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Overactivation of the mitogen-activated protein kinase (MAPK) pathway is a critical driver of many human cancers. However, therapies directly targeting this pathway lead to cancer drug resistance. Resistance has been linked to compensatory RAS overexpression, but the mechanisms underlying this response remain unclear. Here, we find that MEK inhibitors (MEKi) are associated with an increased translation of the KRAS and NRAS oncogenes through a mechanism involving dissolution of processing body (P-body) biocondensates. This effect is seen across different cell types and is extremely dynamic since removal of MEKi and ERK reactivation result in reappearance of P-bodies and reduced RAS-dependent signaling. Moreover, we find that P-body scaffold protein levels negatively impact RAS expression. Overall, we describe a new feedback loop mechanism involving biocondensates such as P-bodies in the translational regulation of RAS proteins and MAPK signaling.

Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy.We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55).We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

Background Mucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and therefore virus shedding. Parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior mucosal infection compared with injections of a parenteral vaccine in a mucosally naive subject. We investigated whether this was also the case for the BNT162b2 coronavirus disease 2019 (COVID-19) messenger RNA vaccine. Methods Twenty recovered COVID-19 subjects (RCSs) and 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–naive subjects were vaccinated with, respectively, 1 and 2 doses of the BNT162b2 COVID-19 vaccine. Nasal epithelial lining fluid (NELF) and plasma were collected before and after vaccination and assessed for immunoglobulin G (IgG) and IgA antibody levels to Spike and for their ability to neutralize binding of Spike to angiotensin-converting enzyme-2 receptor. Blood was analyzed 1 week after vaccination for the number of Spike-specific antibody-secreting cells (ASCs) with a mucosal tropism. Results All RCSs had both nasal and blood SARS-CoV-2–specific antibodies at least 90 days after initial diagnosis. In RCSs, a single dose of vaccine amplified preexisting Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including Delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2–naive subjects after 2 vaccine doses compared with RCSs after 1 dose. Conclusions Mucosal immune response to the SARS-CoV-2 Spike protein is higher in RCSs after a single vaccine dose compared with SARS-CoV-2–naive subjects after 2 doses.

Reliable biomarkers to predict the outcome and treatment response of estrogen receptor (ER)-negative breast cancer (BC) are urgently needed. Since immune-related signaling plays an important role in the tumorigenesis of ER-negative BC, we asked whether Notch genes, alone or in combination with other immune genes, can be used to predict the clinical outcome and immune checkpoint blockade (ICB) for this type of cancer.We analyzed transcriptome data of 6918 BC samples from five independent cohorts, 81 xenograft triple-negative BC tumors that respond differently to ICB treatment and 754 samples of different cancer types from patients treated with ICB agents.We found that among four Notch genes, the expression levels of NOTCH1 and NOTCH4 were positively associated with recurrence of ER-negative BC, and that combined expression of these two genes (named Notch14) further enhanced this association, which was comparable with that of the Notch pathway signature. Analysis of 1182 immune-related genes revealed that the expression levels of most HLA genes, particularly HLA-DMA and -DRA, were reversely associated with recurrence in ER-negative BC with low, but not high Notch14 expression. A combined expression signature of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA was more prognostic for ER-negative and triple-negative BCs than previously reported immune-related signatures. Furthermore, we found that the expression levels of these four genes were also synergistically associated with T cell exclusion score, infiltration of specific T cells and ICB efficacy in ER-negative BC, thereby providing a potential molecular mechanism for the synergistic effect of these genes on BC.Our data indicate that a gene signature composed of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA may serve as a potential promising biomarker for predicting ICB therapy efficacy and recurrence in ER-negative/triple-negative BCs.

BACKGROUND: Immunotherapy deeply changed the treatment paradigm of advanced non-small cell lung cancer (NSCLC) in the past years. However, the objective response rate (ORR) after immunotherapy is about 20–30% of NSCLC patients. Therefore, identification of predictive biomarkers is crucial for selecting patients with NSCLC who would most benefit from programmed cell death receptor protein 1 (PD-1) inhibitor-based immunotherapy. METHODS: We retrospectively collected medical records and thioredoxin reductase (TrxR) data from 90 patients with a NSCLC who received PD-1 inhibitor-based combination therapy. Serum biomarkers were also measured at 6- and 12-week post-treatment and compared with their baseline values. Associations between changes in serum biomarkers, clinical characteristics and treatment efficacy were evaluated using univariate tests. The patients who were still alive were followed up remotely by phone or email to assess survival. The association between serum biomarkers and TrxR with overall survival (OS) and progression-free survival (PFS) were assessed by univariate and multivariate Cox proportional hazard regression. Nomogram prediction models were constructed using factors associated with PFS and OS, respectively. RESULTS: The median follow-up time among the 90 patients was 19.7 (range, 13.6 to 25.8) months. Median PFS and OS were 13.6 [95% confidence interval (CI): 13.5 to 13.7] and 19.7 (95% CI: 13.6 to 25.8) months, respectively. Patients with decreased carcinoembryonic antigen (CEA), albumin (Alb), and TrxR values at 6- and 12-week post-treatment compared to baseline had statistically significantly improved disease remission rates (P

At a time when complex diseases affect globally 280 million people and claim 14 million lives every year, there is an urgent need to rapidly increase our knowledge into their underlying etiologies. Though critical in identifying the people at risk, the causal environmental factors (microbiome and/or pollutants) and the affected pathophysiological mechanisms are not well understood. Herein, we consider the variations of autophagy-related (ATG) genes at the heart of mechanisms of increased susceptibility to environmental stress. A comprehensive autophagy genomic resource is presented with 263 single nucleotide polymorphisms (SNPs) for 69 autophagy-related genes associated with 117 autoimmune, inflammatory, infectious, cardiovascular, neurological, respiratory, and endocrine diseases. We thus propose the term ‘autophagopathies’ to group together a class of complex human diseases the etiology of which lies in a genetic defect of the autophagy machinery, whether directly related or not to an abnormal flux in autophagy, LC3-associated phagocytosis, or any associated trafficking. The future of precision medicine for common diseases will lie in our ability to exploit these ATG SNP x environment relationships to develop new polygenetic risk scores, new management guidelines, and optimal therapies for afflicted patients. Abbreviations: ATG, autophagy‐related; ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementia; ccRCC, clear cell renal cell carcinoma; CD, Crohn disease; COPD, chronic obstructive pulmonary disease; eQTL, expression quantitative trait loci; HCC, hepatocellular carcinoma; HNSCC, head and neck squamous cell carcinoma; GTEx, genotype-tissue expression; GWAS, genome-wide association studies; LAP, LC3-associated phagocytosis; LC3-II, phosphatidylethanolamine conjugated form of LC3; LD, linkage disequilibrium; LUAD, lung adenocarcinoma; MAF, minor allele frequency; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSCLC, non-small cell lung cancer; OS, overall survival; PtdIns3K CIII, class III phosphatidylinositol 3 kinase; PtdIns3P, phosphatidylinositol-3-phosphate; SLE, systemic lupus erythematosus; SNPs, single-nucleotide polymorphisms; mQTL, methylation quantitative trait loci; ULK, unc-51 like autophagy activating kinase; UTRs, untranslated regions; WHO, World Health Organization.

Supplementary Figure from Cancer Induces a Stress Ileopathy Depending on β-Adrenergic Receptors and Promoting Dysbiosis that Contributes to Carcinogenesis

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of β-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies.Significance:Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a β-adrenergic receptor–dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.–related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy.This article is highlighted in the In This Issue feature, p. 873

Purpose: To identify genetic changes that could drive cancer pathogenesis in never and ever smokers with lung adenocarcinoma.Experimental Design: We analyzed the copy number and gene expression profiles of lung adenocarcinomas in 165 patients and related the alterations to smoking status. Having found differences in the tumor profiles, we integrated copy number and gene expression data from 80 paired samples.Results: Amplifications at 8q24.12 overlapping MYC and ATAD2 were more frequent in ever smokers. Unsupervised analysis of gene expression revealed two groups: in the group with mainly never smokers, the tumors expressed genes common to normal lung; in the group with more ever smokers, the tumors expressed “proliferative” and “invasive” gene clusters. Integration of copy number and gene expression data identified one module enriched in mitotic genes and MYC targets. Its main associated modulator was ATAD2, a cofactor of MYC. A strong dose–response relationship between ATAD2 and proliferation-related gene expression was noted in both never and ever smokers, which was verified in two independent cohorts. Both ATAD2 and MYC expression correlated with 8q24.12 amplification and were higher in ever smokers. However, only ATAD2, and not MYC, overexpression explained the behavior of proliferation-related genes and predicted a worse prognosis independently of disease stage in a large validation cohort.Conclusions: The likely driving force behind MYC contribution to uncontrolled cell proliferation in lung adenocarcinoma is ATAD2. Deregulation of ATAD2 is mainly related to gene amplification and is more frequent in ever smokers. Clin Cancer Res; 18(20); 5606–16. ©2012 AACR.

Quantification of organotypic invasion assay screening.

PDF file, 63K, Enrichment q-values of ATAD2-associated modules in the LG cohort for cluster f genes, MYC target genes and GO term 'cell cycle process'.

Purpose: Despite research efforts to develop more effective diagnostic and therapeutic approaches, malignant pleural mesothelioma (MPM) prognosis remains poor. The assessment of tumor response to therapy can be improved by a deeper phenotypical classification of the tumor, with emphasis on its clinico-biological heterogeneity. The identification of molecular profiles is a powerful approach to better define MPM subclasses and targeted therapies.Experimental Design: Molecular subclasses were defined by transcriptomic microarray on 38 primary MPM cultures. A three-gene predictor, identified by quantitative reverse transcription PCR, was used to classify an independent series of 108 frozen tumor samples. Gene mutations were determined in BAP1, CDKN2A, CDKN2B, NF2, and TP53. Epithelial-to-mesenchymal transition (EMT) markers were studied at the mRNA and protein levels.Results: Unsupervised hierarchical clustering on transcriptomic data defined two robust MPM subgroups (C1 and C2), closely related to prognosis and partly to histologic subtypes. All sarcomatoid/desmoplastic MPM were included in the C2 subgroup. Epithelioid MPM were found in both subgroups, with a worse survival prognosis in the C2 subgroup. This classification and its association with histologic subtypes and survival were validated in our independent series using the three-gene predictor. Similar subgroups were found after classification of other MPM series from transcriptomic public datasets. C1 subgroup exhibited more frequent BAP1 alterations. Pathway analysis revealed that EMT was differentially regulated between MPM subgroups. C2 subgroup is characterized by a mesenchymal phenotype.Conclusions: A robust classification of MPM that defines two subgroups of epithelioid MPM, characterized by different molecular profiles, gene alterations, and survival outcomes, was established. Clin Cancer Res; 20(5); 1323–34. ©2014 AACR.

XLS file - 11306KB, Pathway analysis.

XLS file - 212KB, Sample annotations and gene expression.

PDF file - 58KB, Primer sequences used for BAP1 and CDKN2B gene analysis.

Supplemental files

PDF file, 64K, Different characteristics between never smokers and ever smokers.

Supplemental data

PDF file - 356KB, This file contains supplementary figures S1-S7. Figure S1: Flowchart depicting the analyses carried out to establish and validate a molecular classification of MPM; Figure S2: Unsupervised molecular subgroups in the discovery series; Figures S3: Recurrent regions of chromosomal alteration in MPM molecular subgroups; Figures S4: Identification of the three-gene predictor; Figure S5: Unsupervised molecular subgroups in the two public series; Figure S6: Distribution of histological subtypes and overall survival differences between MPM molecular subgroups in the two public series; Figure S7: Differential mRNA expression of epithelial-to-mesenchymal transition (EMT) markers in MPM molecular subgroups.

PDF file, 44K, LitVAn significant terms for gene clusters f and i in the unsupervised analysis of gene expression.

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PDF file, 528K, Overview of module 1 to 27 profiles. Samples are sorted according to the regulatory programs found with CONEXIC in the LG cohort.

XLS file 669K, This file contains supplementary table S4: Convergence between microRNA and mRNA deregulation

In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo. These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg. Cancer Res; 78(18); 5229–42. ©2018 AACR.

Purpose: Pathologic TNM staging is currently the best prognostic factor for non‐small cell lung carcinoma (NSCLC). However, even in early-stage NSCLC, the recurrence rates after surgery range from 25% to 50%. The preoperative detection of circulating tumor cells (CTC) could be useful to tailor new therapeutic strategies in NSCLC. We assessed the presence of CTC in NSCLC patients undergoing surgery, using cytologic analyses, after their isolation by size of epithelial tumor cells (ISET method). The presence and the number of CTCs were considered and correlated with clinicopathologic parameters including patient follow-up.Experimental Design: Of the 247 blood samples tested, 208 samples were from patients with resectable NSCLC and 39 from healthy subjects. The mean follow-up was 24 months. An image of detected cells with presumably nonhematologic features [initially defined as “circulating nonhematologic cells” (CNHC)] was recorded. The presence of CNHC was assessed blindly and independently by 10 cytopathologists, using cytologic criteria of malignancy on stained filters. The count of detected CNHCs was made for each filter.Results: One hundred two of 208 (49%) patients showed CNHCs corresponding to CNHC with malignant cytopathologic features in 76 of 208 (36%) cases. CNHCs were not detected in the control group. A level of 50 or more CNHCs corresponding to the third quartile was associated with shorter overall and disease-free-survival, independently of disease staging, and with a high risk of recurrence and death in early-stage I + II-resectable NSCLC.Conclusion: A high percentage of NSCLC patients show preoperative detection of CNHC by the ISET method. The presence and level of 50 or more CNHCs are associated with worse survival of patients with resectable NSCLC. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

PDF file, 188K, Module network views of the original and the modified module 62 in the 68 lung adenocarcinomas from the Ding cohort (GSE12667) and of the modified module 62 in the LG cohort.

Legends of Supplementary Figures and Tables PDF file 134K, This file contains the legends for supplementary figures S1-S7 and tables S1-S4

PDF file, 686K, Heat maps of genes of the proliferative cluster (cluster f) according to ATAD2 expression, smoking status-associated discrete variables and disease stage in the LG cohort.

Supplementary Figures S1-S2; Supplementary Table S1.

PDF file, 40K, Correlation of cluster f mitotic spindle genes with ATAD2 or MYC in the LG cohort.

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Supplementary Figures 4-5 from Microphthalmia-Associated Transcription Factor Controls the DNA Damage Response and a Lineage-Specific Senescence Program in Melanomas

Supplementary Figures. Fig.S1: Additional confocal images showing that expression of EFA6Bvsvg in MCF7 cells restores normal apico-basal polarity and promotes lumen formation Fig.S2: Analysis of the specificity of EFA6B repression using siRNAs Fig.S3: Additional confocal images showing that repression of EFA6B in MCF7 cells abrogates the assembly of TJ and leads to unorganized aggregates Fig.S4: Exogenous expression of EFA6Bvsvg abrogates the TGFβ-induced EMT in MDCK cells Fig.S5: Analysis of EFA6B/PSD4 mutations and copy number alterations from the cBioPortal website

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Supplementary Figure 9 from Microphthalmia-Associated Transcription Factor Controls the DNA Damage Response and a Lineage-Specific Senescence Program in Melanomas

Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance. Cancer Res; 73(14); 4311–22. ©2013 AACR.