Chronic exposure to cigarette smoke may reprogram different populations of lung stem cells, which maintain and repair lung tissue, in ways that make them vulnerable to specific cancer-causing gene alterations and ultimately helps determine the type of lung cancer that develops.
Using laboratory-grown lung organoids, researchers at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center followed changes in lung cells over six months of exposure to cigarette smoke condensate, a collection of chemicals and particles contained in cigarette smoke. They found that chronic exposure altered both the epigenetic programming that regulates gene activity and the genes being expressed in different populations of lung stem cells, creating distinct precancerous cell states. When researchers subsequently introduced two genetic alterations commonly associated with smoking-related lung cancer, KRAS and TP53, the altered stem cell populations responded differently. KRAS mutations drove lung adenocarcinomas primarily from cells derived from bronchioalveolar stem cells, while loss of the tumor suppressor gene TP53 produced squamous cell carcinomas derived from basal stem cells.
The findings, reported Oct. 5 in the Proceedings of the National Academy of Sciences , help explain how environmental exposure, epigenetic changes and genetic mutations may work together during the earliest stages of non-small cell lung cancer (NSCLC), the most common type of lung cancer.
Epigenetic alterations are chemical changes that affect how genes are turned on or off without changing the DNA sequence, while genetic alterations, such as mutations, permanently alter the DNA sequence. Both can affect the activity or function of genes, including cancer-promoting oncogenes, such as KRAS, and tumor suppressor genes, such as TP53.
"What we have tried to do is model lung cancer from its very earliest stages," says Michelle Vaz, Ph.D. , instructor in oncology and senior author of the study. "Using these organoid models, which are rich in stem cells, we were able to follow the cells over six months of exposure and see the epigenetic and gene expression changes occurring in these different stem cell types."
Researchers have long known that mutations in genes such as KRAS and TP53 can drive NSCLC. However, such mutations may also be found in normal or precancerous tissue, suggesting that mutations alone may not be sufficient to start cancer. Cigarette smoke also causes chronic inflammation and epigenetic and genetic changes, so the researchers wanted to understand their combined role in transforming normal lung cells into lung cancer.
The researchers created lung organoids — tiny 3D laboratory models that contain multiple lung cell types — from normal mouse lung tissue and exposed them to cigarette smoke condensate for up to six months. They also confirmed key early findings in organoids made from normal human lung tissue.
Over time, cigarette smoke exposure changed the balance and behavior of cells in the organoids. The exposed cells increasingly acquired characteristics associated with a precancerous, tumor-permissive state. At the same time, the researchers found progressive changes in DNA methylation (chemical tags added to regions of DNA that help control gene activity) and chromatin accessibility (how tightly or loosely DNA is packaged within a cell). These two mechanisms regulate gene activation and changes in gene expression.
Among the notable changes was suppression of inflammatory and immune signaling pathways and genes involved in programmed cell death. One gene affected was ZBP1, a key regulator of inflammatory cell death.
The investigators next tested whether cigarette smoke exposure made the organoids more susceptible to transformation by cancer-driving mutations. They introduced either mutant KRAS or knocked out TP53 in organoids that had and had not undergone six months of smoke exposure and implanted the cells in mice.
Only the smoke-exposed organoids containing one of the genetic alterations formed tumors. Cigarette smoke exposure alone did not produce tumors, nor did introducing the genetic alterations into unexposed control organoids.
Silencing ZBP1 and related interferon signaling (part of the immune response) was particularly pronounced in KRAS-mutant tumors, suggesting that reducing these normal cell-death defenses may help KRAS-driven cancer cells survive. Analysis of human lung adenocarcinoma data also showed significantly lower expression of ZBP1 and related interferon pathway genes in tumors with KRAS mutations compared with those with TP53 mutations.
"What this tells us is that the genetic event alone is not sufficient," says Vaz. She 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 emphasize that the cells first need to undergo changes from the chronic cigarette smoke exposure, which then makes them susceptible to transformation by these mutations.
More strikingly, the genetic changes produced two different forms of NSCLC. Smoke-exposed organoids with mutant KRAS developed poorly differentiated cancers with features of lung adenocarcinoma. Smoke-exposed organoids lacking TP53 developed squamous cell cancers.
Single-cell RNA sequencing allowed the researchers to trace these tumors back toward distinct stem cell populations. The KRAS-driven adenocarcinomas appeared to arise from an altered cell state derived from bronchioalveolar stem cells, which are found where the lung airways meet the alveoli, the tiny air sacs in the lungs. TP53-deficient squamous cell carcinomas were traced to an altered state derived from basal stem cells, which maintain and repair the lining of airways, passages that carry air into and through the lungs.
"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 says. "We did not engineer the genetic event into a particular cell type. This is what the mutations selected for."
The findings suggest that the type of lung cancer that ultimately develops depends both on which genetic mutation occurs and also on the molecular state of the cell in which that mutation takes hold. Chronic cigarette smoke exposure appears to reshape those cellular states through a combination of inflammatory, epigenetic and gene-expression changes, effectively creating different environments in which particular cancer-driving mutations can thrive.
The researchers say the altered stem cell states identified in the study also could eventually provide molecular markers for recognizing the earliest evolution of different NSCLC subtypes. Additional experimental validation is needed, but these markers could potentially point toward strategies for identifying people at increased risk and preventing cancer development.
The findings also raise questions about whether some of the pathways altered by cigarette smoke could eventually be therapeutically targeted.
"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 says. "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 could also uncover new therapeutic vulnerabilities, she says.
In addition to Vaz and Baylin, other researchers involved in the study were Na Wang, Raksha Padaki, Sara-Jayne Thursby, Ray-Whay Chiu Yen, Leslie Cope, Malcolm Brock, Edward Gabrielson and Hariharan Easwaran.
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, the National Institutes of Health grant R01ES011858, National Cancer Institute grants R01CA229240 and R01CA230995, the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Support Grant P30CA006973, and the Van Andel Research Institute through the Van Andel Research Institute–Stand Up To Cancer Epigenetics Dream Team.
