Development of Novel Therapeutic Agents for Herceptin Resistance Breast Cancer Cells through Targeting Nicotinic Acetylcholine Receptors

Our recent study (J Natl Cancer Inst. 2010, 102, 1322-1335) demonstrated that the a9-nicotinic receptor (a9-nAChR) was detected at a higher level in breast tumor tissues (tumor/normal >8 fold, n=276). The result showed that higher levels of a9-nAChR were detected in advanced (stages 3-4) tumor and HER-2 overexpressed tissues. The recent preliminary data using Immuno-Precipitation (IP), Forster Resonance Energy Transfer (FRET) and Split luciferase illustrated that an a9-nAChR/HER-2 complex can be formed in breast cancer cells and this might correlate to Herceptin-induced drug resistance. Such results motivated us to study the potential a9-nAChR antagonists and a specific antibody targeting at a9-nAChR/HER-2 complex formation in Herceptin resistant tumor cells, which could potentially be used to overcome drug resistance in Herceptin-treated patients. Our five-year proposal will be completed in accordance to the following aims: Aim 1: Investigation of a9-nAChR/HER-2 complex formation in drug resistance breast cancers In order to clarify the a9-nAChR/HER-2 complex formation which mediates Herceptin resistant breast cancers, we will first correlate the a9-nAChR expressions and breast cancer malignancy in 20 breast cancer cell lines. Secondly, we will observe the a9-nAChR/HER-2 complex formation in nicotine exposure under live cell of FRET and FLIM microscopy. Thirdly, we will use Split luciferase in xenografted-tumor to study a9-nAChR/HER-2 complex formation under IVIS Imaging System. Fourthly, we have been cooperating with Prof. Jinn-Moon Yang (National Chiao Tung University / Institute of bioinformatics and systems biology) to pinpoint the specific domain of the a9-nAChR that interacts with HER-2 for novel Herceptin resistant breast cancer therapy. Aim 2: Investigation of how a9-nAChR point mutation mediates breast cancer malignancy The Nature journal has published an article showing that the a9-nAChR mutation caused high potency of breast cancer metastasis. We will use iPLEX and LCM techniques to analyze the a9-nAChR mutation with breast cancer metastasis and HER2 expression status in clinical breast cancers. We have been cooperating with Prof. Gen-Yun Zhang (BGI Company) to detect a9-nAChR mutation in blood stream for metastasis breast cancer cells by One Cell Sequencing. Aim 3: Investigation of how a9-nAChR phosphorylation mediates drug resistance breast cancers First, we will investigate if potential phosphorylation sites (Y421、Y430) of a9-nAChR could be activated by nicotine and NNK exposure and to regulate downstream signals (AKT and ERK) in breast cancers. Second, we will analyze the mechanism of a9-nAChR phosphorylation in mediating a9-nAChR/HER-2 complex formation. Third, the model of a9-nAChR phosphorylation in breast cancers may be applied to a7-nAChR in lung cancers. Aim 4: Investigation of how the mechanism and functional regulation of a9-nAChR could mediate breast cancer stem cells First, we will develop side population and CD44+/CD24- systems for breast cancer stem cells (CSCs) detection. Second, we will analyze the correlation of a9-nAChR expression and breast cancer stem cell formation. Third, we will inspect the influences of a9-nAChR antagonists and specific antibody treatments on the mediation of CSCs Herceptin resistance breast cancers. Finally, we will use iPS technique to investigate the mechanism of a9-nAChR expression and how CSCs mediates Herceptin resistance in breast tumors. Aim 5: Investigation of cell and animal models can be applied for studying the mechanisms of a9-nAChR in mediating Herceptin resistance To establish an experimental animal platform to study the dynamic mechanisms of a9-nAChR/HER-2 complex formation, the a9-nAChR and HER-2 genes will be constructed into fluorescence protein and split luciferase plasmids. These cells will be established and transplanted into nude mice as a screening tool to evaluate the a9-nAChR /HER-2 complex formation in vivo. We will also search for agents (RglA and antibody) that might interfere with a9-nAChR /HER-2 complex formation, thereby attenuating Herceptin-resistance. Aim 6: Investigation of the biological impact of a9-nAChR during development Previous studies found multiple functions of a9-nAChR expression in various organs, such as hearing in inner ear, blood cell attachment and oncogenic ability in breast cells. First, we will develop conditional a9-nAChR gene knock-in mice to evaluate the pathologic changes in dimensions of horizontal axis (organs) and vertical axis (development). Second, we will cooperate with BGI company use data mining for construction the whole atlas of a9-nAChR expression in every organ of distinct human races. We hope to draw out the a9-nAChR expression during development for drug design and unexpected applications.