The BRAF–MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells

The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAFV600E). MEK inhibitor U0126 or RNA interference (RNAi) for BRAFV600E decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor α by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAFV600E RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAFV600E and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system

Factors Associated with Cancer-Related Pain Requiring High-Dose Opioid Use in Palliative Cancer Patients

Background:There are no universal tools to predict the necessity of high-dose opioid use for cancer-related pain. Early recognition and interventions for intractable cancer pain could minimize the distress of palliative patients.Objective:We sought to identify the clinical factors associated with high-dose opioid use in advanced cancer patients to recognize palliative patients who would develop intractable cancer pain, as early as possible.Setting/Subjects:Among 385 in-hospital cancer patients from April 1, 2014 to July 31, 2019, who were referred to the palliative care team for cancer-related pain, clinical factors significantly correlated to high-dose opioid use were retrospectively analyzed.Measurements:We conducted a multiple logistic regression analysis to identify variables significantly related to high-dose opioid use (>120 mg/day oral morphine equivalent dose).Results:Independent factors of high-dose opioid use included younger age (odds ratio [OR] 0.965, 95% confidence interval [CI] 0.944-0.986, p = 0.001), respiratory cancers (OR 1.882, 95% CI 1.069-3.312, p < 0.001), and opioid switch (OR 2.869, 95% CI 1.497-5.497, p = 0.001). The percentage of correct classifications of the regression equation was 86.9%.Conclusions:Younger age, respiratory cancers, and opioid switch were related to high-dose opioid use. Our findings may help palliative caregivers to deal with intractable cancer pain in palliative patients, and thus relieve their distress

Theoretical Study of the Reaction Mechanism of Phenol–Epoxy Ring-Opening Reaction Using a Latent Hardening Accelerator and a Reactivity Evaluation by Substituents

The mechanism of the phenol–epoxide ring-opening reaction using tetraphenylphosphonium-tetraphenylborate (TPP-K) was investigated using the density functional theory (DFT) method. The reaction was initiated by breaking the P-B bond of TPP-K. The generated tetraphenylborate (TetraPB−) reacted with phenol to form a phenoxide ion, which combined with tetraphenylphosphonium (TPP+) to produce the active species, i.e., tetraphenylphosphonium phenolate (TPP-OPh). The phenoxide ion in TPP-OPh nucleophilically attacked the epoxide. Simultaneously, the H atom in the phenolic OH group moved to the O atom of the ring-opened epoxide. The formed phenoxide ion bound to TPP+ again, and TPP-OPh was regenerated. The rate-determining steps in the reaction were the cleavage of the P-B bond and the triphenylborane-forming reaction. The free energies of activation were calculated to be 36.3 and 36.1 kcal/mol, respectively. It is also suggested that these values in the rate-determining steps could be manipulated by substituents introduced on the Ph group of TetraPB−. Based on these results, it is possible to construct new design guidelines for latent hardening accelerators such as TPP-K

RAS-Mitogen-Activated Protein Kinase Signal Is Required for Enhanced PD-L1 Expression in Human Lung Cancers.

Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers

Oncogenic epidermal growth factor receptor signal-induced histone deacetylation suppresses chemokine gene expression in human lung adenocarcinoma

Abstract Epidermal growth factor receptor (EGFR)-mutated (mt) lung adenocarcinoma (LA) is refractory to immune checkpoint inhibitors (ICIs). However, the mechanisms have not been fully elucidated. CD8+ T cell infiltration was significantly lower in EGFR-mt than in EGFR-wild-type LA, which was associated with suppression of chemokine expression. Since this T cell-deserted tumor microenvironment may lead to the refractoriness of ICIs against EGFR-mt LA, we investigated the mechanism by focusing on the regulation of chemokine expression. The expression of C-X-C motif ligand (CXCL) 9, 10 and 11, which constitute a gene cluster on chromosome 4, was suppressed under EGFR signaling. The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) revealed open chromatin peaks near this gene cluster following EGFR-tyrosine kinase inhibitor (TKI) treatment. The histone deacetylase (HDAC) inhibitor recovered the expression of CXCL9, 10 and 11 in EGFR-mt LA. Nuclear HDAC activity, as well as histone H3 deacetylation, were dependent on oncogenic EGFR signaling. Furthermore, the Cleavage Under Targets and Tagmentation (CUT & Tag) assay revealed a histone H3K27 acetylation peak at 15 kb upstream of CXCL11 after treatment with EGFR-TKI, which corresponded to one of the open chromatin peaks detected by ATAC-seq. The data suggest that EGFR-HDAC axis mediates silencing of the chemokine gene cluster through chromatin conformational change, which might be relevant to the ICI resistance by creating T cell-deserted tumor microenvironment. Targeting this axis may develop a new therapeutic strategy to overcome the ICI resistance of EGFR-mt LA

Towards the Development of Synthetic Routes Using Theoretical Calculations: An Application of In Silico Screening to 2,6-Dimethylchroman-4-one

This study describes an attempt to develop a synthetic route using theoretical calculations, i.e., in silico synthesis route development. The KOSP program created four potential synthetic routes for generating 2,6-dimethylchroman-4-one. In silico screening of these four synthetic routes was then performed. In silico screening involves theoretical analysis of synthetic routes prior to actual experimental work. A synthetic route using the Mitsunobu reaction had already been reported by Hoddgets et al. Theoretical investigations were also conducted on two SNAr reactions as well as a Michael reaction before they were examined experimentally. In silico screening using DFT calculations indicated that only the Michael reaction was likely to produce the target. Experimental work confirmed that the target was obtained in a yield of 76.4% using the Michael reaction. The other two routes, except for the Mitsunobu reaction, failed to generate the target. Our results demonstrate that theoretical calculations can be used to narrow down the number of experiments that need to be conducted when developing novel synthetic routes