Yuqing Chen

Effect of hypoxia-inducible factor-1α on transcription of survivin in non-small cell lung cancer

BackgroundSurvivin is a structurally and functionally unique member of the inhibitor of apoptosis protein (IAP) family. It plays an important role, not only in regulating mitosis but also in inhibiting apoptosis. The current literature contains few reports on the transcriptional regulation of survivin expression in lung cancer.MethodsIn this study, we investigated the effect of hypoxia-inducible factor-1α (HIF-1α) on the transcriptional activity of the survivin promoter in non-small cell lung cancer (NSCLC). Immunohistochemical staining was used to detect the expression of survivin and HIF-1α in the lung tissue of 120 patients with non-small cell lung cancer (NSCLC) and 40 patients with benign pulmonary disease. We also performed experiments with the lung adenocarcinoma cell line A549 cells, which were cultured under hypoxic conditions. The expression of survivin and HIF-1α was detected by real-time RT-PCR and Western blotting. In the survivin promoter the putative binding-site for HIF-1α, is -19 bp~-16 bp upstream of TSS. We performed site-directed mutagenesis of this binding site, and used luciferase reporter plasmids to determine the relative activity of the survivin promoter in A549 cells. We also studied the effect of HIF-1α on the expression of survivin by dsRNA targeting of HIF-1α mRNA.ResultsHIF-1α (58.33%) and survivin (81.60%) were both over-expressed in NSCLC and their expressions correlated with one another. They were also expressed in A549 cells under normal and hypoxic conditions, with a significant increase under hypoxic conditions. Site directed mutagenesis of the putative binding site for HIF-1α in the survivin promoter significantly decreased the activity of the survivin promoter in A549 cells. Inhibition of HIF-1α by RNAi decreased the expression of survivin in A549 cell lines.ConclusionOur results indicate that the binding of HIF-1α to the survivin promoter increases transcription of the survivin gene. Thus, HIF-1α is an important transcriptional regulator of survivin expression

Notch-1 signaling facilitates survivin expression in human non-small cell lung cancer cells

The oncogenic potential of Notch activation is observed in many instances including lung tumorigenesis, but the associated molecular regulatory mechanism has not been thoroughly defined. It has been demonstrated that hypoxia can act as one of the major stimuli in the progression of many types of tumorigenesis. This study was designed to examine the activation of Notch-1 signaling by hypoxia and its contribution to survivin expression in human lung carcinomas. Western-blot and PCR analysis showed that Notch-1 signaling is activated by hypoxia in the human non-small cell lung cancer (NSCLC) cell line, A549, through the upregulation of Notch-1, along with its intracellular domain (N1ICD). The activity of Hes-1, a crucial target molecule of N1ICD, was also increased under hypoxia. Interestingly, blockade of the Notch-1 pathway by a γ-secretase inhibitor or small interfering RNA (siRNA) inhibited survivin expression. Conversely, activation of Notch-1 signaling by N1ICD or stimulation with the Jagged1 ligand enhanced survivin levels in A549 cells. Notably, HIF-1α cooperated with Notch-1 signaling to increase survivin expression through its direct association with N1ICD, consequently accelerating survivin transcription. Overall, our findings suggest that Notch-1 signaling is involved in the upregulation of survivin expression in lung cancer cells, which is synergized by HIF-1α.

HIF-1α knockdown by miRNA decreases survivin expression and inhibits A549 cell growth in vitro and in vivo

The present study examined the downregulation of survivin expression by hypoxia-inducible factor-1α (HIF-1α) miRNA and its effect in the inhibition of A549 cell growth in vitro and in vivo. Survivin expression, apoptosis, proliferation and migration under normoxic and hypoxic conditions were assessed by standard methods. Cotransfection and chromatin immunoprecipitation were used to observe the effects of HIF-1α on survivin transcription. HIF-1α knockdown in A549 cells were injected into nude mice to examine survivin expression and suppression of tumorigenicity. Transfection of A549 cells with HIF-1α miRNA led to decreased expression of HIF-1α and survivin mRNA and protein. Survivin overexpression is mediated by HIF-1α by direct binding to a putative binding site in the survivin core promoter. HIF-1α-miRNA induced apoptosis and inhibited proliferation of A549 cells under hypoxic, but not normoxic, conditions, whereas transfection by survivin expression vectors partly rescued the apoptotic phenotype and revived cell proliferation under hypoxic conditions. However, cell migration was substantially suppressed by HIF-1α silencing under normoxic and hypoxic conditions. After A549 cells were xenografted in nude mice, survivin expression in mice treated with HIF-1α miRNA was downregulated, and tumor growth was significantly inhibited. Silenced HIF-1α gene expression induced apoptosis and suppressed growth of A549 cells by downregulating survivin expression in vitro and in vivo. Our results also provide a basis to target the HIF-1α pathway in lung cancer therapy.

Sp1 Upregulates Survivin Expression in Adenocarcinoma of Lung Cell Line A549

Survivin has been implicated in tumor genesis, progression, and resistance to anticancer agents. However, the precise regulatory mechanism for survivin expression is not thoroughly defined. In this study, we showed that Sp1 was co‐overexpressed with survivin in adenocarcinoma of lung cells A549, but not in differentiated human bronchial epithelial cells 4F0439 or small airway epithelial cells 3F1584. Subsequently, transfection experiments demonstrated that the inhibition of Sp1 signaling suppressed survivin expression in A549 cells, whereas Sp1 overexpression increased the level of survivin protein as well as its mRNA. We also found that Sp1 could decrease capase‐9 activity, which is shown to be suppressed by survivin during apoptosis inhibition. Finally, Luciferase activity and ChIP assays revealed that Sp1 activated survivin promoter by direct interaction with it. Taken together, our data suggest Sp1 plays a potent role in the upregulation of survivin expression in lung cancer cells at the transcriptional level. Anat Rec, 2011.

The DEAD-box RNA helicase 51 controls non-small cell lung cancer proliferation by regulating cell cycle progression via multiple pathways.

The genetic regulation of cell cycle progression and cell proliferation plays a role in the growth of non-small cell lung cancer (NSCLC), one of the most common causes of cancer-related mortality. Although DEAD-box RNA helicases are known to play a role in cancer development, including lung cancer, the potential involvement of the novel family member DDX51 has not yet been investigated. In the current study we assessed the role of DDX51 in NSCLC using a siRNA-based approach. DDX51 siRNA-expressing cells exhibited a slower cell proliferation rate and underwent arrest in S-phase of the cell cycle compared with control cells. Microarray analyses revealed that DDX51siRNA expression resulted in the dysregulation of a number of cell signalling pathways. Moreover, injection of DDX51 siRNA into an animal model resulted in the formation of smaller tumours compared with the control group. We also assessed the expression of DDX51 in patients with NSCLC, and the data revealed that the expression was correlated with patient age but no other risk factors. Overall, our data suggest for the first time that DDX51 aids cell cancer proliferation by regulating multiple signalling pathways, and that this protein might be a therapeutic target for NSCLC.

CDK16 overexpressed in non-small cell lung cancer and regulates cancer cell growth and apoptosis via a p27-dependent mechanism

Cyclin-dependent kinase 16 (CDK16, PCTAIRE1) expression is upregulated in a wide variety of human malignancies. However, the function(s) of CDK16 in non-small cell lung cancer (NSCLC) remain unknown. Therefore, here we investigated the role of CDK16 in NSCLC. From 43 NSCLC tumors and matching healthy control lung tissues, immunohistochemistry revealed significantly greater CDK16 and phospho-p27Ser10 staining levels in NSCLC samples relative to healthy controls. The NSCLC cell line EKVX was transfected with a control siRNA, a CDK16-siRNA, or CDK16-siRNA + p27-siRNA. We found significantly decreased proliferation levels and significantly increased apoptosis levels in CDK16-silenced NSCLC cells. However, these effects were abrogated in cells treated with both the CDK16-siRNA and the p27-siRNA. In CDK16-silenced NSCLC cells, we found upregulated p27 and downregulated phospho-p27Ser10 protein expression but downregulated ubiquitinated p27 and ubiquitinated phospho-p27Ser10 protein expression. Cycloheximide-treated CDK16-silenced NSCLC cells displayed a much milder reduction in p27 protein expression over time relative to untreated CDK16-silenced NSCLC cells. In summary, CDK16 is significantly upregulated in human NSCLC tumor tissue and plays an oncogenic role in NSCLC cells via promoting cell proliferation and inhibiting apoptosis in a p27-dependent manner. Moreover, CDK16 negatively regulates expression of the p27 via ubiquination and protein degradation.

The diagnostic value of narrow-band imaging for early and invasive lung cancer: a meta-analysis

This study aimed to compare the ability of narrow-band imaging to detect early and invasive lung cancer with that of conventional pathological analysis and white-light bronchoscopy. We searched the PubMed, EMBASE, Sinomed, and China National Knowledge Infrastructure databases for relevant studies. Meta-disc software was used to perform data analysis, meta-regression analysis, sensitivity analysis, and heterogeneity testing, and STATA software was used to determine if publication bias was present, as well as to calculate the relative risks for the sensitivity and specificity of narrow-band imaging vs those of white-light bronchoscopy for the detection of early and invasive lung cancer. A random-effects model was used to assess the diagnostic efficacy of the above modalities in cases in which a high degree of between-study heterogeneity was noted with respect to their diagnostic efficacies. The database search identified six studies including 578 patients. The pooled sensitivity and specificity of narrow-band imaging were 86% (95% confidence interval: 83–88%) and 81% (95% confidence interval: 77–84%), respectively, and the pooled sensitivity and specificity of white-light bronchoscopy were 70% (95% confidence interval: 66–74%) and 66% (95% confidence interval: 62–70%), respectively. The pooled relative risks for the sensitivity and specificity of narrow-band imaging vs the sensitivity and specificity of white-light bronchoscopy for the detection of early and invasive lung cancer were 1.33 (95% confidence interval: 1.07–1.67) and 1.09 (95% confidence interval: 0.84–1.42), respectively, and sensitivity analysis showed that narrow-band imaging exhibited good diagnostic efficacy with respect to detecting early and invasive lung cancer and that the results of the study were stable. Narrow-band imaging was superior to white light bronchoscopy with respect to detecting early and invasive lung cancer; however, the specificities of the two modalities did not differ significantly.

Cigarette smoke enhances initiation and progression of lung cancer by mutating Notch1/2 and dysregulating downstream signaling molecules

Lung cancer is the primary cause of cancer related deaths in the western world and smoking significantly increases the risk of developing lung cancer. Smoking enhances lung cancer initiation and progression. The effects of cigarette smoke on lung cancer are mediated by the presence of highly mutagenic substances, including nicotine, leading to mutations in oncogenes and tumor suppressor genes. An emerging pathway in cancer is the Notch signaling pathway which is essential for embryonic lung development and tissue homeostasis. The role of Notch signaling in lung cancer remains controversial and no studies have directly linked cigarette exposure to mutations in Notch. Therefore, we investigated the direct effect of Notch signaling pathways on cigarette-induced lung tumors and the correlation between smoking and mutations in Notch leading to altered downstream signaling. Human cell lines, mouse models and clinical lung cancer samples were utilized in this study. Cigarette-induced in vitro human lung cancer models and in vivo mouse models demonstrated strong effects of cigarette exposure on the Notch signaling pathway. Immunohistochemistry (IHC) of 50 clinical samples collected from smokers and non-smokers with and without lung cancer also demonstrated a link between smoking and changes in Notch signaling. Finally, 34 lung cancer samples analyzed through direct sequencing indicated smoking significantly increased small nucleotide polymorphisms (SNPs) in Notch 1 and 2 and specific SNPs significantly modulated expression levels of downstream signaling pathway molecules. Taken together, these results demonstrate a direct effect of smoking on the Notch signaling pathway leading to lung cancer initiation and progression.