Non-small Cell Lung Cancer Clinical Samples Research Articles

Targeting DNAJC19 overcomes tumor growth and metastasis in NSCLC by regulating AKT1 signaling.

e15024 Background: Some driver oncogenes are still unknown in non-small cell lung cancer (NSCLC). DNAJC19, known as a major component of the translocation machinery of mitochondrial membranes, is a disease-associated protein. Herein, we reported the role of DNAJC19 in NSCLC cell growth and metastasis. Methods: Immunohistochemistry (IHC) was performed to investigate DNAJC19 expression in NSCLC clinical samples. For knockdown or overexpression assays in A549 or NCI-H1299 lung cancer cells, lentiviral vectors were used. After assessment of cell functions, DNAJC19-knockdown A549 cells were further applied to establish mice xenograft and metastasis tumor models. STRING database, western, PCR, immunoprecipitation and IHC were performed for the mechanistic study. Results: Expression of DNAJC19 was higher in tumors than non-cancerous adjacent tissues. Survival analysis found that low DNAJC19 levels were correlated with increased progression-free survival rate. ShRNA-mediated knockdown of DNAJC19 markedly inhibited cell growth, viability, migration and invasion. Moreover, RNA-seq analysis explored that PI3K/AKT signaling pathway was involved in molecular events when A549 cells treated with shDNAJC19. And then DNAJC19 knockdown also decreased AKT1 expression in A549 cells. In addition, cellular functions were greatly rescued in DNAJC19-knockdown A549 cells by exotic overexpression of AKT1. Furthermore, tumor xenograft growth and metastasis were markedly repressed in the shDNAJC19 group than control group. As expected, DNAJC19 and AKT1 expression levels in xenograft mice samples were also lower in the shDNAJC19 group than in the shCtrl group. Conclusions: DNAJC19 greatly promotes NSCLC cell growth and metastasis via regulating AKT1 signaling, providing a novel therapeutic target for NSCLC patients.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355.

SATB1 protein is associated with the epithelial‑mesenchymal transition process in non‑small cell lung cancers.

Lung cancer is one of the most frequently diagnosed neoplasms and the leading cause of cancer-related mortality worldwide. Its predominant subtype is non-small cell lung cancer (NSCLC), which accounts for over 80% of the cases. Surprisingly, the majority of lung cancer-related deaths are caused not by a primary tumour itself, but by its metastasis to distant organs. Therefore, it becomes especially important to identify the factors involved in lung cancer metastatic spread. Special AT-rich binding protein 1 (SATB1) is a nuclear matrix protein that mediates chromatin looping and plays the role of global transcriptional regulator. During the past decade, it has received much attention as a factor promoting tumour invasion. In breast, colorectal and prostate cancers, SATB1 has been shown to influence the epithelial-mesenchymal transition (EMT) process, which is thought to be crucial for cancer metastasis. The aim of this study was to analyse the possible correlations between the expression of SATB1 and major EMT-associated proteins in NSCLC clinical samples. Additionally, the impact of EMT induction in NSCLC cell lines on SATB1 mRNA expression was also investigated. Immunohistochemistry was used to assess the expression of SATB1, SNAIL, SLUG, Twist1, E-cadherin, and N-cadherin in 242 lung cancer clinical samples. EMT was induced by TGF-β1 treatment in the A549 and NCI-H1703 lung cancer cell lines. Changes in gene expression profiles were analyzed using real-time PCR and Droplet Digital PCR. SATB1 expression was positively correlated with the expression of SNAIL (R=0.129; P=0.045), SLUG (R=0.449; P<0.0001), and Twist1 (R=0.264; P<0.0001). Moreover, SATB1 expression significantly increased after in vitro EMT induction in A549 and NCI-H1703 cell lines. The results obtained may point to the role of SATB1 as one of the regulators of EMT in NSCLC.

CircUBAP2 exacerbates malignant capabilities of NSCLC by targeting KLF4 through miR-3182 modulation.

Chemo-resistance and refractoriness remain challenges for Non-small cell lung cancer (NSCLC) patients and the underlying molecular mechanisms haven’t been fully explained. In this study, we investigated the influence of circUBAP2 on the NSCLC tumor cells. This study might provide novel therapeutic targets for NSCLC treatment. Clinical samples and NSCLC cell lines were used to investigate circUBAP2 expressions and their impact on tumor cell chemo-resistance. CCK8 and transwell assays were conducted to explore the differences of NSCLC tumor proliferation and migration capabilities affected by circUBAP2. Dual-luciferase reporter gene assay was performed to explore the detailed molecular mechanism of circUBAP2 regulation network. circUBAP2 exhibited significantly elevated average level in our clinical samples of NSCLC, compared with normal tissues. CircUBAP2 level was positively correlated with disease stage and metastatic status. circUBAP2 significantly enhanced the migration, proliferation and chemo-resistance of NSCLC cell lines. Further experiments indicated that circUBAP2 promoted malignant biological behavior of NSCLC tumor cells by targeting KLF4 through modulating miR-3182 expression. Our study demonstrated for the first time that circUBAP2 played an important role exacerbating malignant capabilities of NSCLC. circUBAP2-miR3182-KLF4 regulative network demonstrated in this study could be a novel therapeutic target for future NSCLC treatment.

P37.26 Comprehensive Comparison of 22C3 and SP263 PD-L1 Expression using Routine Clinical and Conditioned Archives of Non-Small-Cell Lung Cancer

Harmonization studies for PD-L1 revealed high correlation of 22C3 and SP263 antibodies in non-small cell lung cancer (NSCLC). However, characteristics of those antibodies in various clinical and analytical setting has yet to be validated. The results of simultaneously performed PD-L1 22C3 and SP263 assays on 431 routine clinical NSCLC samples were reviewed and both assays were performed on 314 archives of surgically resected NSCLCs and assessed the PD-L1 expression according to FFPE block age, and the storage duration and the temperature of FFPE sections. In routine clinical samples, 22C3 showed highest concordance rate with 94.5% of SP263 tumor proportion score (TPS) ≥50% and 98.9% 78.8% of SP263 TPS 1-49%, while SP263 showed concordance rate with 79.6% of 22C3 TPS ≥50% and 82.4% of 22C3 TPS 1-49%. In archives, both 22C3 and SP263 high TPS (versus TPS <1%) were significantly associated with more recent block (<3 years versus ≥3 years) (p = 0007 and p = 0.009, respectively). When FFPE sections were stored at room temperature, only 22C3 TPS was more decreased in longer stored sections. However, the expression was not affected by the section storage duration in both assays when stored at 4°C. For 22C3 TPS 1-49% and ≥50%, there was a significantly higher probability of preserved 22C3 expression in resent section in room temperature (OR = 1.73, p = 0.006 and OR = 1.98, p = 0.002, respectively) but SP263 showed preserved expression in longer section duration in room temperature. Our results showed the high correlation between PD-L1 22C3 and SP263 in routine clinical samples but FFPE block older than 3 years and sections stored at room temperature more than 1 week may lead to underestimation of PD-L1 status especially with 22C3 assay.

CircMED13L_012 promotes lung adenocarcinoma progression by upregulation of MAPK8 mediated by miR-433-3p

BackgroundMetastasis and disease refractoriness remain as major challenges for non-small cell lung cancer (NSCLC) treatment and understanding the underlying molecular mechanisms is of scientific and clinical value. Therefore, in this study, we aimed to explore the effects of circMED13L_012 on the proliferation, migration, invasion and drug-resistance of NSCLC tumor cells.MethodsIn this study, we utilized clinical samples and NSCLC cell lines to explore the association between circMED13L_012 expressions and tumor cell metastasis and chemo resistance. CCK8 and transwell assay were conducted to explore the impact of circMED13_012 on NSCLC tumor proliferation and migrative capabilities. Dual-luciferase reporter gene assay was conducted to validate the circMED13L_012 interaction network.ResultsOur results demonstrated that circMED13L_012 exhibited significantly elevated average level in our clinical samples of NSCLC, compared with normal tissues. circMED13L_012 level was positively correlated with disease stage and metastatic status. Increased circMED13L_012 expression was associated with the enhanced migration, proliferation and chemo resistance of NSCLC cell lines. Further experiments indicated that circMED13L_012 promoted malignant behavior of NSCLC tumor cells by targeting MAPK8 through modulation miR-433-3p expression.ConclusionsOur study for the first time demonstrated that circMED13L_012–miR-433-3p–MAPK8 axis played important role for NSCLC pathogenesis, which could be potential therapeutic target for the development of future NSCLC treatment.

The use of COLD-PCR and pyrosequencing for sensitive detection of EGFR T790M mutation

A sensitive and convenient method for the detection of epidermal growth factor receptor (EGFR) T790M mutation in non-small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs) would be desirable to guide treatment strategy. Consequently, studies have focused on sensitive characterization of EGFR T790M mutation. Herein, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and pyrosequencing were combined (COLDPCR/ pyrosequencing) for detecting EGFR T790M mutation. Evaluation of mutation-containing dilutions revealed that the sensitivities of COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing assays for the detection of the T790M mutation were 0.1 and 5%, respectively, indicating a 50-fold increase in sensitivity. When the T790M mutation in 20 clinical NSCLC samples who had relapsed under firstgeneration EGFR TKI were further determined using COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing, the detection rates were 35% (7/20) and 25% (5/20), respectively. All patients who were positive for the T790M mutation with conventional PCR/pyrosequencing were also found to be positive with COLD-PCR/pyrosequencing. The discordant cases were 2 samples with no T790M mutation detected with conventional PCR/pyrosequencing, but which were positive with COLD-PCR/pyrosequencing. COLD-PCR/pyrosequencing is a sensitive and cost-effective tool for detecting the T790M mutation which will permit an improvement of therapeutic management.

Overcoming of Radioresistance in Non-small Cell Lung Cancer by microRNA-320a Through HIF1α-Suppression Mediated Methylation of PTEN.

BackgroundRadioresistance is a major challenge in the use of radiotherapy for the treatment of lung cancer while microRNAs (miRs) have been reported to participate in multiple essential cellular processes including radiosensitization. This study was conducted with the main objective of investigating the potential role of miR-320a in radioresistance of non-small cell lung cancer (NSCLC) via the possible mechanism related to HIF1α, KDM5B, and PTEN.MethodsFirstly, NSCLC radiosensitivity-related microarray dataset GSE112374 was obtained. Then, the expression of miR-320a, HIF1α, KDM5B, and PTEN was detected in the collected clinical NSCLC samples, followed by Pearson’s correlation analysis. Subsequently, ChIP assay was conducted to determine the content of the PTEN promoter fragment enriched by the IgG antibody and H3K4me3 antibody. Finally, a series of in vitro and in vivo assays were performed in order to evaluate the effects of miR-320a on radioresistance of NSCLC with the involvement of HIF1α, KDM5B, and PTEN.ResultsThe microarray dataset GSE112374 presented with a high expression of miR-320a in NSCLC radiosensitivity samples, which was further confirmed in our clinical samples with the use of reverse transcription-quantitative polymerase chain reaction. Moreover, miR-320a negatively targeted HIF1α, inhibiting radioresistance of NSCLC. Interestingly, miR-320a suppressed the expression of KDM5B, and KDM5B was found to enhance the radioresistance of NSCLC through the downregulation of PTEN expression. The inhibition of miR-320a in radioresistance of NSCLC was also reproduced by in vivo assay.ConclusionTaken together, our findings were suggestive of the inhibitory effect of miR-320a on radioresistance of NSCLC through HIF1α-suppression mediated methylation of PTEN.

Kinesin family member 3A inhibits the carcinogenesis of non-small cell lung cancer and prolongs survival.

Kinesin family member 3A (KIF3A) plays a crucial role in the carcinogenesis of different types of human cancer. The present study aimed to identify the role of KIF3A in the carcinogenesis of non-small cell lung cancer (NSCLC). KIF3A protein expression was determined in 163 patients with NSCLC using immunohistochemistry staining. The prognosis of patients with NSCLC was determined using Kaplan-Meier survival and Cox regression analyses. The function of KIF3A on the carcinogenesis and metastasis of NSCLC was determined in vitro. Furthermore, a protein-protein interaction (PPI) network of KIF3A was constructed and the potential interacting molecules were identified using bioinformatic analysis. The protein expression levels of KIF3A were significantly lower in the NSCLC tissues compared with that in the adjacent tissues, and low KIF3A expression level was associated with unfavorable survival outcomes in patients with NSCLC. Furthermore, KIF3A knockdown increased proliferation, invasion and metastasis, and inhibited apoptosis of NSCLC cells. KIF3A was demonstrated to interact with intraflagellar transport 57 (IFT57) in the PPI network. In addition, validation analyses indicated that KIF3A mRNA expression levels were positively correlated with IFT57 mRNA expression levels in clinical NSCLC samples and NSCLC cell lines. Taken together, the results of the present study suggested that KIF3A is a key tumor suppressor gene for carcinogenesis and metastasis of NSCLC, it may also function as a biomarker and interacts with IFT57 in the progression of NSCLC.

High levels of MESP1 expression in non-small cell lung cancer can facilitate cell proliferation, metastasis and suppresses cell apoptosis.

BackgroundThere are growing number of researches have shown that mesoderm posterior basic helix-loop helix (BHLH) transcription factor 1 (MESP1) plays a crucial role in the development of tumors. However, its expression pattern and function in non-small cell lung cancer (NSCLC) are still unknown.MethodsMESP1 expression and biological process were investigated in NSCLC based on bioinformatics analysis. The mRNA or protein expression levels of MESP1 were measured by quantitative real-time PCR (qRT-PCR) and western blot (WB) assay in NSCLC cells and clinical tissue samples. Then, we used small interfering RNA (siRNA) interference to knocking down expression of gene in NSCLC cells. Cell proliferation was performed using cell counting kit-8 (CCK-8), colony forming assay and real-time cell analyzer (RTCA); transwell chambers and RTCA were used to analysis cell migration and invasion. Besides, analyses of the cell cycle progression and apoptosis were measured via BD JAZZ flow cytometric analysis. All the experiments were repeated ≥3 times. And analyses were performed using SPSS software version 21.0 and GraphPad Prism 6.0. P<0.05 was considered statistically significant.ResultsDetection of MESP1 showed that mRNA was up-regulated in NSCLC cells and patients compared with the normal controls (P<0.05). And high expression of MESP1 were correlated with increasingly cell proliferation, metastasis, cycle and apoptosis. Besides, through WB experiments, it was found that knocking down MESP1 mainly activated the caspase-3/PARP1 signal pathway. Furthermore, it was also verified from clinical samples that MESP1 was highly expressed on both mRNA and protein aspect.ConclusionsOur study suggested that MESP1 is indeed highly expressed in NSCLC, and MESP1 high expression obviously promote cell proliferation, migration, invasion. What’s more, it has good sensitivity to the occurrence and development of NSCLC patients. This can be used as a novel potential therapeutic target for NSCLC.

Loss of histone lysine methyltransferase EZH2 confers resistance to tyrosine kinase inhibitors in non-small cell lung cancer.

Tyrosine kinase inhibitor (TKI) treatment is the first-line therapy for non-small cell lung cancer (NSCLC) caused by activating mutations of epidermal growth factor receptor (EGFR). However, acquired resistance to EGFR-TKI occurs almost inevitably. Aberrant activation of proto-oncogene MET has been known to confer EGFR-TKI resistance; however, the mechanisms involved remains unclear. Recent evidence implicates epigenetic heterogeneity as playing roles in cancer drug resistance, whereas links involving epigenetic heterogeneity and MET in NSCLC remain poorly understood. We found that expression of EZH2, a histone methyltransferase, was negatively correlated with MET activation and EGFR-TKI resistance in NSCLC cells and clinical samples, suggesting the potential for EZH2 to be used as a biomarker for EGFR-TKI sensitivity. Knockdown or inhibition of EZH2 up-regulated MET expression and phosphorylation, and elevated proliferation and EGFR-TKI resistance of cells in vitro. Meanwhile, inhibition of MET or PI3K/AKT enhanced EZH2 levels and restored sensitivity to EGFR-TKI. These findings indicate a "MET-AKT-EZH2" feedback loop regulating EGFR-TKI-resistance. Furthermore, combination therapy of PI3K/AKT inhibition and EGFR-TKI, which interrupts the loop, enhanced tumor-suppressive effects in an EGFR-TKI-resistant xenograft model, indicating a potential approach against drug resistance in NSCLC.

KIAA1522 potentiates TNF\u03b1-NF\u03baB signaling to antagonize platinum-based chemotherapy in lung adenocarcinoma

BackgroundThe platinum-based chemotherapy is the first-line regimen for the treatment of Non-small cell lung cancer (NSCLC). However, the therapeutic efficiency is largely limited by tenacious chemo-insensitivity that results in inferior prognosis in a cohort of patients. It has been known that KIAA1522 is aberrantly expressed and implicated in several types of solid tumors including NSCLC. Nowadays, knowledge about this gene is quite limited. Here, we aimed to identify the role of KIAA1522 in lung adenocarcinomas, and the molecular events that underlie KIAA1522-mediated chemoresistance to the platinum.MethodsImmunohistochemistry were used to detect KIAA1522 expression in clinical NSCLC samples. Then, the survival analyses were performed to assess the link between KIAA1522 expression and overall survival or therapeutic outcome. In vivo depletion of KIAA1522 in adenocarcinoma cells were achieved by adeno-associated virus-mediated sgRNA/Cre delivery into the conditional KrasG12D/Cas9 expressed mice, which were designated to identify the roles of KIAA1522 in tumorigenesis and/or chemotherapy responses. The effects of KIAA1522 and downstream molecular events were studied by pharmacology in mice model and assays using in vitro cultured cells. The clinical relevance of our findings was examined by data-mining of online datasets from multiple cohorts.ResultsThe clinical evidences reveal that KIAA1522 independently predicts both the overall survival and the outcome of platinum-based chemotherapy in lung adenocarcinomas. By using a KrasG12D-driven murine lung adenocarcinoma model and performing in vitro assays, we demonstrated that KIAA1522 is a critical positive regulator of lung adenocarcinoma and a modulator of cisplatin response. KIAA1522 potentiates the TNFα-TNFR2-NFκB signaling which in turn intensifies recalcitrance to cisplatin treatment. These results were further manifested by integrative bioinformatic analyses of independent datasets, in which KIAA1522 is tightly associated with the activity of TNFα-NFκB pathway and the cisplatin-resistant gene signatures. More strikingly, overexpression of KIAA1522 counteracts the cisplatin-induced tumor growth arrest in vivo, and this effect can be remarkably diminished by the disruption of NFκB activity.ConclusionHigh expression of KIAA1522 is turned out to be an indicator of dismal effectiveness of platinum-based therapy in lung adenocarcinomas. KIAA1522 hyperactivates TNFα-NFκB signaling to facilitate resistance to platinum reagents. Targeting NFκB signaling through small molecule inhibitors may be a rational strategy to conquer chemoresistance and synergize platinum-based chemotherapy in KIAA1522 overexpressed lung adenocarcinomas.

Next Generation Sequencing for Gene Fusion Analysis in Lung Cancer: A Literature Review.

Gene fusions have a pivotal role in non-small cell lung cancer (NSCLC) precision medicine. Several techniques can be used, from fluorescence in situ hybridization and immunohistochemistry to next generation sequencing (NGS). Although several NGS panels are available, gene fusion testing presents more technical challenges than other variants. This is a PubMed-based narrative review aiming to summarize NGS approaches for gene fusion analysis and their performance on NSCLC clinical samples. The analysis can be performed at DNA or RNA levels, using different target enrichment (hybrid-capture or amplicon-based) and sequencing chemistries, with both custom and commercially available panels. DNA sequencing evaluates different alteration types simultaneously, but large introns and repetitive sequences can impact on the performance and it does not discriminate between expressed and unexpressed gene fusions. RNA-based targeted approach analyses and quantifies directly fusion transcripts and is more accurate than DNA panels on tumor tissue, but it can be limited by RNA quality and quantity. On liquid biopsy, satisfying data have been published on circulating tumor DNA hybrid-capture panels. There is not a perfect method for gene fusion analysis, but NGS approaches, though still needing a complete standardization and optimization, present several advantages for the clinical practice.

MiR-1299 Impedes the Progression of Non-Small-Cell Lung Cancer Through EGFR/PI3K/AKT Signaling Pathway.

BackgroundNon-small-cell lung cancer (NSCLC) is one of the most malignant tumors. In which, numerous miRNAs had been reported to participate in the pathogenesis. However, the expression and function of miR-1299 in NSCLC are not clear.MethodsTo explore the roles of miR-1299 in NSCLC, we detected the levels of miR-1299 in clinical samples of NSCLC and investigated the role of miR-1299 in the regulation of the NSCLC cells proliferation, metastasis, and EMT. Luciferase reporter assay was employed to verify the target of miR-1299. Additionally, the proliferation, metastasis, and EMT of A549 and H1299 cells were analyzed after the overexpression and knockdown of miR-1299.ResultsWe found that the miR-1299 expression negatively corresponded with the clinical stage and overall survival in NSCLC patients. Overexpression of miR-1299 inhibited the migration, invasion, and EMT of A549 and H1975 cells. Meanwhile, we proved that miR-1299 is the sponge of EGFR. Besides, our results suggested that miR-1299 inhibits the progression of NSCLC cells through the PI3K/Akt signal pathway.ConclusionWe demonstrated that miR-1299 inhibits the progression of NSCLC through the EGFR/PI3K/Akt signal pathway. Therapeutic intervention targeting the miR-1299 may provide a potential strategy for the treatment of NSCLC.

LncRNA FTX activates FOXA2 expression to inhibit non-small-cell lung cancer proliferation and metastasis.

Lung cancer leads to the highest mortality among all cancer types in the world, and non–small‐cell lung cancer (NSCLC) occupies over 80% of the lung cancer cases. Numerous studies have demonstrated that long non‐coding RNA (lncRNA) is involved in various human diseases including cancer. LncRNA FTX was firstly identified in Xist gene locus and was dysregulated in many human cancers. However, the function of FTX in NSCLC is still unclear. Here, we report that long non‐coding RNA FTX expression level is down‐regulated in NSCLC clinical tissue samples and cell lines. Ectopic expression of FTX inhibits proliferation and metastasis of lung cancer cells in vitro and in vivo. Furthermore, we find that FTX overexpression activates the expression of transcription factor FOXA2, an important regulator in lung cancer progression, and we reveal a novel FTX/miR‐200a‐3p/FOXA2 competing endogenous RNA regulatory axis in lung cancer cells. Our results provide new insights and directions for exploring the function of FTX in lung cancer progression.

Ultrasound-targeted microbubble destruction-mediated miR-767 inhibition suppresses tumor progression of non-small cell lung cancer.

MicroRNAs (miRNAs/miRs) have important roles in tumor progression in various human cancers. Ultrasound-targeted microbubble destruction (UTMD)-mediated gene transfection has been considered a useful tool for improving cancer treatment. The present study aimed to investigate the role of miR-767 in non-small cell lung cancer (NSCLC) and further analyze the effects of UTMD-mediated miR-767 inhibition on tumor progression. The expression of miR-767 was measured by reverse transcription-quantitative PCR. UTMD-mediated miR-767 inhibition was achieved by the co-transfection of microbubbles and miR-767 inhibitor in NSCLC cells. Cell proliferation was assessed by a CCK-8 assay and cell migration and invasion were examined by a Transwell assay. The expression of miR-767 was increased in NSCLC serum, tissues and cells compared with controls. The reduction of miR-767 in NSCLC cells led to the inhibition of cell proliferation, migration and invasion. UTMD increased the transfection efficiency of the miR-767 inhibitor in NSCLC cells, and UTMD-mediated miR-767 inhibition resulted in a more significant suppressive effect on tumor cell proliferation, migration and invasion. Taken together, the results indicated that miR-767 expression is upregulated in both NSCLC clinical samples and cells. The downregulation of miR-767 can inhibit tumor cell proliferation, migration and invasion, and these effects are further promoted by UTMD-mediated miR-767 inhibition, which indicated the potential of a UTMD-mediated miR-767 inhibition as a novel therapeutic strategy for NSCLC treatment.

Enhanced histone H3 acetylation of the PD-L1 promoter via the COP1/c-Jun/HDAC3 axis is required for PD-L1 expression in drug-resistant cancer cells

BackgroundDrug resistance is a major obstacle to treating cancers because it desensitizes cancer cells to chemotherapy. Recently, attention has been focused on changes in the tumor immune landscape after the acquisition of drug resistance. Programmed death-ligand-1 (PD-L1) is an immune suppressor that inhibits T cell-based immunity. Evidence has shown that acquired chemoresistance is associated with increased PD-L1 expression in cancer cells. However, the underlying mechanism is still largely unknown.MethodsPD-L1 expression in three drug-resistant A549/CDDP, MCF7/ADR and HepG2/ADR cell lines was detected by qRT-PCR, western blotting and flow cytometry, and a T cell proliferation assay was performed to test its functional significance. Then, the potential roles of JNK/c-Jun, histone H3 acetylation, histone deacetylase 3 (HDAC3) and the E3 ligase COP1 in the PD-L1 increase were explored through ChIP assays and gain- and loss-of-function gene studies. Furthermore, murine xenograft tumor models were used to verify the role of JNK/c-Jun and HDAC3 in PD-L1 expression in A549/CDDP cells in vivo. Finally, the correlations of PD-L1, c-Jun and HDAC3 expression in clinical cisplatin-sensitive and cisplatin-resistant non-small cell lung cancer (NSCLC) tissues were analyzed by immunohistochemistry and Pearson’s correlation coefficient.ResultsPD-L1 expression was significantly increased in A549/CDDP, MCF7/ADR and HepG2/ADR cells and was attributed mainly to enhanced JNK/c-Jun signaling activation. Mechanistically, decreased COP1 increased c-Jun accumulation, which subsequently inhibited HDAC3 expression and thereby enhanced histone H3 acetylation of the PD-L1 promoter. Furthermore, PD-L1 expression could be inhibited by JNK/c-Jun inhibition or HDAC3 overexpression in vivo, which could largely reverse inhibited CD3+ T cell proliferation in vitro. PD-L1 expression was significantly increased in the cisplatin-resistant clinical NSCLC samples and positively correlated with c-Jun expression but negatively correlated with HDAC3 expression.ConclusionsEnhanced histone H3 acetylation of the PD-L1 promoter via the COP1/c-Jun/HDAC3 axis was crucial for the PD-L1 increase in drug-resistant cancer cells. Our study reveals a novel regulatory network for the PD-L1 increase in drug-resistant cancer cells and that combined PD-L1-targeting strategies could improve T cell-based immunity in drug-resistant cancers.

Association between interleukin-36γ and tumor progression in non-small cell lung cancer.

Immunotherapy is effective in improving the survival and prognosis of patients with non-small cell lung cancer (NSCLC), and identifying effective immunomarkers is important for immunotherapy. Interleukin (IL)-36γ is a novel immunomarker that has an important function in the antitumor immune response. The present study investigated the association between IL-36γ and NSCLC to provide novel insight into immunotherapy for patients with NSCLC. Tissue microarrays of lung adenocarcinoma and squamous cell carcinoma were purchased for immunohistochemical analysis of IL-36γ expression levels and clinical parameters. In addition, fresh clinical NSCLC and adjacent normal tissue samples were collected to analyze IL-36γ mRNA expression levels using quantitative PCR. IL-36γ protein was primarily located in the cytoplasm, with a small quantity in the nucleus, and IL-36γ mRNA and protein expression levels in lung cancer tissues were significantly higher compared with those in adjacent normal tissues. Elevated IL-36γ protein expression levels were significantly associated with a higher tumor grade of lung adenocarcinoma; however, IL-36γ mRNA expression levels were inversely associated with the clinical Tumor-Node-Metastasis stage in patients with lung squamous cell carcinoma. In addition, patients with adenocarcinoma with high IL-36γ protein expression levels tended to longer post-operative survival times. These findings indicate that IL-36γ may have potential as an immunomarker for prediction of tumor progression and survival in patients with NSCLC.

Circ_0000376 enhances the proliferation, metastasis, and chemoresistance of NSCLC cells via repressing miR-384.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. Circular RNAs (circRNAs) are recently identified as important gene regulators with critical roles in cancer biology. In this study, we focus on the effect of circ_0000376 targeting miR-384 on malignant phenotypes of NSCLC cells. Circ_0000376 and miR-384 expression in NSCLC tissue samples were measured using qRT-PCR. The association between pathological parameters and the circ_0000376 expression was analyzed as well. Human NSCLC cell lines A549 and NCI-H460 were used as cell models. CCK-8 and BrdU assay were used to assess the effect of circ_0000376 on NSCLC cell line proliferation and drug sensitivity. Transwell assay was conducted to detect the effect of circ_0000376 on migration and invasion. Further, luciferase reporter assay was employed to validate the targeting of miR-384 by circ_0000376. Circ_0000376 expression in NSCLC clinical samples was up-regulated and this was linked to unfavorable pathological parameters. Circ_0000376 markedly accelerated the proliferation and metastasis, and enhanced chemoresistance of NSCLC cells. Mechanically, circ_0000376 overexpression could bind with miR-384 and repress its expression. Circ_0000376 is a newly discovered oncogenic circRNA in NSCLC, and can be potentially regarded as a diagnostic biomarker and therapy target.

MiR-501-3p functions as a tumor suppressor in non-small cell lung cancer by downregulating RAP1A

MicroRNA-501-3p (miR-501-3p) has been reported to play tumor-suppressive roles in different cancers; however, its expression pattern and biological function in non–small cell lung cancer (NSCLC) remain unknown. In this study, we noted downregulation of miR-501-3p in NSCLC tissues and cell lines. Functional assays showed that overexpression of miR-501-3p suppressed NSCLC cell proliferation, clonogenicity, migration, and invasion. Moreover, miR-501-3p overexpression attenuated in vivo tumor growth in a nude mouse model. In terms of the mechanism, RAP1A was identified as a novel target of miR-501-3p. Overexpression of RAP1A strongly attenuated the inhibitory effects of miR-501-3p on the capacity of NSCLC cells for proliferation and motility. In the clinical samples of NSCLC, miR-501-3p levels negatively correlated with RAP1A expression, which was upregulated in NSCLC. Collectively, these results indicate that miR-501-3p acts as a tumor suppressor in NSCLC by directly targeting RAP1A mRNA and may serve as a theranostic biomarker for patients with NSCLC.