The overall goal of this K01 proposal is to allow Harold I. Saavedra, Ph.D., under the mentorship of Gustavo Leone, Ph.D. to transition from a postdoctoral fellowship to the establishment of his own independent laboratory in an academic institution. The study of the Rb/E2F pathway is crucial to the understanding of cancer, since components of this pathway are de-regulated in almost all human tumors. One of the central components of the Rb/E2F pathway is the E2F3 transcription factor, which plays a key role in cell cycle progression. The E2F3 locus codes for two transcripts, E2F3a and E2F3b, whose individual functions are presently unknown. We show that gene knock-out of E2F3, which included knocking-out of E2F3a and E2F3b, leads to slower growth rates, centrosome amplification and aneuploidy. Our hypothesis is that the uncoupling of the centrosome cycle and the cell cycle, triggered by the loss of E2F3 function, may ultimately lead to cancer. To explore this hypothesis we propose the following specific aims: 1. To unravel the individual contributions of E2F3a and E2F3b towards ceil cycle control, suppression of centrosome amplification and aneuploidy. SiRNAs will be introduced into mouse embryonic fibroblasts to knock-down the expression of E2F3a and E2F3b. We will determine the growth characteristics of those knock-downs, and their individual contributions towards centrosome amplification and aneuploidy. 2. To explore, using a centrin-GFP transgenic mouse, whether loss of E2F3 leads to centrosome amplification in vivo. We will introduce a conditional allele of E2F3 into centrin-GFP mice. Conditional deletion of E2F3 in mammary epithelial cells will be achieved by crossing those mice with WAP-CRE mice. Frequencies of centrosome amplification will be calculated from sections of those mammary glands. 3. To explore the potential role of the myclE2F pathway in centrosome amplification and mammary carcinogenesis. We will generate WAP-myc/centrin-GFP mice and determine progression from expression of myc to centrosome amplification, aneuploidy and tumorigenesis. We will also explore whether loss of E2F3 cooperates with myc to accelerate mammary tumorigenesis. The results obtained from this K01 proposal will be invaluable to the understanding of the relationship between centrosome amplification, aneuploidy and carcinogenesis in vivo, and to whether genomic instability is a cause or a consequence of tumorigenesis.

K01CA104079

2004-05-01

2010-04-30