Molecular Studies of the p53 Pathway in Human Cancer

Columbia Univ New York Morningside, New York NY

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Abstract

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