The research team, led by senior author Michelle Vaz, Ph.D., instructor in oncology, and co-senior author Stephen Baylin, M.D., Virginia and D.K. Ludwig Professor of Cancer Research and co-director of the Cancer Genetics and Epigenetics Program, set out to model lung cancer from its very earliest stages. Rather than starting with fully malignant cells, they used laboratory-grown lung organoids, tiny three-dimensional structures containing multiple lung cell types, created from normal mouse lung tissue. These organoids were exposed to cigarette smoke condensate, a concentrated collection of the chemicals and particles found in cigarette smoke, for up to six months. Key early findings were then confirmed in organoids generated from normal human lung tissue, lending the work translational weight.

What the researchers observed over those six months was a gradual, coordinated shift in the biology of the stem cells. The exposed cells increasingly acquired characteristics associated with a precancerous, tumor-permissive state. At the same time, the team documented progressive changes in two epigenetic mechanisms that govern gene activity without altering the underlying DNA sequence. The first was DNA methylation, the addition of chemical tags to regions of DNA that help control whether genes are switched on or off. The second was chromatin accessibility, a measure of how tightly or loosely DNA is packaged within the cell nucleus, which determines how readily the cellular machinery can reach and read specific genes. Together, these mechanisms reshaped gene expression across the different stem cell populations.

Among the most notable changes was the suppression of inflammatory and immune signaling pathways, along with genes involved in programmed cell death, the built-in suicide program that normally eliminates damaged or dangerous cells. One gene affected in particular was ZBP1, a key regulator of inflammatory cell death. Silencing this defensive machinery, the researchers found, was especially pronounced in cells that would later prove susceptible to KRAS-driven transformation, suggesting that dampening these normal cell-death defenses may help incipient cancer cells survive long enough to become fully malignant.

The critical question was whether this smoke-induced reprogramming actually made the cells more vulnerable to cancer-causing mutations. To test this, the investigators introduced either mutant KRAS, one of the most common oncogenes in smoking-related lung cancer, or knocked out TP53, a crucial tumor suppressor gene, in organoids that had and had not undergone six months of smoke exposure. The engineered cells were then implanted into mice. The result was striking: only the smoke-exposed organoids carrying one of the genetic alterations formed tumors. Cigarette smoke exposure alone did not produce tumors, and neither did introducing the mutations into unexposed control organoids. Neither factor was sufficient on its own; the two had to act in sequence.

“What this tells us is that the genetic event alone is not sufficient,” Vaz explained. She and Baylin emphasize that cells must first undergo changes wrought by chronic cigarette smoke exposure before mutations like KRAS or TP53 loss can drive them into full transformation. This helps resolve a long-standing puzzle in cancer biology: mutations in genes such as KRAS and TP53 are known drivers of non-small cell lung cancer, the most common form of the disease, yet the same mutations can also be found in normal or precancerous tissue, implying that mutations by themselves are not enough to ignite cancer.

Even more surprising was what happened when the smoke-primed cells transformed. The genetic alterations produced two entirely different forms of non-small cell lung cancer, and each appeared to select for a different stem cell population. Smoke-exposed organoids with mutant KRAS developed poorly differentiated cancers with features of lung adenocarcinoma, and single-cell RNA sequencing traced these tumors back to an altered cell state derived from bronchioalveolar stem cells, which reside where the lung airways meet the alveoli, the tiny air sacs where gas exchange occurs. Smoke-exposed organoids lacking TP53, by contrast, developed squamous cell carcinomas, which traced to an altered state derived from basal stem cells, the cells that maintain and repair the lining of the airways.

“It was very interesting that when we introduced KRAS or loss of TP53, each seemed to select for a particular type of stem cell state,” Vaz said. “We did not engineer the genetic event into a particular cell type. This is what the mutations selected for.” In other words, the chronic smoke exposure had created distinct precancerous cellular environments, and each cancer-driving mutation thrived in a different one. The type of lung cancer that develops, the findings suggest, depends on both the mutation that occurs and the molecular identity of the cell in which it takes hold, a convergence of environmental exposure, epigenetic change and genetics at the earliest stages of tumor evolution.

The human data lent further support to the mechanism. Analysis of lung adenocarcinoma samples from patients showed significantly lower expression of ZBP1 and related interferon pathway genes, part of the immune response, in tumors carrying KRAS mutations compared with those carrying TP53 mutations. This mirrors what the organoids revealed in the laboratory and points to a possible shared signature between the mouse and human disease. The researchers note that the altered stem cell states identified in the study could eventually serve as molecular markers for recognizing the earliest evolution of different NSCLC subtypes, potentially pointing toward strategies for identifying people at elevated risk and intervening before cancer develops, though additional experimental validation is still needed.

The work also opens therapeutic questions. “What’s exciting as a next step is whether we can learn more about these cell death pathways and whether some can be targeted, specifically in the KRAS versus TP53 setting,” Vaz said. “Can we combine that with epigenetic therapy or immunotherapy and help tumors that currently do not respond to treatment, respond better?” Further study of the epigenetic changes occurring within individual stem cell states, she added, could uncover new therapeutic vulnerabilities. The research was supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, The Hodson Trust, an American Lung Association Lung Cancer Discovery Award, the Evelyn Grollman Glick Scholar Award, a Johns Hopkins University Discovery Award, TEDCO, the National Institute of Environmental Health Sciences, National Institutes of Health grant R01ES011858, National Cancer Institute grants R01CA229240 and R01CA230995, the Sidney Kimmel Comprehensive Cancer Center Support Grant P30CA006973, and the Van Andel Research Institute through the Van Andel Research Institute–Stand Up To Cancer Epigenetics Dream Team. In addition to Vaz and Baylin, the research team included Na Wang, Raksha Padaki, Sara-Jayne Thursby, Ray-Whay Chiu Yen, Leslie Cope, Malcolm Brock, Edward Gabrielson and Hariharan Easwaran.

Subject of Research: How cigarette smoke-induced epigenetic reprogramming of lung stem cells determines susceptibility to specific lung cancer-driving mutations

Article Title: Cigarette smoke may prime lung stem cells for different types of lung cancer

Article References: Cigarette smoke may prime lung stem cells for different types of lung cancer. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: lung cancer, cigarette smoke, stem cells, epigenetics, KRAS, TP53, organoids, non-small cell lung cancer, DNA methylation, adenocarcinoma, squamous cell carcinoma, ZBP1