Genetic Events Contributing to OncogenesisMy laboratory is interested in the genetic events that contribute to the development of cancer. The focus of the work is a series of mouse strains in which we have engineered mutations in genes known to be involved in human cancer.
Mouse models for human familial cancer syndromes: It has recently been shown that a number of human familial cancer syndromes ( in which affected individuals have a greatly increased risk of developing particular types of cancer) are caused by the inheritance of a mutant allele of a tumor suppressor gene. These genes are thought to normally negatively regulate cell growth and they contribute to carcinogenesis when mutated or lost. Thus, individuals who carry only one functional copy of a given tumor suppressor gene are predisposed to cancer because all of their cells are just one mutational event from lacking an important negative growth regulator. Examples of diseases (and genes) in this class are: familial retinoblastoma (RB), neurofibromatosis type I and type II (NF1, NF2), Li-Fraumeni syndrome (p53), and familial adenomatous polyposis (APC).
Over the past several years, we have used gene targeting in mouse embryonic stem cells to create novel mouse strains with mutations in the murine homologues of several tumor suppressor genes. To date, we have constructed strains with germline mutations in Rb, Nf1, Nf2, p53 and Apc. Animals that are heterozygous for these mutations mimic (at least genetically) humans with one of the familial cancer syndromes mentioned above. The effects of some of these mutations in the mouse are consistent the human disease phenotypes, and in other cases there are clear species-specific differences. For example, humans who are heterozygous for an RB mutation have a 90% likelihood of developing retinoblastoma (a tumor of the eye), while we have not observed a single case of this tumor in several hundred Rb heterozygous mice examined. Instead, the Rb mutant mice are highly predisposed to pituitary tumors, with a penetrance of nearly 100%. In contrast, heterozygous mutation of the p53 gene causes predisposition to a similar spectrum of tumors in humans and mice. Through interbreeding of the different tumor suppressor gene-deficient strains, we are also examining possible synergistic effects of the multiple mutations.
The role of tumor suppressor genes in development: In addition to studying the effects of heterozygosity for tumor suppressor gene mutations, we are interested in the developmental consequences of homozygosity for these mutations. An understanding of the homozygous phenotype may provide clues to the function of these genes in normal cells and indicate why their loss contributes to carcinogenesis. We have carried out the heterozygous crosses for all of the mutant strains described above and determined that Rb, Nf1, Nf2, and Apc are all required for normal mouse development. Deficiency for Rb function leads to defective erythropoiesis and neurogenesis and the eventual death of the embryo by days 14-15 of gestation. The survival of Rb homozygotes to mid-gestation was somewhat surprising, and we have gone on to mutate the Rb-related genes, p107 and p130, to investigate possible functional redundancy in this gene family. Nf1 and Apc homozygotes show defects in cardiac and neural development, respectively, while Nf2-deficient embryos fail prior to day 8 of gestation.
Use of cell lines derived from mutant mice to probe the function of tumor suppressor genes in vitro: In addition to studying the effects of mutations of different tumor suppressor genes in the context of the whole animal, we are using cells isolated from the mutant mice to begin to investigate the function of these genes in vitro. Primary embryo fibroblast cultures have been isolated from embryos that are homozygous for a mutation in Rb, Nf1, or p53 as well as from the appropriate heterozygous and wild type controls. Since these cells are isogenic except for the mutations in the tumor suppressor genes, any phenotypic differences observed between the homozygotes and controls can be attributed to the known mutation and should reflect a function of the tumor suppressor gene. These experiments have focused to date on the role of Rb in transcriptional control during the cell cycle and on the importance of p53 in the normal cellular responses to DNA damage and other adverse conditions. For example, we have shown that p53-deficient fibroblasts fail to arrest their growth during the G1 phase of the cell cycle in response to gamma irradiation. Also, immature thymocytes which lack p53 function do not undergo programmed cell death (apoptosis) following irradiation. In addition, we have shown that p53 function is also required for the execution of the apoptotic pathway in response to the expression of the adenovirus E1A oncogene. This observation suggests that another mechanism by which p53 can effect tumor suppression is through the elimination of cells that have already acquired oncogenic mutations. Finally, p53-dependent apoptosis is also an important determinant of the sensitivity of tumor cells to various anti-cancer agents. We are currently examining the functional domains of p53 required for these various biological effects as well as constructing a mouse strain carrying a mutation in the p21 gene, which encodes a cyclin-cdk inhibitor thought to be an important downstream effector of p53 function.
Oncogene mutations. We have complemented our research on tumor suppressor gene mutations with one oncogene project. We have created two germline mutations of the K-ras gene. The first of these is a loss-of-function mutation in the gene. Embryos lacking K-ras function die with associated defects in liver function and generalized developmental delay; thus, of the three mammalian Ras proteins (K-, N-, and H-Ras), K-Ras is the only one essential for development. We have also used a modified "hit-and-run" gene targeting protocol to create an allele of K-ras that can be activated to an oncogenic state upon somatic mutation. The tumorigenic consequences of this mutation are currently under study.