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HOXA11 hypermethylation is associated with progression of non-small cell lung cancer.

Hwang JA, Lee BB, Kim Y, Park SE, Heo K, Hong SH, Kim YH, Han J, Shim YM, Lee YS, Kim DH - Oncotarget (2013)

Bottom Line: HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs.HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively).HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival.

View Article: PubMed Central - PubMed

Affiliation: Cancer Genomics Branch, Research Institute, National Cancer Center, Goyang-si, Korea.

ABSTRACT
This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.

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Analysis of the methylation and expression of HOXA11 in vitro(A) Gene map shows location of CpGs that were studied by different methods in this study. (B) The methylation status of 90 CpGs at the promoter region of HOXA11 was analyzed using the EpiTYPER™ assay in six lung cancer cell lines (H23, H226, H460, H520, H1650, and A549), a bronchial epithelial cell line (HBE135-E6E7), and in a normal human dermal fibroblast (HDF). Two-way cluster analysis shows the methylation status of HOXA11 in eight cell lines. Levels of methylation are depicted in color change on a continuous scale from red (0% methylated) to light yellow (100% methylated). X-axis and Y-axis indicate CpG sites and cell lines, respectively. (C & D) The mRNA levels of HOXA11 were analyzed by real-time PCR (C), and protein levels were determined using western blotting (D). Error bars indicate one standard deviation.
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Figure 1: Analysis of the methylation and expression of HOXA11 in vitro(A) Gene map shows location of CpGs that were studied by different methods in this study. (B) The methylation status of 90 CpGs at the promoter region of HOXA11 was analyzed using the EpiTYPER™ assay in six lung cancer cell lines (H23, H226, H460, H520, H1650, and A549), a bronchial epithelial cell line (HBE135-E6E7), and in a normal human dermal fibroblast (HDF). Two-way cluster analysis shows the methylation status of HOXA11 in eight cell lines. Levels of methylation are depicted in color change on a continuous scale from red (0% methylated) to light yellow (100% methylated). X-axis and Y-axis indicate CpG sites and cell lines, respectively. (C & D) The mRNA levels of HOXA11 were analyzed by real-time PCR (C), and protein levels were determined using western blotting (D). Error bars indicate one standard deviation.

Mentions: The locations of CpGs that were analyzed using EpiTYPER™, MS-HRM, and MSP are indicated in Figure 1A. The HOXA11 promoter sequence was obtained from Transcription Element Search System (http://www.cbil.upenn.edu/cgi-bin/tess/tess), and methylation statuses of 90 CpGs at the promoter region of HOXA11 were first analyzed quantitatively using the EpiTYPER™; some of the CpGs were partially methylated in H23, H520, and H1650 cells (Fig. 1B). HOXA11 expression, analyzed using quantitative real-time PCR (Fig. 1C) and western blotting (Fig. 1D), correlated well with these methylation statuses. The mRNA and protein levels of six lung cancer cell lines were downregulated compared to HBE135-E6E7 bronchial epithelial cells, except weak expression in H460. This result suggests that HOXA11 hypermethylation may be responsible for silencing of HOXA11.


HOXA11 hypermethylation is associated with progression of non-small cell lung cancer.

Hwang JA, Lee BB, Kim Y, Park SE, Heo K, Hong SH, Kim YH, Han J, Shim YM, Lee YS, Kim DH - Oncotarget (2013)

Analysis of the methylation and expression of HOXA11 in vitro(A) Gene map shows location of CpGs that were studied by different methods in this study. (B) The methylation status of 90 CpGs at the promoter region of HOXA11 was analyzed using the EpiTYPER™ assay in six lung cancer cell lines (H23, H226, H460, H520, H1650, and A549), a bronchial epithelial cell line (HBE135-E6E7), and in a normal human dermal fibroblast (HDF). Two-way cluster analysis shows the methylation status of HOXA11 in eight cell lines. Levels of methylation are depicted in color change on a continuous scale from red (0% methylated) to light yellow (100% methylated). X-axis and Y-axis indicate CpG sites and cell lines, respectively. (C & D) The mRNA levels of HOXA11 were analyzed by real-time PCR (C), and protein levels were determined using western blotting (D). Error bars indicate one standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926829&req=5

Figure 1: Analysis of the methylation and expression of HOXA11 in vitro(A) Gene map shows location of CpGs that were studied by different methods in this study. (B) The methylation status of 90 CpGs at the promoter region of HOXA11 was analyzed using the EpiTYPER™ assay in six lung cancer cell lines (H23, H226, H460, H520, H1650, and A549), a bronchial epithelial cell line (HBE135-E6E7), and in a normal human dermal fibroblast (HDF). Two-way cluster analysis shows the methylation status of HOXA11 in eight cell lines. Levels of methylation are depicted in color change on a continuous scale from red (0% methylated) to light yellow (100% methylated). X-axis and Y-axis indicate CpG sites and cell lines, respectively. (C & D) The mRNA levels of HOXA11 were analyzed by real-time PCR (C), and protein levels were determined using western blotting (D). Error bars indicate one standard deviation.
Mentions: The locations of CpGs that were analyzed using EpiTYPER™, MS-HRM, and MSP are indicated in Figure 1A. The HOXA11 promoter sequence was obtained from Transcription Element Search System (http://www.cbil.upenn.edu/cgi-bin/tess/tess), and methylation statuses of 90 CpGs at the promoter region of HOXA11 were first analyzed quantitatively using the EpiTYPER™; some of the CpGs were partially methylated in H23, H520, and H1650 cells (Fig. 1B). HOXA11 expression, analyzed using quantitative real-time PCR (Fig. 1C) and western blotting (Fig. 1D), correlated well with these methylation statuses. The mRNA and protein levels of six lung cancer cell lines were downregulated compared to HBE135-E6E7 bronchial epithelial cells, except weak expression in H460. This result suggests that HOXA11 hypermethylation may be responsible for silencing of HOXA11.

Bottom Line: HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs.HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively).HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival.

View Article: PubMed Central - PubMed

Affiliation: Cancer Genomics Branch, Research Institute, National Cancer Center, Goyang-si, Korea.

ABSTRACT
This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.

Show MeSH
Related in: MedlinePlus