scholarly journals Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms

2020 ◽  
Vol 8 (1) ◽  
pp. e000441 ◽  
Author(s):  
Alexander J Neuwelt ◽  
Abigail K Kimball ◽  
Amber M Johnson ◽  
Benjamin W Arnold ◽  
Bonnie L Bullock ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Nsclc Cell ◽  
Erk Signaling ◽  
Mhc I ◽  
Mek Inhibitors ◽  
Mhc Ii ◽  
Programmed Death ◽  
Mitogen Activated Protein

BackgroundProgrammed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression.MethodsResponsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis.ResultsSingle-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone.ConclusionsThese studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.

scholarly journals Identification and Exploration of Serine Peptidase Inhibitor Kazal Type 1 (SPINK1) as a Potential Biomarker Correlated with the Progression of Non-Small Cell Lung Cancer

2020 ◽  
Author(s):  
Xinxin Zhang ◽  
Rongshuang Ai ◽  
Zhiqiang Ding ◽  
Zimeng Wang ◽  
Qian He ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Lines ◽  
Small Cell ◽  
Nsclc Cell ◽  
Erk Signaling ◽  
Small Cell Lung ◽  
Potential Biomarker ◽  
Mrna And Protein Expression ◽  
Nsclc Patients ◽  
Normal Controls

Abstract Background Lung cancer is one of the most common malignancies worldwide and is the leading cause of cancer-related death. Approximately 85% of lung cancer patients represent a group of histological subtypes collectively known as non-small cell lung cancer (NSCLC).Methods To explore the molecular mechanisms underlying tumorigenesis and progression of NSCLC, mRNA expression profiles were downloaded from GEO database (GSE19804, GSE18842, GSE27262, and GSE43458) and differentially expressed genes (DEGs) in NSCLC tissues were analyzed by GEO2R. DEGs in NSCLC tissues were further analysed in TCGA (The Cancer Genome Atlas), GTEx (The Genotype-Tissue Expression Project) and IST (In Silico Transcriptomics) online databases. Serum of NSCLC patients and normal controls were collected and serum concentration of SPINK1 were analysed by ELISA. mRNA and protein expression levels of SPINK1 were analysed by qRT-PCR and western assays. siRNA targeting SPINK1 and normal controls were used for the silence of SPINK1 and GAPDH. CCK-8 assays were employed for cell proliferation detection. Flow cytometric analysis and western blot assays were conducted to assess cell cycle distribution and apoptosis. Western blot assays were performed for the evaluation of cell autophagy and signaling pathways.Results Among these DEGs, SPINK1 was distinctively up-regulated in NSCLC tissues, which was further validated in TCGA, GTEx and IST databases. Furthermore, serum SPINK1 concentration notably increased in NSCLC patients compared with normal controls. Besides, both mRNA and protein expression levels of SPINK1 significantly increased in human NSCLC cell lines A549 and H1299 compared with normal human bronchial epithelial cell line HBE. Silence of SPINK1 significantly inhibited proliferation of NSCLC cell lines, and exogenous addition of rhSPINK1 partially rescued proliferation suppressed by endogenous knockdown of SPINK1. Mechanistically, silence of SPINK1 could inhibit MEK/ERK signaling pathway, while rhSPINK1 could activate MEK/ERK signaling pathway and then promote proliferation of NSCLC cell lines. In addition, silence of SPINK1 also could activate cell autophagy and apoptosis.Conclusions Overall, our results suggested that SPINK1 may be a potential biomarker correlated with the progression of NSCLC.

Characteristics of NSCLCs harboring NRAS mutations.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7532-7532
Author(s):  
Kadoaki Ohashi ◽  
Lecia V. Sequist ◽  
Martin Sos ◽  
Xi Chen ◽  
Charles M. Rudin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lung Cancer ◽  
Cell Lines ◽  
Expression Profiles ◽  
Nsclc Cell ◽  
Disease Stage ◽  
Smoking History ◽  
Egfr Mutations ◽  
Targeted Agents ◽  
Mek Inhibitors

7532 Background: We sought to determine the frequency and clinical characteristics of patients with non-small cell lung cancers (NSCLCs) harboring NRAS mutations. We used preclinical models to identify targeted therapies likely to be of benefit against NRAS mutant lung cancer cells. Methods: We reviewed data in the Catalogue of Somatic Mutations in Cancer (COSMIC) and clinical history from patients with NSCLC whose tumors underwent systematic screening for driver mutations including NRAS. Patient characteristics examined included age, gender, race, smoking history, disease stage, treatment history, and overall survival (OS). 6 NSCLC cell lines with NRAS mutations were screened for sensitivity against multiple targeted agents. Gene expression was profiled using Affymetrix U133A arrays in 5 NRAS mutant NSCLC cell lines, 8 with EGFR mutations and 17 with KRAS mutations. Results: Among 4524 patients with NSCLC tested, NRAS mutations were present in 29 (0.64%). The types of substitutions found were Q61H/K/L/R and G12A/C/D/R/S, with NRAS Q61L the most common (n=14; 48%). One tumor had a concurrent KRAS mutation. 83% had adenocarcinoma histology, with no significant differences in gender. While 90% of patients were former or current smokers, smoking-related G:C>T:A transversions were significantly less frequent in NRAS than in KRAS-mutant NSCLC (KRAS: 66%, NRAS: 13%, p<0.05). Systemic chemotherapy showed limited efficacy in 7 patients with metastatic disease (median OS 7 mos). 5 of 6 NRAS mutant lung cancer cell lines were sensitive to the MEK inhibitors, AZD6244 and GSK1120212, while other targeted agents (against EGFR, ALK, MET, IGF-1R, PIK3CA, BRAF) were minimally effective. Gene expression profiles of NRAS mutant cell lines were distinct from those with KRAS or EGFR mutations. Conclusions: NRAS mutations define a distinct subset of NSCLCs (~1%) with potential sensitivity to MEK inhibitors. While NRAS gene mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking.

scholarly journals Cytotoxic activity of IMMUNEPOTENT CRP against non-small cell lung cancer cell lines

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7759 ◽  
Author(s):  
Ana Carolina Martinez-Torres ◽  
Luis Gomez-Morales ◽  
Alan B. Martinez-Loria ◽  
Ashanti Concepcion Uscanga-Palomeque ◽  
Jose Manuel Vazquez-Guillen ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Death ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cancer Cell ◽  
Dna Degradation ◽  
Cancer Cell Lines ◽  
Nsclc Cell ◽  
Ros Production

Background IMMUNEPOTENT-CRP® (I-CRP) is a bovine dialyzable leukocyte extract containing transfer factor. It is a cost-effective, unspecific active immunotherapy that has been used in patients with non-small cell lung cancer (NSCLC) as an adjuvant to reduce the side-effects of chemotherapy and radiotherapy, and has shown cytotoxic activity in vitro on different cancer cell lines. However, its mechanism of action against lung cancer cells has not been assessed. Therefore, the objective of this work was to assess the cytotoxic mechanism of I-CRP on lung cancer cell lines. Methods We assessed cell viability through MTT assay on the NSCLC cell lines A549, A427, Calu-1, and INER-51 after treatment with I-CRP. To further understand the mechanisms of cell viability diminution we used fluorescence-activated cell sorting to evaluate cell death (annexin-V and propidium iodide [PI] staining), cell cycle and DNA degradation (PI staining), mitochondrial alterations (TMRE staining), and reactive oxygen species (ROS) production (DCFDA staining). Additionally, we evaluated caspase and ROS dependence of cell death by pretreating the cells with the pan-caspase inhibitor Q-VD-OPH and the antioxidant N-acetylcysteine (NAC), respectively. Results Our data shows that I-CRP is cytotoxic to NSCLC cell lines in a dose and time dependent manner, without substantial differences between the four cell lines tested (A549, A427, Calu-1, and INER-51). Cytotoxicity is induced through regulated cell death and cell cycle arrest induction. I-CRP-induced cell death in NSCLC cell lines is characterized by DNA degradation, mitochondrial damage, and ROS production. Moreover, cell death is independent of caspases but relies on ROS production, as it is abrogated with NAC. Conclusion Altogether, these results improve the knowledge about the cytotoxic activity of I-CRP on NSCLC cells, indicating that cell death, cell cycle arrest, DNA degradation and mitochondrial damage are important features, while ROS play the main role for I-CRP mediated cytotoxicity in lung cancer cells.

scholarly journals Mesoporous silica particles functionalized with newly extracted fish oil (Omeg@Silica) inhibit lung cancer cell growth

2021 ◽  
Author(s):  
Caterina Di Sano ◽  
Claudia D’Anna ◽  
Antonino Scurria ◽  
Claudia Lino ◽  
Mario Pagliaro ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Mesoporous Silica ◽  
Fish Oil ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Nsclc Cell ◽  
Lung Cancer Cell ◽  
Antitumor Effects

AbstractOmeg@Silica microparticles consisting of whole fish oil rich in omega-3 lipids, vitamin D3 and zeaxanthin extracted with biobased limonene from anchovy fillet leftovers (AnchoisOil) encapsulated within mesoporous silica particles are highly effective in modulating oxidative stress, mitochondrial damage or in promoting antitumor effects in lung cancer cells. A panel of three different human non-small cell lung cancer (NSCLC) cell lines (A549, Colo 699 and SKMES) was used. Cancer cells were treated with AnchoisOil dispersed in ethanol (10 and 15 μg/ml) or encapsulated in silica, and cell cycle, reactive oxigen species (ROS) and mitochondrial stress (MitoSOX) assessed by flow cytometry. The effects on long-term proliferation (clonogenic assay) were also evaluated. The sub-micron Omeg@Silica microparticles were more effective than fish oil alone in increasing ROS and mitocondrial damage, in altering cell cycle as well as in reducing colony formation ability in the tested lung cancer cell lines. These results suggest that Omeg@Silica mesoporous silica functionalized with whole fish oil has antitumor effects in NSCLC cell lines and support its investigation in lung cancer therapy.

scholarly journals Increased EGFR Phosphorylation Correlates with Higher Programmed Death Ligand-1 Expression: Analysis of TKI-Resistant Lung Cancer Cell Lines

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Kenichi Suda ◽  
Leslie Rozeboom ◽  
Koh Furugaki ◽  
Hui Yu ◽  
Mary Ann C. Melnick ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Molecular Mechanisms ◽  
Cancer Cell Lines ◽  
Lung Cancer Cell ◽  
Programmed Death Ligand 1 ◽  
Programmed Death ◽  
L1 Protein

Despite the recent development of immunotherapies that target programmed death-1 (PD-1) or programmed death ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) treatment, these therapies are less effective in NSCLC patients with epidermal growth factor receptor (EGFR) mutations. However, the molecular mechanisms underlying this lower efficacy of immunotherapies in EGFR mutant lung cancers are still unclear. In this study, we analyzed PD-L1 protein expression in lung cancer cell lines with EGFR mutations prior to and after acquisition of resistance to EGFR tyrosine kinase inhibitors (TKIs). We found that parental lung cancer cell lines harboring EGFR mutations showed negative (PC9 and H3255 cells) and positive (HCC827 cells) staining for PD-L1 by immunohistochemistry. Comparing PD-L1 expression between EGFR-TKI resistant cell lines and their parental cells, we found that increased phosphorylation of EGFR was related to increased expression of PD-L1. Increased phosphorylation of EGFR was accompanied by the T790M secondary mutation. Acquired resistance cells with MET amplification or EGFR loss both showed decreased phosphorylation of EGFR and decreased PD-L1 expression. Our results indicate that lung cancer cell lines with EGFR mutations (parental cells) do not harbor high PD-L1 protein expression. In addition, EGFR phosphorylation affects PD-L1 expression after acquisition of resistance to EGFR-TKIs.

The Key microRNAs Regulated the Development of Non-small Cell Lung Cancer by Targeting TGF-β-induced epithelial–mesenchymal Transition

2019 ◽  
Vol 22 (4) ◽  
pp. 238-244 ◽  
Author(s):  
Gang Chen ◽  
Bo Ye
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lines ◽  
Cell Lung Cancer ◽  
Small Cell ◽  
Nsclc Cell ◽  
Normal Lung ◽  
Migration And Invasion ◽  
Small Cell Lung ◽  
Mesenchymal Transition

Purpose: Epithelial-to-Mesenchymal Transition (EMT) was reported to play a key role in the development of Non-Small Cell Lung Cancer (NSCLC). The process of EMT is regulated by the changes of miRNAs expression. However, it is still unknown which miRNA changed the most in the process of canceration and whether these changes played a role in tumor development. Methods: A total of 36 SCLC patients treated in our hospital between 11th, 2015 and 10th, 2017 were enrolled. The samples of cancer tissues and paracancer tissues of patients were collected and analyzed. Then, the miRNAs in normal lung cells and NSCLC cells were also analyzed. In the presence of TGF-β, we transfected the miRNA mimics or inhibitor into NSCLC cells to investigate the role of the significantly altered miRNAs in cell migration and invasion and in the process of EMT. Results: MiR-330-3p was significantly up-regulated in NSCLC cell lines and tissues and miRNA- 205 was significantly down-regulated in NSCLC cell lines and NSCLC tissues. Transfected miRNA-205 mimics or miRMA-330-3p inhibitor inhibited the migration and invasion of NCIH1975 cell and restrained TGF-β-induced EMT in NSCLC cells. Conclusion: miRNA-330-3p and miRNA-205 changed the most in the process of canceration in NSCLC. Furthermore, miR-330-3p promoted cell invasion and metastasis in NSCLC probably by promoting EMT and miR-205 could restrain NSCLC likely by suppressing EMT.

scholarly journals Protein citrullination as a source of cancer neoantigens

2021 ◽  
Vol 9 (6) ◽  
pp. e002549
Author(s):  
Hiroyuki Katayama ◽  
Makoto Kobayashi ◽  
Ehsan Irajizad ◽  
Alejandro Sevillarno ◽  
Nikul Patel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Immune Response ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Family Members ◽  
Immunohistochemical Analysis ◽  
Cancer Cell Lines ◽  
Mhc Ii

BackgroundCitrulline 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.MethodsProtein 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.ResultsProteomic 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).ConclusionsAn immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

scholarly journals Nicotine promotes the development of non-small cell lung cancer through activating LINC00460 and PI3K/Akt signaling

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Hongying Zhao ◽  
Yu Wang ◽  
Xiubao Ren
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Akt Signaling ◽  
Small Cell ◽  
Nsclc Cell ◽  
Expression Level ◽  
In Vitro Experiments ◽  
Small Cell Lung

Abstract Objective: Nicotine, the main ingredient in tobacco, is identified to facilitate tumorigenesis and accelerate metastasis in tumor. Studies in recent years have reported that long intergenic non-protein coding RNA 460 (LINC00460) is strongly associated with lung cancer poor prognosis and nicotine dependence. Nonetheless, it is unclear whether nicotine promotes the development of lung cancer through activation of LINC00460. Methods: We determined that LINC00460 expression in lung cancer tissues and the prognosis in patients with non-small cell lung carcinoma (NSCLC) using Gene Expression Profiling Interactive Analysis (GEPIA) website and The Cancer Genome Atlas (TCGA) database. Through in vitro experiments, we studied the effects of nicotine on LINC00460 in NSCLC cells lines using Cell Counting Kit-8 (CCK-8), transwell test, flow cytometry, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot assays. Results: We identified the significant up-regulated expression level of LINC00460 in NSCLC tissues and cell lines, especially, the negative correlation of LINC00460 expression level with overall survival (OS). In in vitro experiments, LINC00460 was overexpressed in NSCLC cell lines under nicotine stimulation. Nicotine could relieve the effect of LINC00460 knockdown on NSCLC cell proliferation, migration and apoptosis. The same influence was observed on PI3K/Akt signaling pathway. Conclusions: In summary, this is the first time to examine the potential roles of LINC00460 in lung cancer cell proliferation, migration and apoptosis induced by nicotine. This may help to develop novel therapeutic strategies for the prevention and treatment of metastatic tumors from cigarette smoke-caused lung cancer by blocking the nicotine-activated LINC00460 pathway.

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