Prognostic Marker In Non-small Cell Lung Cancer Research Articles

Dual-specificity phosphatase 23 functions as a promising prognostic biomarker in non-small cell lung cancer.

The mechanical remodeling of tumor microenvironment is critical for non-small cell lung cancer (NSCLC) progression. Dual-specificity phosphatase 23 (DUSP23) has been previously identified as a mechano-responsive gene, but its role in NSCLC progression remains unknown. We aim to elucidate the clinical significance of DUSP23 in NSCLC progression. We analyzed the expression of DUSP23 in cancer using polyacrylamide hydrogels designed to mimic the stiffness of normal (soft; ~0.5kPa) and cancerous (stiff; ~40kPa) tissues. The prognostic significance of DUSP23 expression in patients was examined using public databases. Additionally, we conducted various cell-based assays and transcriptomic analyses in DUSP23-silenced NSCLC cell lines. A risk score prognosis model was constructed using univariate Cox regression and Kaplan-Meier analysis. Our findings show that DUSP23 is upregulated in stiff matrices and is highly associated with poor prognosis in patients with solid cancers such as NSCLC and breast cancer. Silencing of DUSP23 resulted in decreased cell proliferation and invasion. Transcriptomic profiling revealed that 182 genes were downregulated, and 230 genes upregulated following DUSP23-depletion. Notably, 182 downregulated genes were enriched in cancer-related pathways, including cell cycle progression and cytoskeleton organization. Through KEGG pathway analysis, we identified 11 cancer-related genes and developed a prognostic risk model. In this model, the high-risk group of NSCLC patients exhibited significantly shorter overall survival compared to low-risk group, based on public datasets. Our study demonstrates the clinical significance of DUSP23 as a prognostic marker in NSCLC and highlights its potential role of DUSP23 in promoting NSCLC progression.

The clinical features and prognostic implications of the co-mutated TP53 gene in advanced non-small cell lung cancer.

TP53 is a frequently mutated oncogene within non-small cell lung cancer (NSCLC). However, the clinical and prognostic significance of co-mutations in TP53 in patients with advanced NSCLC has not been fully elucidated. A total of 174 patients with advanced NSCLC were enrolled in this study. All patients were subjected to sequencing analysis of tumor-related genes and information such as PD-L1 expression, TMB, and co-mutation changes were collected. Patients were categorized into TP53 mutant and TP53 wild-type groups according to their TP53 mutation status and then statistically analyzed. TP53 mutations were the most common among all patients, accounting for 56.32%, followed by epidermal growth factor receptor mutations at 48.27%. The most common mutation sites in the TP53 mutation group were exons 5-8.TP53 mutations were significantly associated with PD-L1 and TMB levels. Univariate Cox analysis showed that gender and EGFR mutation affected the prognosis of TP53-mutated NSCLC patients, and multivariate Cox regression analysis identified EGFR mutation as an independent risk factor. The OS of NSCLC patients in the TP53 mutation group was significantly shorter than that of the TP53wt group. Survival curves in the TP53/EGFR combined mutation group showed that patients with combined EGFR mutation had a lower survival rate. TP53 mutations are associated with different clinical indicators and have important implications in clinical treatment. TP53 is a poor prognostic factor for NSCLC patients, and TP53/EGFR co-mutation will affect the survival time of patients. TP53/EGFR co-mutation may be a new prognostic marker for NSCLC.

Epidermal growth factor receptor and programmed cell death-1 expression levels in peripheral T cell subsets of patients with non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths, in part due to its late diagnosis. Increased epidermal growth factor receptor (EGFR) expression in cancer cells is associated with a poor prognosis, and EGFR tyrosine kinase inhibitors are widely used in cancer treatment. This study aimed to clarify the relationship between EGFR expression on T cells and cancer prognosis in patients with non-small cell lung cancer (NSCLC). Forty patients with NSCLC and 40 healthy volunteers were included in this study. Peripheral CD4+T helper (Th1, Th2, Th9, Th17, Th1Th17, follicular and peripheral Th) and cytotoxic T lymphocyte (CD8+follicular and peripheral T) subsets were identified with flow cytometry according to their chemokine receptors. EGFR expression on T lymphocytes in relation to overall survival (OS) was investigated in patients with NSCLC. The patients [mean age (min-max) = 64.03 (45-83); 20 stage I-III and 20 stage IV] had increased EGFR expression on CD3+T, CD4+Th, Th1, Th2, and Th17 cells compared to the controls (p < 0.05). High EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells was associated with poor OS. Also, PD-1 expression on lymphocytes, CD3+T, and Th cells was increased in patients with NSCLC compared to controls. The high expression of EGFR and PD-1 on Th cells and the reduced percentage of lymphocytes and Th cells, especially in stage IV patients with NSCLC, revealed that increased EGFR activity may trigger apoptosis of Th cells and promote the development of metastases, while high EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells may be an independent poor prognostic marker in NSCLC.

The role of leptin, resistin, and irisin levels as prognostic indicators of lung cancer in Iraqi patients

ABSTRACT Lung cancer is the most lethal malignancy and is often associated with a poor prognosis because of a lack of prognostic markers. Thus, this study aimed to determine whether irisin, resistin, and leptin could be useful biomarkers for lung cancer prognosis. The study is designed on 100 lung cancer patients, (66) patients with non-small cell lung cancer (NSCLC) and (34) patients with small cell lung cancer (SCLC). For the purpose of comparison, (66) samples as control group. Using the ELISA technique to estimate leptin, resistin, and irisin levels in patients and controls. The results indicate a significant increase in mean leptin in NSCLC and SCLC groups (10.71 ± 0.30 and 10.13 ± 0.51) ng/ml, respectively, in contrast to the control (8.26 ± 0.47) ng/ml. The mean of irisin and resistin had been significantly increased in SCLC group (5.86 ± 0.13 pg/ml and 7.25 ± 0.38 ng/ml, respectively) and NSCLC group (5.08 ± 0.09 pg/ml and 6.35 ± 0.13 ng/ml, respectively) in contrast to the control (4.13 ± 0.09 pg/ml and 3.96 ± 0.17 ng/ml, respectively). The higher levels of leptin in NSCLC patients could serve as a prognostic marker for NSCLC. The variations in Resistin and Irisin levels across different stages of lung cancer suggest that they might be useful in predicting the prognosis of lung cancer.

Detection of Oncogene Hotspot Mutations in Female NSCLC Tumor DNA and Cell-Free DNA.

Non-small-cell lung cancer (NSCLC) is the most prevalent type of lung cancer, with extensively characterized mutational spectra. Several biomarkers (such as EGFR, BRAF, KRAS gene mutations, etc.) have emerged as predictive and prognostic markers for NSCLC. Unfortunately, the quality of the available tumor biopsy and/or cytology material is not always adequate to perform the necessary molecular testing, prompting the search for alternatives. Cell-free DNA (cfDNA) found in plasma is emerging as a highly promising avenue or a supplementary method for assessing the efficacy of cancer treatments. This is especially valuable in instances where conventional biopsy specimens, like formalin-fixed, paraffin-embedded (FFPE), or freshly frozen tumor tissues prove inadequate for conducting molecular pathology analyses subsequent to the initial diagnostic procedures. By leveraging cfDNA from plasma, clinicians gain an additional tool to gauge the effectiveness of cancer therapies, thereby enhancing their ability to optimize tailored treatment strategies. In this study, 51 Lithuanian females with NSCLC were analyzed, with adenocarcinoma being the predominant pathology diagnosis in 40 cases (78%). Target mutations were identified in 38 out of 51 patients (74.5%) in tumor tissue samples, while in plasma samples, they were identified in only 10 patients' samples (19.6%). Even though we did not have enough voluminous plasma samples in our study, gene mutations were detected in plasma from ten women, three of whom were diagnosed with early stages of lung cancer (stages I and II). For these patients, the following mutations were detected: deletion in exon 19 of the EGFR gene and single nucleotide polymorphisms in the TP53 and MET genes. All other women were diagnosed with stages III or IV of lung cancer. This indicates that the later stages of cancer contribute more cfDNA in plasma, making extraction less complicated.

The value of lung function assessment and Testin expression detection in clinicopathological features and prognosis of NSCLC patients.

The aim of this study is to investigate the clinical value and potential prognostic significance of lung function assessment and Testin expression in non-small cell lung cancer (NSCLC) patients. The NSCLC patients were classified into three groups according to lung function: group of normal lung function, group of PRISm (preserved ratio impaired spirometry) (FEV1, forced expiratory volume during the first second < 80% predicted and FEV1/FVC (forced vital capacity) ≥ 70%) and group of COPD (chronic obstructive pulmonary disease) (FEV1/FVC < 70%). The pre-operational clinicopathological characteristics of these patients were recorded and the markers of systemic inflammatory response, including neutrophil to lymphocyte ratio (NLR), lymphocyte to monocyte ratio (LMR), platelet to lymphocyte ratio (PLR) and eosinophils (EOS), were compared between three groups. The expression of Testin in NSCLC samples was detected by IHC and we further explored the correlation between Testin expression and clinicopathological characteristics and prognosis of NSCLC patients. Finally, Cox regression analysis was conducted to study the prognostic factors of NSCLC patients. Of the 158 NSCLC patients, percentages of normal lung function, PRISm and COPD were 41.4%, 22.8% and 36.1%, respectively. Patients with tumor in the left lung were more likely to have pulmonary dysfunction (PRISm and COPD) than the right lung. The markers of systemic inflammatory response showed differences to various degree in the three groups and NSCLC patients with PRISm or COPD presented more unfavorable prognosis than patients with normal function. The expression of Testin correlated with lymph node metastasis, TNM stage and tumor invasion of NSCLC patients. Moreover, patients with low Testin expression exhibited poorer disease-free survival and overall survival than those with high Testin expression. In Cox regression analysis, we found that PRISm, COPD and Testin expression served as prognostic factors in NSCLC patients. The presence of COPD or PRISm influenced systemic inflammatory response and prognosis of NSCLC patients. Testin expression correlated with clinicopathological features and could be potentially used as a prognostic marker in NSCLC.

Regulatory role of the miR-142-3p/CDC25C axis in modulating autophagy in non-small cell lung cancer.

With its diverse genetic foundation and heterogeneous nature, non-small cell lung cancer (NSCLC) needs a better comprehension of prognostic evaluation and efficient treatment targeting. Bioinformatics analysis was performed of The Cancer Genome Atlas (TCGA)-NSCLC and GSE68571 dataset. Overlapping differentially expressed genes (DEGs) were used for functional enrichment analysis and constructing the protein-protein interaction (PPI) network. In addition, key prognostic genes were identified through prognostic risk models, and their expression levels were verified. The phenotypic effects of cell division cycle 25C (CDC25C) regulation on NSCLC cell lines were assessed by in vitro experiments using various techniques such as flow cytometry, Transwell, and colony formation. Protein levels related to autophagy and apoptosis were assessed, specifically examining the impact of autophagy inhibition [3-methyladenine (3-MA)] and the miR-142-3p/CDC25C axis on this regulatory system. CDC25C was identified as a key prognostic marker in NSCLC, showing high expression in tumor samples. In vitro experiments showed that CDC25C knockdown markedly reduced the capacity of cells to proliferate, migrate, invade, trigger apoptosis, and initiate cell cycle arrest. CDC25C and miR-142-3p displayed a reciprocal regulatory relationship. CDC25C reversed the inhibitory impacts of miR-142-3p on NSCLC cell cycle proliferation and progression. The synergy of miR-142-3p inhibition, CDC25C silencing, and 3-MA treatment was shown to regulate NSCLC cell processes including proliferation, apoptosis, and autophagy. MiR-142-3p emerged as a key player in governing autophagy and apoptosis by directly targeting CDC25C expression. This emphasizes the pivotal role of the miR-142-3p/CDC25C axis as a critical regulatory pathway in NSCLC.

DUSP22 suppresses tumor progression by directly dephosphorylating AKT in non-small cell lung cancer.

Protein kinase B (AKT) plays a pivotalin regulating cell migration, proliferation, apoptosis, and survival, making it a prominent target for anticancer therapy. While the kinase activity of AKT has been extensively explored, its dephosphorylation have largely remained uncharted. Herein, we aimed to unravel the molecular mechanisms governing AKT dephosphorylation, with a specific emphasis on dual-specificity phosphatases DUSP22. Our investigation sought to shed light on the potential of DUSP22 as a potential therapeutic target for non-small cell lung cancer (NSCLC). To determine the expression level of DUSP22 in NSCLC cell lines, the gene expression profiling interactive analysis (GEPIA) and Oncomine database were searched. Additionally, the effect of DUSP22 on patient survival was analyzed with Kaplan-Meier database. Antitumor effects of DUSP22 were tested in A549 and H1299 cell lines. Experiments are based on: (1) cell viability determined by the cell counting kit-8 assay and colony-formation assay; (2) cell migratory ability assessed throughthe scratch assay and the transwell migration assay; (3) the mechanism behind the antitumor effects of DUSP22 dissected with co-immunoprecipitation (Co-IP) and in vitro kinase assays. Our study revealed a significant downregulation ofDUSP22 in both NSCLC cell lines and tissues. Meanwhile, survival rate analysis results demonstratedthat reduced DUSP22 expression was correlated with pooreroverall survival in lung cancer patients. Moreover, DUSP22 exhibited an inhibitory effect on the cell viability and migratory capacity of A549 and H1299 cells. This inhibition was accompanied by the decrease in the phosphorylation of AKT and p38. Mechanistically, the phosphatase domain of DUSP22 interacted with AKT, resulting in theinhibitionof AKT phosphorylation. This inhibitory effect was contingent upon the phosphatase activity of DUSP22. These findings provide compelling evidence that DUSP22 directly interacted with AKT, leading to the dephosphorylation ofAKT at S473 and T308 residues, ultimately curbing the proliferation and migration of lung cancer cells. Additionally, our results also highlight a preclinical rationale for utilizing DUSP22 as a prognostic marker in NSCLC.

Analysis of survival outcomes in patients with non–small-cell lung cancer with mutations in STK11/LKB1 receiving chemoimmunotherapy.

e15043 Background: Many patients with non-small-cell lung cancer (NSCLC) do not benefit from first-line treatment with immune checkpoint inhibitors (ICI) alone or in combination with chemotherapy due to resistance or toxicity. In NSCLC, significant research has been undertaken to provide prognostic markers for single agent immune checkpoint inhibitor therapy. Co-alterations in TP53, STK11/LKB1 and KRAS have a low reported prevalence in NSCLC, however, potentially may represent a distinct molecular subtype of NSCLC that conveys primary resistance to single-agent ICI. Data continues to accumulate regarding resistance mechanisms to ICI, but limited data exists exploring these alterations and the corresponding survival outcomes in patients that receive chemoimmunotherapy. The primary objective of this study was to assess the association between alterations and co-alterations in STK11/LKB1 and survival outcomes in patients with NSCLC receiving chemo-immunotherapy. Methods: This was a retrospective study of patients ≥18 years old who had completed NGS testing and had a diagnosis of non-small-cell lung cancer that received chemoimmunotherapy from January 2016 to November 2022. NGS reports were reviewed to assess the status of STK11/LKB1, KRAS, KEAP1, and TP53 alterations. Additional markers assessed were PD-L1 status, TPS, CPS, TMB, and MSI if available. Results: Sixty-five patients were identified who had received chemoimmunotherapy for NSCLC, had NGS testing results, and were considered to be evaluable for retrospective review. Four patients were identified with co-altered KRAS, STK11, and TP53, and this alone was not significant for PFS (p= 0.48) or OS (p = 0.52) compared to patients without the alterations, however patients with the co-alterations and a positive PD-L1 showed an improvement in PFS (p =0.053). Patients with altered STK11, including those classified as VUS, had a poorer OS (p = 0.088). Our analysis also showed that PD-L1 negative patients had poorer OS (p = 0.088). Conclusions: The addition of chemotherapy to ICI may overcome resistance previously described in NSCLC patients with co-alterations in KRAS, STK11, and TP53, who received single-agent ICI and these genomic findings should not deter from the use of chemoimmunotherapy based on our limited experience. Patients with alterations in STK11 did have poorer outcomes overall, which is in line with previously published data describing STK11 as a poor prognostic marker in NSCLC. STK11 alterations have previously been described to be associated with PD-L1 negativity which may explain, at least in part, the poorer responses in the STK11 mutated subgroup.

The identification of hub biomarkers and pathways in lung cancer and prognostic evaluation.

BackgroundLung cancer is the most frequently diagnosed malignant tumor and the highest mortality worldwide, and can be divided into two differential histologic subtypes, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). However, there are significant differences in diagnosis and prognosis between NSCLC and SCLC. We aimed to identify hub differentially expressed genes (DEGs) and pathways for diagnostic and prognostic prediction in NSCLC and SCLC.MethodsThree expression profiles (GSE43346, GSE40275 and GSE18842) were obtained through GEO2R tools from Gene Expression Omnibus (GEO) database. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to investigate functional enrichment of the DEGs. The protein–protein interaction network was constructed by the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape. Kaplan-Meier analysis was performed using Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA).ResultsWe have identified 84 overlap DEGs that may play an important role in SCLC & NSCLC. However, we also found some genes were only significantly differential expressed in SCLC or NSCLC. There were 87 DEGs unique to SCLC tissues and 28 DEGs unique to NSCLC ones. Functional analysis results indicated that these DEGs had different biological functions and were significantly enriched in different pathways. Hub DEGs were identified via protein-protein interaction network and cross-validated using Kaplan-Meier plotter and GEPIA. The 14 hub DEGs were highly correlated with the overall survival of NSCLC. Kyoto Encyclopedia of Genes and Genome (KEGG) re-analysis of 14 hub DEGs showed that RRM2, CHEK1 and SERPINB5 enriched in the p53 signaling pathway, RRM2 and TYMS enriched in pyrimidine metabolism pathway maybe play a key role in SCLC&NSCLC and were significantly related to overall survival in patients with NSCLC.ConclusionsRRM2, CHEK1, TYMS and SERPINB5, which are mainly enriched in the p53 signaling pathway and pyrimidine metabolism pathway, were significantly associated with the overall survival of NSCLC patients. These genes could serve as potential prognostic markers in NSCLC and therapeutic target in lung cancer for personalized oncology.

EGFR amplification is a putative resistance mechanism for NSCLC-LM patients with TKI therapy and is associated with poor outcome.

BackgroundLeptomeningeal metastases (LM) have become increasingly common in non-small cell lung cancer (NSCLC) patients who harbor epidermal growth factor receptor (EGFR) mutation treated with EGFR-TKI and are correlated with inferior prognosis. Evidence in prior research demonstrated that EGFR amplification was more likely presented in advanced clinical stages and was associated with worse survival. However, whether EGFR amplification is a prognostic marker in NSCLC–LM is still inconclusive.MethodsThis study enrolled patients diagnosed with NSCLC–LM from June 2019 to September 2021 and who had received previous EGFR-TKI at Guangdong Sanjiu Brain Hospital. Cerebrospinal fluid (CSF) samples were collected and subjected to targeted next-generation sequencing of 168 cancer-related genes. Clinical characteristics and overall survival (OS) were compared in patients with and without EGFR amplification.ResultsThis study enrolled 53 NSCLC–LM patients, all of whom had EGFR mutations. TP53 and EGFR amplifications are the two most frequent mutations in the study cohort, presenting at 72% (38 of 53) and 40% (21 of 53), respectively. The rate of EGFR amplification was much higher at the time of leptomeningeal progression than at initial diagnosis (p < 0.01). Karnoskfy performance status was poorer (p = 0.021), and CSF pressure was higher (p = 0.0067) in patients with EGFR amplification than those without. A multivariable Cox proportional hazard regression model showed that EGFR amplification was an independent prognostic factor for poorer OS (8.3 vs. 15 months; p = 0.017). The median OS was shorter in NSCLC–LM patients with mutated TP53 than those with wild-type TP53, but the difference was not statistically significant (10 vs. 17.3 months, p = 0.184).Conclusions EGFR gene amplification could be a potential resistance mechanism to EGFR-TKI failure in NSCLC–LM and is associated with inferior clinical outcomes.

Overall survival of patients with chronic obstructive pulmonary disease receiving immunotherapy for non-small cell lung cancer: A population-based analysis in Ontario, Canada.

e21172 Background: Outside of clinical trial eligibility criteria, there is limited data to guide the selection of patients with non-small cell lung cancer (NSCLC) for immune checkpoint inhibitor (ICI) therapy. Chronic obstructive pulmonary disease (COPD) and lung cancer are associated, independent of smoking history, with a common background of chronic inflammation. Previous studies have demonstrated that COPD is a negative prognostic marker for NSCLC, but the clinical benefit of ICI in patients with NSCLC and COPD is unknown. Methods: A population-level administrative data analysis of Ontario patients was performed through the Institute of Clinical Evaluative Sciences (ICES) Data Analytic Services. All patients with NSCLC diagnosed between Jan 2010 and Dec 2020 and treated with immune-checkpoint inhibitors (pembrolizumab, nivolumab, atezolizumab) were included. Demographics, comorbidity and marginalization scores, and COPD status were extracted along with outcome information. Overall survival (OS) was estimated using the Kaplan-Meier method, and compared between patients with or without COPD using Cox proportional hazards regression. The frequency of patients requiring hospitalization and duration of treatment was also estimated and compared using the chi-square and Wilcoxon rank-sum test. Results: 73331 NSCLC patients were identified, of which 4.5% (n = 3285) patients received ICI. COPD patients were less likely to receive immunotherapy (3.8% vs. 5.1%, p &lt; 0.001). Among those receiving an ICI, 41% (n = 1362) of patients had a diagnosis of COPD prior to NSCLC diagnosis. Median (95% CI) OS was 17.3 (16.6 to 18.2) months for patients with COPD and 16.9 (16.2 to 17.8) for patients with no known COPD, which was not significantly different in univariate (hazard ratio = 0.96, 95% CI = 0.89 to 1.04, p = 0.35) or multivariate analysis (HR = 0.96, 95% CI = 0.89 to 1.05, p = 0.40). The 5-year survival was also similar between both groups (6.7% vs. 6.5%). The rate of hospitalization within 6 months (18.4% vs 18.0%, p = 0.82) and the duration of immunotherapy treatment (median = 80 vs 71 days p = 0.23) did not differ for the COPD vs. non-COPD groups. Conclusions: Despite an expectation of frailty, our data suggest that NSCLC patients with COPD receiving ICI maintained similar durations of treatment and similar rates of hospitalization, with no significant difference in survival time, compared with those without COPD. While a treatment selection bias cannot be excluded in this non-randomized dataset, our data suggest that a diagnosis of COPD itself should not be considered a contraindication to immune checkpoint inhibitor use in NSCLC.

Choline Kinase Alpha2 Promotes Lipid Droplet Lipolysis in Non-Small-Cell Lung Carcinoma.

BackgroundRapid tumor growth inevitably results in energy stress, including deficiency of glutamine, a critical amino acid for tumor cell proliferation. However, whether glutamine deficiency allows tumor cells to use lipid droplets as an energy resource and the mechanism underlying this potential regulation remain unclear.MethodsWe purified lipid droplets from H322 and H358 human non-small-cell lung cancer (NSCLC) cells under glutamine deprivation conditions and performed immunoblotting to determine the binding of choline kinase (CHK) α2 to lipid droplets. Immunofluorescence was used to quantify lipid droplet numbers and sizes. Immunoprecipitation and immunoblotting were performed to examine AMPK activation and CHKα2 phosphorylation. Cellular fatty acid levels, mitochondrial acetyl coenzyme A and ATP production, and cell apoptosis and proliferation were measured. Immunohistochemical analyses were performed to determine the expression levels of ACC pS79 and CHKα2 pS279 in tumor specimens from NSCLC patients. The prognostic value of ACC pS79 and CHKα2 pS279 was assessed using the Kaplan-Meier method and Cox regression models.ResultsGlutamine deficiency induces AMPK-mediated CHKα2 S279 phosphorylation, which promotes the binding of CHKα2 to lipid droplets, resulting in recruitment of cytosolic lipase ATGL and autophagosomes and subsequent lipolysis of lipid droplets to sustain tumor cell survival and proliferation. In addition, the levels of ACC pS79 and CHKα S279 were much higher in human NSCLC specimens than in their adjacent normal tissues and positively correlated with each other. Notably, ACC pS79 and CHKα pS279 expression levels alone were associated with poor prognosis of NSCLC patients, and combined values of both phosphorylation levels were correlated with worse prognosis of the patients.ConclusionCHKα2 plays a critical role in lipolysis of lipid droplets in NSCLC. ACC pS79 and CHKα2 pS279 alone or in combination can be used as prognostic markers in NSCLC.

Gene Expression Levels of the Prolyl Hydroxylase Domain Proteins PHD1 and PHD2 but Not PHD3 Are Decreased in Primary Tumours and Correlate with Poor Prognosis of Patients with Surgically Resected Non-Small-Cell Lung Cancer.

Simple SummaryHypoxia correlates with poor prognosis in several cancer types, including lung cancer. Prolyl hydroxylase domain proteins (PHDs) belong to an evolutionarily conserved superfamily of dioxygenases that play a role in cell oxygen sensing and homeostasis. In this study, we evaluated PHD1, PHD2 and PHD3 mRNA expression in 60 NSCLC tumours and compared it to that in normal lungs and evaluated the prognostic significance of these differences for distinguishing the survival of NSCLC patients treated with radical surgery. Our results showed that the mRNA expression PHD1 and PHD2 in NSCLC primary tumours was decreased, which correlated with larger tumour size and poor prognosis of patients. PHD1 also showed borderline independent prognostic value in multivariate analysis. In contrast, we found no associations between PHD3 expression and any of the observed parameters. Our results suggest that loss of PHD1 and PHD2 expression is associated with the development and progression of NSCLC, whereas PHD1 could be further assessed as a prognostic marker in NSCLC.Background: Hypoxia correlates with poor prognosis in several cancer types, including lung cancer. Prolyl hydroxylase domain proteins (PHDs) play a role in cell oxygen sensing, negatively regulating the hypoxia-inducible factor (HIF) pathway. Our study aim was to evaluate PHD1, PHD2 and PHD3 mRNA expression levels in primary tumours and normal lungs of non-small-cell lung cancer (NSCLC) patients and to correlate it with selected regulators of HIF signalling, with clinicopathological characteristics and overall survival (OS). Methods: Tumour tissue samples were obtained from 60 patients with surgically resected NSCLC who were treated with radical surgery. In 22 out of 60 cases, matching morphologically normal lung tissue was obtained. PHD1, PHD2 and PHD3 mRNA expressions were measured using RT-qPCR. Results: The PHD1 and PHD2 mRNA levels in primary tumours were significantly decreased compared to those in normal lungs (both p < 0.0001). PHD1 and PHD2 expression in tumours was positively correlated (rs = 0.82; p < 0.0001) and correlated well with HIF pathway downstream genes HIF1A, PKM2 and PDK1. Decreased PHD1 and PHD2 were associated with larger tumour size, higher tumour stage (PHD1 only) and squamous cell carcinoma. Patients with low PHD1 and patients with low PHD2 expression had shorter OS than patients with high PHD1 (p = 0.02) and PHD2 expression (p = 0.01). PHD1 showed borderline independent prognostic values in multivariate analysis (p = 0.06). In contrast, we found no associations between PHD3 expression and any of the observed parameters. Conclusions: Our results show that reduced expression of PHD1 and PHD2 is associated with the development and progression of NSCLC. PHD1 could be further assessed as a prognostic marker in NSCLC.

EHF enhances malignancy by modulating AKT and MAPK/ERK signaling in non‑small cell lung cancer cells.

Overexpression of ETS-homologous factor (EHF) in non-small cell lung cancer (NSCLC) is associated with poor patient prognosis. To explore the mechanism of the effect of EHF in NSCLC, EHF expression was examined in NSCLC and its role in cell proliferation, invasion, cell cycle, and apoptosis of NSCLC cells was evaluated by overexpressing EHF and/or knocking down EHF expression in NSCLC cells in vitro and in cancer cell grafted mice in vivo. The results revealed that the knockdown of EHF expression in NSCLC with siRNA significantly inhibited cell proliferation and invasion, arrested the cell cycle at the G0/G1 phase, and induced apoptosis, whereas overexpression of EHF in NSCLC promoted cell proliferation, tumor growth, and cancer cell migration in vitro. The in vivo experiments demonstrated that siRNA-mediated downregulation of EHF expression in NSCLC cells significantly suppressed tumor growth in xenografted nude mice as compared to cancer progression in the mice grafted with NSCLC cells transfected with non-specific control siRNA. The biochemical analyses revealed that EHF promoted NSCLC growth by regulating the transcription of Erb-B2 receptor tyrosine kinase 2/3 (ERBB2, ERBB3) and mesenchymal-epithelial transition (MET) factor tyrosine kinase receptors and modulating the AKT and ERK signaling pathways in the NSCLC cells. The present findings indicated that EHF could be used as a prognostic marker for NSCLC, and tyrosine kinase receptors of ERBB2, ERBB3 and MET could be drug targets for NSCLC treatment.

Plausible Role of Estrogens in Pathogenesis, Progression and Therapy of Lung Cancer.

Malignant neoplasms are among the most common diseases and are responsible for the majority of deaths in the developed world. In contrast to men, available data show a clear upward trend in the incidence of lung cancer in women, making it almost as prevalent as breast cancer. Women might be more susceptible to the carcinogenic effect of tobacco smoke than men. Furthermore, available data indicate a much more frequent mutation of the tumor suppressor gene-p53 in non-small cell lung cancer (NSCLC) female patients compared to males. Another important factor, however, might lie in the female sex hormones, whose mitogenic or carcinogenic effect is well known. Epidemiologic data show a correlation between hormone replacement therapy (HRT) or oral contraceptives (OCs), and increased mortality rates due to the increased incidence of malignant tumors, including lung cancer. Interestingly, two types of estrogen receptors have been detected in lung cancer cells: ERα and ERβ. The presence of ERα has been detected in tissues and non-small-cell lung carcinoma (NSCLC) cell lines. In contrast, overexpression of ERβ is a prognostic marker in NSCLC. Herein, we summarize the current knowledge on the role of estrogens in the etiopathogenesis of lung cancer, as well as biological, hormonal and genetic sex-related differences in this neoplasm.

Role of tesmin expression in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the most frequent cause of cancer-associated mortality worldwide. Tesmin (MTL5) is a 60 kDa protein which has cysteine rich motifs, characteristic of metallothioneins. Tesmin expression was first observed in germ cells during spermatogenesis. Increased tesmin expression in NSCLC has been described previously. Minichromosome maintenance proteins (MCMs) serve a critical role in replication and cell cycle progression, i.e. in NSCLC. The aim of the present study was to evaluate the localization and intensity of tesmin, MCM5 and MCM7 protein expression in NSCLC and their association with the clinicopathological data of patients. Archival paraffin blocks of 243 cases of NSCLC and 104 non-cancerous tissue samples from the surgical margin (control) were obtained from patients treated at the Clinic of Thoracic Surgery of Wroclaw Medical University (Wroclaw, Poland) between 2010 and 2016, and were used for tissue microarrays and immunohistochemical (IHC) experiments. Laser capture microdissection was used for the isolation of cancer cells from 36 frozen samples of NSCLC and 8 control samples, and subsequently, MTL5, MCM5 and MCM7 mRNA expression was detected separately by reverse transcription-quantitative PCR. Positive cytoplasmic and nuclear tesmin, as well as nuclear MCM5 and MCM7 IHC expression were observed in 95.1, 83.67, 95.51 and 100% of the NSCLC cases, respectively. MTL5, MCM5 and MCM7 mRNA expression was observed in 91.66% of the cancer cases for all genes. The statistical analysis revealed increased tesmin IHC expression in cancer cells compared with the control. A positive correlation was observed between the IHC expression of nuclear tesmin and MCM5 proteins (r=0.33; P<0.0001) and nuclear tesmin and MCM7 proteins (r=0.315; P<0.0001). In addition, a positive correlation between the mRNA expression levels of MTL5 and MCM5 (r=0.421; P<0.05), MTL5 and MCM7 (r=0.557; P<0.01) was demonstrated. The survival analysis revealed that the presence of IHC cytoplasmic tesmin expression was a positive prognostic marker in NSCLC (P=0.0524). Furthermore, in vitro experiments performed on the NCI-H1703 cell line revealed that silencing of MTL5 mRNA and tesmin caused the downregulation of the expression levels of MCM5 and MCM7 and decreased the number of cells in the G2 phase. A positive association among tesmin, MCM5 and MCM7 could indicate a possible role of tesmin in the proliferation of NSCLC cancer cells.

CNOT4 enhances the efficacy of anti-PD-1 immunotherapy in a model of non-small cell lung cancer.

The use of immune checkpoint inhibitors that target programmed cell death-1 (PD-1) has been proposed for the treatment of advanced non-small cell lung cancer (NSCLC). However, in clinical trials, cumulative response rates to anti-PD-1 treatment were approximately 20% in patients with NSCLC. CCR4-NOT transcription complex, subunit 4 (CNOT4) is a RING finger protein with E3 ubiquitin ligase activity. We previously reported that CNOT4 may act as a tumor suppressor in NSCLC. Here, we examined whether CNOT4 can enhance the efficacy of anti-PD-1 immunotherapy in a model of NSCLC. The association of CNOT4 and overall survival was analyzed using datasets from The Cancer Genome Atlas (TCGA). Tumor models were established by subcutaneously implanting tumor cells line (A549 cell) into mice. CNOT4 was observed to be positively associated with relapse-free survival and overall survival in patients with NSCLC. CNOT4 overexpression suppressed tumor growth invivo and enhanced the effect of anti-PD-1 immunotherapy, which was accompanied by increased CD3+ and CD8+ T lymphocyte infiltration and higher interferon-γ and tumor necrosis factor-α levels. In conclusion, CNOT4 may enhance the efficacy of anti-PD-1 immunotherapy and may have potential as a prognostic marker for NSCLC, or as a combinational target with anti-PD-1 treatment for patients with NSCLC.

Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients.

BackgroundVarious methods of liquid biopsy through the sampling of blood in cancer patients allow access to minuscule amounts of tumor that can easily be sampled repeatedly throughout therapy. Circulating tumor cells (CTCs) represent shed tumor cells that can be characterized by imaging or molecular techniques using an amenable enrichment platform. Here we validate the Hitachi Chemical Micro Cavity Array (MCA) for the enrichment of CTCs from the blood of patients diagnosed with stage III non-small cell lung cancer (NSCLC). MCA is a semi-automated filtration system that enriches CTCs on the basis of size and membrane deformability rather than a biased selection of surface antigens.MethodsCTCs were enriched from the peripheral blood of 38 patients diagnosed with stage III NSCLC at the start of chemoradiation. Two tubes of EDTA blood were collected from each patient and processed through MCA in parallel. In the first tube, CTCs were identified as pan-cytokeratin (CK)+ CD45− nucleated cells and enumerated. The second tube was depleted of leukocytes using CD45 antibody-coated magnetic microbeads before enrichment by MCA, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to interrogate CTC-enriched lysates for expression of 16 target mRNAs from a panel of epithelial, mesenchymal, stem-like, and cancer signaling-related genes. CTC-enriched lysates from similarly prepared peripheral blood samples from 18 healthy donors were used to define positive gene expression.ResultsCTCs were identified by imaging in 30 of 38 patient samples (79%). At least 1 target gene was positively expressed in 23 of 25 (92%) patient samples that was subjected to molecular characterization. A CTC count of ≥7 was associated with poor progression-free survival (PFS) [hazard ratio (HR) 4.24, 95% confidence interval (CI), 1.73–10.40, P=0.020] and poor overall survival (HR 8.17, 95% CI, 2.87–23.26, P<0.001). Expression of BCL2 by MCA-enriched CTCs was associated with poor PFS (HR 3.11, 95% CI, 1.18–8.22, P=0.022). Individually, CTC count and expression of BCL2 each remained statistically significant predictors of disease progression and overall survival in multivariate analysis.ConclusionsThis is the first demonstration that lysates of MCA-enriched CTCs are amenable to molecular characterization. CTCs enriched by MCA are an independent prognostic marker in NSCLC.

E2F1-induced ferritin heavy chain 1 pseudogene 3 (FTH1P3) accelerates non-small cell lung cancer gefitinib resistance

Long noncoding RNAs (lncRNAs) have been identified to be critical regulator for various human diseases and emerging evidence illustrate the essential function of lncRNAs in the non-small cell lung cancer (NSCLC). Here, our research team tried to identify the roles of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) in the NSCLC, as well as its molecular mechanism. LncRNA microarray analysis revealed that ferritin heavy chain 1 pseudogene 3 (FTH1P3) was up-regulated in the gefitinib-resistant cells (PC9/GR). Clinically, lncRNA FTH1P3 high-expression was closely correlated with NSCLC patients’ unfavorable prognosis. Gain and loss of functional experiments revealed that FTH1P3 promoted the proliferation and invasion of NSCLC cells in vitro, and FTH1P3 knockdown repressed the tumor growth in vivo. Mechanistically, transcription factor E2F1 accelerated the transcription of FTH1P3. RNA immunoprecipitation and chromatin immunoprecipitation experiments showed that FTH1P3 can recruit lysine-specific demethylase 1 (LSD1) and epigenetically repress the TIMP3, thereby accelerating the tumorigenesis of NSCLC. In summary, these findings suggest that FTH1P3 plays a critical role in the gefitinib resistance and progression of NSCLC, providing a potential novel prognostic marker for NSCLC.