Cigarette smoke causes epigenetic changes in lung cells that prime them to develop cancer, and researchers can now observe how these changes unfold over time. Reporting September 11 in the journal Cancer Cell, Johns Hopkins University School of Medicine investigators show that healthy lung cells in a dish exposed to cigarette smoke condensate for 10-15 months--the equivalent of someone smoking 20-30 years--have accumulated epigenetic abnormalities associated with abnormal "turning off" of multiple genes, which are otherwise needed to help protect normal cells from developing cancer.
These changes are accompanied by increased activity of cell signals that would indicate the presence of a mutation in a key oncogene, KRAS; yet no such mutation was present in this or other genes frequently mutated in lung cancer. However, if the investigators inserted a KRAS mutation into the cells with the epigenetic abnormalities, they became cancerous with just this one step and having this one mutation. Introduction of mutant KRAS into healthy cells without chronic exposure to cigarette smoke condensate was not enough to induce tumors.
Studies in The Cancer Genome Atlas and other databases have shown that about 30 % of lung adenocarcinomas, the most frequent type of lung cancer, have KRAS mutations. These tumors also frequently harbor the epigenetic abnormalities produced by the cigarette smoke condensate exposure used in the above lab studies.
"When you're smoking, you are building up a substrate of epigenetic changes that we hypothesize are increasing your mathematics for developing lung cancer, because if you're not a smoker, your risk of lung cancer is very low," says senior author Stephen B. Baylin, co-director of the Cancer Biology program at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins and Hopkins scientist, co-corresponding author Hari Easwaran. "If you are a smoker, however, while you still have 8 or 9 out of 10 chances not to get lung cancer, the mathematics are now still very robustly much more going against you, and maybe it's because there are these epigenetic changes that build up."
Baylin and his colleagues show that lung cells--engineered for these experiments to survive in a laboratory dish in the presence of cigarette smoke condensate--begin to behave abnormally within 10 days of smoke exposure. The first changes are cellular signs of DNA damage and tighter binding to DNA of proteins that help establish the above epigenetic changes. However, only after 10 to 15 months, but not earlier, the DNA is covered in a dramatic number of abnormal methylation marks at the start sites of multiple genes, which is associated with the silencing of the protective genes discussed earlier. DNA methylation is an example of an epigenetic change, as it modifies gene expression rather than altering the basic DNA sequence of the gene.
The researchers identified that the epigenetic changes in the chronically exposed lung cells are responding to smoking by regressing into a more stem-cell like state, meaning that they are priming themselves to self-renew. Mature cells normally repress the types of cell signaling pathways in which KRAS is a key player to help prevent uncontrolled growth. Multiple genes that are silenced with the abnormal epigenetic changes induced by the cigarette smoke exposure would normally act to turn down KRAS signaling.
To test their theory for the role of the epigenetic abnormalities observed, Baylin and his team introduced a single KRAS mutation in lung cells, both exposed and not exposed to cigarette smoke extract, and then transplanted them into mice. The cells exposed for only 6 months did not become cancerous, but, indeed, those exposed for 15 months, which harbored the epigenetic abnormalities, did become cancerous and form lung adenocarcinomas in the mice.
"This work suggests the possibility that unlike mutations, which are harder to reverse, if you stop smoking at a certain time and duration, then you have a chance to decrease your likelihood of getting cancer that might be due to the buildup of epigenetic changes," says first author Michelle Vaz, a postdoctoral researcher at Johns Hopkins University School of Medicine. "The hypothesis is that there are potentially reversible changes that are contributing to a certain set of lung cancers."
Baylin, Easwarn, Vaz, and their collaborators are now exploring the possibility of using therapies that target the specific epigenetic changes that occur in the lung cells of smokers.
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The research was supported by the National Cancer Institute, the National Institutes of Health, and the National Institute of Environmental Health Sciences.
Cancer Cell, Vaz et al: "Chronic Cigarette Smoke-Induced Epigenomic Changes Precede Sensitization of Bronchial Epithelial Cells to Single Step Transformation by KRAS Mutations." http://www.cell.com/cancer-cell/fulltext/S1535-6108(17)30349-5
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