Cigarette smoke induces methylation of the tumor suppressor gene NISCH

Abstract

We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (<20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients' plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer.

Keywords: Nisch; lung cancer; methylation; smoking; tobacco.

Figure 1. Bar graph of quantitative fluorogenic real-time PCR analysis of the NISCH gene promoter. Normal human oral keratinocytes (HOK) cells were treated with 0.1% side stream smoke extract (SSE) and 0.1% mainstream smoke extract (MSE). Cells exposed to smoke showed hypermethylation of the NISCH gene promoter while untreated cells did not.

Figure 2. Scatter plot of quantitative fluorogenic real-time PCR analysis of the NISCH gene promoter in the plasma DNA of the control group. Hypermethylation was detected in controls who smoked 20+ pack/year. *LNS, life time non-smokers; **py, pack/year.

Figure 3. Scatter plot of quantitative fluorogenic real-time PCR analysis of the NISCH gene promoter in plasma DNA from lifetime non-smokers and light smokers: control group vs. cancer cases. *LNS, life time non-smokers; **py, pack/year