Hunting for the 'dark matter' of human carcinogenesis

In the 13 December 2023 issue of Atlantic magazine, medical oncologist and Pulitzer Prize winning author Siddhartha Mukherjee employs a metaphor from physics and cosmology, the existence of poorly understood dark matter and energy to relate a puzzle in contemporary oncology – just what causes a cancer to form in the first place, a process termed carcinogenesis. Analogous to the problem cosmologists face in a shortfall in their understanding the forces governing galaxies and an expanding universe, so too do oncologists now realize how our belief that carcinogenesis was the sole product of successive gene mutations has proven inadequate to account for many of the actual circumstances preceding some cancers encountered in the clinic and studied in the lab.

Dr. Mukherjee begins his story in California in the 1970s, which at the time was a hotbed of medical research into the molecular basis of cancer. Pivotal then was the discovery at the University of California at San Francisco of how a normal gene governing cellular growth could undergo mutation causing enhanced gene-mediated growth by promoting the enzymatic activity of that gene when introduced into a subject organism, in this first case, a chicken. This transfection of a strategic regulatory gene by a retrovirus led to development of a rare tumor, a sarcoma, occurring in the infected animals. A normal, ‘proto’ oncogene had the potential when mutated to become a cancer causing gene, an active oncogene.

At the same time, right across the Bay, investigators at UC Berkley further demonstrated the importance of mutation by the development of a test, the Ames test, named after its inventor, correlating the potency of a mutagenic chemical measured by its effect on bacterial growth. The rate of bacterial mutation proved to correlate with the likelihood of that chemical being capable of causing a cancer. The higher the likelihood that a chemical could induce a mutation, the more likely a cancer would result. Carcinogens then were mutagens. This research tied together epidemiologic observations going back to the 18^th century with the finding of scrotal cancer occurring in chimney sweeps exposed to soot from the combustion products from household fireplaces analogous in modern times to the highly mutagenic properties of hydrocarbon combustion products from tobacco smoke.

Establishing such a dose-response relationship between mutagen potency of a chemical and chance for cancer occurrence following exposure provided strong evidence for a cause and effect relationship between the two. This led to a ‘multi-hit’ model of carcinogenesis in which cancer development was the result of a series of mutational ‘hits’, each hit lowering the threshold for subsequent neoplastic transformation. This model was influential in leading to surveys of the vast world of chemicals in our world to determine their cancer causing risk, for example that industrial workers would be subjected to following exposure and pointing to the need for environmental protection. Not only are chemicals mutagenic but physical agents too prove to be mutagenic including electromagnetic radiation such as ultraviolet light as a cause for skin cancer and X-Rays used in medical imaging causing iatrogenic cancers such as thyroid cancer.

Mukherjee interviewed some cancer researchers, however, who detected anomalous findings from newer experiments examining the carcinogenic potential of well-established mutagens in  certain experimental systems. Unexpectedly these studies examining mutagenic exposure did not yield a high rate of cancer following such exposure. The investigators found though that the efficiency of a carcinogen in yielding cancer could be enhanced by subsequent exposure to non-mutagenic chemicals, particularly those capable of causing inflammation. Neither exposure to the inflammatory non-mutagen alone or the carcinogen alone would of produce many tumors. Nor would preexposure of the inflammogen in a sequence of administration prior to the carcinogen exposure be effective. It was the order of exposure demonstrated in these experiments, that of an initial exposure to a mutagenic chemical followed by inflammation which proved effective in cancer inducement. That finding was an indication of how timing of exposure is a crucial factor in carcinogenesis.

The recognition then of a dual requirement for genetic mutation followed by accompanying inflammation would prove pivotal for understanding a modern paradox in oncology, the development of lung cancer in those who are non-smokers. Although the great majority of cases of lung cancer are attributable to the effects of inhaled tobacco smoke, about 10% of cases do not reveal such exposure history. Importantly, a subset of these patient’ cancers are found to harbor an activating mutation in a gene expressing a membrane-bound receptor/enzyme, the epidermal growth factor receptor (EGFR). When activated this receptor-enzyme complex ordinarily acts to maintain epithelial integrity through a process of growth initiation to guide recovery from epithelial injury such as healing from a laceration of the skin. Following certain special mutations however it may become constitutively (constantly) activated resulting in a sustained signal for growth of the affected epithelial cells, including those which line the airways of the lung.  

Although mutation in the EGFR gene occurs on a random basis, the development of lung cancer from such a mutation is not, instead demonstrating a worldwide distribution corresponding with regions of the world suffering from levels of exposure to fine particle air pollution, referred to as Particulate Matter 2.5 micrometers (PM2.5). Further investigations demonstrated a direct relationship between the rate of occurrence of lung cancer and the degree of exposure to PM2.5 which a population is subject to. These special air pollution particles are small enough to evade normal lung defenses and can reach the distal airways of the lung. The microscopic particles are not mutagenic themselves but instead prove to be potent activators of one particular arm of the cellular immune system, macrophages.

These scavenger cells ordinarily provide us with tissue waste management services, disposing of dying cells while at the same time working in concert to protect us from invading microorganisms. Under the corrupting influence of chronic inflammation though these normally protective cells may turn on their host, acting instead to release a profile of imbalanced chemical mediators including certain interleukins, chemical signals which ordinarily stimulate immune cell growth. In the airway of the lung, exposure of lung epithelium to PM2.5 triggers a surge in macrophage-produced interleukin, IL-1 beta. In experiments examining EGFR-related lung cancer, it was only the combination of a preexistent mutation in EGFR together with subsequent PM2.5 exposure-induced macrophage reversal that proved potent for inducing lung cancer development. It seems likely that it was the combination of the earlier activating mutation in a gene governing epithelial cell growth brought together with the subsequent non-mutagenic environmental influence of air pollution-stimulated chronic immune activation that proved to be the deadly combination.

The role of time in understanding neoplasia is illustrated by the clonal nature of cancer and the process of clonal evolution. A clone represents a group of cells all having identical genetic makeup, an indication of those cells having one single cell from which they originated. Because of the imperfect nature of gene replication during cell growth, variations in the genetic characteristic of clonal cells occur leading to the emergence of subclones with slightly altered genetic composition derived from the original clone. This inevitable process accounts for the development of genetic and subsequent phenotypic divergence that define the process of clonal evolution. This change over time of a clonal population provides a basis for understanding the emergence of divergent neoplastic behavior within a cancer, particularly with regard to sensitivity to anticancer medication and the development of treatment resistance.

Contemporary oncology then needs to consider both the timing of exposure to a carcinogen as well as the context of the organ system being affected to help best understand cancer occurrence. The oncologist should consider the health of the ‘neighborhood’, that is the surrounding tissue environment, the microenvironment, in conjunction with oncogenic mutation to best predict the likelihood for neoplastic transformation. Increasingly, we must view oncogenes as being mostly narrowly oncogenic, constricted by the cell state* of a particular tissue along with the influence of accompanying ‘dark matter’, such as chronic inflammation which together drive carcinogenesis. Investigations of both time as well as place, cancer evolution and cancer ecology then provide a broad framework for understanding carcinogenesis. Dr. Mukherjee’s article challenges us to consider what other yet undiscovered and untoward non-mutagenic influences we may be missing in searching for such knowledge, a call for a new Ames test for our times.

 

Jim Cunningham

 

*Cell state refers to the degree of differentiation of that cell, ranging from pluripotent stem cells which have no differentiating features to that of mature cells having undergone differentiation. Cell state reflects the overlying influence of the epigenome, the collection of chromatin and DNA ligands which regulate gene expression. Study of this regulation is of central importance in understanding the plasticity of cell subtypes and their accompanying vulnerability to neoplastic transformation, a subject I will tackle in an upcoming blog post.