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    Research Grants Awarded

    Investigation Of Oncogenic Properties And Therapeutic Application Of Pik3Ca Mutations In Breast Cancer

    Grant Mechanism:
    Career Catalyst Research

    Scientific Abstract:
    Title of Project: Investigation of oncogenic properties and the therapeutic application of PIK3CA mutations in breast cancer Principal Investigator: Guojun Wu Ph.D Background: PIK3CA, a phosphatidylinositol 3-protein kinase, is an essential component in the EGFR/PI3K/AKT signaling pathway. Our group, as well as several others, found a high frequency of somatic mutation of the PIK3CA in breast cancer. Moreover, mutant PIK3CAs, harboring different levels of kinase activity, contribute to cell proliferation, and can activate the downstream signaling pathway. Inhibition of PIK3CA can significantly induce apoptosis and inhibit the cell growth rate in cells with somatic mutation of PIK3CA. This information makes the PIK3CA a promising therapeutic target for breast cancer treatment. So far, the oncogenic properties of mutant PIK3CAs have not been confirmed in vivo and very few studies have been performed with the aim of targeting mutant PIK3CAs. On the other hand, a protesome inhibitor disulfiram (DSF) has been shown to induce breast cancer cell apoptosis in vitro and inhibit tumorigenesis in vivo. We recently found that DSF treatment could lead to activation of pAKT and degradation of PTEN which in turn will lead to cell more dependent on AKT signaling pathway. These findings triggered us to propose a combinational treatment of breast cancer cells with mutant PIK3CAs using a protesome inhibitor and the PI3K inhibitor. Hypothesis: We hypothesize that 1) PIK3CA mutations have oncogenic properties in vivo. 2) A proteasome inhibitor (Disulfiram-copper complex) can synergize with the PI3K inhibitor (LY294002) to target breast cancer cells with mutant PIK3CA in vitro and to inhibit breast tumorigenesis in vivo. Specific Aims: Aim 1) To study the oncogenic properties of PIK3CA mutation in breast tumorigenesis using transgenic mouse, Aim 2) To evaluate the combinational treatment of cultured human breast cancer cells with a proteasome inhibitor (Disulfiram-copper complex) and a PI3K inhibitor (LY294002) in vitro; and Aim 3) To investigate the combinational treatment effects of a proteasome inhibitor (Disulfiram-copper complex) and a PI3K inhibitor (LY294002) in breast tumorigenesis in vivo using xenograft and transgenic mouse model. Study design: For Aim 1, we will test the oncogenic property of the somatic mutation of PIK3CA using transgenic mouse models. We plan to generate about 10 transgenic mice for the mutant and wild type form of PIK3CA. These transgenic mice will be used to evaluate the oncogenic properties of the PIK3CA mutation by comparing them to non- transgenic mice. We will then statistically analyze the latency, tumor loci and tumor size between each group of transgenic mice. For Aim 2, cell viability and apoptosis screen assay will be used to study the effect of a specific proteasome inhibitor (Disulfiram-copper complex) in combination with a PI3K inhibitor (LY294002) in our established model cell lines which are known for their genetic alterations (PIK3CA mutation). The following treatments will be applied: (i) Control vehicle; (ii) the proteasome inhibitor DSF-Cu alone; (iii) PI3K inhibitor alone; (iv) Pre-treatment with PI3K inhibitor, followed by co-treatment with DSF-Cu; (v) The authentic proteasome inhibitor MG132 alone at 1 ?M; (vi) Pre-treatment with 1 ?M MG132, followed by co-treatment with the PI3K inhibitor. Groups (v) and (vi) will be used as positive controls. For Aim3, the identified combinational treatment will then be applied and validated in our xenografted mouse model and transgenic mouse model. The healthy mice bearing tumors will be randomized into test groups of 10 mice each. Four different treatments will be performed as follows: (i) Control vehicle (PBS+ DMSO); (ii) DSF (which will interact with tumor tissue Cu); (iii) PI3K inhibitor (LY294002); (iv) DSF plus PI3K inhibitor LY294002. Statistic analysis will be performed to evaluate the significance. Significances: Our study will provide direct evidence of the oncogenic role of the PIK3CA mutation in vivo and provide a novel strategy to target breast cancer with PIK3CA mutations. Therefore, our project is directly related to the main focus of the breast cancer research field-- the identification of specific drugs to target the important signaling pathways that contribute to cell growth, cell survival and proliferation of breast cancer. The result of this study will have a profound impact on the prevention and therapeutics of breast cancer.

    Lay Abstract:
    Title of Project: Investigation of oncogenic properties and the therapeutic application of PIK3CA mutations in breast cancer Principal Investigator: Guojun Wu Ph.D The PI3K/AKT signaling pathway, a pathway involved in cell growth, cell survival, and cell proliferation, has been firmly established as a critical contributor to tumorigenesis in the last two decades. The components in this pathway are often altered in different kinds of human tumors, making them promising targets for the development of cancer therapeutics. More recently, mutations of PIK3CA, the gene encoding the 110-kDa subunit of PI3K, were identified in different human cancers including colon, brain, gastric, and lung cancers, respectively. The follow-up studies, including ours, indicated that the mutation frequency of the somatic mutation PIK3CA gene was 20-35% in breast cancer. Several groups initiated the investigation of the phenotypic effects of these PIK3CA mutations in various human cancers and only two reports studying the PIK3CA oncogenic properties in breast cancer. These studies focused on the two most common variants, E545K and H1047R, in the MCF10A and HMEC immortalized breast epithelial cell lines. But so far, no in-vivo data directly showed the oncogenic properties of mutant PIK3CA in breast cancer and very few studies have been performed with the aim of targeting mutant PIK3CA as a strategy to cure breast cancer. Recently, our group performed a systematic functional analysis of mutant PIK3CA genes (wild type and mutant) based on the human mammary epithelial cell MCF10A. We found that nine different PIK3CA mutants harbor different abilities to promote cell proliferation and EGF independent growth. In addition, most PIK3CA mutants had the ability to change the morphogenesis of the MCF10A cell in the 3D Matrigel assay. Moreover, different PIK3CA mutants have different abilities to promote colony formation and cell invasion. We further observed that most of the PIK3CA mutants could activate p-AKT and p-p70-S6K in the absence of EGF stimulation. Finally, LY294002, a PI3K inhibitor, can effectively inhibit cell growth in cell lines with different PIK3CAs. Our results suggest exploration of the therapeutic application of these mutations will benefit the breast cancer patients with the PIK3CA mutations. On the other hand, Dr. Q. Ping Dou?s group in Karmanos Cancer Institute, reported that the protesome inhibitor disulfiram (DSF) can induce apoptosis of breast cancer cell in vitro and inhibit tumor growth in vivo. We recently found that DSF treatment will lead to activation of p-AKT and degradation of PTEN in breast cancer cells, which in turn will lead cells more dependent on AKT signaling pathway. In this project, we propose to analyze the oncogenic properties of the PIK3CA somatic mutation in vivo using a transgenic mouse model. Moreover, we will investigate the effect of the combinational use of a proteasome inhibitor (DSF) and a PI3K inhibitor and identify whether a specific proteasome inhibitor can synergize the effect of PI3K inhibition in our established model cell lines using high throughput cell viability and apoptosis screen assay. The combinational treatment effect of the proteasome inhibitor and the PI3K inhibitor will also be validated using the xenograft mouse model and the transgenic mouse model. We aim at finding the combination of the PI3K inhibitor and the proteasome inhibitor which has maximum efficacy and minimum side effects. There is a crucial need for information about a target gene which contributes to breast tumorigenesis and the possibility of using this target to design specific drugs to block breast tumorigenesis. Our proposal focuses on investigating the oncogenic properties of the PIK3CA mutation in vitro and in vivo and the combinational treatment effect of a proteasome inhibitor and a PI3K inhibitor in breast cancer with PIK3CA mutation. Our work specifically addresses the need in the breast cancer research field. The outcome of this research is expected to have an important impact on the practice of breast cancer medicine.