The transcription activity of OTX2 on p16 expression is significantly blocked by methylation of CpG shore in non-promoter of lung cancer cell lines.

Background: The aberrant expression of the classical tumor suppressor gene p16 is a frequent event in lung cancer mainly due to the hypermethylation of its 5'-cytosine-phosphate-guanine-3' island (Cgi). However, whether methylation happens in other regions and how p16 expression and function are affected are largely unknown. Methods: Clustered Regularly Interspaced Short Palindromic Repeats/dCas9 (CRISPR/dCas9) technology was used for methylation editing at specific site of p16. The effects of methylation editing were detected by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS), transwell migration and wound healing tests. Chromatin immnoprecipitation-quantitative polymerase chain reaction (CHIP-qPCR) was performed to explore the impact of Cgi shore methylation on the binding abilities of transcription factors (TFs) including YY1, SP1, ZNF148 and OTX2 to p16 gene. A rescue experiment was performed to verify the regulatory effect of OTX2 on p16. The negative relationship between p16 expression and the methylation level of Cgi shore in non-promoter region was further verified with datasets from The Cancer Genome Atlas (TCGA) program and lung adenocarcinoma (LUAD) patients' samples. Results: The suppressive effect of p16 Cgi shore methylation on its expression was demonstrated in both HEK293 and A549 cells using CRISPR/dCas9-mediated specific site methylation editing. Methylation of the Cgi shore in the p16 non-promoter region significantly decreased its expression and promoted cell growth and migration. The ability of OTX2 bound to p16 was significantly reduced by 19.35% after methylation modification. Over-expression of OTX2 in A549 cells partly reversed the inhibitory effect of methylation on p16 expression by 19.04%. The verification results with TCGA and LUAD patients' samples supported that the p16 Cgi shore is a key methylation regulatory region. Conclusions: Our findings suggested that methylation of the Cgi shore in the p16 non-promoter region can hamper the transcriptional activity of OTX2, leading to a reduction in the expression of p16, which might contribute to the development of lung cancer.

CRISPR-based DNA methylation editing of NNT rescues the cisplatin resistance of lung cancer cells by reducing autophagy.

Cisplatin is recommended as a first-line chemotherapeutic agent against advanced non-small cell lung cancer (NSCLC), but acquired resistance substantially limits its clinical efficacy. Recently, DNA methylation has been identified as an essential contributor to chemoresistance. However, the precise DNA methylation regulatory mechanism of cisplatin resistance remains unclear. Here, we found that nicotinamide nucleotide transhydrogenase (NNT) was silenced by DNA hypermethylation in cisplatin resistance A549 (A549/DDP) cells. Also, the DNA hypermethylation of NNT was positively correlated to poor prognosis in NSCLC patients. Overexpression of NNT in A549/DDP cells could reduce their cisplatin resistance, and also suppressed their tumor malignancy such as cell proliferation and clone formation. However, NNT enhanced sensitivity of A549/DDP cells to cisplatin had little to do with its function in mediating NADPH and ROS level, but was mainly because NNT could inhibit protective autophagy in A549/DDP cells. Further investigation revealed that NNT could decrease NAD+ level, thereby inactivate SIRT1 and block the autophagy pathway, while re-activation of SIRT1 through NAD+ precursor supplementation could antagonize this effect. In addition, targeted demethylation of NNT CpG island via CRISPR/dCas9-Tet1 system significantly reduced its DNA methylation level and inhibited the autophagy and cisplatin resistance in A549/DDP cells. Thus, our study found a novel chemoresistance target gene NNT, which played important roles in cisplatin resistance of lung cancer cells. Our findings also suggested that CRISPR-based DNA methylation editing of NNT could be a potential therapeutics method in cisplatin resistance of lung cancer.

LINC01224/ZNF91 Promote Stem Cell-Like Properties and Drive Radioresistance in Non-Small Cell Lung Cancer.

BACKGROUND: Radioresistance is the main reason for the failure of radiotherapy in non-small-cell lung cancer (NSCLC); however, the molecular mechanism of radioresistance is still unclear. METHODS: An RNA-Seq assay was used to screen differentially expressed long non-coding RNAs (lncRNAs) and genes in irradiation-resistant NSCLC cells. RT-PCR and Western blotting assays were performed to analyze the expressions of lncRNAs and genes. The chromosome conformation capture (3C) assay was performed to measure chromatin interactions. Cell cytotoxicity, cell apoptosis, sphere formation and Transwell assays were performed to assess cellular function. RESULTS: In this study, it was found that LINC01224 increased during the induction of radioresistance in NSCLC cells. LINC01224 was located within the enhancer of ZNF91, and LINC01224 could affect the transcription of ZNF91 by regulating the long-range interactions between the ZNF91 enhancer and promoter. Moreover, upregulation of LINC01224 and ZNF91 could promote irradiation resistance by regulating the stem cell-like properties of NSCLC cells. In addition, high expression levels of LINC01224 and ZNF91 in tissue samples were associated with radioresistance in NSCLC patients. CONCLUSION: Our findings demonstrated that LINC01224/ZNF91 drove radioresistance regulation by promoting the stem cell-like properties in NSCLC.

Comparative analysis of first-line treatment regimens for advanced EGFR-mutant non-small cell lung cancer patients with stable brain metastases.

BACKGROUND: To compare the survival outcomes of first-line treatment regimens for advanced epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) patients with stable brain metastases. METHODS: We conducted a systematic review of available data from randomized controlled trials (RCTs) of first-line treatment regimens of NSCLC patients with stable brain metastases. Progression free survival (PFS) and overall survival (OS) were extracted and analysed from the RCT subgroups. A network meta-analysis was constructed using the Bayesian statistical model to synthesize the survival outcomes of all the treatments. RESULTS: The analysis included 6 eligible RCT subgroups with 417 patients and 7 treatment regimens osimertinib, afatinib, first-generation EGFR-TKI (gefitinib or erlotinib), erlotinib + bevacizumab, gefitinib + pemetrexed + carboplatin, gemcitabine + cisplatin, and pemetrexed + cisplatin. Of these seven treatment regimens, gefitinib + pemetrexed + carboplatin had the highest potential for favorable PFS and OS, followed by osimertinib, in the treatment of advanced EGFR-mutant NSCLC patients with stable brain metastases. None of the results met the predetermined statistical significance of P<0.05. CONCLUSIONS: The regimens of "Gefitinib + pemetrexed + carboplatin" and "Osimertinib" were associated with the most favorable PFS and OS compared to the other therapies in advanced EGFR-mutant NSCLC patients with stable brain metastases, although the difference between these regimens and the others was not statistically significantly different.

Cell adhesion molecule 2 (CADM2) promotes brain metastasis by inducing epithelial-mesenchymal transition (EMT) in human non-small cell lung cancer.

BACKGROUND: To investigate the effect of CADM2 on brain metastasis in non-small cell lung cancer (NSCLC). METHODS: Human transcriptome-wide microarray analysis was used to identify gene expression in lung tissue of NSCLC patients with or without brain metastasis, which indicated that CADM2 was significantly up-regulated. Quantitative real-time PCR (qRT-PCR) was used to confirm the CADM2 up-regulation further. SiRNA was used to knock down the expression of CADM2 in NSCLC cell lines and a Transwell assay was performed to determine the effects of CADM2 knockdown on cell migration and invasion. The expressions of Vimentin and E-cadherin were detected by western blot assay. RESULTS: The result of microarray analysis and qRT-PCR showed that CADM2 was significantly up-regulated in NSCLC patients with brain metastasis than in those without brain metastasis. The result of the Transwell assay showed that the migration and invasion abilities of NSCLC cells were inhibited after CADM2 knockdown. Also, the expression of Vimentin was reduced while E-cadherin was increased, followed by CADM2 knockdown. CONCLUSIONS: The results showed that CADM2 might promote brain metastasis by inducing epithelial-mesenchymal transition (EMT) in human NSCLC. We propose that CADM2 can be used as a novel molecular target for the prevention and treatment in NSCLC with brain metastasis patients.

Epigenetic activation of FOXF1 confers cancer stem cell properties to cisplatin­resistant non­small cell lung cancer.

The underlying molecular mechanisms of cisplatin resistance in non­small cell lung cancer (NSCLC) are unclear. In this study, a novel differential methylation region located in the upstream regulatory region of the forkhead box F1 (FOXF1) gene was identified. The abnormal hypomethylation of FOXF1 increased the expression of FOXF1, and the high expression of FOXF1 promoted cell proliferation and inhibited cell apoptosis induced by cisplatin, which resulted in cisplatin resistance in NSCLC cells. In addition, FOXF1 promoted the expression of stem cell markers and self­renewal capability, indicating that FOXF1 regulated cisplatin resistance by promoting cancer stem cell properties in NSCLC cells. Moreover, a strong association was observed between FOXF1 upregulation and the presence of platinum­based chemotherapy resistance in patients with NSCLC. On the whole, the findings of this study indicate the regulatory mechanisms of cisplatin resistance by FOXF1 in NSCLC, and suggest that FOXF1 may be used as a prognostic biomarker of platinum­based chemotherapy resistance in NSCLC.

HGF/MET Regulated Epithelial-Mesenchymal Transitions And Metastasis By FOSL2 In Non-Small Cell Lung Cancer.

BACKGROUND: HGF/MET has been found to be associated with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms of HGF/MET involved in regulating the metastasis of NSCLC remain unclear. METHODS: The effect of HGF/MET and FOSL2 on cell migration and invasion were assessed by transwell and scratch assays. HGF/MET-induced phosphorylation and upregulation of FOSL2 was analyzed by RT-PCR and Western blotting. Regulatory effects of FOSL2 on SNAI2 transcription were detected by chromatin immunoprecipitation (ChIP) and dual-Luciferase reporter assays. The correlations of FOSL2 expression with clinical outcomes were assessed in 56 NSCLC patients. RESULTS: HGF/MET induced the phosphorylation and upregulation of FOSL2 by ERK1/2 kinase, FOSL2 promoted the transcription of SNAI2 by binding with the SNAI2 promoter, and SNAI2 subsequently promoted the epithelial-mesenchymal transition (EMT), invasion, and migration of NSCLC cells. According to the clinical correlation analysis in NSCLC, high expression of FOSL2 correlated with advanced tumor stage and metastasis. CONCLUSION: Our studies propose that the regulatory mechanisms of the HGF/MET-induced cascade pathway is mediated by FOSL2 in NSCLC metastasis and suggested that FOSL2 could potentially be employed as a prognostic biomarker and potential therapeutic target of NSCLC metastasis.

Epigenetic activation of hepatocyte growth factor is associated with epithelial-mesenchymal transition and clinical outcome in non-small cell lung cancer.

Hepatocyte growth factor (HGF) expression is repressed in normal differentiated lung epithelial cells, but its expression is aberrantly upregulated in non-small cell lung cancer (NSCLC) and acts as a poor prognostic factor. The underlying molecular mechanisms of aberrant HGF expression are unclear. In this study, a novel differential methylation region located in the HGF promoter was identified, which was associated with aberrant HGF expression in NSCLC. The correlations of HGF promoter methylation detected by methylation specific PCR and HGF expression detected by immunohistochemistry with clinical outcomes were assessed in NSCLC patients. DNA methylation of the HGF promoter was correlated with the activation of HGF expression, which induced epithelial-mesenchymal transition, cell migration and invasion. According to the clinical correlation analysis in 63 NSCLC patients, those with high methylation were more likely to have stages III and IV (51.6% vs. 25.0%, P<0.05) and metastasis (57.5% vs. 16.7%, P<0.05) than patients with low methylation. In addition, compared with the protein marker of HGF expression, the DNA methylation marker of the HGF promoter had higher specificity for prognostic analysis of metastases in NSCLC. Our study indicated the regulatory mechanisms related to DNA methylation of the HGF promoter for HGF expression in NSCLC epithelial cells, and suggested that the DNA methylation signature of the HGF promoter could potentially be employed as a biomarker to improve the prognostic accuracy of NSCLC.

let­7 and miR­17 promote self­renewal and drive gefitinib resistance in non­small cell lung cancer.

Epidermal growth factor receptor­tyrosinase kinase inhibitor (EGFR­TKI) resistance represents a major obstacle in the therapy of non­small cell lung cancer (NSCLC), and the underlying molecular mechanisms are unknown. In this study, it was found that let­7 family expression was downregulated and miR­17 family expression was upregulated in gefitinib­resistant PC9/GR cells compared with gefitinib­sensitive PC9 cells. The downregulation of let­7 and upregulation of miR­17 have significant clinical relevance to gefitinib resistance in NSCLC. Moreover, it was shown that downregulation of let­7 and upregulation of miR­17 promoted resistance to gefitinib by regulating the self­renewal capability of NSCLC cells. In addition, let­7 participated in the maintenance of stem cell characteristics by regulating the target gene MYC, and miR­17 participated in regulation of the cell cycle by regulating the target gene CDKN1A. In NSCLC cells, low expression of let­7 increased MYC expression to help maintain the undifferentiated status, and high expression of miR­17 decreased CDKN1A expression to help maintain the proliferative potential. Thus, both let­7 and miR­17 promoted self­renewal, which is typical of stem cell­like characteristics and resulted in gefitinib resistance. Therefore, this study demonstrated that let­7 and miR­17 were involved in the regulation of EGFR­TKI resistance, and could be used as predictive biomarkers of EGFR­TKI resistance in NSCLC.

ANKRD22 promotes progression of non-small cell lung cancer through transcriptional up-regulation of E2F1.

Lung cancer is the leading cause of death among all malignancies due to rapid tumor progression and relapse; however, the underlying molecular mechanisms of tumor progression are unclear. In the present study, we identified ANKRD22 as a novel tumor-associated gene in non-small cell lung cancer (NSCLC). According to the clinical correlation analysis, ANKRD22 was highly expressed in primary cancerous tissue compared with adjacent cancerous tissue, and high expression levels of ANKRD22 were significantly correlated with relapse and short overall survival time. Knockdown and overexpression analysis revealed that ANKRD22 promoted tumor progression by increasing cell proliferation. In xenograft assays, knockdown of ANKRD22 or in vivo treatment with ANKRD22 siRNA inhibited tumor growth. Furthermore, ANKRD22 was shown to participate in the transcriptional regulation of E2F1, and ANKRD22 promoted cell proliferation by up-regulating the expression of E2F1 which enhanced cell cycle progression. Therefore, our studies indicated that ANKRD22 up-regulated the transcription of E2F1 and promoted the progression of NSCLC by enhancing cell proliferation. These findings suggest that ANKRD22 could potentially act as a novel therapeutic target for NSCLC.

Disturbance of the let-7/LIN28 double-negative feedback loop is associated with radio- and chemo-resistance in non-small cell lung cancer.

Radio- and chemo-resistance represent major obstacles in the therapy of non-small-cell lung cancer (NSCLC) and the underlying molecular mechanisms are not known. In the present study, during induction of radio- or chemo-resistance in NSCLC cells, dynamic analyses revealed that decreased expression of let-7 induced by irradiation or cisplatin resulted in increased expression of its target gene LIN28, and increased expression of LIN28 then contributed to further decreased expression of let-7 by inhibiting its maturation and biogenesis. Moreover, we showed that down-regulation of let-7 and up-regulation of LIN28 expression promoted resistance to irradiation or cisplatin by regulating the single-cell proliferative capability of NSCLC cells. Consequently, in NSCLC cells, let-7 and LIN28 can form a double-negative feedback loop through mutual inhibition, and disturbance of the let-7/LIN28 double-negative feedback loop induced by irradiation or chemotherapeutic drugs can result in radio- and chemo-resistance. In addition, low expression of let-7 and high expression of LIN28 in NSCLC patients was associated significantly with resistance to radiotherapy or chemotherapy. Therefore, our study demonstrated that disturbance of the let-7/LIN28 double-negative feedback loop is involved in the regulation of radio- and chemo-resistance, and that let-7 and LIN28 could be employed as predictive biomarkers of response to radiotherapy or chemotherapy in NSCLC patients.

The regulatory and predictive functions of miR-17 and miR-92 families on cisplatin resistance of non-small cell lung cancer.

BACKGROUND: Chemotherapy is an important therapeutic approach for non-small cell lung cancer (NSCLC). However, a successful long-term treatment can be prevented by the occurring of chemotherapy resistance frequently, and the molecular mechanisms of chemotherapy resistance in NSCLC remain unclear. In this study, abnormal expressions of miR-17 and miR-92 families are observed in cisplatin-resistant cells, suggesting that miR-17 and miR-92 families are involved in the regulation of cisplatin resistance in NSCLC. METHODS: miRNA microarray shows that miR-17 and miR-92 families are all down-regulated in cisplatin-resistant A549/DDP cells compared with cisplatin-sensitive A549 cells. The aim of this study is to investigate the regulatory functions of miR-17 and miR-92 families on the formation of cisplatin resistance and the predictive functions of them as biomarkers of platinum-based chemotherapy resistance in NSCLC. RESULTS: The low expressions of miR-17 and miR-92 families can maintain cisplatin resistance through the regulation of CDKN1A and RAD21. As a result of high expressions of CDKN1A and RAD21, the inhibition of DNA synthesis and the repair of DNA damage are achieved and these may be two major contributing factors to cisplatin resistance. Moreover, we demonstrate that the expressions of miR-17 and miR-92 families in NSCLC tissues are significantly associated with platinum-based chemotherapy response. CONCLUSION: Our study indicates that miR-17 and miR-92 families play important roles in cisplatin resistance and can be used as potential biomarkers for better predicting the clinical response to platinum-based chemotherapy in NSCLC.

MiRNA 17 family regulates cisplatin-resistant and metastasis by targeting TGFbetaR2 in NSCLC.

MicroRNAs (miRNAs) have been proven to play crucial roles in cancer, including tumor chemotherapy resistance and metastasis of non-small-cell lung cancer (NSCLC). TGFß signal pathway abnormality is widely found in cancer and correlates with tumor proliferation, apoptosis and metastasis. Here, miR-17, 20a, 20b were detected down-regulated in A549/DDP cells (cisplatin resistance) compared with A549 cells (cisplatin sensitive). Over-expression of miR-17, 20a, 20b can not only decrease cisplatin-resistant but also reduce migration by inhibiting epithelial-to-mesenchymal transition (EMT) in A549/DDP cells. These functions of miR-17, 20a, 20b may be caused at least in part via inhibition of TGFß signal pathway, as miR-17, 20a, 20b are shown to directly target and repress TGF-beta receptor 2 (TGFßR2) which is an important component of TGFß signal pathway. Consequently, our study suggests that miRNA 17 family (including miR-17, 20a, 20b) can act as TGFßR2 suppressor for reversing cisplatin-resistant and suppressing metastasis in NSCLC.

Colonic metastasis from primary lung adenocarcinoma: case report and review of the literature.

We report a rare case of a 38-year-old woman with metastatic colonic adenocarcinoma from primary lung adenocarcinoma detected by PET/CT. She underwent colonoscopy and adenocarcinoma was diagnosed in the pathology report, which was the same as that for lymph node biopsy from a left supraclavicular lymph node. We used immunohistochemistry to diagnose primary adenocarcinoma of the lung with colonic metastasis. Owing to mutation in exon 19 of EGFR gene, targeted therapy was given to her with a prescription of oral gefitinib for 1 month as first-line treatment. It was chosen to further treat the patient with chemotherapy and radiotherapy. As the patient was suffering from increasing coughing and sputum, radiotherapy and chemotherapy were subsequently cancelled. Since the general condition of the patient was relatively poor, Tarceva was therefore prescribed. The patient had lived for 5 more months since the diagnosis of metastatic colonic adenocarcinoma.