DNA hypermethylation as prognosticator of NSCLC survival/ hTERT promotor in NSCLC/znd EGFR in MSCLC<all Roth papers>
Abstract Number: 28
Presentation Title: Aberrant promoter methylation profile associated with survival in patients with non-small cell lung cancer
Presentation Start/End Time: Sunday, Apr 02, 2006, 8:00 AM -12:00 PM
Location: Exhibit Hall, Washington Convention Center
Poster Section: 1
Poster Board Number: 28
Author Block: Jian Gu, Margaret R. Spitz, Jack A. Roth, Xifeng Wu. The University of Texas M.D. Anderson Cancer Center, Houston, TX
Purpose: Epigenetic gene silencing plays important roles in cancer initiation and progression. The aim of the current study was to investigate the prognostic value of DNA hypermethylation in patients with non-small cell lung cancer (NSCLC). Experimental Design: We examined the methylation status of a panel of 9 genes p16, RAS association domain family 1A (RASSF1A), E-cadherin (CDH1), tissue inhibitor of metalloproteinase-3 (TIMP3), adenomatous polyposis coli (APC), death-associated protein kinase (DAPK), fragile histidine triad (FHIT), O6-methylguanine-DNA-methyltransferase (MGMT), glutathione S-transferase P1 (GSTP1) using quantitative real-time methylation-specific PCR in 155 tumors from patients with NSCLC. We analyzed the associations between gene methylation status and patient survival.
Results: Among 155 patients with NSCLC (56.1% stage I, 20.0% stage II, 25.9% stage III), the median survival time was 29.6 months and the 5-year survival rate was 54.8%. Methylation frequencies of the genes analyzed were 21.9% for p16, 27.1% for RASSF1A, 47.1% for TIMP-3, 32.9% for CDH1, 67.1% for APC, 53.5% for DAPK, 31.6% for FHIT, 17.4% for MGMT, and 5.8% for GSTP1. In the Cox proportional hazards model, hypermethylation of p16 was associated with a significantly poorer survival (HR=1.95, 95% CI=1.12-3.39) and the association was more evident in squamous carcinoma than in adenocarcinoma, and in older patients than in younger patients. Kaplan-Meier survival curves showed that patients with hypermethylated p16 had significantly shorter survival (median = 21.7 months) than patients without p16 hypermethylation (median = 62.5 months, p = 0.0001, log-rank test). Hypermethylation of CDH1 and TIMP3 were associated with significantly better survival with HRs of 0.51 (95% CI=0.29-0.90) and 0.59 (95% CI=0.36-0.97), respectively. Patients with hypermethylated CDH1 or TIMP3 exhibited significantly longer survival than those without hypermethylation (p= 0.0002 and 0.01, respectively). Joint analysis of these three genes showed a significant trend for poorer survival as the number of unfavorable methylation status increased (P for trend = 0.002). Conclusions: Hypermethylation of multiple genes exhibited significant differential impact on NSCLC patient survival. The individual gene methylation and the combination of multiple gene methylations may become valuable prognostic markers of clinical outcome of NSCLC patients. Supported by NCI CA 55769, CA 111646, CA 70907, DAMD17-02-1-0706 and Flight Attendant Medical Research Institute.
Abstract Number: 3948
Presentation Title: Tumor-specific Activation of hTERT Promoter Activityity by the AP-2ß Transcription Factor in Human Lung Cancer Cells
Presentation Start/End Time: Tuesday, Apr 04, 2006, 10:40 AM -10:55 AM
Location: Room 144, Washington Convention Center
Author Block: Wuguo Deng, Gitanjali Jayachandran, Kai Xu, Jack A. Roth, Lin Ji. The University of Texas M.D. Anderson Cancer Center, Houston, TX
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is active in immortalized cells and most human cancers but is quiescent in most normal somatic cells. The hTERT promoter can promote the expression of both constitutive and transgenic hTERT in tumor cells but not in normal cells. However, little is known about how tumor cells activate hTERT transcription and induce telomerase activity. Hypothesizing that transcription factors or regulatory elements that are expressed differently in tumor cells vs normal cells can associate with the hTERT promoter to result in cell-selective activation or repression of hTERT expression, we used ProteinChip array-based mass spectrometry analysis to profile and identify transcription factors or cellular proteins that are differentially expressed and specifically bind to the hTERT promoter in normal lung cells and in non-small cell lung cancer (NSCLC) cells. One of several such proteins identified was the transcription factor AP-2ß that was detected only in hTERT promoter DNA-protein complexes in nuclear extracts from NSCLC cells, not in extracts from normal bronchial epithelial or lung fibroblast cells. A computer-aided transcription factor binding site analysis revealed a consensus AP-2ß binding site in the hTERT promoter and a chromatin immunoprecipitation assay with an anti-AP-2ß antibody demonstrated tumor cell-specific binding of AP-2ß to the hTERT promoter in living cells. We also detected tumor-specific upregulation of AP-2ß transcription (by RT-PCR) and protein expression (by Western blotting) in various NSCLC cell lines relative to normal cells. To study the specific function of AP-2ß in the activation of hTERT promoter-mediated gene expression, we cotransfected normal lung and NSCLC cells with plasmids expressing wt-AP-2ß (driven by a cytomegalovirus [CMV] promoter) and a green fluorescence protein (GFP) reporter (driven by the hTERT promoter). Exogenous expression of AP-2ß reactivated hTERT promoter-mediated GFP expression in normal cells and greatly enhanced GFP expression in tumor cells cotransfected with CMV-AP-2ß and hTERT-GFP plasmids; no hTERT promoter activity was seen in cells cotransfected with CMV-LacZ and hTERT-GFP. Finally, inhibiting AP-2ß expression with AP-2ß-specific siRNA significantly attenuated hTERT promoter-driven GFP expression in NSCLC cells, confirming the AP-2ß-mediated tumor-specific activation of the hTERT promoter. Because hTERT activity is involved in tumorigenesis and is tightly regulated at the transcriptional level, our discovery of AP-2ß as a tumor-specific hTERT promoter activator suggests that targeting AP-2ß has therapeutic potential by its ability to inhibit hTERT activity and tumor cell growth. This work was supported by NIH/NCI SPORE grant P50 CA70907.
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