Glucosamine-phosphate N-acetyltransferase 1 and its DNA methylation can be biomarkers for the diagnosis and prognosis of lung cancer.

OBJECTIVE: Lung cancer ranking high in the cancer-related list has long perplexed patients, in which glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be highly expressed. Besides, DNA methylation is perceived as a biomarker to assess the prognosis of patients with various cancers. However, the correlation between GNPNAT1 and DNA methylation and the role of GNPNAT1 in lung cancer remain vague. METHODS: Principal component analysis (PCA), heatmap, volcano map, Venn diagram, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to screen out the candidate genes. The viability, migration, and invasion of lung cancer cells were detected by CCK-8 and Transwell assays. An xenograft tumor mouse model was established. The relative expressions of GNPNAT1, E-cadherin, vimentin, Matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), E2F1, and cyclin D1 in cells or xenograft tumor tissues were quantified by Western blot, RT-qPCR, or immunohistochemistry assay. RESULTS: GNPNAT1 was screened as the research object. GNPNAT1 methylation was downregulated, while GNPNAT1 expression was upregulated in lung cancer tissues. The methylation and mRNA levels of GNPNAT1 were correlated with the patient prognosis. GNPNAT1 increased cell viability, migration and invasion, and promoted the xenograft tumor volume and weight, whereas shGNPNAT1 acted oppositely. Moreover, expressions of Vimentin, MMP-2, E2F1, and cyclin D1 were increased, but E-cadherin and TIMP-2 expressions were decreased by overexpressed GNPNAT1, whilst GNPNAT1 knockdown ran conversely. CONCLUSION: GNPNAT1 and methylated GNPNAT1 coverage are biomarkers for the diagnosis and prognosis of lung cancer.

SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the Wnt/ß-catenin pathway.

BACKGROUND: The distant metastasis of cancer cells is a risk factor for tumor lethality and poor prognosis in non-small-cell lung carcinoma (NSCLC). Increased SOX9 expression has been associated with clinical stage and poor prognosis in NSCLC, but the molecular mechanisms by which SOX9 promotes metastasis in NSCLC are still unknown. METHODS: The relationship between SOX9 expression and T, N, M classification was assessed using the χ2 test and Spearman's analysis in 142 immunohistochemically diagnosed specimens of NSCLC. We also generated SOX9-overexpression and SOX9-knockdown cells lines and their corresponding control cell lines by transfection with lentiviral constructs. In vivo assay, SOX9-overexpressing and SOX9-knockdown NSCLC cells were injected in zebrafish to examine distance metastasis. Gene set enrichment analysis (GSEA) was applied to analysis the correlation between SOX9 overexpression and Wnt/ß-catenin pathway. Luciferase assay was used to check transcriptional activity of TCF/LEF and western blot and immunofluorescence was employed to detect ß-catenin translocation in SOX9-overexpression, SOX9-knockdown and their corresponding control cell lines. RESULTS: We found that SOX9 overexpression correlates with the T, N and M stage significantly (p = 0.03, 0.000, and 0.032 respectively) in 142 immunohistochemically diagnosed specimens of NSCLC. SOX9 overexpression was found to decrease the expression of the epithelial cell markers E-cadherin and γ-catenin and increase the expression of the mesenchymal cell markers N-cadherin and vimentin. An in vivo assay showed distant metastasis of the SOX9-overexpressing cells, which was not observed in the SOX9-knockdown cells. These findings indicate that SOX9 promotes distant metastasis by promoting EMT in NSCLC cells. GSEA showed that SOX9 overexpression was significantly correlated with the Wnt/ß-catenin pathway which was corroborated by the expression of EMT-associated proteins in this pathway and its downstream target genes. SOX9 overexpression was also found to enhance the transcriptional activity of TCF/LEF, promote the nuclear translocation of ß-catenin and increase the phosphorylation of GSK3ß at Ser9. Further, inhibition of ß-catenin suppressed the metastasis-promoting effects of SOX9 overexpression. CONCLUSIONS: This study is the first to report that SOX9 is associated with clinical TNM stage and indicates that SOX9 promotes migration, invasion and the EMT process through the Wnt/ß-catenin pathway.

Potential Impact of Cancer Susceptibility Genes on Lung Cancer Metastasis.

Background: Studies of prognosis-related molecular markers are an important tool to uncover the mechanism of tumour metastasis. Cancer susceptibility gene testing is an important tool for genetic counselling of cancer risk. However, the impact of lung cancer susceptibility genes (LCSGs) on lung cancer metastasis and prognosis has not been well studied. Methods: The list of lung cancer susceptibility genes was retrospectively analysed and updated. After expression profiling and functional analysis, LCSG-based signatures for prognosis were identified by Cox regression and LASSO regression analyses. For translational purposes, nomograms integrating LCSGs and clinical characteristics were constructed. Results: A total of 301 LCSGs were employed for modelling. For lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), 10-gene and 7-gene signatures were created and independently validated. The LCSG-based risk score could stratify LUAD survival (univariate: hazard ratio (HR) = 1.076, 95% confidence interval (CI) = 1.049-1.103, P < 0.001; multivariate: HR = 1.066, 95% CI = 1.037-1.095, P < 0.001) and LUSC survival (univariate: HR = 1.149, 95% CI = 1.066-1.239, P < 0.001; multivariate: HR = 1.129, 95% CI = 1.038-1.228, P = 0.005). One of the processes affected by differentially expressed genes in both LUAD and LUSC was the negative regulation of epithelial cell differentiation. Conclusions: Overall, novel LCSG-based gene signatures for LUAD and LUSC were constructed. These findings could expand the understanding of the impact of LCSG expression on cancer metastasis and prognosis.

LINC00355 promoted the progression of lung squamous cell carcinoma through regulating the miR-466/LYAR axis.

LINC00355 has been reported aberrantly over-expressed and associated with poor prognosis in various types of cancer. However, reports regarding the effect of LINC00355 on lung squamous cell carcinoma (SCC) are rare. This study aimed to explore the function of LINC00355 in the development and progression of lung SCC and reveal the underlying mechanism. The expression and subcellular location of LINC00355 were determined by qRT-PCR and RNA-FISH, respectively. The lung SCC cell growth was analyzed by CCK-8 assay, transwell invasion, wound healing, colony formation, and flow cytometry assays. Reactive oxygen species level was evaluated by DCFH-DA probes. Bioinformatics online websites, luciferase reporter assay, RNA binding protein immunoprecipitation (RIP), and RNA pull-down assays were utilized to investigate the interaction among LINC00355, miR-466, and Ly-1 antibody reactive clone (LYAR). The results showed that LINC00355 was upregulated in lung SCC and was positively associated with poor overall survival in lung SCC patients. LINC00355 was mainly located in the cytoplasm of SCC cells. Additionally, LINC0035 functioned as a competing endogenous RNA (ceRNA) to target miR-466, and LYAR was identified as a direct target of miR-466. LINC00355 expression negatively correlated with miR-466 level, and positively correlated with LYAR level. Mechanistically, knockdown of LINC00355 inhibited cell proliferation, migration and invasion, promoted cell apoptosis in vitro, and suppressed tumor growth in vivo through targeting miR-466, and thus down-regulated LYAR expression. These findings provide a new sight for understanding the molecular mechanism of lung SCC and indicate that LINC00355 may serve as a potential biomarker for the diagnosis and treatment of lung SCC.

Correction: FTSJ1 regulates tRNA 2'-O-methyladenosine modification and suppresses the malignancy of NSCLC via inhibiting DRAM1 expression.

The original version of this article contained an error in the spelling of the author Yuchen Chen, which was incorrectly given as Yuhuan Chen. This has now been corrected in both the PDF and HTML versions of the article.

FTSJ1 regulates tRNA 2'-O-methyladenosine modification and suppresses the malignancy of NSCLC via inhibiting DRAM1 expression.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. The mechanisms underlying NSCLC tumorigenesis are incompletely understood. Transfer RNA (tRNA) modification is emerging as a novel regulatory mechanism for carcinogenesis. However, the role of tRNA modification in NSCLC remains obscure. In this study, HPLC/MS assay was used to quantify tRNA modification levels in NSCLC tissues and cells. tRNA-modifying enzyme genes were identified by comparative genomics and validated by qRT-PCR analysis. The biological functions of tRNA-modifying gene in NSCLC were investigated in vitro and in vivo. The mechanisms of tRNA-modifying gene in NSCLC were explored by RNA-seq, qRT-PCR, and rescue assays. The results showed that a total of 18 types of tRNA modifications and up to seven tRNA-modifying genes were significantly downregulated in NSCLC tumor tissues compared with that in normal tissues, with the 2'-O-methyladenosine (Am) modification displaying the lowest level in tumor tissues. Loss- and gain-of-function assays revealed that the amount of Am in tRNAs was significantly associated with expression levels of FTSJ1, which was also downregulated in NSCLC tissues and cells. Upregulation of FTSJ1 inhibited proliferation, migration, and promoted apoptosis of NSCLC cells in vitro. Silencing of FTSJ1 resulted in the opposite effects. In vivo assay confirmed that overexpression of FTSJ1 significantly suppressed the growth of NSCLC cells. Mechanistically, overexpression of FTSJ1 led to a decreased expression of DRAM1. Whereas knockdown of FTSJ1 resulted in an increased expression of DRAM1. Furthermore, silencing of DRAM1 substantially augmented the antitumor effect of FTSJ1 on NSCLC cells. Our findings suggested an important mechanism of tRNA modifications in NSCLC and demonstrated novel roles of FTSJ1 as both tRNA Am modifier and tumor suppressor in NSCLC.

RNA sequencing of exosomes revealed differentially expressed long noncoding RNAs in early-stage esophageal squamous cell carcinoma and benign esophagitis.

Aim: To explore the roles of exosomal long noncoding RNAs (lncRNAs) in early-stage esophageal squamous cell carcinoma (ESCC) and benign esophagitis. Materials & methods: Exosomal lncRNAs were analyzed using RNA-seq and validated by quantitative real-time PCR, loss-of-function, co-culture and RNA pulldown assays. Results: Exosomal lncRNAs displayed tighter tissue-specificity, higher expression level and lower splicing efficiency than that of mRNAs. A total of 152 exosomal lncRNAs were differentially expressed between ESCC and controls. A total of 124 exosomal lncRNAs were dysregulated between ESCC and esophagitis. Knockdown of 13 ESCC-associated lncRNAs modified proliferation, migration, and apoptosis of ESCC cells. A novel lncRNA RP5-1092A11.2 was highly expressed in ESCC-derived exosomes, ESCC cells and tumor tissues. Exosomes released from RP5-1092A11.2-knockdown cells inhibited ESCC cell proliferation. Conclusion: Dysregulated exosomal lncRNAs were functionally associated with different disease status in esophagus.

Case report: HER2 amplification as a resistance mechanism to crizotinib in NSCLC with MET exon 14 skipping.

Patients with non-small cell lung cancer (NSCLC) harboring MET exon 14 skipping can benefit from crizotinib treatment. Currently, the main resistance mechanisms to crizotinib are MET D1228N and Y1230C mutations. We reported a case of a Chinese NSCLC patient with MET exon 14 skipping detected by targeted next-generation sequencing (NGS) achieved clinical and imaging remission after crizotinib treatment. Then, amplification of multiple genes such as erb-b2 receptor tyrosine kinase 2 (HER2) was detected when disease progressed, indicating novel resistance mechanisms to crizotinib. Ultimately the patient died from cancer-related factors. This was the first NSCLC case with MET exon 14 skipping which reported the HER2 gene amplification at the time of progression during crizotinib treatment, indicating that bypass mechanisms contribute to the development of acquired resistance to MET inhibitors.

EGFR and Ras regulate DDX59 during lung cancer development.

Oncogenes EGFR and ras are frequently mutated and activated in human lung cancers. In this report, we found that both EGFR and Ras signaling can upregulate RNA helicase DDX59 in lung cancer cells. DDX59 can be induced through the mitogen activated protein kinase (MAPK) pathway after EGFR or Ras activation. Inhibitors for Ras/Raf/MAP pathway significantly decreased DDX59 expression at both protein and mRNA levels. Through immunohistochemistry, we found that DDX59 protein expression correlated with Ras and EGFR mutation status in human lung adenocarcinoma. Finally, through a xenograft nude mice model, we demonstrated that DDX59 is pivotal for EGFR mutated lung cancer cell growth in vivo. Our study identified a novel protein downstream of Ras and EGFR, which may serve as a potential therapeutic drug target for lung cancer patients.

Efficacy generated by afatinib in a lung adenocarcinoma patient harboring HER2 S310Y mutation.

Afatinib exhibits therapeutic efficacy for lung adenocarcinoma patients harboring HER2 exon 20 insertions. HER2 S310Y single site substitution was discovered in recent years and afatinib efficacy for adenocarcinoma patients harboring S310Y mutation has not been reported. We presented a case of a 41-year-old male patient with lung adenocarcinoma harboring the HER2 S310Y mutation obtained clinical response to the treatment of afatinib, an oral HER family blocker. After the treatment of afatinib, the patient achieved partial response (PR) in chest lesions and almost complete response (CR) in intracranial lesions. He experienced progressive disease (PD) with liver metastasis and achieved a progression-free survival (PFS) of 5 months. He continually treated with afatinib after CT guided percutaneous radiofrequency ablation to eradicate the hepatic tumor cells and achieved stable disease (SD). In this study, we reported the first clinical evidence of efficacy generated by afatinib, the irreversible HER family inhibitor, targeting HER2 S310Y single site mutation in lung adenocarcinoma.

LINC00355 promoted the progression of lung squamous cell carcinoma through regulating the miR-466/LYAR axis

LINC00355 has been reported aberrantly over-expressed and associated with poor prognosis in various types of cancer. However, reports regarding the effect of LINC00355 on lung squamous cell carcinoma (SCC) are rare. This study aimed to explore the function of LINC00355 in the development and progression of lung SCC and reveal the underlying mechanism. The expression and subcellular location of LINC00355 were determined by qRT-PCR and RNA-FISH, respectively. The lung SCC cell growth was analyzed by CCK-8 assay, transwell invasion, wound healing, colony formation, and flow cytometry assays. Reactive oxygen species level was evaluated by DCFH-DA probes. Bioinformatics online websites, luciferase reporter assay, RNA binding protein immunoprecipitation (RIP), and RNA pull-down assays were utilized to investigate the interaction among LINC00355, miR-466, and Ly-1 antibody reactive clone (LYAR). The results showed that LINC00355 was upregulated in lung SCC and was positively associated with poor overall survival in lung SCC patients. LINC00355 was mainly located in the cytoplasm of SCC cells. Additionally, LINC0035 functioned as a competing endogenous RNA (ceRNA) to target miR-466, and LYAR was identified as a direct target of miR-466. LINC00355 expression negatively correlated with miR-466 level, and positively correlated with LYAR level. Mechanistically, knockdown of LINC00355 inhibited cell proliferation, migration and invasion, promoted cell apoptosis in vitro, and suppressed tumor growth in vivo through targeting miR-466, and thus down-regulated LYAR expression. These findings provide a new sight for understanding the molecular mechanism of lung SCC and indicate that LINC00355 may serve as a potential biomarker for the diagnosis and treatment of lung SCC.