Mutational Activation of the NRF2 Pathway Upregulates Kynureninase Resulting in Tumor Immunosuppression and Poor Outcome in Lung Adenocarcinoma.

Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of NFE2L2, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of KEAP1 or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.

Plasma Based Protein Signatures Associated with Small Cell Lung Cancer.

Small-cell-lung cancer (SCLC) is associated with overexpression of oncogenes including Myc family genes and YAP1 and inactivation of tumor suppressor genes. We performed in-depth proteomic profiling of plasmas collected from 15 individuals with newly diagnosed early stage SCLC and from 15 individuals before the diagnosis of SCLC and compared findings with plasma proteomic profiles of 30 matched controls to determine the occurrence of signatures that reflect disease pathogenesis. A total of 272 proteins were elevated (area under the receiver operating characteristic curve (AUC) ≥ 0.60) among newly diagnosed cases compared to matched controls of which 31 proteins were also elevated (AUC ≥ 0.60) in case plasmas collected within one year prior to diagnosis. Ingenuity Pathway analyses of SCLC-associated proteins revealed enrichment of signatures of oncogenic MYC and YAP1. Intersection of proteins elevated in case plasmas with proteomic profiles of conditioned medium from 17 SCLC cell lines yielded 52 overlapping proteins characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition. Among samples collected more than one year prior to diagnosis there was a predominance of inflammatory markers. Our integrated analyses identified novel circulating protein features in early stage SCLC associated with oncogenic drivers.

Protein citrullination as a source of cancer neoantigens.

BACKGROUND: Citrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response. METHODS: Protein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12-34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls. RESULTS: Proteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER- tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen-antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012). CONCLUSIONS: An immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

Lead-Time Trajectory of CA19-9 as an Anchor Marker for Pancreatic Cancer Early Detection.

BACKGROUND & AIMS: There is substantial interest in liquid biopsy approaches for cancer early detection among subjects at risk, using multi-marker panels. CA19-9 is an established circulating biomarker for pancreatic cancer; however, its relevance for pancreatic cancer early detection or for monitoring subjects at risk has not been established. METHODS: CA19-9 levels were assessed in blinded sera from 175 subjects collected up to 5 years before diagnosis of pancreatic cancer and from 875 matched controls from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. For comparison of performance, CA19-9 was assayed in blinded independent sets of samples collected at diagnosis from 129 subjects with resectable pancreatic cancer and 275 controls (100 healthy subjects; 50 with chronic pancreatitis; and 125 with noncancerous pancreatic cysts). The complementary value of 2 additional protein markers, TIMP1 and LRG1, was determined. RESULTS: In the PLCO cohort, levels of CA19-9 increased exponentially starting at 2 years before diagnosis with sensitivities reaching 60% at 99% specificity within 0 to 6 months before diagnosis for all cases and 50% at 99% specificity for cases diagnosed with early-stage disease. Performance was comparable for distinguishing newly diagnosed cases with resectable pancreatic cancer from healthy controls (64% sensitivity at 99% specificity). Comparison of resectable pancreatic cancer cases to subjects with chronic pancreatitis yielded 46% sensitivity at 99% specificity and for subjects with noncancerous cysts, 30% sensitivity at 99% specificity. For prediagnostic cases below cutoff value for CA19-9, the combination with LRG1 and TIMP1 yielded an increment of 13.2% in sensitivity at 99% specificity (P = .031) in identifying cases diagnosed within 1 year of blood collection. CONCLUSION: CA19-9 can serve as an anchor marker for pancreatic cancer early detection applications.

Plasma-Derived Extracellular Vesicles Convey Protein Signatures that Reflect Pathophysiology in Lung and Pancreatic Adenocarcinomas.

Using a combination of mass-spectrometry and aptamer array-based proteomics, we characterized the protein features of circulating extracellular vesicles (EVs) in the context of lung (LUAD) and pancreatic ductal (PDAC) adenocarcinomas. We profiled EVs isolated from conditioned media of LUAD and PDAC cell lines to identify EV-associated protein cargoes released by these cancer cell types. Analysis of the resulting data identified LUAD and PDAC specific and pan-adenocarcinoma EV protein signatures. Bioinformatic analyses confirmed enrichment of proteins annotated to vesicle-associated processes and intracellular compartments, as well as representation of cancer hallmark functions and processes. Analysis of upstream regulator networks indicated significant enrichment of TP53, MYC, TGFB1 and KRAS-driven network effectors (p = 1.69 × 10-77-2.93 × 10-49) manifest in the adenocarcinoma sEV protein cargoes. We extended these findings by profiling the proteome of EVs isolated from lung (N = 15) and pancreatic ductal (N = 6) adenocarcinoma patient plasmas obtained at time of diagnosis, along with EVs derived from matched healthy controls (N = 21). Exploration of these proteomic data revealed abundant protein features in the plasma EVs with capacity to distinguish LUAD and PDAC cases from controls, including features yielding higher performance in the plasma EV isolates relative to unfractionated plasmas.

Mediator function in plant metabolism revealed by large-scale biology.

Mediator is a multisubunit transcriptional co-regulator that is involved in the regulation of an array of processes including plant metabolism. The pathways regulated by Mediator-dependent processes include those for the synthesis of phenylpropanoids (MED5), cellulose (MED16), lipids (MED15 and CDK8), and the regulation of iron homeostasis (MED16 and MED25). Traditional genetic and biochemical approaches laid the foundation for our understanding of Mediator function, but recent transcriptomic and metabolomic studies have provided deeper insights into how specific subunits cooperate in the regulation of plant metabolism. In this review, we highlight recent developments in the investigation of Mediator and plant metabolism, with particular emphasis on the large-scale biology studies of med mutants.

Mutation of Mediator subunit CDK8 counteracts the stunted growth and salicylic acid hyperaccumulation phenotypes of an Arabidopsis MED5 mutant.

The Mediator complex functions as a hub for transcriptional regulation. MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semidominant mutation (ref4-3) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. By contrast, loss of MED5 results in increased concentrations of phenylpropanoids. We used a reverse genetic approach to identify suppressors of ref4-3 and found that ref4-3 requires CDK8, a kinase module subunit of Mediator, to repress plant growth. The genetic interaction between MED5 and CDK8 was further characterized using mRNA-sequencing (RNA-seq) and metabolite analysis. Growth inhibition and suppression of phenylpropanoid metabolism can be genetically separated in ref4-3 by elimination of CDK8 kinase activity; however, the stunted growth of ref4-3 is not dependent on the phosphorylation event introduced by the G383S mutation. In addition, rather than perturbation of lignin biosynthesis, misregulation of DJC66, a gene encoding a DNAJ protein, is involved in the dwarfism of the med5 mutants. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.

Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition.

Lignin, a polymer found in the plant secondary cell wall, is a major contributor to biomass' recalcitrance toward saccharification. Because of this negative impact toward the value of lignocellulosic crops, there is a special interest in modifying the content and composition of this important plant biopolymer. For many years this endeavor has been hindered by the plant growth inhibition that is often associated with manipulations to phenylpropanoid metabolism. Although the actual mechanism by which dwarfism arises remains unknown, recent advances in tissue-specific lignin complementation and better understanding of phenylpropanoid transcriptional regulation has made it possible to disentangle lignin modification from perturbations in plant development.

[Analysis of the osteogenetic effects exerted on mesenchymal stem cell strain C3H10T1/2 by icariin via MAPK signaling pathway in vitro].

OBJECTIVE: To investigate the effects of icariin, an effective extract from traditional Chinese medicine Epimedium pubescens with the function of tonifying kidney, in promoting osteogenesis of mesenchymal stem cell line C3H10T1/2, and to explore the underlying mechanism. METHODS: After culture with icariin (0, 10(-7), 10(-6), 10(-5), and 10(-4) mol/L) and osteogenic supplement for 26 d in vitro, osteogenic differentiation of C3H10T1/2 cells was detected by alkaline phosphatase (ALP) assay. The RNA was extracted from cells cultured with 10(-5) mol/L icariin for 2, 8, 24 and 48 hours, and mRNA expressions of p38, p42 and p44 were measured using real-time reverse transcription-polymerase chain reaction (PCR) method. Three main proteins of MAPK signaling pathway (p38, and extracellular signal-regulated protein kinase (ERK), also named p42/44) and c-Jun N-terminal kinase (JNK) and their phospho-products were examined using Western blotting after icariin treatments of 10, 30, 60 and 120 min. RESULTS: Icariin at a dose of 10(-5) mol/L, when combined with the osteogenic supplement, had the best ability to promote osteogenic differentiation on C3H10T1/2 cells. Based on real-time PCR, the authors found that after two-hour ICA treatment, the gene expression of p38 revealed a significant decline compared with the control group (P<0.01). The levels of p42 and p44 mRNAs were decreased greatly after two-hour ICA treatment, while increased after 48-hour ICA treatment (P<0.05, P<0.01). There was no significant difference at other time points (P>0.05). Phospho-p42 was decreased after 10-minute icariin treatment, while phospho-p38 expression displayed an increase after 10- and 30-minute of treatment with icariin. There was no notable difference in phospho-JNK expression at these four time points. CONCLUSION: Icariin promotes differentiation of the mesenchymal stem cells C3H10T1/2 into osteoblasts, and its effect is related to the restraining of ERK expression and activation of p38 expression in the MAPK signaling pathway.

[Prognosis and its affecting factors in children with acute respiratory distress syndrome].

OBJECTIVE: To study the prognosis and the factors affecting the prognosis in children with acute respiratory distress syndrome (ARDS). METHODS: Seventy-eight children with ARDS were enrolled. The states of their survival within 30 days were followed-up. RESULTS: Of the 78 children with ARDS, 51 cases demised, 27 cases survived, with a 30-days survival rate of 35%. The average survival time was 14.4 days (median: 8 days). The peak of death appeared within 3 days after ARDS. There were significant differences in aspects of age, primary disease, percentage of neonatal hyaline membrane disease, pediatric critical illness score (PCIS), duration of mechanical ventilation, oxygenation index (PaO(2)/FiO(2)), white blood cell count and number of involved organs between the died and survived children (P<0.05 or 0.01). The Cox multiple factors analysis showed that the age (HR 3.924~3.938), primary disease (HR=1.817) and PCIS (HR=0.469) were the risk factors of death. CONCLUSIONS: The peak of death usually appears within 3 days after ARDS. Age, primary disease and PCIS are the independent factors of prognosis in children with ARDS.