Studies on the Mechanisms of Histamine N-Methyltransferase (Hnmt)-Mediated Herceptinr Drug-Resistance in Breast Cancer Cells

Social impact of this research project: • HER2+ Breast cancer (BC): HER2 overexpression in BC patients has been associated with poor response to hormone therapy and chemotherapy. • HER2+ BC targeted therapy: One-third of the BC patients were HER2 positive. Compared with adjuvant chemotherapy alone, the recurrence rate was reduced by 46% after using combined targeted therapy plus chemotherapy. However, this therapy costs 50000 uSd per year. Only about one-third of BC cases that overexpress HER2 demonstrate tumor regression with Herceptin® monotherapy. • Histamine N-methyltransferase (HNMT): HNMT was overexpressed in tumor tissues (n=440, T/N >25-fold). HNMT and HER2 were detected simultaneously (*p=0.001) in the same patients. In vitro studies (FRET and FLIM) also demonstrated that HNMT/HER2 complex formation in BC cells may affect therapeutic efficacy. • HNMT is potentially a predictive biomarker that can help clinicians to more accurately identify responders or nonresponders to improve the therapeutic index for Herceptin® monotherapy. Year-1: Validation of HNMT as a predictive marker for Herceptin-Resistance (HR) in breast cancer patients Aim 1: Determination of the HNMT and HER2 mRNA expression level in BC cells 1. Establishment of the BC bio-bank for determination of HNMT/HER2 expression. 2. Isolation of the HR-and Herceptin sensitive (HS)-tumor cells from the BC tissues for genotyping analysis by laser capture microdissection (LCM). Aim 2: Study the possible mechanisms of HNMT-mediated HR in BC cells 1. Use Luciferase reporter system for determination of transcriptional factors that regulate HER2 expression. 2. Use chip-sequencing analysis for detection of the regulatory regions in the HER2 promoter induced by Herceptin® treatment. 3. Study the action of Herceptin-induced HNMT overexpression and the mechanisms that cause HR. Aim 3: Establish HNMT inhibition (siRNA) and overexpression cell lines to clarify the HR-related signaling pathways Year-2 proposal: Study the mechanisms of HNMT-mediated gene regulation in the HR cells Aim 1: Establish the HR cells for transcriptome sequencing analysis 1. HR cell model establishment. 2. RNA-quantification (or transcriptome) sequencing analysis in HR cells during different Herceptin® training-passages (p1〜p12). 3. Development of the Chip-array diagnosis kit to help clinicians to select responders for Herceptin therapy. Aim 2: Validate whether the HNMT/HER2 complex formation can be used as a predictor for HR 1. Observation of HNMT/HER2 complex formation in live cell models by Split Luciferase, FRET, and FLIM analysis to prove its association with HR. 2. Evaluate HNMT/HER2 complex formation in tumor tissues as a predictor for HR in BC patients. Aim 3: Study the role of HNMT-phosphorylation involved in HR 1. Tissue-array analysis in HR and HS tumor tissues to prove the correlation between HR and the p-STAT3/p-HNMT expression level. 2. Animal experimental model to investigate the changes of p-STAT3/p-HNMT in a time-dependent manner to prove their possible applications in the HR process. Year-3 proposal: Animal studies on the mechanisms of HNMT-mediated gene regulation in the HR cells Aim 1: Establish the HNMT-HR cell-xenografted tumor model 1. Establish the primary cultured cell lines from the HR cell-xenografted tumor tissues during different stages of Herceptin® treatment. 2. Transcriptome sequencing analysis for these primary cultured HR cell lines. 3. Tissue microarray analysis for the HNMT/STAT3 expression in the animal samples. Aim-2: Establish the HNMT-HR cell-xenografted tumor model for drug screening Herceptin-induced the HNMT/HER2 complex can be used as a predictor for clinical applications. 1. To test whether this phenomenon is detectable in the HR primary cultured cells. 2. The HR-cell xenografted tumor animals will be used for FRET or Split Luciferase analysis. Aim-3: HNMT as a predictive biomarker to accurately select responders for Herceptin® Tissue microarray analysis will be performed to determine the following: 1. HNMT/STAT3 phosphorylation, 2. HNMT/HER2 complex formation, and 3. Genomic analysis by transcriptome sequencing to predict the outcomes in HR patients.