Genetic Interaction Between the pRB and Warts Tumor Suppressor Pathways

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DESCRIPTION (provided by applicant): The retinoblastoma protein (pRB) is very important tumor suppressor that is often mutated or inactivated during the formation of cancer. As a tumor suppressor pRB regulates the activity of the E2F transcription factor family. The E2F family is characterized by "activators" and "repressors", both of which traditionally form a heterodimer with the DP protein at the promoters of target genes. The activator E2Fs, in the absence of pRB, can drive the cell into the cell cycle by activating the transcription of the G1 cyclins. Thus, in cancers in which pRB function is lost, E2F activity is high, and therefore, the tumor continues to grow and divide. Due to the high incidence in which pRB function is lost during tumor formation it has been suggested that targeted inhibition of the E2F family could be a promising anti-cancer therapeutic. However, to carry out these studies in the mammalian model system has proven challenging due to the overwhelming number of E2F, pRB, and Dp, family members. Fortunately, the fruit fly, Drosophila melanogaster, has less complexity between the E2F, pRB, and DP families. There is one activator E2F(dE2F1) and one repressor E2F(dE2F2), and when the activator E2F is lost due to mutation or targeted inhibition it results in a severe proliferation block. Thus, reinforcing the idea that loss of E2F activity could help slow cancerous growths. However, when all E2F activity is lost, both in vivo and in vitro, cells can proliferate with relative normality. Recently, a new tumor suppressor pathway referred to'as the Hippo pathway has been delineated in Drosophila. All members of this pathway have orthologs or homologs in humans, and are found to be mutated in human cancers. When this pathway becomes inactive, cells gain a proliferative advantage over neighboring wildtype tissue and are resistant to apoptotic stresses. This study aims to test what effects the loss of the E2Fs, in all combinations, has in tissue that has an inactive Hippo tumor suppressor pathway. This study will use a combination of new and classical Drosophila genetics, molecular biology, and cell biology to address this issue. Immunohistochemistry analysis will be used to determine in vivo effects upon proliferation and apoptosis Hippo mutant tissue in the absence of the E2Fs. Furthermore, any requirement for dE2F dependent transcription will be confirmed via ChIP analysis. Due to the evolutionary conservation between both of these pathways (pRB/E2F and SWH) in Drosophila and humans, this study will provide insight into the functional relationship between two important tumor suppressor pathways. Thus, the outcome of this study could demonstrate that targeted inhibition of activator E2Fs could prove to be a productive anticancer therapy.