The Molecular Biology of Thoracic Malignancies

Environmental Exposure

First, most thoracic malignancies are the end result of the effect of an environmental exposure (e.g., tobacco smoke, alcohol use, chronic gastroesophageal reflux, asbestos) on a cell's genome. Such exposures can cause alterations in DNA structure (e.g., pyrimidine dimers and double-strand breaks), which can alter DNA replication and transcription. Environmental exposures can also induce mutations if errors are introduced by DNA repair mechanisms while attempting to correct mistakes in DNA structure. Other carcinogens (e.g., asbestos), however, cause chromosomal loss, gain, and/or rearrangements before causing genetic mutations.

Genetic Mutations

Second, mutated genes contribute to oncogenesis by selectively conferring a growth advantage to cancer cells. Such mutations can be either inherited (germline) mutations or acquired (somatic) mutations. Proto-oncogenes are dominant genes that encode proteins that are responsible for cell-cycle progression, cell differentiation, or regulation of apoptosis (programmed cell death). When proto-oncogenes are mutated (by point mutations, translocations, gene amplifications, gene insertions, or gene deletions), they are called oncogenes, which are either constitutively active or abnormally active (under conditions that the normal wild-type gene is not).

Tumor-suppressor genes are the genetic brakes on aberrant cell proliferation by regulating cell division, DNA repair, and apoptosis. In contrast to oncogenes, tumor-suppressor genes are recessive genes—both copies need to be inactivated for their effect to be lost. One allele is typically lost through loss of heterozygosity, and the other is typically lost by a mutation or epigenetic modification (discussed later). Mutations in tumor-suppressor genes confer a growth advantage by removing a normal checkpoint in cellular proliferation.

Mismatch repair genes are recessive genes involved in repairing mistakes occurring primarily during DNA replication. Because microsatellites (tandem repeats in DNA sequences) are especially vulnerable to DNA replication mistakes, microsatellite instability is a hallmark of defective DNA repair. To appreciate the interaction among proto-oncogenes, tumor suppressor genes, and mismatch repair genes, these genes have also been subclassified as gatekeepers, caretakers, and landscapers.

Gatekeepers are oncogenes and tumor suppressor genes that control cell growth and differentiation. In contrast, caretakers (i.e., tumor suppressor genes and mismatch repair genes) are responsible for maintaining the genomic integrity of the cell. Mutations in caretaker genes promote oncogenesis indirectly by inciting genetic instability, which increases the rate of mutations of other genes (including gatekeepers). If these mutations result in a selective advantage toward cell proliferation (or repression of apoptosis), tumor formation may ensue. Finally, landscapers are not directly involved in cellular growth. Rather, they foster a stromal environment conducive to unregulated cell proliferation. Indeed, although normal stroma creates an environment with innate antitumor properties, inherited or acquired mutations in landscaper genes facilitate tumor growth by removing the natural antagonism of the stroma toward oncogenesis (e.g., by altering cell adhesions, cellular signaling, and altered innate immunity) or by altering the microenvironment to a more oncogenic state (e.g., through matrix metalloproteinases and angiogenesis).

Heterogeneity of Mutations

Third, not all genetic mutations that accrue during tumorigenesis are equipotent—some mutations involved in cancer progression are more significant than others. As oncogenesis progresses, cancer cell proliferation becomes increasingly reliant on these more potent (driver) mutations, a phenomenon known as oncogene addiction . Because of a tumor's reliance on the products of these critical oncogenes for growth and survival, this dependence is a point of weakness (an Achilles heel in the seemingly impenetrable armor of unrestrained genetic activity) and, therefore, a potential therapeutic target.

Driver mutations, which comprise a minority of mutations in cancer, are mutations that have been positively selected during oncogenesis by conferring a growth advantage to the cancer cell. They “drive” cancer cell proliferation. In contrast, passenger mutations, which comprise the majority of mutations in cancer cells, confer no such advantage. They are simply the expected functional consequence of normal clonal and subclonal expansion. In other words, passenger mutations are simply “along for the ride.” To have a positive therapeutic effect on tumor biology, one must sort through the passenger mutations and devise means to target the driver mutations effectively.

Most genetic mutations found in tumors are clonal (they are present in all cells within the tumor). However, subclonal mutations are an important source of intratumoral, intermetastatic, intrametastatic, and interpatient tumoral heterogeneity. This source of heterogeneity has significant treatment implications.

Intratumoral heterogeneity refers to heterogeneity of tumor cells within the same tumor. Each time a tumor cell divides, the progeny cells acquire new mutations that distinguish them from their common progenitor cells. Therefore, in the process of cell replication down the genetic tree of oncogenesis, cells that are generationally divergent will also be more genetically distinct. From a practical standpoint, this form of heterogeneity is usually not clinically significant in patients with localized disease because the primary tumor is often surgically removed. However, this divergence down the genetic tree of oncogenesis spawns the seeds of intermetastatic heterogeneity.

Intermetastatic heterogeneity refers to heterogeneity among spatially distinct metastatic lesions in the same patient. Most patients who die from cancer die from an overwhelming tumor burden in metastatic sites that cannot be removed surgically and that fail systemic chemotherapy or radiation therapy. As such, this form of heterogeneity has significant clinical implications. At one end of the spectrum, if each metastatic site is sufficiently genetically distinct to respond differently to chemotherapeutic agents, then long-term survival is nearly impossible to achieve. Fortunately, most intermetastatic heterogeneity is confined to passenger mutations. The mutations required for metastasis are present in the progenitor cells before the seeds of metastasis are released.

Similar to the heterogeneity within the primary tumor that develops during the process of tumor cell propagation (intratumoral heterogeneity), intrametastatic heterogeneity is the end result of genetic mutations that occur as a founding cell of a metastatic site divides. Such mutations are the grounds for drug resistance. Metastatic deposits may initially respond to treatment that targets mutations that occurred in progenitor cells before metastases occurred, because many of the cells in a metastatic lesion have the same susceptibility to chemotherapy, evidenced by a decrease in the size (or complete resolution) of the lesion noted on posttreatment medical imaging. However, hundreds or thousands of the cells with acquired drug resistance are likely still present, perhaps below the resolution of medical imaging or clinical examination. Recurrence in such situations is inevitable.

There is also heterogeneity of the same tumor type between different patients (interpatient heterogeneity) . This form of heterogeneity is responsible for the clinical observation that “no two cancer patients are the same.” As a result, uniformly applying the same systemic treatment to all patients with the same type of tumor does not result in the same response rates for each patient. Even if the end result is the same in two different patients (e.g., a particular driver mutation that leads to lung cancer), the mutations in each patient are likely sufficiently distinct such that they alter the coding sequence of the gene and hence the translated protein product of the gene in unique ways. Even small differences in a protein product can affect the conformation of the protein and hence its susceptibility to treatment. This heterogeneity is likely the result of host factors (e.g., inherited mutations that predispose to developing cancer, mutations that affect the pharmacokinetics of chemotherapeutic agents, environmental factors within a host that facilitate tumor propagation) and distinct mutations between different tumors that develop during clonal and subclonal expansion.

DNA Aneuploidy

One of the most common observations in solid tumors is DNA aneuploidy (abnormal chromosome number and content). Aneuploidy can result from aberrant mitotic divisions, chromosome cohesion defects, improper attachments of chromosomes to microtubules, weakened mitotic checkpoint signaling, or mutated mitotic checkpoint genes. The contribution of aneuploidy to oncogenesis is controversial. Aneuploidy may simply facilitate (rather than directly cause) oncogenesis. For instance, populations of cells that are capable of redistributing chromosomes such that there is an increased probability of loss of heterozygosity (and hence loss of function of tumor-suppressor genes) or duplication of a mutated oncogene allele would confer a growth advantage. Alternatively, aneuploidy (generated either spontaneously or by a carcinogen) could catalyze a chain reaction of genetic instability and therefore initiate carcinogenesis. Regardless of its precise role in oncogenesis, aneuploidy is clinically significant, because it may be a mechanism of acquired resistance to chemotherapy.

Dysregulation of Cellular Proliferation

Mutations in proto-oncogenes and tumor-suppressor genes promote uncontrolled cellular growth if they lead to increased cellular proliferation, decreased apoptosis, or both. Cell proliferation is directly controlled by cyclins, a group of proteins that interact with cyclin-dependent kinases (CDKs) and are controlled by cyclin-dependent kinase inhibitors (CDKIs). Increased expression of cyclins and decreased activity (or loss) of CDKIs contribute to cell cycle dysregulation.

Cell proliferation is also controlled by telomeres, noncoding, tandemly repeated DNA sequences at the ends of chromosomes that allow DNA to be completely replicated all the way to the end of the coding sequence, thereby preventing enzymatic degradation and stabilizing the chromosome. A small amount of telemetric DNA is lost with cell cycle. Once this DNA reaches a critical point, a signal prevents further cell division. Telomerase is a ribonucleoprotein enzyme complex (containing a reverse transcriptase subunit and an RNA subunit) that maintains telomere length and hence evades replicative senescence. In contrast to most somatic cells (which have no telomerase activity), many cancer cells have increased telomerase activity. Consequently, telomerase activity may be a marker of progression for a benign to malignant process and may be a therapeutic target.

There are multiple interconnected complex pathways of cell-cycle control. Noncoding ribonucleic acids (ncRNA) are functional RNA molecules that are not translated into proteins and are important mediators of these pathways. One such ncRNA is microRNA (miRNA), single-stranded, short (about 22-nucleotide) ncRNAs that are important regulators of mRNA translation and degradation by binding at partially complementary sites in the untranslated regions of target mRNAs. miRNAs contribute to tumorigenesis by directly and indirectly (through epigenetic regulation) influencing translation of tumor-suppressor genes and oncogenes and by triggering a toll-like receptor–mediated tumorigenesis inflammatory response. miRNAs are in turn regulated by endogenous RNAs, which share miRNA response elements with mRNA, thereby vying for miRNA binding. As such, endogenous RNAs can remove the repressive effect of miRNAs on gene expression. The result is a complex network of classic genetic and epigenetic regulation of gene expression by miRNAs and endogenous RNAs. Dysfunctional endogenous RNAs can promote oncogenesis by removing the inhibitory effect of miRNA on oncogenes.

Cancer Stem Cells

Most cancers arise from a single cell that has undergone genetic mutations. Such cells, known as cancer stem cells, have the ability to self-renew, proliferate, and produce the heterogeneous lineages of cancer cells within a tumor. It is unclear whether cancer stem cells are true pluripotent stem cells, adult tissue-specific stem cells that acquire oncogenic mutations, or mature cells influenced by their microenvironment to undergo de-differentiation. The process of tumor progression from cancer stem cells is also unclear. It may involve aberrant differentiation of cancer stems cells or it may involve cell-cell fusion between cancer stem cells and differentiated heterogeneous somatic cells, which can then undergo further cell division into various permutations of phenotypically unique cancer stem cells, mutated differentiated cells, or fused cells. Both pathways account for cellular heterogeneity and aneuploidy noted in tumors.

There is growing evidence of lung cancer stem cells. Normal stem cells and lung cancer cells share several proliferative cell signaling pathways (e.g., K-ras, hedgehog, phosphate and tensin homolog [PTEN], Akt, and phosphoinositide 3-kinase [PI3K]). Furthermore, lung cancers often share cell-surface markers with lung stem cells, which may explain the distribution of lung cancer histologies along the tracheal-bronchiole-alveoli axis. For instance, squamous cell carcinomas, which tend to occur in the central airways, and stem cells of the pseudostratified epithelium of the central airways both contain keratin-expressing basal cells.

There is also growing evidence of esophageal cancer stem cells. Several studies on murine models demonstrated evidence of phenotypic stem-cell surface markers (e.g., α-6 integrin, p75ntr, and CD44 ). Based on evidence found in murine models, pluripotent bone marrow progenitor cells contribute to Barrett esophagus. Given the relative low turnover of lung and esophageal epithelial tissue compared with hematopoietic cells and keratinocytes, stem cells research in lung and esophageal basic science research has been somewhat limited.

The cancer stem cell theory has important therapeutic implications. The success of existing systemic chemotherapy relies on a stochastic model in which all cells in a tumor have the same oncogenic potential. However, if there is only a small subset of cells with proliferative potential (i.e., cancer stem cells), treatment failure is likely. In fact, non–small-cell lung cancer (NSCLC) patients with CD133 ⁺ lung cancer stem cells are less responsive to platinum treatment. Potential mechanisms for resistance of cancer stem cells to chemoradiation therapy include their relative dormant nature compared with nontumorigenic cancer cells (which tend to replicate more rapidly), ability to survive in hypoxic microenvironments, upregulation of chemotherapy efflux mechanisms, and upregulation of DNA repair pathways.

Detection in Tumor Tissue