AKT1(Q79K) conferred BRAFi resistance via amplification of BRAFi-elicited PI3K-AKT signaling. In addition, mitogen-activated protein kinase pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy.

Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy.

BRAF inhibitors elicit rapid antitumor responses in the majority of patients with BRAF(V600)-mutant melanoma, but acquired drug resistance is almost universal. We sought to identify the core resistance pathways and the extent of tumor heterogeneity during disease progression. We show that mitogen-activated protein kinase reactivation mechanisms were detected among 70% of disease-progressive tissues, with RAS mutations, mutant BRAF amplification, and alternative splicing being most common. We also detected PI3K-PTEN-AKT-upregulating genetic alterations among 22% of progressive melanomas. Distinct molecular lesions in both core drug escape pathways were commonly detected concurrently in the same tumor or among multiple tumors from the same patient. Beyond harboring extensively heterogeneous resistance mechanisms, melanoma regrowth emerging from BRAF inhibitor selection displayed branched evolution marked by altered mutational spectra/signatures and increased fitness. Thus, melanoma genomic heterogeneity contributes significantly to BRAF inhibitor treatment failure, implying upfront, cotargeting of two core pathways as an essential strategy for durable responses.