| Summary: | <i>Iroquois homeobox</i> (IRX) encodes members of homeodomain containing genes which are involved in development and differentiation. Since it has been reported that the <i>IRX1</i> gene is localized in a lung cancer susceptibility locus, the epigenetic regulation and function of IRX1 was investigated in lung carcinogenesis. We observed frequent hypermethylation of the <i>IRX1</i> promoter in non-small cell lung cancer (NSCLC) compared to small cell lung cancer (SCLC). Aberrant <i>IRX1</i> methylation was significantly correlated with reduced <i>IRX1</i> expression. In normal lung samples, the <i>IRX1</i> promoter showed lower median DNA methylation levels (<10%) compared to primary adenocarcinoma (ADC, 22%) and squamous cell carcinoma (SQCC, 14%). A significant hypermethylation and downregulation of <i>IRX1</i> was detected in ADC and SQCC compared to matching normal lung samples (<i>p </i>< 0.0001). Low <i>IRX1</i> expression was significantly correlated with impaired prognosis of ADC patients (<i>p </i>= 0.001). Reduced survival probability was also associated with higher <i>IRX1</i> promoter methylation (<i>p </i>= 0.02). Inhibition of DNA methyltransferase (DNMT) activity reactivated <i>IRX1</i> expression in human lung cancer cell lines. Induced DNMT3A and EZH2 expression was correlated with downregulation of <i>IRX1</i>. On the cellular level, IRX1 exhibits nuclear localization and expression of IRX1 induced fragmented nuclei in cancer cells. Localization of IRX1 and induction of aberrant nuclei were dependent on the presence of the homeobox of IRX1. By data mining, we showed that <i>IRX1</i> is negatively correlated with oncogenic pathways and <i>IRX1</i> expression induces the proapoptotic regulator <i>BAX</i>. In conclusion, we report that <i>IRX1</i> expression is significantly associated with improved survival probability of ADC patients. <i>IRX1</i> hypermethylation may serve as molecular biomarker for ADC diagnosis and prognosis. Our data suggest that <i>IRX1</i> acts as an epigenetically regulated tumor suppressor in the pathogenesis of lung cancer.
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