The objective of the proposed studies is to determine whether data on a patient's p53 status aids clinical management of bladder cancer. Our working hypothesis is that mutations and altered expression of the TP53 gene, or those affecting certain regulatory genes involved in the p53 pathway, produce a selective advantage for tumor growth and aggressive behavior in cancer patients.
Our specific aims are as follows:
Aim 1. To conduct molecular and functional studies of the p53 pro-apoptotic response and its clinical significance in bladder cancer. We will determine the clinical relevance of detecting TP53 mutations and altered patterns of p53 expression using a combination of methods. To assess the consequences of TP53 mutations, we will clone p53 mutants in expression vectors and ascertain their activities. Regulatory events of the p53 pathway will also be analyzed, centering on genetic and expression studies of HDM2 (collaboration with Project by Levine). We will also define the frequency and clinical significance of altered Bax, PUMA and Noxa expression, mainly in relation to treatment response (working with Project by Lowe).
Aim 2. To define the clinical and biological implications of p53 DNA damage response in bladder cancer. We and others have observed that early bladder cancer, but not normal tissues, express markers associated with an activated DNA damage response, such as phosphorylated Chk2. We have also identified CHK2 mutations in primary bladder tumors (collaborating with Project by Prives). We will assess the frequency of CHK2 mutations in bladder cancer, and their association with increased genetic instability and tumor progression. The phosphorylation status of Chk2, ATM, and p53 will be investigated in bladder cancer cell lines and primary tumor samples. The consequences of CHK2 mutations will be studied by cloning Chk2 mutants in expression vectors and determining their response to gamma-radiation.
Aim 3. To ascertain the role of p53 in senescence triggered by Pten inactivation in bladder cancer. We have recently reported the involvement of p53 at inducing senescence in response to inactivation of the Pten pathway. We also found that p53 and Pten have cooperative tumor suppressor roles in bladder cancer, their concomitant inactivation being associated with tumor progression and poor outcome. Using a combination of techniques, we will determine the clinical relevance of detecting PTEN abnormalities. In addition, the oncogenic potential of Akt constitutive activation will be investigated (working with Project by Lowe). Mechanistic studies will be aimed at further defining the crosstalk between these two pathways (e.g., silencing PTEN and/or p53 expression by shRNA to pheno-copy the ablation of the senescent status, followed by gene expression profiling analyses). Identified target genes will be validated in bladder cancer cell lines and primary bladder tumors of known Pten and p53 status. The main goal is to translate basic research findings into clinically applied studies.
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