Yongwen Li

Clinical Significance of EML4-ALK Fusion Gene and Association with EGFR and KRAS Gene Mutations in 208 Chinese Patients with Non-Small Cell Lung Cancer

The EML4-ALK fusion gene has been recently identified in a small subset of non-small cell lung cancer (NSCLC) patients who respond positively to ALK inhibitors. The characteristics of the EML4-ALK fusion gene in Chinese patients with NSCLC are poorly understood. Here, we report on the prevalence of EML4-ALK, EGFR status and KRAS mutations in 208 Chinese patients with NSCLC. EGFR mutations were found in 24.5% (51/208) of patients. In concordance with previous reports, these mutations were identified at high frequencies in females (47.5% vs 15.0% in males; P<0.05); never-smokers (42.3% vs 13.9% in smokers; P<0.05), and adenocarcinoma patients (44.2% vs 8.0% in non-adenocarcinoma patients; P<0.05). There were only 2.88% (6/208) patients with KRAS mutations in our study group. We identified 7 patients who harbored the EML4-ALK fusion gene (3.37%, 7/208), including 4 cases with variant 3 (57.1%), 2 with variant 1, and 1 with variant 2. All positive cases corresponded to female patients (11.5%, 7/61). Six of the positive cases were non-smokers (7.69%, 6/78). The incidence of EML4-ALK translocation in female, non-smoking adenocarcinoma patients was as high as 15.2% (5/33). No EGFR/KRAS mutations were detected among the EML4-ALK positive patients. Pathological analysis showed no difference between solid signet-ring cell pattern (4/7) and mucinous cribriform pattern (3/7) in ALK-positive patients. Immunostaining showed intratumor heterogeneity of ALK rearrangement in primary carcinomas and 50% (3/6) of metastatic tumors with ALK-negative staining. Meta-analysis demonstrated that EML4-ALK translocation occurred in 4.84% (125/2580) of unselected patients with NSCLC, and was also predominant in non-smoking patients with adenocarcinoma. Taken together, EML4-ALK translocations were infrequent in the entire NSCLC patient population, but were frequent in the NSCLC subgroup of female, non-smoker, adenocarcinoma patients. There was intratumor heterogeneity of ALK rearrangement in primary carcinomas and at metastatic sites.

MicroRNA-449a inhibits cell growth in lung cancer and regulates long noncoding RNA nuclear enriched abundant transcript 1

OBJECTIVE Lung cancer has become the primary cause of cancer-related death now. New therapies targeting the molecular regulatory machinery were required imperatively. MicroRNAs and long noncoding RNAs can respectively or cooperatively function as oncogenes or tumor suppressor genes in human cancers. The present study identified that miR-449a was down-regulated in tissue of human lung cancer. In this study, we aimed to investigate the function of miR-449a in NL9980 and L9981 lung carcinoma cells lines and the relationship with lncRNA nuclear enriched abundant transcript 1 (NEAT1). MATERIALS AND METHODS miR-449a was profiled in several lung carcinoma cell lines by quantitative reverse transcription-polymerase chain reaction RT-PCR. We analyzed the effects of miR-449a overexpression on proliferation, apoptosis and cell cycle in L9981 cells. The regulatory relationship between miR-449a and NEAT1 was predicted in silico and further studied by miR-449a inhibitor and mimics assay. RESULTS miR-449a was expressed in four cell lines, which we selected, however miR-449a was in high level in NL9980 and in low level in L9981 (P < 0.05). When the miR-449a was the overexpression in L9981 cells, the cell growth was suppressed, and the apoptosis cells were promoted compared with the control group (P < 0.05). The G1/G0 became longer and S, G2/M became shorter (P < 0.05) by miR-449a overexpression. Further study of the interaction between miR-449a and NEAT1 show that NEAT1 was up-regulated when cells were transfected with miR-449a inhibitor, and NEAT1 was down-regulated when cells transfected with miR-449a mimics. CONCLUSIONS Our data indicate that miR-449a may function as a suppressor of lung cancer, and affects the expression of NEAT1 in lung cancer cells.

Expression levels of microRNA-145 and microRNA-10b are associated with metastasis in non-small cell lung cancer

ABSTRACT Although metastasis remains the overwhelming cause of death for patients with non-small cell lung cancer (NSCLC), the underlying mechanisms of metastasis remain unknown. Accumulating evidence suggests that microRNAs (miRNAs) are key players in the regulation of tumor cell invasion and metastasis. Expression of miR-9, miR-10b, miR-145, and miR-155, 4 miRNAs previously shown to play roles in metastasis in other tumor types, was compared in lymph node (LN)-positive NSCLC versus LN-negative NSCLC. Expression of miR-145 was significantly lower in LN-positive NSCLC (P < 0.05), while expression of miR-10b was significantly higher (P < 0.05). Expression of both miR-145 and miR-10b was correlated with lymph node metastasis in NSCLC (both Ps < 0.001). In addition, miR-10b facilitated the migration and invasion of lung cancer cell line A549, while miR-145 suppressed the migration and invasion capacity of A549 in vitro. These results suggest that miR-10b and miR-145 may act as an oncogene or tumor suppressor gene, respectively, in NSCLC metastasis.

MiR-146b-5p functions as a suppressor miRNA and prognosis predictor in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. However, science has not yet been able to substantially improve the prognosis of lung cancer patients. Accumulating evidence suggests that microRNAs (miRNAs) are key players in the regulation of tumor development and metastasis. Expression of six miRNAs previously shown to play roles in tumor development (miR-146b-5p, miR-128b, miR-21, miR-221, miR-34a, and Let-7a) in other tumor types was examined using real-time RT-PCR in 78 specimens of NSCLC. The results revealed that patients with low expression of miR-146b-5p had significant shorter median and mean survival time than those with high miR-146b-5p expression (33.00 and 30.44 months versus 42.0 and 36.90 months, respectively; log-rank test P=0.048), thus low miR-146b-5p expression level was associated with poor prognosis in NSCLC patients. Univariate Cox hazard regression analysis demonstrated that miR-146b-5p expression levels tended to be a significant prognostic indicator of NSCLC (adjusted hazard ratio=0.482, 95% CI: 1.409- 29.593, P=0.016). Multivariate Cox proportional hazard regression analysis showed that miR-146b-5p expression levels were an independent prognostic factor for NSCLC patients (hazard ratio=0.259, 95% CI: 0.083-0.809, P=0.020). Furthermore, the effects of miR-146b-5p and miR-146b-3p on NSCLC cell growth and invasion in vitro were investigated. Our findings demonstrate that ectopic expression of miR-146b-5p suppressed cell proliferation, clonogenicity, migration/ invasion and also induced G1 arrest in vitro, but did not induce cell apoptosis; whereas enforced expression of miR-146b-3p did not have a significant effect on cell growth and metastasis. Further experiments indicated that miR-146b-5p could reduce mRNA levels of MMP16 and TRAF6 in vitro and was negatively related to the expression of TRAF6 in human NSCLC tissues. In a mouse model, Ago-miR-146b-5p could significantly inhibit the growth of lung cancer xenografts in nude mice. In conclusion, our findings demonstrate that miR-146b-5p functions as a suppressor miRNA and prognosis predictor in NSCLC.

Cloning and Functional Analysis of FLJ20420: A Novel Transcription Factor for the BAG-1 Promoter

BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5′ cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ∼26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix), except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski). Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA –transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p = 0.0006). The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p = 0.0001). Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal expression may be involved in the oncogenesis of human malignancies such as lung cancer.

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, sensitizes lung cancer cells to treatment with epidermal growth factor receptor tyrosine kinase inhibitors

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge to targeted therapy for non-small cell lung cancer (NSCLC). We investigated whether a cyclin D kinase 4/6 (CDK4/6) inhibitor, PD 0332991, could reverse EGFR-TKI resistance in human lung cancer cells and explored the underlying mechanisms. We found that PD 0332991 potentiated gefitinib-induced growth inhibition in both EGFR-TKI-sensitive (PC-9) and EGFR-TKI-resistant (PC-9/AB2) cells by down-regulating proliferation and inducing apoptosis and G0/G1 cell cycle arrest. Tumor xenografts were then used to verify the effects of PD 0332991 in vivo. Mice treated with a combination of PD 0332991 and gefitinib had the fastest tumor regression and delayed relapse. Tumors from mice receiving the combination treatment exhibited down-regulated proliferation, up-regulated apoptosis, and less angiogenesis. Finally, lung adenocarcinoma patients with acquired resistance to EGFR-TKIs were given an exploratory treatment of PD 0332991. One patient with gefitinib resistance exhibited clinical remission after treatment with PD 0332991. These findings suggest PD 0332991 reverses acquired EGFR-TKI-resistance in NSCLC cells, and may provide a novel treatment strategy for NSLSC patients with EGFR-TKI resistance.

MiR-182 inhibits the epithelial to mesenchymal transition and metastasis of lung cancer cells by targeting the Met gene

The microRNA miR‐182, belonging to the miR‐183 family, is one of the most frequently studied cancer‐related oncogenic miRNAs that is dysregulated in various cancer tissues, and it plays a crucial role in tumorigenesis and tumor progression. Studies revealed that miR‐182 might function as an oncogenic or tumor suppressor miRNA in different tissues. However, the role of miR‐182 in the development of lung cancer remains largely unknown. miR‐182 expression in tumor samples from 58 patients, normal lung tissue samples, and lung cancer cell lines were evaluated by qRT‐PCR. Survival curves were analyzed using the Kaplan‐Meier method and compared with a log‐rank test. Our study demonstrated that miR‐182 is frequently downregulated in metastatic NSCLC cells compared with primary tumor tissues. Over‐expression of miR‐182 significantly inhibited the migration and invasion of lung cancer cells and promoted the expression of the epithelial marker (E‐cadherin) in addition to reducing the levels of Snail in lung cancer cells. Further studies demonstrated that miR‐182 negatively regulated Met via direct binding to the Met 3′‐untranslated region (3′‐UTR). Furthermore, we found that miR‐182 suppressed the phosphorylation of AKT and the nuclear accumulation of Snail, a transcription factor that promotes the epidermal to mesenchymal transition (EMT). Moreover, miR‐182 could repress cell migration, invasion, and EMT of lung cancer cells induced by hepatocyte growth factor (HGF). miR‐182 might suppress the EMT and metastasis via inactivation of Met/AKT/Snail in non‐small cell lung cancer (NSCLC) cells, which implicates miR‐182 may be useful as a new therapeutic target in NSCLC.

Activating transcription factor 3 promotes malignance of lung cancer cells in vitro

Lung cancer remains the most common cause of cancer‐related death, with high rates of recurrence and poor outcomes. An abnormally high expression of activating transcription factor 3 (ATF3) in various cancers suggests an oncogenic role; however, its function in lung cancer is largely unknown.

[A highly efficient in vitro site-directed mutagenesis protocol for introducing multiple-site mutations into target genes].

BACKGROUND The methods for introducing point mutations into target genes are important for dissecting the relationship of gene structure and function. Up to date, there are numbers of protocols available for the purpose. However, many of them are suited for introducing single site mutation into the target gene. For introducing multiple-site mutations simultaneously into the target genes, it is required to further improve the related methods. METHODS In this report, we describe an improvement on the type IIs restriction enzyme-dependent site-directed mutagenesis method and the improved protocol is highly efficient for multiple-site mutagenesis. In our method, a pair of mutagenic primers are synthesized for each desired site and each mutagenic primer contains a selected type IIs restriction site. The DNA fragments between two neighboring sites are amplified with PCR. All amplified fragments are then digested by the selected Type IIs restriction enzyme. The expected mutant is eventually generated by ligation of these digested DNA fragments. RESULTS The improved protocol is very easy and can be achieved in just one day. As a proof of principle, we have introduced multiple-site, i.e., 3-site or 4-site mutations into the fusion gene of nm23 and EGFP (enhanced green fluorecence protein). The mutagenic frequencies are almost reached 100%. CONCLUSIONS Our protocol provides a useful tool for gene function research.

Abstract 4966: Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective: Lung cancer has become one of the leading causes of cancer-related mortality in the world. Metastasis remains the overwhelming causes of death for lung cancer patients. 80-90% of lung cancer deaths are caused by metastasis. However, an ideal in vitro model to explore the regulation and the molecules that control the lymph node metastasis of lung cancer is required. Methods: A gene expression profiling for both of the cell lines were generated by microarray. Further investigations were carried out in the lung cancer cell lines and paired tissues by migration and invasion assays and immunohistochemistry. Results: Firstly, we established a paired human lung adenocarcinoma cell lines from primary tumor site (named DC cells) and metastatic paratracheal lymph nodes (named DL cells). The two cell lines have different biological characteristics which DC cells grow clump easily and DL cells grow more scattered. The doubling time of DC and DL cell lines were 32.6h and 45.2h, respectively. The detection of the human D1S80 and D17S30 VNTR locus verified the same individual for DC and DL cell lines. Secondary, Microarray analysis revealed 232 upregulated probe sets and 739 downregulated probe sets in DL cell line compared to DC cell line. Compared to DC cells, the transcriptional expression of 27 genes were dramatically increased (p < 0.05), including AIM2, IGFBP4, IL6 and IL6R in DL cells, while expression of 55 genes was clearly decreased (p < 0.05), including FGF13, FGF19, IL1A, IL1B, MCAM, MMP15, MMP7, and TGFB2 by real-time PCR analysis. Furthermore, we selected 32 genes and screened the expression of those genes in 10-paired human NSCLC primary lung cancer tissues and matched metastasis lymph nodes tissues by real-time PCR. The results showed: 8 out of 10 metastasis lymph node tissues got up-regulated of RGS7, VNN2, and 7 out of 10 metastasis lymph node tissues got up-regulated of PECAM1, VNN1, CLIC2 expression. On the other hand, 8 of 10 metastasis lymph node tissues got down-regulated expression of CEACAM6, and 7 out of 10 got down-regulated expression of PIAS3. At mean time, none of metastasis lymph node tissues got decreased expression of TNFRSF9, which only was seen in DL cells. The further study in other lung cancer cell lines indicated that CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes influenc lung cancer cell migration ability in vitro. And these genes were also analyzed in more lung cancer tissues by immunohistochemistry staining. Conclusion: We established a paired human lung adenocarcinoma cell lines from primary tumor site and metastatic paratracheal lymph nodes. The CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes involve in the lung cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Jun Chen, Min Wang, Lingling Zu, Yongwen Li, Weiqiang Wang,Ying Li, Hongyu Liu. Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2014-4966