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Genomic Consequences of Restoring p53 Transactivating Function with PRIMA-1 in Breast Cancer
Genomic Consequences of Restoring p53 Transactivating Function With PRIMA-1 in Breast Cancer
a) Background: Mutations in p53 genes are common in human cancers and more than half of breast tumors harbor mutations in p53 protein. Various cellular stresses such as DNA damage, hypoxia and oncogene activation result in stabilization of p53 that culminates either in apoptosis or cell cycle arrest due to enhanced transcriptional activation of p53 target genes. As tumor cells become more refractory to chemotherapy and radiotherapy, considerable scientific efforts are directed to restore normal function of mutant p53 to promote tumor growth inhibition either by the induction of apoptosis or cell cycle arrest. From such studies, PRIMA-1 (p53-reactivation and induction of massive apoptosis) was identified in an in vitro screen of drugs that enable mutant p53 to restore its sequence specific DNA binding confirmation and transcriptional activity of target genes. We showed that PRIMA-1 restored p53 transcriptional activity of p21 and PUMA (p53-dependent pro-apoptotic gene) in breast cancer cells. Therefore, we propose to use PRIMA-1 as an adjuvant therapy with DNA damaging agent such as adriamycin for the treatment of breast cancer, because it (1) restores p53 transcriptional transactivation function in breast cancer cells, (2) has antitumor activity in its own right (3) is not a substrate (small molecule) for multidrug resistance. In addition, we will identify protein-binding partners of p53 protein (p53 complexes) with tools available for proteomics application.
b) Objective/Hypothesis: We propose that the restoration of transcriptional transactivation function of mutant p53 with PRIMA-1 provides an effective adjuvant therapy for breast cancer when used in combination with DNA damaging agents such as adriamycin. This approach allows for the use of lower doses of adriamycin to achieve higher tumor cell killing. Furthermore, we hypothesize that protein-protein interactions critical for p53 function restoration will be identified using tools for proteomics analysis.
c) Specific Aims: (1) To test the ability of PRIMA-1 to facilitate apoptosis of breast cancer cells treated in vitro and in a mouse model of breast cancer with adriamycin. In vitro testing will involve the use of normal mammary epithelial cells (HMEC), human MCF-7 breast carcinoma cells (p53 wild-type), human GI101A and MDA-231 breast carcinoma cells (both p53-mutant), and human MDA-157 breast carcinoma cells (p53 null), as we previously reported. The in vivo testing will involve the use of our nude mice model developed for breast cancer. (2) To identify protein-binding partners of p53 with tools available for proteomics analysis. We will use immunoprecipitated p53 complexes obtained from cytoplasmic and nuclear fractions of normal and various breast cancer cells and determine potential binding partners (protein-protein interactions) as well as protein expression profiling by 2D-gel electrophoresis and mass spectrometry.
d) Study Design: In specific aim 1, we will test the ability of PRIMA-1 to facilitate apoptosis of adriamycin on breast cancer cells. Mitochondrial involvement as target for p53-produced apoptosis will be investigated. We will also examine the effects of various treatments on normal mammary epithelial cells and select the treatment combination for in vivo experiments that induces maximal apoptosis with minimal toxicity in our mouse model of breast cancer. In specific aim 2, we will identify p53-protein partners following treatment with PRIMA-1 by co-immunoprecipitation and LC/MS/MS-based proteomics analysis. Furthermore, we will characterize protein expression profiling of cytoplasmic and nuclear fractions of normal and tumor cells treated with PRIMA-1 with 2D-gel electrophoresis and Ion exchange chromatography. Our aim is to use various mass spectrometry-based proteomic tools such as MALDI-TOF, quadrupole ion trap, and FTICR-MS with both MALDI and ESI combined with chromatographic separations to maximize our ability to identify functionally relevant proteins for better understanding of p53 function restoration with PRIMA-1.
e) Potential Outcomes and Benefits of the Research: The proposed application is designed to selectively target tumor cells by virtue of their defects in p53 signaling. The proposed experiments are important because they should enhance the likelihood that new, less toxic, apoptosis-based therapies will be developed for the treatment of breast cancer in women. Furthermore, we anticipate that our functional proteomics approach will lead to the identification of protein-based, breast cancer-specific targets.
Genomic Consequences of Restoring p53 Transactivating Function With PRIMA-1 in Breast Cancer
Sometimes called the "guardian of the genome", the tumor suppressor protein p53 responds to DNA damage by either shutting down cell division or causing cells to commit suicide, a process known as apoptosis. Either way, p53's action helps short-circuit tumor formation by preventing cells that have suffered malignant mutations from continuing to grow. Yet the p53 gene itself is susceptible to damage, which is thought to contribute to the majority of all human cancers including more than 50% of breast cancer. Tumor cells defective in p53 gene tend also to be resistant to the effect of chemo- and radiotherapy. Therefore, considerable research effort has centered on how to restore the normal function to mutant p53 protein to halt cell division and to trigger cell suicide (apoptosis) mechanisms. PRIMA-1 (p53-reactivation and induction of massive apoptosis) emerged from an in vitro screen for drugs that may be able to restore the normal function of some mutated p53 proteins. Recently we showed that PRIMA-1 restored the p53 function in breast cancer cells through transcriptional transactivation of p53-target genes such as p21 and PUMA (p53 up-regulated modulator of apoptosis). Therefore, we propose to use PRIMA-1 as an adjuvant therapy in combination with clinically useful anticancer drugs such as adriamycin for the treatment of breast cancer, because it (a) restores p53 functional activity in breast cancer cells, (b) has anticancer activity in its own right, and (c) is not involved (small molecule) in drug resistance similar to adriamycin. Our preliminary data provided in this application showed the existence of marked synergism between PRIMA-1 and adriamycin on breast cancer cells, especially when adriamycin is given first followed by PRIMA-1. These observations indicate that combined treatment of adriamycin + PRIMA-1 may achieve better therapeutic outcome than the use of each drug individually.
In specific aim 1, we will test the ability of PRIMA-1 to facilitate the induction of apoptosis in breast cancer cells with mutant p53 when treated in vitro with adriamycin, a clinically approved drug for the treatment of primary and metastatic breast cancer. Tumor cells mutated for p53 tend to be resistant to this drug. We will examine the ability of the treatment regimen to inhibit growth, induce apoptosis and increase survival of human breast tumors inoculated into immunocomparimised mice that will not reject human breast tumors. If successful, these experiments will provide a valuable model in which to investigate the optimal dose and schedule for drug administration. In turn, this approach will provide critical information for the subsequent application to human clinical trials.
In specific aim 2, we will identify partner proteins that tend to interact with p53 as a function of restored confirmation with PRIMA-1. To halt cell division or trigger apoptosis mechanisms, p53 protein needs to regulate the activity of many other proteins (protein-protein interactions) that requires its binding first to the DNA of the target gene regulatory sequences. To identify how precisely PRIMA-1 modulates this protein-protein interactions, we will use an approach called Functional Proteomics. It consists of selective purification and enrichement of a bait protein (p53) and its interactors from cell lysate, protein separation by gel electrophoresis, excision of protein spots and in-gel protein digestion followed by mass spectrometric analysis for protein identification and characterization. We used this approach and successfully identified the heat shock protein-90 (HSP90) as one of p53-partner proteins that highly expressed upon PRIMA-1 treatment. Our further investigation of this type of protein-protein interactions will identify additional proteins that show altered levels of interaction with p53 upon PRIMA-1 treatment. This approach will not only reveal how potential anticancer agents such as PRIMA-1 affect p53-protein interactions, but more importantly, how p53 mutation can modulate p53 function. Thus, our proteomics approach could potentially provide oppotunities for rational drug discovery and individualization of therapy for breast cancer patients through the identification of protein-based, breast cancer-specific targets.