Abstract
(1) The TBX2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) are markedly down-regulated in human non-small cell lung cancer (NSCLC) and exert tumor suppressor effects in lung malignancy. Yet, mechanisms underlying suppressed expression of the TBX2 subfamily in NSCLC are elusive. Here, we interrogated probable epigenetic mechanisms in suppressed expression of the TBX2 subfamily in human NSCLC. (2) TBX2 subfamily gene expression and methylation levels in NSCLC and normal lung tissues were surveyed using publicly available RNA-sequence and genome-wide methylation datasets. Methylation β-values of the four genes were statistically compared between NSCLCs and normal lung tissues, correlated with gene expression levels, and interrogated with clinicopathological variables. Expression and methylation levels of TBXs were quantified in NSCLC cells using real-time PCR and methylation-specific PCR assays, respectively. Effects of the DNA methyltransferase inhibitor 5-azacytidine (Aza) on TBX2 subfamily expression were assessed in NSCLC cells. Impact of TBX2 subfamily expression on Aza-treated cells was evaluated by RNA interference. (3) All four TBXs were significantly hypermethylated in NSCLCs relative to normal lung tissues (p < 0.05). Methylation β-values of the genes, with exception of TBX2, were significantly inversely correlated with corresponding mRNA expression levels (p < 0.05). We found no statistically significant differences in hypermethylation levels of the TBX2 subfamily by clinicopathological features including stage and tobacco history. Expression levels of the TBX genes were overall suppressed in NSCLC cells relative to normal alveolar cells. Members of the subfamily were significantly hypermethylated in all tested NSCLC cell lines relative to normal alveolar cells. Treatment with Aza induced the expression of the TBX2 subfamily concomitant with NSCLC cell growth inhibition. Further, simultaneous knockdown of the four TBX genes markedly reduced anti-growth effects of Aza in NSCLC cells. (4) Our study sheds light on new epigenetic profiles in the molecular pathogenesis of human NSCLC.
Highlights
Lung cancer is the leading cause of cancer-related deaths in both men and women worldwide and accounts for more deaths than breast, colorectal, and prostate cancers combined [1]
We demonstrated that the TBX2 subfamily of evolutionarily conserved transcription factors, TBX4 and TBX5, are epigenetically suppressed by hypermethylation in human non-small cell lung cancer (NSCLC)
We showed that TBX2 subfamily hypermethylation is a common attribute in NSCLC tumors and cell lines
Summary
Lung cancer is the leading cause of cancer-related deaths in both men and women worldwide and accounts for more deaths than breast, colorectal, and prostate cancers combined [1]. While significant advances have been made in lung cancer therapy, the overall 5-year survival rates of this malignancy remain very poor, not exceeding 15% across all stages [2]. These low survival rates are largely due to late diagnosis at advanced stages in which tumors have already metastasized [2]. NSCLC, the most common type of lung cancer, accounts for approximately 85% of all lung malignancies and is histologically subdivided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [3]. Despite the significant functions the TBX2 subfamily members play in murine lung development and organogenesis, their roles in malignancies of the lung are otherwise very poorly understood. Over-expression of each of the four members of the TBX2 subfamily independently inhibited cell growth and proliferation as well as induced apoptosis in NSCLC cell lines [10]
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