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Study Sees Cigarette Smoke-Related Epigenetic and miRNA Links in Lung Cancer

NEW YORK (GenomeWeb News) – A study online today in the Journal of Clinical Investigation suggests cigarette smoke may exert some of its negative, cancer-promoting effects through an epigenetic pathway that curtails the activity of a microRNA called miR-487b.

Researchers with the National Cancer Institute narrowed in on miR-487b following PCR array-based analyses of normal and cancerous lung cell samples grown under different conditions in the lab. There they saw lower-than-usual levels of miR-487b following exposure to cigarette smoke and in respiratory tract cells from current or former smokers.

Through a series of follow-up experiments — including methylated DNA immunoprecipitation, bisulfite sequencing, and several other sorts of analyses — the group found evidence that the dip in miR-487b activity is related to shifts in DNA methylation and other epigenetic features in and around the miR-487b coding locus in the genome.

This ultimately loosens the reins on miR-487b's usual targets, investigators explained, including cancer-associated genes such as MYC and KRAS.

"Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis," senior author David Schrump, with NCI's thoracic oncology section, and colleagues postulated, "and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy."

Past studies suggest that the expression of certain miRNAs is dialed up or down in lung cancers, particularly in response to cigarette smoke, the researchers noted. These miRNA shifts have been implicated in cell signaling changes that ramp up cell proliferation, for instance, though the details of such relationships are still somewhat murky.

"Despite recent studies demonstrating miRNA expression profiles correlating with tumor histology as well as smoking status, response to therapy, and overall survival of patients with primary lung cancers," Schrump and co-authors explained, "limited information is available regarding mechanisms by which miRNA alterations directly contribute to initiation and early progression of these malignancies."

For their new analysis, researchers focused on cell lines generated using normal respiratory tract tissue and lung cancer samples from smokers or never smokers.

After growing these lines with or without adding cigarette smoke condensate, the team did PCR array-based miRNA profiling on each, uncovering a drop in miR-487b expression in the smoke condensate-exposed cells.

Researchers verified the link between lung cancer and lower-than-usual miR-487b levels using quantitative RT-PCR analyses on several more cell lines, including a panel of 51 primary lung tumors and nearby normal tissue.

Consistent with the notion that cigarette smoke can exacerbate the decline in miR-487b expression, their analyses pointed to more modest miR-487b repression in lung tumors from individuals who had never smoked compared to those who had. On the other hand, even apparently normal lung tissue from smokers had lower miR-487b levels than did similar tissues taken from those who had never smoked.

From there, the team turned to a combination of computer-guided analyses and cell biology experiments to carefully teas apart the players and processes contributing to the miR-487b effect.

Together, the analyses revealed roles for both miRNA and epigenetic pathways in this process, while at once identifying apparent targets for miR-487b — a gene set that includes MYC, KRAS, WNT5A, BMI1, and SUZ12.

In lung cells exposed to cigarette smoke condensate, for example, the investigators found a jump in expression for the TGF-beta family gene TGFB1. The TGFB1 gene product, in turn, appeared to prompt enhanced methylation in suspected regulatory sequences near the miR-487b coding sequence.

Coupled with decreased promoter occupancy by a histone H2A variant called H2AZ and a drop in binding by a transcription factor called TCF1, such epigenetic alterations seem to contribute to the lower miR-487b levels found in cigarette smoke-exposed lung cells.

Curbing the expression of miR-487b, meanwhile, frees up genes normally targeted by it, researchers explained, leading to cell proliferation and more invasive lung cancer cells.

Given the apparent contribution that cigarette smoke makes to this process, study authors explained, the new findings "not only provide insight regarding the pathogenesis of human lung cancers, but also suggest a possible mechanism by which smoking status at diagnosis or during treatment affects survival of lung cancer patients."

In addition, they said the apparent epigenetic ties hint that certain lung cancers could potentially be susceptible to treatments that dial DNA methylation back down in the vicinity of the miR-487b gene, losing the expression of this miRNA.

For instance, normal and cancerous cell lines did show a jump in miR-487b expression after treatment with a demethylating agent known as 5-aza-2'-deoxycytidine, or DAC, even when these cells had been exposed to the cigarette smoke mimic.