Final Report Summary - P63 PROSTUMOR (Investigating the role of p63 in PROstate STem cells and tUMOR development) Background P53 is one of the best-characterized tumor suppressor genes, whose expression is lost or mutated in approximately 50% of all human cancers. Although much is known about the mechanisms of action of p53, the recent discovery of two other family members, p63 and p73, has generated much interest as to whether these may function similarly to p53. Determining the function of the p63 gene has been complicated by the expression of six different isoforms of the p63 protein. Transcription from two separate promoters generates two main subclasses of protein - those possessing a full-length transactivation domain (TA) and those that have a truncated N-terminus (deltaN). Alternative splicing at the C-terminus further generates three subtypes (alfa, beta, gamma) within each group. Among the better characterized roles for p63, the deltaNp63alfa isoform is suggested to be required for the normal proliferation of stem cells in many epithelial tissues, including the skin and the prostate gland, though the mechanisms by which it does this remain unknown. With regards to tumor development, however, the role of p63 remains unclear. Unlike p53, which is frequently lost during tumor development, p63, and specifically the deltaNp63 alfa isoform, is overexpressed in a variety of human tumors, particularly squamous cell carcinoma (SCC). We have recently demonstrated for the first time, that the deltaNp63alfa isoform is an oncogene in the skin that is capable of forming SCC in cooperation with oncogenic Ras. We found that this isoform of p63 is capable of promoting tumor development specifically by inhibiting the tumor suppressive mechanism of oncogene-induced senescence. This data, in agreement with many human studies, supports a role for deltaNp63alfa in initiating epithelial tumors. With regards to prostate cancer however, the role of p63 is less clear. The deltaNp63alfa isoform is suggested to be the predominant isoform that can be detected in the normal tissue. Indeed, data from mouse models have shown that p63 is ultimately required for the normal development of the prostate gland, as p63-/- mice fail to develop this organ [3, 4]. As such, it has been suggested that p63 is necessary for both the initial commitment of cells to the prostate cell lineage during development and for the proliferation of the prostate stem cell population. In prostate cancer however, the role of p63 is also unknown. Unlike the overexpression of p63 that is seen in SCC, during the development of adenocarcinoma, the most common prostate tumor type, p63 expression is actually lost from the cells that are undergoing malignant conversion, in a process that is thought to favor epithelial-mesenchymal transformation and malignant invasion.As such, there are few specific studies that functionally address the role of p63 and it’s isoforms in normal prostate biology or during the development of prostate cancer.Specific Aims 1. To determine the expression of p63-isforms in prostate and prostate stem cells. a. To determine the expression pattern of p63 isoforms in normal prostate tissue. b. To determine the expression of p63 isoforms in prostate stem cells. 2. To determine the effects of loss of p63 on normal prostate and during prostate tumor development. a. To determine the effects of loss-of-function studies with p63-ablation in the normal prostate. b. To determine the effects of loss-of-function studies with p63-ablation in prostate tumors. 3. Identification of novel p63 target genes in the prostate.We have successfully completed all aims or modifications therein. Initially, we have identified that stem cells in mouse and human prostate predominantly express the deltaNp63alfa isoform. Functionally, we have found that deltaNp63alfa is required for maintaining these stem cells, through isoform-specific knockdown in human cells, and preliminary loss-of-function studies in wildtype mice. And finally, we have identified direct targets of p63 and the pathways that are regulated by p63 in the prostate, by combining microarray on cells that have been infected with a shRNA for deltaNp63 alfa and CHIP-Sequencing in the human cells lines. Together our work has revealed a critical role for deltaNp63alfa in maintaining the proliferative capacity of prostate stem cells. By taking a multidisciplinary approach, using functional genetics, in vivo animal models and high-throughput genomic screens, we have identified important mechanisms that drive prostate stem cell growth.