Tumors of the colon and rectum are a major health problem: in 2006 alone, a million new cancer cases occurred in the world, resulting in ~590,000 deaths. Half of the population of the United States will develop at least one benign colorectal tumor, and in one-tenth of these, the tumors will eventually become malignant. Our research is aimed at understanding the molecular basis of colorectal neoplasia, in the hope that this knowledge can be used to improve the diagnosis and therapy of this disease.
Pathways That Control Tumorigenesis
A pathway is defined by a set of genes that regulate a specific cellular function. Two kinds of pathways have been shown to drive the process of colorectal neoplasia. One kind contains oncogenes and tumor-suppressor genes that directly regulate cell birth and cell death. In the normal colon, the rate of cell birth precisely equals that of cell death, resulting in a constant number of colon cells. When a particular growth-controlling pathway gene is altered through mutation, the rate of cell birth exceeds that of cell death, and a tumor is initiated. These tumors progress, becoming larger and more dangerous as mutations in other growth-controlling pathway genes accumulate during the tumorigenic process. When several such pathways are altered by mutation, a malignancy is likely to form (see figure).
Genes that participate in the second kind of pathway, called stability genes, do not directly control cell birth or cell death, but rather control the rate of mutations of other genes, including growth-controlling genes. When stability genes are genetically altered, the cell accumulates mutations at a high rate and the tumorigenic process is accelerated. Members of our laboratory and others have discovered several stability genes that appear to be important for colorectal neoplasia. These include the mismatch repair genes MSH2, MSH6, MLH1, and PMS2, whose alterations result in subtle DNA changes, and BUBR1, MRE11, and CDC4, whose alterations can result in gross chromosomal abnormalities.
Initiation and Progression of Colorectal Neoplasia
The APC (adenomatous polyposis coli) pathway must be altered for colonic tumors to form. Patients with inherited mutations of the APC gene develop thousands of benign tumors of the colon, called adenomas. Our group has helped discover the mechanisms through which APC regulates colon cell growth. APC binds to another protein, called β-catenin, and stimulates its phosphorylation. In cells with an APC mutation, this phosphorylation does not take place and β-catenin is activated. The intracellular location of β-catenin changes upon APC mutation; it migrates to the nucleus, interacts with transcription factors, and induces the expression of genes that stimulate cell birth.
Mutations in APC or β-catenin are sufficient to initiate the growth of a small benign tumor but are not sufficient to make such tumors progress to more advanced forms. Several other pathways participate in this progression. One of these pathways involves transforming growth factor β (TGFβ) family members, a group of small polypeptide hormones that negatively control colon cell growth through regulation of transcription factors such as SMAD4. A second critical pathway involved in tumor progression involves TP53, a gene that is inactivated not only in colorectal cancers but also in most other cancer types. Activation of the normal TP53 gene inhibits cell growth by blocking the cell cycle and by stimulating cellular suicide (apoptosis). Our group has discovered several of the genes that mediate these effects. The p21WAF1 and 14-3-3σ genes control cell birth by regulating passage through various phases of the cell cycle. The PUMA gene is a powerful stimulator of apoptosis whose product is located in mitochondria and binds to homologous proteins, like BAX, that control cell death.
The studies described above were generally performed through the analysis of single genes that appeared useful on the basis of functional evidence or gross chromosomal changes in cancers. Recently we have developed technologies that allow detailed analysis of all of the protein-coding genes within the human genome. We have found that a typical solid tumor (such as that of the colon, breast, pancreas, or brain) contains 50–100 genetic alterations with major effects on one or more genes. The complex picture that emerges from these studies can be simplified by the realization that the most important of these alterations can be grouped into a smaller number of pathways. The dozen or so pathways that are genetically altered in each tumor are apparently responsible for most of the known properties of tumors. Although detailed understanding of these pathways will require much future research, their recognition is already providing novel approaches to diagnosis, prognosis, and treatment.
Practical Applications
Knowledge of the pathogenesis of tumors has already had a significant impact on the management of patients. For example, our group has developed sensitive blood tests to identify patients with inherited mutations of APC or of the mismatch repair genes. These tests are now routinely used in the clinic to help advise families with hereditary forms of colorectal cancer predisposition. When combined with genetic counseling and surveillance measures, these diagnostic tests can ameliorate much of the anxiety and suffering previously encountered by these families and help affected members achieve normal life spans.
Deaths from colorectal cancer can almost always be prevented if the tumors are detected prior to the onset of metastasis. We have designed new approaches for such early detection that employ DNA purified from stool specimens or blood samples. Our most recent advance in this area is the development of an assay that can efficiently query each of tens of thousands of single DNA molecules for mutations in APC and other genes. This assay, called BEAMing for its four principal components (beads, emulsions, amplifications, magnetics), has been used to show that more than 60 percent of patients with early, presumably curable forms of colorectal cancers can be identifiedthrough the analysis of routine blood samples. Moreover, the number of mutant molecules in the blood of patients with colorectal cancer following treatment provides an objective measure of residual disease.
In addition to improving diagnosis, the new understanding of cancer can guide therapeutic development. We are attempting to develop chemotherapeutic drugs that inhibit the lipid kinase activity of the PIK3CA gene product. Mutations in this gene were discovered through the unbiased genomic analyses methods described above. Additionally, we are attempting to develop C. novyi-NT, a genetically modified strain of Clostridium novyi, as a new therapeutic agent. These bacteria are exquisitely sensitive to oxygen. When C. novyi-NT spores are injected into animals with human tumors, the spores germinate in the poorly oxygenated regions of cancerous tissues but not in normal tissues. This poor oxygenation results from aberrations of the signaling pathways that are genetically altered in the cancers. We have recently begun a clinical trial of C. novyi-NT in patients with advanced cancers.
