Background: Muscle-invasive bladder cancer is one of the major clinical challenges with high mortality resulting in the proximal cause of ~14,000 deaths in the U.S. Recently we established a novel mouse model of invasive bladder cancer based on the combinatorial deletion of p53 and Pten in bladder epithelium using an adenovirus expressing Cre recombinase (Adeno-Cre) delivered to the bladder lumen of p53flox/flox;Ptenflox/flox mouse (Puzio-Kuter Gene Dev 2009). We found marked upregulation of tumor suppressor ARF in tumor tissue compared to normal epithelium in our mouse model.Hypothesis: We hypothesize that ARF has a previously unknown function in promoting bladder tumorigenesis in the context of p53- and Pten-deficiency, which can be a potent therapeutic target in bladder cancer treatment.Rationale: Invasive bladder cancer in our mouse model shows histological and genetic features highly similar to those of aggressive human invasive bladder cancer. Additionally, we have demonstrated the usefulness of our mouse model in preclinical study (Seager Cancer Prev Res 2009). In terms of functional significance of ARF, we found that tumors induced in Arfflox/flox; p53flox/flox; Ptenflox/flox triple flox (TKO) mice grew more slowly and allowed longer survivals than those induced in p53flox/flox;Ptenflox/flox double flox (DKO) mice. Our preliminary data showed that some tumor suppressors including TSC2 and p16 were upregulated in TKO tumors in correlation with decreased proliferation status as assessed by Ki67 expression.Specific aims: We aim (1) to elucidate molecular pathways through which ARF suppresses TSC2 an p16 expression and promotes tumorigenesis, and (2) to investigate clinical significance of role of ARF in human bladder cancer.Study design: We will investigate key regulators of tumor-promoting pathways involving ARF by master regulator analysis in which differential gene expression profiles of TKO and DKO mouse tumors are compared with a large-scale human bladder gene regulation maps (interactome). Simultaneously, we will investigate the transcriptional activity of specific transcription factors that potentially bind to the TSC2 promoter region preserved beyond species. Then we will validate the hypothetical pathways functionally using human bladder cancer cells. Clinical significance of the activation of the molecular pathways will be interrogated by high throughput immunohistochemical analysis on tissue microarray. Finally we will utilize our mouse model to examine whether the molecules involved in the tumor-promoting pathway can be a predictive factor or therapeutic target in the treatment of invasive bladder cancer.Relevance to cancer: Since previously known p53-independent function of ARF is mostly tumor-suppressive, novel tumor-promoting functions of ARF would have a significant impact on the entire field of cancer research. Additionally, our research will provide promising treatment strategies for bladder cancer treatment.