Tzu Chin Wu

Association of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation with p53 mutation occurrence in non-small cell lung cancer with different histology, gender, and smoking status

BackgroundO6-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been demonstrated to associate with the G:C→A:T transition mutation in the p53 gene of lung tumors. The purpose of this study is to clarify whether MGMT promoter methylation is not only associated with the shift from the G:C→A:T mutation in the p53 gene but also whether MGMT increases other mutation patterns in lung tumors.Materials and MethodsTo further verify whether a different prevalence of MGMT promoter methylation is observed in lung tumors with a different tumor histology, gender, and smoking status, 220 lung tumors were collected to evaluate the status of MGMT promoter methylation and p53 mutation using methylation-specific PCR (MSP) and direct sequencing, respectively.ResultsThe data shows that a higher prevalence of MGMT promoter methylation was observed in tumors with the G:C→A:T transition or other p53 mutation patterns compared with those with p53 wild-type (P < 0.001 for G:C→A:T; P = 0.015 for other mutation patterns), and this prevalence was more pronounced in tumors from male than from female patients. MGMT promoter methylation in p53 mutation patterns had a different effect on squamous cell carcinomas (SCC) and adenocarcinomas (ADC). Interestingly, the highest prevalence of MGMT promoter methylation was found in male nonsmokers followed by male smokers and female nonsmokers. This may be a partial explanation for the reason why male nonsmokers had a higher p53 mutation occurrence than female nonsmokers.ConclusionsMGMT promoter methylation may associate with increased occurrence of p53 mutation including the G:C→A:T transition and other p53 mutation patterns in lung cancer, especially among male nonsmokers.

Loss of telomerase activity may be a potential favorable prognostic marker in lung carcinomas

Many cancer and immortal cells exhibit telomerase activity that stabilizes telomere lengths, possibly contributing to cell immortality and carcinogenesis. The aim of this study was to elucidate the clinicopathological relationship between telomerase activity and telomerase reverse transcriptase subunit (hTERT) status in non small cell lung cancer. hTERT status in non small cell lung cancer using telomeric repeat amplification protocol (TRAP) and RT-PCR assay, respectively. Telomerase activity and hTERT were detected in 85.7 and 80.3% of cancerous tissues, respectively. Telomerase activity does not correlate with clinicopathological variables. However, there was an association between p53-correlated expression and hTERT negative status. Lung cancer patients without telomerase activity survived for a significantly longer period than those with telomerase activity. In addition, hTERT was not associated with the prognosis. TERT expression did not correlate well with any clinical parameter. Reactivated telomerase activity may be a poor prognostic factor in NSCLCs.

IL-10 Promotes Tumor Aggressiveness via Upregulation of CIP2A Transcription in Lung Adenocarcinoma

Purpose: Interleukin-10 (IL-10) determines virus persistent infection and promotes viral-associated tumor progression via tumor immune escape. However, the role of IL-10 in tumor progression and prognosis in lung adenocarcinoma remains controversial. Experimental Design: To investigate how IL-10 is regulated by HPV E6, IL-10 promoter was constructed to understand which transcriptional factor could be responsible for its transcription. To verify which molecule could be responsible for IL-10–mediated soft agar growth and invasion capability, PCR array and mechanistic strategies were conducted. IL-10 and CIP2A mRNA levels in lung tumors from patients with lung cancer were determined by real-time reverse transcription PCR. The prognostic value of both molecules on survival was estimated by Cox regression model. Results: Mechanistic studies showed that IL-10 protein and mRNA expression was decreased in E6 knockdown TL1 cells and increased in E6- overexpressing TL4 cells. In addition, IL-10 transcription was predominantly regulated by E6-mediated phosphorylation of cAMP response element-binding protein (CREB) and C/enhancer-binding protein β (C/EBPβ) via phosphoinositide 3-kinase (PI3K) signaling pathway. IL-10–mediated tumor aggressiveness in vitro and in vivo occurs through increased CIP2A expression via PI3K signaling pathway. Among patients, IL-10 mRNA expression in lung tumors was positively correlated with CIP2A mRNA expression. Cox-regression analysis showed that IL-10 and CIP2A mRNA levels may independently predict survival in patients with lung adenocarcinoma, especially in patients with E6-positive tumors. Conclusion: IL-10 production from lung tumors and immune cells promotes lung adenocarcinoma aggressiveness and patients with poor survival. We thus suggest that PI3K inhibitor combined with chemotherapy may potentially enhance tumor regression and improve patients’ outcome and life quality. Clin Cancer Res; 19(15); 4092–103. ©2013 AACR.

Human Papillomavirus Type 16/18 Up-Regulates the Expression of Interleukin-6 and Antiapoptotic Mcl-1 in Non–Small Cell Lung Cancer

Purpose: Human papillomavirus (HPV) 16/18 infection is reported to be associated with nonsmoking Taiwanese female lung cancer. In this study, we attempted to further reveal the association between HPV infection with Mcl-1 and interleukin (IL)-6 expressions and to elucidate the roles of HPV infection in lung tumorigenesis. Experimental Design: IL-6 and Mcl-1 expressions were investigated in 79 tumor tissues from lung cancer patients by immunohitochemistry. Secreting IL-6 levels and Mcl-1 expressions were examined by ELISA and Western blot, respectively, in HPV 16/18 E6- and E7-transfected A549 human lung cancer cells, as well as in the HPV16-infected TL-1 lung cancer cells established from lung cancer patients. Results: Lung tumors (70.9% and 57.0%) had positive IL-6 and Mcl-1 immunostainings, respectively. Significant correlation between IL-6 and Mcl-1 expression were observed (P < 0.0001). Both IL-6 and Mcl-1 expression were significantly associated with HPV 16/18 infection (P = 0.014 and P = 0.004, respectively). IL-6 and Mcl-1 protein levels were not only elevated in HPV 16/18 E6- and E7-transfected A549 cells but also in TL-1 cells. Phosphatidylinositol-3-OH kinase pathway was the major pathway contributing to the up-regulation of Mcl-1 by IL-6 in HPV-infected lung cancer cells. Conclusions: The up-regulating effects of HPV 16/18 E6 and E7 to IL-6 and Mcl-1 expressions were observed in E6- and E7-transfected A549 cells and in HPV16-infected TL-1 cells, mainly through the phosphatidylinositol-3-OH kinase pathway. The involvement of HPV infection in lung tumorigenesis may be partly through a concomitant increased expression of autocrine and/or paracrine IL-6 and the downstream Mcl-1.

MnSOD promotes tumor invasion via upregulation of FoxM1-MMP2 axis and related with poor survival and relapse in lung adenocarcinomas

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme responsible for the elimination of superoxide radical. The role of MnSOD in tumor progression in different human cancers is still controversial. In the present study, MnSOD expression in lung cancer cells was explored by knockdown or overexpression using transfection of a short hairpin RNA (shRNA) or an expression vector, respectively, to determine whether MnSOD expression mediates lung cancer cell migration, invasion, and oncogenic potential by increasing FoxM1 and MMP2 expression. Western blotting showed that FoxM1 and MMP2 expression was dependent on MnSOD expression, suggesting that FoxM1 could be upregulated by MnSOD. Three FoxM1 promoters were constructed to verify this activation of FoxM1 by MnSOD and to determine the transcription factors responsible. Luciferase reporter and chromatin immunoprecipitation assays indicated that MnSOD overexpression in lung cancer cells promoted binding of E2F1 and Sp1 to their putative FoxM1 promoter-binding sites and activated FoxM1 reporter activity. MnSOD also enhanced the potential for cell migration, invasion, and anchorage-independent colony growth on soft-agar plates, again via upregulation of FoxM1 and MMP2 expression. In patients with lung cancer, evaluation of MnSOD expression in lung tumors by immunohistochemistry indicated a positive correlation between FoxM1 and MMP2 mRNA expressions. Kaplan–Meier and Cox regression analysis revealed a poorer overall survival (OS) and relapse-free survival (RFS) in patients with MnSOD-positive tumors than with MnSOD-negative tumors. We conclude that MnSOD may promote tumor aggressiveness via upregulation of the FoxM1–MMP2 axis, and that MnSOD expression can independently predict survival and relapse in patients with resected lung adenocarcinoma. Mol Cancer Res; 11(3); 261–71. ©2012 AACR.

Human telomerase reverse transcriptase activated by E6 oncoprotein is required for human papillomavirus-16/18-infected lung tumorigenesis.

Purpose: Our recent report indicates that human papillomavirus (HPV)-16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether human telomerase reverse transcriptase (hTERT) transcription is up-regulated by E6 and contributes to lung tumor development. Experimental Design: Immunohistochemistry detected HPV-16 E6 oncoprotein in 135 lung tumors, and hTERT mRNA was evaluated by real-time reverse transcription-PCR and in situ hybridization, respectively. A small RNA interference (RNAi), Western blotting, and chromatin immunoprecipitation analysis were used to clarify whether hTERT transcription was regulated by c-Myc and Sp1. The telomerase activity and oncogenic potential of TL-1 with or without E6- or hTERT-RNAi was determined by real-time quantitative telomeric repeat amplification protocol analysis and soft-agar assay, respectively. Results: hTERT mRNA levels in E6-positive tumors, which were prevalent in females, nonsmokers, and adenocarcinomas, were significantly higher than in E6-negative tumors. In addition, hTERT mRNA levels in early tumors (stage I) were greater than levels in advanced tumors (stages II and III). Chromatin immunoprecipitation assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells, which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6- or hTERT-RNAi. A soft-agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6-RNAi. Moreover, a colony of TL-1 cells could not form after transfection with hTERT-RNAi. Conclusion: Transcriptional activation of hTERT by E6 oncoprotein is required for HPV-16/18-infected lung tumorigenesis.

Interleukin-10 Haplotype May Predict Survival and Relapse in Resected Non-Small Cell Lung Cancer

IL-10 is associated with tumor malignancy via immune escape. We hypothesized that IL-10 haplotypes categorized by IL-10 promoter polymorphisms at –1082A>G, –819C>T, and –592C>A might influence IL-10 expression and give rise to non-small cell lung cancer (NSCLC) patients with poor outcomes and relapse. We collected adjacent normal tissues from 385 NSCLC patients to determine IL-10 haplotypes by direct sequencing and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 385 tumors, 241 were available to evaluate IL-10 mRNA expression levels by real-time RT-PCR. The influence of IL-10 haplotypes on overall survival (OS) and relapse free survival (RFS) were determined by Kaplan-Meier and multivariate Cox regression analysis. The results showed that IL-10 mRNA levels were significantly higher in tumors with the non-ATA haplotype than with the ATA haplotype (P = 0.004). Patients with the non-ATA haplotype had shorter OS and RFS periods than did patients with the ATA haplotype. This may be associated with the observation that the number of tumor-infiltrating lymphocytes was decreased in the tumors with higher levels of IL-10. Consistently, T cells from the peripheral blood of the patients with non-ATA haplotype were more susceptible to apoptosis and less cytotoxic to tumor cells, compared to those from the patients with ATA haplotype. The results suggest that IL-10 can promote tumor malignancy via promoting T cell apoptosis and tumor cell survival, and IL-10 haplotype evaluated by PCR-RFLP or direct sequencing may be used to predict survival and relapse in resected NSCLC, helping clinicians to make appropriate decisions on treatment of the patients.

Up-regulation of interleukin-17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

Promoter methylation of O6-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.

Activation of NF-κB by SOD2 promotes the aggressiveness of lung adenocarcinoma by modulating NKX2-1-mediated IKKβ expression

Magnesium superoxide dismutase (SOD2) has been shown to cause dysfunction of p53 transcriptional activity, whereas, in turn, SOD2 expression is regulated by p53 to modulate lung tumorigenesis. In this study, we found that the level of SOD2 expression in a panel of lung cancer cells was negatively correlated with that of NK2 homeobox 1 (NKX2-1) but was not associated with p53 status. Mechanistic studies indicated that a decrease in NKX2-1 caused by SOD2-activated IKKβ transcription was achieved by derepression of binding of Sp1 to the IKKβ promoter. Immunoprecipitation, glutathione S-transferase pull-down experiments and electrophoretic mobility shift assays demonstrated a direct interaction between NKX2-1 and Sp1, blocking Sp1-mediated IKKβ transcription. SOD2-mediated nuclear factor-kappaB activation, via elevation of IKKβ transcription, promoted anchorage-independent soft-agar growth, invasion and xenograft tumor formation, because of development of the epithelial-to-mesenchymal transition. The expression level of NKX2-1 messenger RNA was negatively associated with the extent of SOD immunostaining and the IKKβ messenger RNA expression level in lung tumors. The extent of SOD2 immunostaining and IKKβ messenger RNA levels may independently predict overall survival and relapse-free survival in lung adenocarcinoma patients. In summary, we found that SOD2 activates nuclear factor-kappaB signaling by increasing IKKβ transcription, which results in progression of lung adenocarcinoma and poorer patient outcomes. We suggest that IKKβ may potentially be targeted to improve outcomes in patients with SOD2-positive tumors.