Genomic Instability in Solid Tumors
Genomic Instability in Solid Tumors
By Daniel L. Stoler, PhD, Rajiv Datta, MD, Bruce M. Brenner, MD, and Garth R. Anderson, PhD
Carcinogenesis represents the accumulation of a small number of specific mutations, resulting in the eventual transition from a normal to a malignant phenotype. Knudson’s ancestral "two-hit" hypothesis asserted that deletion and/or mutation of both alleles of a single gene could cause cancer.1 Vogelstein described a staged, linear model of colon cancer progression in which multiple "hits" in tumor suppressor genes and oncogenes are required for malignancy to occur.2 It now appears that 4-10 events are necessary for the development of sporadic solid tumors.2-4 Loeb calculated that the normal baseline rate of mutation within a cell would be insufficient to account for the number of events required.5 Early inactivation of genes that maintain genomic stability, however, could result in a mutator phenotype that would significantly destabilize the genome, increase the mutation rate, and lead to tumor progression. Such genomic instability, reflecting the propensity and susceptibility of the genome to acquire multiple alterations, is believed to be the driving force behind multistep carcinogenesis. Thus, humans carrying defective genes which produce genomic instability are more susceptible to cancer, as has been observed in patients with Werner’s and Bloom’s syndrome and xeroderma pigmentosum, where defects exist in DNA repair proteins.
Forms of Genomic Instability
Three basic types of genomic instability have been described in solid tumors.
Aneuploidy. Aneuploidy, the first known type, involves the gain or loss of an entire chromosome(s), and has been reported in a wide variety of malignancies.6 This process involves aberrant segregation of chromosomes during cell division, resulting from improper centrosome amplification.7 Normally, a centrosome is replicated once during the cell cycle so that two centrosomes are found in a dividing cell, and to these the spindle apparatus is attached. When more than two centrosomes are present, aberrant segregation, that is spindle formation and segregation of chromosomes to more than two centrosomes, occurs and results in cells with gross karyotypic abnormalities. Evidence from tissue culture cells suggests a role for the p53 tumor suppressor gene as aneuploidy occurs concomitantly with inactivation of p53. However it is unclear if p53 participates in this process in vivo.8
Microsatellite Instability. The second and perhaps the best-characterized form of instability is microsatellite instability (MSI). MSI, first recognized by Perucho et al,9 involves the insertion or deletion of one or two base pairs in simple repeat sequences. These errors result from inherited or somatically acquired defects in DNA mismatch repair (MMR) genes, most commonly hMSH2 and hMLH1. This phenomenon has been widely observed in hereditary non-polyposis colorectal cancer (HNPCC) and in approximately 15% of sporadic colorectal cancers, as well as endometrial and gastric cancers, and to a lesser extent in other types of cancer.10 Individuals from HNPCC families who have inherited a single normal copy of one of the MMR genes require only a single "hit" to inactivate their second allele and allow the subsequent accumulation of errors. Therefore, these individuals are at greater risk for developing colorectal cancer. What is the extent of the accumulation of errors in these tumors? Perucho estimates that defects in MMR genes result in approximately 100,000 sequence alterations in the tumor genome. Although only a few genes altered by these defects have been identified, some cancer-causing candidates shown to be altered in tumors include the antiapoptotic gene Bax, transforming growth factor (TGF) B receptor type II (which normally functions to suppress tumor growth), and other mismatch repair proteins such as hMSH3.10 It should be pointed out that not all families who meet the clinical criteria for HNPCC would have defects in their mismatch repair genes, indicating that multiple pathways lead to this syndrome.
Intrachromosomal Instability. Intrachromosomal instability is the major form of instability seen in sporadic tumors and is manifested as deletions, amplifications, insertions, inversions, and translocations events which are believed to be initiated by DNA strand breaks.11 Various techniques to measure intrachromosomal instability, such as comparative genomic hybridization, allelotyping, arbitrarily primed PCR, and inter-simple-sequence-repeat (inter-SSR) PCR have demonstrated that tumor genomes contain alterations in numbers far greater than the model suggested by Vogelstein and in far greater numbers than is likely to be necessary to achieve malignancy.2,12,13 Estimates based on inter-SSR PCR place the number of genome events at greater than 10,000 per genome.13 If genomic instability is a driver of transformation, its onset should occur early in tumor progression. Conversely, if instability is the result of malignancy, then it should be restricted to carcinomas. Measurements of genomic instability in benign colonic polyps by inter-SSR PCR and by allelotyping indicate that intrachromosomal instability precedes the adenoma-carcinoma transition and is therefore likely to be an effector of carcinogenesis.13 The mechanism(s) that underlie this highly important type of instability are still unknown.
Experiments by Kahlenberg and colleagues comparing genomic instability in colorectal tumors as measured by inter-SSR PCR, to presence of mutation in the p53 gene, suggest that this tumor suppressor gene is not associated with the type of events prinicipally detected by this methodology.14 Also, the observation of instability in adenomas highlights the fact that p53 cannot be the principal facilitator of tumor progression, as mutation in p53 is a late event closely associated with the transition to carcinoma. But these experiments definitely do not rule out any p53 involvement in genomic instability as multiple pathways leading to intrachromosomal destabilization of the tumor genome are almost certain to exist. Other evidence for mechanisms involving deregulation of cell cycle checkpoints, chromosome bridge-breakage-fusion, and nuclease activation has been repeatedly described, and more than one of these pathways may be active within any given tumor.
Tumor Evolution Mediated by Genomic Instability: Clinical Implications
With early, massive disruption of the genome, why do we see the ordered stages of the Vogelstein model of colorectal tumor progression? Our belief is that cancer represents accelerated evolution in favor of the tumor, directed only toward proliferation and spread of the tumor cells, and occurring at the ultimate expense of the host. Genomic instability activated in a normal cell results in destabilization of the genome and disruption of those processes that regulate coordinated growth and differentiation of the cell that evolved over millions of years. Just as natural selection is a powerful force in the evolution of species, it is also presumably the major force in tumor evolution. We see the advantageous events described in the Vogelstein pathway as emerging from genomic chaos.
What are the implications of genomic instability for the tumor? The multitude of genomic events occurring during tumor progression will inevitably produce a heterogeneous tumor. This concept of tumor heterogeneity is not a new one, although its underpinnings at the genomic level have been enigmatic. It is likely that among the many genotypes in the tumor, there will be some present at the time of diagnosis already capable of resisting standard methods of chemotherapy. Similarly, selection for mechanisms of evading the immune system must have taken place for the tumor to have grown. In addition, multiple genotypes lead to heterogeneous expression of cell surface antigens. So what is the significance of such a genomically heterogeneous tumor mass at the clinical level? Therapies that are directed at the individual tumor cells or tumor cell antigens, as occurs with chemotherapy and immunotherapy, have a low probability of permanent success. In general, treatment of solid tumors with such therapies leads infrequently to cures, more often yielding partial responses followed by the outgrowth of subpopulations of cells that are drug-resistant or resistant to immunotherapy. On the other hand, therapies directed at the tumor as whole may meet with greater success. Surgery, for example, is such an approach. It has been successful when cancers are detected early, underscoring the need for new and more sensitive screening methodologies. Suppressing genomic instability to slow progression to cancer or targeting the genomically stable tumor vasculature necessary for tumor survival with antiangiogenic drugs are additional approaches that now appear to have particularly great potential. (Drs. Datta and Brenner are Surgical Oncology Fellows, and Drs. Stoler and Anderson are Cancer Research Scientists at Roswell Park Cancer Institute, Buffalo, NY.)
References
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