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    Research Grants Awarded

    Targeting Aberrant Wnt Signaling In Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    The Wnt/beta-catenin pathway initiates a signaling cascade critical in both normal development and the initiation and progression of cancer. Although the role of Wnt signaling in breast cancer is far from being fully understood, its importance and significance has been frequently reported in the last 5 years. In human breast cancer, there are many reports of inactivation of negative regulators of the Wnt signaling pathway. Similarly, there are numerous studies that have documented the amplification or overexpression of positive regulators of components of this pathway. In mice, it has long been known that misexpression of Wnt-1, -3 or -10 induces mammary adenocarcinomas. The APCMin has also been shown to exhibit an enhanced incidence (10%) of spontaneous mammary cancer and a greatly increased susceptibility (90%) to carcinogen-induced mammary cancer. Additionally, there is now clear evidence that crosstalk exists between the Wnt pathway, the estrogen receptor (ER) and epidermal growth factor receptor (EGFR/Her) signaling pathways - pathways where aberrant regulation is often associated with breast cancer. Studies have shown that ectopic expression of the Wnt-1 proto-oncogene is sufficient to induce mammary hyperplasia and tumorigenesis in both female and male ER-alpha knockout mice, although requiring a longer time to develop than in a wild-type background. Wnt-1 has been found to be up regulated by estradiol in MCF-7 cells, which in part explains the results in the ER-alpha knockout. Recently, Katzenellenbogen and colleagues showed that upon treatment of MCF7 cells with estrogen, there was a significant increase in survivin message. This data implies that estrogen signaling can affect Wnt/TCF/beta-catenin transcription as well as cancer development and progression. Additionally, Kouzmenko et al. have demonstrated that Wnt/beta-catenin signaling and estrogen signaling converge in vivo. It has also been shown that the ErbB signaling pathway cooperates with the Wnt signaling pathway to promote the induction of mammary tumors through the unvarying association between beta-catenin and the epidermal growth factor receptor/c-Neu (ErbB1/ErbB2 heterodimer) tyrosine kinase. We have recently developed a small molecule inhibitor that specifically inhibits the TCF/CBP/beta-catenin interaction, but not the TCF/p300/beta-catenin interaction. Despite their high degree of homology and similar patterns of expression, CBP and p300 play unique and distinct roles in gene regulation. Our chemogenomic studies with ICG-001 present strong evidence that CBP and p300 have distinct functions in regulating the transcription of Wnt/beta-catenin genes including, survivin, EphB2, S100A4, connexin 43 and cyclin D1. We have developed a model, which proposes that TCF/beta-catenin/CBP-mediated transcription is critical for proliferation without differentiation (e.g. in cancer and stem cells). However, the switch of coactivators by the TCF/beta-catenin complex is the essential first step in the initiation of differentiation. The TCF/beta-catenin/p300 complex then drives the transcription of the Wnt/beta-catenin regulated genes associated with normal cellular differentiation. Aberrant regulation of the balance between these two related transcriptional programs may be associated with a wide array of diseases including cancer. Survivin belongs to the Inhibitor of Apoptosis (IAP) gene family that counteracts cell death and controls mitotic progression. It is a 16KDa protein that is present during embryonic development, but is virtually undetectable in terminally differentiated adult tissues and not required for normal cell viability. Overexpression of survivin has been reported in many types of cancers, including breast cancer. Expression of the survivin gene is associated with poor prognosis in most human cancers. In breast cancer, replication-deficient adenoviral targeting of survivin has been shown to be as effective in tumor reduction as Taxol, and more effective than Adriamycin, in 3 xenograft breast cancer models in nude mice. These data suggest that targeting survivin gene expression is a valid therapeutic approach for breast cancer. We recently demonstrated utilizing both pharmacologic inhibition with the CBP/beta-catenin specific inhibitor ICG-001 and also with CBP and p300 specific siRNA that survivin transcription via Wnt/beta-catenin signaling is critically dependent on the coactivator CBP in a variety of cancer cell types including breast cancer. Furthermore, utilizing chromatin immunoprecipitation assay (ChIP), we demonstrated that coactivator switching from CBP to p300 at the survivin promoter is associated with the recruitment of transcriptionally repressive elements to the survivin promoter. Our central hypothesis is that increased beta-catenin/CBP-mediated transcription at the expense of beta-catenin/p300-mediated transcription is associated with breast cancer development and metastasis. We propose that disrupting the CBP/beta-catenin interaction and inhibiting the transcription of the survivin, S100A4 and other Wnt/CBP/beta-catenin-dependent genes, will lead to the ability to selectively induce apoptosis in breast cancer cells and block tumor metastasis but NOT affect normal breast epithelium. The results of these studies should enhance our understanding of the role of beta-catenin/CBP transcription in general, and survivin gene expression in particular, and their applications to therapeutic strategies for breast cancer.

    Lay Abstract:
    Breast cancer is the major cause of cancer death in women. A major challenge of breast cancer research is the development of novel therapeutic strategies. In many respects, breast cancer is the result of normal cellular events being misregulated, for example (a) uncontrolled cell proliferation, (b) abnormal resistance to cell death, and (c) abnormal development of new blood vessels. Aberrant regulation of multiple signaling pathways has been shown to be associated with breast cancer. The most successful therapies developed to date, inhibit upstream targets in these pathways; for example, Tamoxifen targets the estrogen receptor (ER), and Herceptin targets the receptor tyrosine kinase HER-2/neu. These target proteins are critical to the survival of only a subset of breast cancers. Often, breast cancer cells become resistant to these therapies. Other existing therapies predominantly target proliferation, either with cytotoxic agents, or ionizing radiation, both of which are toxic to normal cells as well. Apart from cytotoxicity to normal cells, many cancer cells, including breast carcinomas, develop broad resistance to these cytotoxic agents as well as chemotherapy and ionizing radiation due to loss of gene expression (e.g. loss of p53), target protein overexpression, multi-drug resistance pumps and increased expression of the anti-apoptotic gene survivin. There is a significant body of data to suggest that targeting survivin gene expression is a valid therapeutic approach for breast cancer. We recently demonstrated that survivin transcription via Wnt/beta-catenin signaling is critically dependent on the coactivator CBP in breast cancer cells. Furthermore, the CBP/beta-catenin specific inhibitor, which we have recently developed ICG-001, inhibits the expression of survivin in both estrogen responsive and non-responsive breast cancers. Despite the clear need for drugs that attenuate the nuclear functions of beta-catenin, to date there is no available therapeutic agent that specifically targets the Wnt/beta-catenin pathway. We have preliminary data, both in vitro and in vivo that ICG-001 is effective in selectively targeting cancerous breast epithelial cells without damaging normal breast tissue or causing systemic toxicity ICG-001 is a small-molecule (MW 548) compound that readily crosses membranes and is stable under physiological conditions. The synthesis of ICG-001 is straightforward requiring only 4 steps and can be readily scaled up. The molecular target and mechanism of action of ICG-001 has been determined and validated. Preliminary data both in vitro and in vivo suggest that ICG-001 is the first pathway specific antagonist of Wnt/beta-catenin signaling. ICG-001 because of its unique mechanism of action is synergistic with a large number of existing chemotherapeutic agents. Furthermore, ICG-001 does demonstrate some degree of oral bioavailability. Importantly, ICG-001 does not adversely affect non-transformed cells either in vitro or in vivo. At dose levels greater than 10 times higher than required to significantly reduce the expression of the direct CBP/beta-catenin target survivin in vivo (mice) there are no detrimental effects even upon prolonged (3 week) treatment. These combination of properties that ICG-001 possesses: readily synthesized orally available small molecule, with a well defined and novel mechanism of action that possesses extremely limited toxicity and has synergistic effects with a large variety of existing chemotherapeutics, augurs well for the ability of specific Wnt/CBP/beta-catenin antagonists like ICG-001 to improve the clinical outcome for advanced and metastatic breast cancers.