Studies on the Potential Antitumor Mechanisms of Hyaluronan Synthase Involved in Early Onset Breast Tumor Tissues

Social impact of this research project: Early-onset breast cancer (BC): The average age of occurrence for BC in Taiwan is approximately 30~50 years old, which is earlier than that in the western world. Hyaluronan (HA) synthase: HA refers to a group of high MW glycosaminoglycan polymers which are produced by three different types of HA synthase (HAS1-3). HAS3 has been demonstrated to be an important ligand for CD44, which is an important cancer stemness marker in BC. It is suggested that HAS3 would be an attracting factor for malignant cancer cells and cause them to move into the stroma. Preliminary results: 1. We found a higher expression level of HAS3 in adjacent normal areas (N>T, n=500) compared to tumor tissues. 2. The HAS3 protein was detected at a lower level in tumor tissue (*p<0.001), especially in younger BC patients. 3. Early-onset BC was detected in the HAS3 KO mice. Rational: We propose that HA is synthesized by HAS3 in the stroma of normal mammary ducts, which promotes an antitumor microenvironment to prevent the metastasis of malignant BC. The fact that HAS3 was detected at very low levels in the adjacent normal region in younger BC patients supports this hypothesis. Year-1: The clinical relevance of HAS3 in early-onset BC tissues Aim 1: Determination of HAS3 levels in early-onset BC and its clinical relevance Detection of HAS3 mRNA and protein levels in a biomaterial bank and tissue array. Determination of HAS3 gene expression and its clinical relevance: statistical analysis of early-onset BC. Aim 2: Study of the tumor-suppressive role of HAS3 using gene KO or transgenic mice Establish over-expressing (Tet-on) and down-regulated (siRNA) cell lines to investigate the role of HAS3. Establish knock-out (KO) animals to investigate the tumor-suppressive role of HAS3. Aim 3: Use of in vitro cells models to investigate the role of HAS3-induced tumor suppressive effects Investigate how HAS3 is involved in the Taxol-induced therapeutic effect. Investigate the role HAS3 plays in the cell growth cycle and in differentiation. Year-2: In vitro study to investigate the role of HAS3 in early-onset BC Aim 1: Study the role of HAS3 in adjacent normal tissues. Establish the characteristics of adjacent normal stromal cells to study the role of HAS3 in earlyonset BC tissues Conduct tumor/adjacent cell culture experiments to study the role of HAS3-mediated anti-tumor effects. Aim 2: Stroma cells from early- vs. late-onset BC patients to validate the HAS3-mediated effects Stroma cells from early- vs. late-onset patients: Analyze the level of HA secretion in BC-adjacent normal tissues. Stroma cells from early- vs. late-onset BC co-cultured with BC cells: Measure HA-induced tumor suppression. Year-3: MMTV++/HAS3-- mice to investigate the effects of HAS3 in early-onset BC patients Aim 1: Establish MMTV++/HAS3-- mice MMTV++ mice can be easily used to generate orthotopic BC. This will be useful for the evaluation of the HAS3-induced tumor-suppressive effects in early-onset BC in mice. Determine the orthotopic BC occurrence rates and age in MMTV++/HAS3-- mice. Aim 2: MMTV++/HAS3-- mice as a research model for early-onset BC Use MMTV++/HAS3-- mice as a drug screening model. Use MMTV++/HAS3-- mice as an early-onset BC model to obtain experimental samples. Study the environmental factors (such as smoking, hormone levels) involved in the formation of early-onset BC. Aim 3: MMTV++/HAS3-- mice as a research model for the genomic study of early-onset BC Conduct an epigenetic study in early-onset BC from MMTV++/HAS3--vs.MMTV++ mice. Conduct a transcriptome study in early-onset BC from MMTV++/HAS3-- vs. MMTV++ mice. Conduct a proteomics study in early-onset BC from MMTV++/HAS3-- vs. MMTV++ mice.