METTL14 enhances the m6A modification level of lncRNA MSTRG.292666.16 to promote the progression of non-small cell lung cancer.

BACKGROUND: m6A modification has close connection with the occurrence, development, and prognosis of tumors. This study aimed to explore the roles of m6A modification and its related mechanisms in non-small cell lung cancer (NSCLC). METHODS: NSCLC tissues and their corresponding para-cancerous tissues were collected to determine the m6A levels of total RNA/lncRNAs and the expression of m6A modification-related genes/lncRNAs. Then, A549 cells were transfected with si-METTL14 or oe-METTL14, and the cell transfection efficiency was assessed. Subsequently, the viability, apoptosis, cell colony formation, migration and invasion of the different cells were determined. Finally, the nude mouse tumorigenicity experiments were performed to observe the effects of METTL14 in vivo. RESULTS: Compared to the para-NSCLC tissues, the m6A level and METTL14 expression were both significantly increased in the NSCLC tissues (P < 0.05). Based on the expression of METTL14 in the different cell lines, A549 cells were chosen for further experiments. Then, the A549 cells with METTL14 knockdown and overexpression were successfully established, as well as it was found that METTL14 knockdown could inhibit the viability, colony formation, migration, and invasion of A549 cells, while facilitate their apoptosis. In vivo experiments also showed that METTL14 knockdown could inhibit tumor formation and growth. Additionally, the m6A level of MSTRG.292666.16 was higher in the NSCLC tissues; and after METTL14 knockdown, the expression and m6A level of MSTRG.292666.16 were both significantly reduced in A549 cells, and vice versa. CONCLUSION: METTL14 may promote the progression of NSCLC through up-regulating MSTRG.292666.16 and enhance its m6A modification level.

DNMT3A governs tyrosine kinase inhibitors responses through IAPs and in a cell senescence-dependent manner in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) treated with tyrosine kinase inhibitors (TKIs) inevitably exhibit drug resistance, which diminishes therapeutic effects. Nonetheless, the molecular mechanisms of TKI resistance in NSCLC remain obscure. In this study, data from clinical and TCGA databases revealed an increase in DNMT3A expression, which was correlated with a poor prognosis. Using NSCLC organoid models, we observed that high DNMT3A levels reduced TKI susceptibility of NSCLC cells via upregulating inhibitor of apoptosis proteins (IAPs). Simultaneously, the DNMT3Ahigh subset, which escaped apoptosis, underwent an early senescent-like state in a CDKN1A-dependent manner. Furthermore, the cellular senescence induced by TKIs was observed to be reversible, whereas DNMT3Ahigh cells reacquired their proliferative characteristics in the absence of TKIs, resulting in subsequent tumour recurrence and growth. Notably, the blockade of DNMT3A/IAPs signals enhanced the efficacy of TKIs in DNMT3Ahigh tumour-bearing mice, which represented a promising strategy for the effective treatment of NSCLC.

Temporal genomic heterogeneity guiding individualized therapy in recurrent non-small cell lung cancer.

Introduction: Despite the benefit of adjuvant systemic therapy for patients with resected non-small cell lung cancer (NSCLC), the risk of postoperative recurrence remains high. Our objective was to characterize temporal genetic heterogeneity between primary resected and recurrent tumors, and its impact on treatment outcomes. Methods: In this study, next-generation sequencing (NGS) testing was performed on tissue specimens and circulating tumor DNA (ctDNA) collected at postoperative recurrence, and results were compared to the genotypes of initial surgical specimens. Results: Of forty-five patients with matched primary and post-operative recurrent tumors, EGFR status switched in 17 patients (37.8%) at post-operative recurrence and 28 patients (62.2%) had no genotype change (17 mutant, 11 wild-type). Based on the changes of EGFR status, patients were divided into 4 groups. Following subsequent treatment with EGFR TKI o chemotherapy: In group A, with sustained sensitive mutation, the percentage achieving partial response (PR) was the highest, at 72.2%, the median progression-free survival (PFS) was 17 months, and the median overall survival (OS) was 44.0 months respectively; In group B, with genotype changed from wild-type to mutant, 50% achieved PR, PFS was 10 months, and OS was 35 months; In group C, in which mutant status shifted to wild-type or new co-mutation emerged, the percentage achieving PR was 30%, PFS was 9 months, and OS was 35 months. In group D, with sustained wild type, the percentage achieving PR was 27.3%, PFS was 8 months, and OS was 22 months. Discussion: Genotypic shift between paired primary and post-operative recurrent tumors was not infrequent, and this temporal genomic heterogeneity substantially impacted subsequent treatment outcomes.

Exosomes derived from M2 type tumor-associated macrophages promote osimertinib resistance in non-small cell lung cancer through MSTRG.292666.16-miR-6836-5p-MAPK8IP3 axis.

BACKGROUND: Osimertinib resistance limits the treatment of epidermal growth factor receptor-(EGFR)-mutated non-small-cell lung carcinoma (NSCLC). The mechanisms of osimertinib resistance need to be elucidated to determine alternative treatment strategies. This study explores the role of M2 type tumor-associated macrophage (TAM)-derived exosomal MSTRG.292666.16 in osimertinib resistance, and its related competing endogenous RNA (ceRNA) mechanism. METHODS: M2 type TAMs were induced with 200 ng/mL phorbol 12-myristate 13-acetate, 20 ng/mL IL-4 and IL-13, and M2 type macrophage markers were measured by RT-qPCR. Next, the exosomes were isolated and characterized. Tumor formation in nude mice was conducted using H1975 cells under different treatment conditions. Small RNA sequencing was performed on exosomes derived from sensitive and resistant plasma, and ceRNA networks were constructed. Fluorescence in situ hybridization was used to observe the localization of MSTRG.292666.16, and a ceRNA network (MSTRG.292666.16-miR-6836-5p-MAPK8IP3) was selected for further validation. RESULTS: M2 type TAMs, and M2 type TAM-derived exosomes were successfully induced and isolated. Nude mice results showed that M2 type TAM-derived exosomes and MSTRG.292666.16 overexpression significantly increased tumor volume after administration of osimertinib for 4 weeks. M2 type TAMs were found in the resistant plasma, and MSTRG.292666.16 localized in the cytoplasm of H1975 cells. In addition, the genes in the ceRNA networks were significantly enriched in eight GO terms and seven KEGG pathways, including the MAPK signaling pathway. Subsequently, the levels of MSTRG.292666.16 and MAPK8IP3 significantly increased in both resistant plasma-derived exosomes and M2 type TAM-derived exosomes, while miR-6836-5p levels were significantly reduced. Finally, MSTRG.292666.16, miR-6836-5p, and MAPK8IP3 were part of the same network. CONCLUSIONS: M2 type TAM-derived exosomes promoted osimertinib resistance in NSCLC by regulating the MSTRG.292666.16/miR-6386-5p/MAPK8IP3 axis.

Exosomal long non-coding RNA MSTRG.292666.16 is associated with osimertinib (AZD9291) resistance in non-small cell lung cancer.

Acquired resistance of osimertinib is encountered in clinic treatment of non-small cell lung cancer (NSCLC). However, the molecular mechanisms of osimertinib resistance are not fully revealed. This study aimed to investigate the roles of exosomes in delivering osimertinib resistance in NSCLC. Exosomes were successfully isolated. LncRNA sequencing identified a total of 123 differentially expressed lncRNAs, including 45 upregulated lncRNAs and 78 downregulated lncRNAs. The relative expression level of lncRNA MSTRG.292666.16 was significantly upregulated in osimertinib-resistant plasma, osimertinib-resistant H1975R cells and their derived exosomes, compared with those in osimertinib- sensitive plasma, H1975 cells and exosomes (P < 0.05). Besides, osimertinib-resistant exosomes could regulate gene expressions induced by osimertinib, including miRNA-21, miRNA-125b, TGFß, ARF6 and c-Kit. Osimertinib-resistant exosomes could be taken up by osimertinib-sensitive H1975 cells and resulting in osimertinib-resistance in vivo. Knockdown of lncRNA MSTRG.292666.16 decreased osimertinib resistance of H1975R cells. Our results suggest that exosomal lncRNA MSTRG.292666.16 might be associated with osimertinib resistance in NSCLC.

Cyclooxygenase-2 mediates gefitinib resistance in non-small cell lung cancer through the EGFR/PI3K/AKT axis.

Gefitinib is a potent inhibitor of EGFR and represents the front-line treatment for non-small cell lung cancer (NSCLC) therapeutics. However, NSCLC patients are prone to develop acquired resistance through as yet, undefined mechanisms of resistance. Here, we investigated the role of COX-2 during gefitinib resistance in NSCLC cells and revealed its underlying mechanism(s) of action. We report the upregulation of COX-2 in gefitinib-resistant NSCLC tissues and cells, which is associated with poor prognosis. In vitro assays in NSCLC cells (PC9/GR) showed that COX-2 facilitates gefitinib resistance in NSCLC cells through its effects on P-gp, MRP1, and BCRP, and cancer cell migration and invasion. In vivo, COX-2 silencing could repress tumor growth. We found that the overexpression of COX-2 enhances the transcription of MMP-2, MMP-7, and MMP-9 which mediates PI3K-AKT activation. In summary, we demonstrate that COX-2 mediates the gefitinib resistance of NSCLC cells through its interaction with EGFR and the PI3K-AKT axis. This highlights COX-2 as a novel molecular target for NSCLC.

A novel virtual barcode strategy for accurate panel-wide variant calling in circulating tumor DNA.

BACKGROUND: Hybrid capture-based next-generation sequencing of DNA has been widely applied in the detection of circulating tumor DNA (ctDNA). Various methods have been proposed for ctDNA detection, but low-allelic-fraction (AF) variants are still a great challenge. In addition, no panel-wide calling algorithm is available, which hiders the full usage of ctDNA based 'liquid biopsy'. Thus, we developed the VBCALAVD (Virtual Barcode-based Calling Algorithm for Low Allelic Variant Detection) in silico to overcome these limitations. RESULTS: Based on the understanding of the nature of ctDNA fragmentation, a novel platform-independent virtual barcode strategy was established to eliminate random sequencing errors by clustering sequencing reads into virtual families. Stereotypical mutant-family-level background artifacts were polished by constructing AF distributions. Three additional robust fine-tuning filters were obtained to eliminate stochastic mutant-family-level noises. The performance of our algorithm was validated using cell-free DNA reference standard samples (cfDNA RSDs) and normal healthy cfDNA samples (cfDNA controls). For the RSDs with AFs of 0.1, 0.2, 0.5, 1 and 5%, the mean F1 scores were 0.43 (0.25~0.56), 0.77, 0.92, 0.926 (0.86~1.0) and 0.89 (0.75~1.0), respectively, which indicates that the proposed approach significantly outperforms the published algorithms. Among controls, no false positives were detected. Meanwhile, characteristics of mutant-family-level noise and quantitative determinants of divergence between mutant-family-level noises from controls and RSDs were clearly depicted. CONCLUSIONS: Due to its good performance in the detection of low-AF variants, our algorithm will greatly facilitate the noninvasive panel-wide detection of ctDNA in research and clinical settings. The whole pipeline is available at https://github.com/zhaodalv/VBCALAVD.

Detection of plasma EGFR mutations for personalized treatment of lung cancer patients without pathologic diagnosis.

INTRODUCTION: Next-generation sequencing (NGS) and digital polymerase chain reaction (PCR) based platforms have been used to detect EGFR mutations in plasma circulating tumor DNA (ctDNA) with high accuracy. Generally, molecular testing is performed after histopathological analysis. However, many patients with suspected advanced nonsmall cell lung cancer are unable to undergo biopsy thus forgoing potential treatment with highly effective tyrosine kinase inhibitors (TKIs) in patients with sensitizing EGFR mutations. We examined the utility of ctDNA testing to detect EGFR mutations in patients' plasma, where tissue biopsy is not feasible. METHODS: We conducted a single-center, prospective study of 30 Chinese patients with suspected advanced lung cancer, who were unable to undergo a biopsy for initial diagnosis due to comorbidities or poor performance status. Patients with plasma EGFR sensitizing mutations were treated with first-generation EGFR TKIs. RESULTS: Twenty of 30 patients enrolled had sensitizing EGFR mutations in ctDNA and were started on EGFR TKIs. After a median follow-up of 12 months, median progression-free survival (PFS) was 10 months and median overall survival (OS) was not reached. The median OS for the 10 untreated patients was 3 months. CONCLUSIONS: In our study, patients with plasma EGFR mutations treated with TKIs showed disease control rate (DCR) and PFS similar to historical controls that were treated based on tissue testing. This is the first prospective study showing that ctDNA genotyping provides a feasible diagnostic approach for frail lung cancer patients who are unable to undergo biopsy, which subsequently leads to EGFR-targeted therapy, and improved outcomes in this subgroup of patients.

Predictive value of unmethylated RASSF1A on disease progression in non-small cell lung cancer patients receiving pemetrexed-based chemotherapy.

BACKGROUND AND OBJECTIVE: Chemotherapy remains the basis of the treatment of lung cancer, and screening biomarkers with predictive value for chemotherapy is of great interest. The present study focused on status of genes methylation in NSCLC patients receiving pemetrexed- or gemcitabine-based chemotherapy. PATIENTS AND METHODS: Promoter methylation of Ras association domain family (RASSF1A) and short stature homeobox 2 (SHOX2) was examined in bronchoalveolar lavage (BAL) from 117 NSCLC patients treated with chemotherapy. Multivariate analysis was used to identify the predictive value of gene methylation. Progression-free survival (PFS) rather than overall survival (OS) was used as the clinical outcome to minimize the impact of chemotherapy on gene methylation. RESULTS: The methylation of RASSF1A and SHOX2 was significantly associated with shorter PFS (RASSF1A: HR = 2.355, 95% CI: 1.533-3.617, P< 0.0001; SHOX2: HR = 2.123, 95% CI: 1.392-3.236, P= 0.0004). After adjusting for confounding factors, RASSF1A methylation was still a predictive factor for PFS (HR = 1.765, 95% CI: 1.064-2.928, P= 0.0278). In the pemetrexed group, unmethylated RASSF1A could be used to predict longer PFS (P= 0.0001), and no predictive value was found in the gemcitabine group. CONCLUSION: Unmethylated RASSF1A is a favorable prognostic indicator for patients receiving pemetrexed doublets. Because of the promoting effect of most chemotherapeutic drugs on gene methylation, unmethylated RASSF1A is not suitable as a predictor for gemcitabine doublets.

Competitive evolution of NSCLC tumor clones and the drug resistance mechanism of first-generation EGFR-TKIs in Chinese NSCLC patients.

PURPOSE: Although many studies have reported on the resistance mechanism of first-generation EGFR TKIs (1st EGFR TKIs) treatment, large-scale dynamic ctDNA mutation analysis based on liquid biopsy for non-small cell lung cancer (NSCLC) in the Chinese population is rare. Using in-depth integration and analysis of ctDNA genomic mutation data and clinical data at multiple time points during the treatment of 53 NSCLC patients, we described the resistance mechanisms of 1st EGFR TKIs treatment more comprehensively and dynamically. The resulting profile of the polyclonal competitive evolution of the tumor provides some new insights into the precise treatment of NSCLC. EXPERIMENTAL DESIGN: A prospective study was conducted in patients with advanced NSCLC with acquired resistance to erlotinib, gefitinib or icotinib. By liquid biopsy, we detected mutations in 124 tumor-associated genes in the context of drug resistance. These 124 genes covered all tumor therapeutic targets and related biological pathways. During the entire course of treatment, the interval between two liquid biopsies was two months. RESULTS: Unlike the common mutations tested in tissue samples, our data showed a higher coverage of tumor heterogeneity (32.65%), more complex patterns of resistance and some new resistance mutation sites, such as EGFR p.V769M and KRAS p.A11V. The major resistance-associated mutations detected were still EGFR p.T790M (45.28%), other point mutations in EGFR (33.9%), and KRAS and NRAS mutations (15.09%). These mutation ratios might be considered as a preliminary summary of the characteristics of Chinese patients. In addition, starting from the two baseline mutations of the EGFR gene (19del vs. L858R), we first described the detailed mutation profile of the EGFR gene. Although there was no significant difference in the number of patients with EGFR p.19del and EGFR p.L858R baseline mutations (24% vs. 16%, P = 0.15), patients from the EGFR p.19del baseline group were much more likely to develop EGFR p.T790M resistance mutations (62.1% vs. 19.3%, P = 0.007). Through careful integration of gene mutation information and clinical phenotype information, an interesting phenomenon was found. Although the variant allele fraction (VAF) of the EGFR p.T790M mutation was significantly linearly correlated with that of the EGFR drug-sensitive mutation (r = 0.68, P = 0.00025), neither VAF was associated with the tumor volume at the advanced stage. It was shown that other tumor clones might contribute more to the resistance to 1st EGFR TKIs treatment than tumor clones carrying the EGFR p.T790M mutation when resistance developed. By further analysis, we found that, in some patients, when the primary tumor clones detected were those carrying EGFR-/- mutations (both types the EGFR p.19del/p.L858R and EGFR p.T790M mutation types were missing), most of them showed a poor prognosis and ineffective late treatment, indicating that EGFR-/- played a more important role than EGFR p.T790M in the process of NSCLC drug resistance in these patients. From the perspective of the clonal evolution of NSCLC tumor cells, these phenomena could be explained by the competitive evolution between different tumor clones. In addition, two new mutations, KRAS p.A11V and EGFR p.V769M, emerged significantly during drug resistance in NSCLC patients and had shown obvious competitive clonal evolution characteristics. Combined with clear clinical drug resistance phenotypic information, we believed that these two new mutations might be related to new drug resistance mechanisms and deserve further study. We have also seen an interesting phenomenon. In some patients undergoing 1st EGFR TKIs treatment, the EGFR p.T790M mutation appeared, disappeared, and reappeared, and this spatial and temporal diversity of the EGFR p.T790M mutation was regulated by targeted drug and chemotherapy and was correlated with the individual tumor mutation profile. CONCLUSIONS: The constitution and competitive evolution of the tumor clones have a decisive influence on treatment and can be regulated by targeted drugs and chemotherapy. Additionally, EGFR p.T790M spatial and temporal diversity during treatment warrants more attention, and this spatial and temporal diversity may be useful for the choice of treatment strategies for certain NSCLC patients. Through longitudinal cfDNA sample analysis, the resistance mechanism and dynamic clinical features of Chinese NSCLC patients are systematically established as reliable and meaningful to understand acquired resistance and make further personalized treatment decisions dynamically. Two new potential drug resistance-associated mutations in EGFR and KRAS have been found and are worthy of further study. Finally, our research shows that the evolutionary process of tumor cloning can be artificially regulated and intervened, possibly providing a new way to treat tumors.

Engineering docetaxel-loaded micelles for non-small cell lung cancer: a comparative study of microfluidic and bulk nanoparticle preparation.

Bulk preparation of micelles has the drawbacks of facile formation of large aggregates and heterogeneous particle size distribution. Microfluidic technology has shown clear potential to address these challenges for robust nanomedicine applications. In this study, docetaxel-loaded PLGA-PEG-Mal-based micelles were prepared by microfluidics and dialysis methods and their physicochemical properties were analyzed. The biological behaviors of these micelles were also investigated in the non-small cell lung cancer (NSCLC) cell line A549 in vitro as well as in vivo. Encouragingly, the mean particle size of the micelles prepared by microfluidics (DMM) was smaller, with an average size of 72 ± 1 nm and a narrow size distribution with a polydispersity index (PDI) of 0.072; meanwhile, micelles prepared by the dialysis method (DMD) had larger particle sizes (range, 102 to 144 nm) and PDIs (up to 0.390). More importantly, significantly high drug loading was achieved using the microfluidic process. The IC50 value of DMM was lower than that of DMD. Whole-body fluorescence imaging of live mice showed that DMM achieved higher accumulation in tumors compared with DMD. DMM showed superior antitumor efficacy, with a tumor inhibition rate of 91.5%. Moreover, pathological histology analysis revealed that no evident biological toxicity was caused by the micelles. In addition, Arg-Gly-Asp (RGD) was employed as a targeting agent on the basis of DMM to prepare targeting micelles, and the targeting micelles exhibited stronger cytotoxicity and obvious antitumor efficacy. In conclusion, DMM may have obvious clinical advantages for the treatment of NSCLC due to its optimized physiochemical properties. Therefore, microfluidic technology-based micelles are a promising platform as an effective drug delivery system for incorporating anticancer agents.

Cancer-Related Triplets of mRNA-lncRNA-miRNA Revealed by Integrative Network in Uterine Corpus Endometrial Carcinoma.

The regulation of transcriptome expression level is a complex process involving multiple-level interactions among molecules such as protein coding RNA (mRNA), long noncoding RNA (lncRNA), and microRNA (miRNA), which are essential for the transcriptome stability and maintenance and regulation of body homeostasis. The availability of multilevel expression data enables a comprehensive view of the regulatory network. In this study, we analyzed the coding and noncoding gene expression profiles of 301 patients with uterine corpus endometrial carcinoma (UCEC). A new method was proposed to construct a genome-wide integrative network based on variance inflation factor (VIF) regression method. The cross-regulation relations of mRNA, lncRNA, and miRNA were then selected based on clique-searching algorithm from the network, when any two molecules of the three were shown as interacting according to the integrative network. Such relation, which we call the mRNA-lncRNA-miRNA triplet, demonstrated the complexity in transcriptome regulation process. Finally, six UCEC-related triplets were selected in which the mRNA participates in endometrial carcinoma pathway, such as CDH1 and TP53. The multi-type RNAs are proved to be cross-regulated as to each of the six triplets according to literature. All the triplets demonstrated the association with the initiation and progression of UCEC. Our method provides a comprehensive strategy for the investigation of transcriptome regulation mechanism.

The Evolution and Expression Pattern of Human Overlapping lncRNA and Protein-coding Gene Pairs.

Long non-coding RNA overlapping with protein-coding gene (lncRNA-coding pair) is a special type of overlapping genes. Protein-coding overlapping genes have been well studied and increasing attention has been paid to lncRNAs. By studying lncRNA-coding pairs in human genome, we showed that lncRNA-coding pairs were more likely to be generated by overprinting and retaining genes in lncRNA-coding pairs were given higher priority than non-overlapping genes. Besides, the preference of overlapping configurations preserved during evolution was based on the origin of lncRNA-coding pairs. Further investigations showed that lncRNAs promoting the splicing of their embedded protein-coding partners was a unilateral interaction, but the existence of overlapping partners improving the gene expression was bidirectional and the effect was decreased with the increased evolutionary age of genes. Additionally, the expression of lncRNA-coding pairs showed an overall positive correlation and the expression correlation was associated with their overlapping configurations, local genomic environment and evolutionary age of genes. Comparison of the expression correlation of lncRNA-coding pairs between normal and cancer samples found that the lineage-specific pairs including old protein-coding genes may play an important role in tumorigenesis. This work presents a systematically comprehensive understanding of the evolution and the expression pattern of human lncRNA-coding pairs.

SPOP promotes SIRT2 degradation and suppresses non-small cell lung cancer cell growth.

SIRT2 is a NAD-dependent deacetylase and inhibition of SIRT2 has a broad anticancer activity. Here we report that SPOP binds to SIRT2 and mediates its degradation by the 26S proteasome, which can be blocked by MG132 treatment. We also found that the levels of SPOP significantly decreased, while the levels of SIRT2 significantly increased in non-small cell lung cancer (NSCLC) cell lines, compared to normal bronchial epithelial cell line and NSCLC specimens, compared to the paired non-tumor lung tissue. Furthermore, SPOP can suppress NSCLC cell growth. Notably, mutations in NSCLC inhibit the abilities of SPOP to degrade SIRT2 and suppress NSCLC cell growth. These results reveal a novel regulation of SIRT2 by SPOP mediated degradation, which is important for the growth of lung tumor cells.

[Relationship between ID1 and EGFR-TKI Resistance 
in Non-small Cell Lung Cancer].

BACKGROUND: Non-small cell lung cancer (NSCLC) presents the highest morbidity and mortality among malignant tumors worldwide. The overall effective rate of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is 30% to 40%, and PFS (progression-free sruvival) is 12 months. However, EGFR-TKI resistance is typical in clinical observations, and this phenomenon significantly affects tumor suppression. To overcome this resistance, a new prognostic factor associated with lung cancer drug resistance should be discovered. This study investigated the relationship between the inhibitor of differentiation 1 (ID1) and non-small cell lung cancer EGFR-TKI resistance in vivo and in vitro to determine any statistical significance and discuss the underlying mechanism. METHODS: Western blot and qRT-PCR were used to quantify the expression of ID1 in lung cancer. IHC was used to detect the expression of ID1 in pathological tissues (lung cancer tissues and adjacent tissues). MTT was used to detect cell proliferation, in which the cells were treated with gefitinib after being transfected by ID1 slow virus vector. Lung cancer cells were inoculated in nude mice until the tumor diameter grew to certain measurement. Gefitinib treatment was started, and the tumor volume was estimated. RESULTS: ID1 was highly expressed in NSCLC (P<0.05). Both ID1 expression and drug resistance of EGFR-TKI in NSCLC were positively correlated (P<0.05). The treatment group with gefitinib showed obviously less expression than the control group. CONCLUSIONS: ID1 is highly expressed in NSCLC. ID1 expression was positively related to drug resistance of EGFR-TKI in NSCLC. Gefitinib can be used to effectively treat NSCLC, and the mechanism may be associated with an increased level of STAT3 phosphorylation.

Overexpression of miR-100 inhibits cancer growth, migration, and chemosensitivity in human NSCLC cells through fibroblast growth factor receptor 3.

Nonsmall cell lung cancer (NSCLC) is a commonly occurring lung cancer. A combination of molecular biological treatments with regular chemotherapy may result in improved therapeutic outcome. Here, we reported significantly higher levels of fibroblast growth factor receptor 3 (FGFR3) and significantly lower levels of miR-100 in the NSCLC specimen, compared to the paired NSCLC-adjacent normal lung tissues. Moreover, the levels of FGFR3 and miR-100 were inversely correlated. Bioinformatics analyses followed by luciferase reporter assay showed that miR-100 bound to the 3'-UTR of FGFR3 messenger RNA (mRNA) to inhibit its translation. Overexpression of miR-100 in NSCLC cells decreased FGFR3 protein levels, whereas inhibition of miR-100 increased FGFR3 protein levels, without affecting FGFR3 mRNA levels. Furthermore, overexpression of miR-100 suppressed cancer growth, migration, and chemosensitivity in NSCLC cells, while inhibition of miR-100 significantly facilitated them. Taken together, our data demonstrate that miR-100 may inhibit NSCLC through FGFR3.

Rapamycin Enhances the Anti-Cancer Effect of Dasatinib by Suppressing Src/PI3K/mTOR Pathway in NSCLC Cells.

Src and the mammalian target of rapamycin (mTOR) signaling are commonly activated in non-small cell lung cancer (NSCLC) and hence potential targets for chemotherapy. Although the combined use of Src inhibitor Dasatinib with other chemotherapeutic agents has shown superior efficacy for cancer treatment, the mechanisms that lead to enhanced sensitivity of Dasatinib are not completely understood. In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis. The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation. Mechanistic investigations demonstrated that FoxO1/FoxO3a and p70S6K/4E-BP1, the molecules at downstream of Src-PI3K-Akt and mTOR signaling, were significantly suppressed by the combined use of Dasatinib and Rapamycin. Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib. In addition, this finding was also validated by a series of assays using another two NSCLC cell lines, NCI-H1706 and NCI-H460. Conclusively, our results suggested that the combinatory application of Src and mTOR inhibitors might be a promising therapeutic strategy for NSCLC treatment.

Shorter EGFR Dinucleotide Repeat Length Predicts Better Response of Patients with Advanced Non-small Cell Lung Cancer to EGFR Tyrosine Kinase Inhibitor.

The purpose of this study is to evaluate the association between intron 1 CA-repeat polymorphisms of the epidermal growth factor receptor gene (EGFR) and the clinical outcome of Chinese patients with advanced non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). We genotyped the intron 1 CA-repeat genetic polymorphisms of EGFR in 84 Chinese patients with NSCLC. The relationship between the length of the CA repeats and EGFR mutations in exons 18-21 in the 84 patients was elucidated. We then analyzed the association between the length of the CA repeats and the clinical outcome of EGFR-TKI-treated patients with NSCLC. EGFR mutations in exon 19 were significantly associated with shorter CA repeats. Patients with shorter CA repeats had a significantly longer progression-free survival with EGFR-TKI treatment than those with longer CA repeats. Our results suggest that shorter CA repeats in intron 1 of EGFR are associated with EGFR mutations and the clinical outcomes of TKI-treated patients with NSCLC.

Mechanisms behind the inhibition of lung adenocarcinoma cell by shikonin.

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, can exert inhibitory effect on tumor cell growth. However, little has been known concerning the effect of shikonin on lung adenocarcinoma cell and underlying mechanisms. In the present study, we investigated the effect of shikonin on the proliferation, cell cycle arrest, and apoptosis in human lung adenocarcinoma cells. We found that shikonin significantly suppressed the proliferation of lung adenocarcinoma cells compared with control in dose- and time-dependent manner (P < 0.05). In the meantime, our results showed that shikonin markedly increased the proportion of A549 cells at stage G1 as well as induced apoptosis in A549 cells. Furthermore, suppressed CCND1 and elevated caspase3 and caspase7 expression levels at mRNA were found in this study, indicating that shikonin may inhibit the growth of lung adenocarcinoma cell by changing cell cycle and promoting cell apoptosis through the regulation of CCND1, caspase3, and caspase7. Although more studies are needed, this study suggests that shikonin has the potential to be used as an anti-cancer agent in the treatment of lung adenocarcinoma.