The functional inactivation of the Rb tumor suppressor is an important event in the genesis of many cancers. pRb is a negative regulator of cell proliferation and it is thought that tumors acquire changes that allow them to escape pRb's ability to suppress cell cycle progression. A key goal at the heart of all Rb research is to understand how cells are changed by the loss of pRb. This information might suggest ways to suppress these changes, or might reveal weaknesses that can be targeted therapeutically. pRb associates with chromatin and regulates transcription. Numerous studies have profiled the transcriptional changes that occur when Rb is lost, and it has generally been assumed that these transcriptional profiles give a meaningful picture of the altered state of Rb-mutant cells. As an alternative approach, in this application we propose to use proteomic profiling to identify proteins whose abundance changes significantly when Rb is removed. Preliminary data from analysis of Rb mutant tissues shows that loss of this tumor suppressor leads to extensive proteomic changes that are strikingly different from the transcriptional signatures that have been studied previously.
In Aim 1 we propose to generate a detailed timecouse of these changes, to identify the types of proteins that are altered and to determine whether the proteomic changes occur before, or after, the changes in transcription. We propose to study one of the largest groups of proteomic changes and to determine which changes promote the survival and proliferation of pRb-deficient cells. One of the surprising features of the proteomic data is that the transcriptional upregulation of classic E2F targets leads to only minor changes in overall protein levels in Rb-mutant issues. We hypothesize that one of the reasons for this is that Rb loss increases the levels of RNA-binding proteins that bind to the 3'UTR sequences of multiple E2F-induced mRNAs and suppress translation.
Aim 2 of the proposal will test this model and will identify transcripts repressed by Nanos and Pumilio in pRb-deficient cells. Collectively these results will provide new insights into the cellular consequences of pRb inactivation, and the ways that cells adapt to cope with deregulated E2F activity.
Cancer is a major cause of mortality in the United States. Tumor suppressor genes protect us against cancer and the loss of these protective shields is a hallmark of tumor progression. This application will characterize the changes that occur in cells following the inactivation of RB1 (Rb), one of the best-known tumor suppressor genes. The experiments exploit a new technology that allows rapid quantitation of large numbers of proteins, and build upon preliminary data showing that the proteins whose levels change the most when Rb is lost are completely different from what was expected. These experiments will provide new insights into the ways that cells are altered by the loss of Rb, and will seek to identify changes that are important for Rb mutant cells to survive and proliferate.
|Guarner, Ana; Morris, Robert; Korenjak, Michael et al. (2017) E2F/DP Prevents Cell-Cycle Progression in Endocycling Fat Body Cells by Suppressing dATM Expression. Dev Cell 43:689-703.e5|
|Dyson, Nicholas J (2016) RB1: a prototype tumor suppressor and an enigma. Genes Dev 30:1492-502|