BSCI-06. Phage display biopanning identifies Amot regulating cell motility in brain metastasis-initiating cells

Abstract

ObjectiveMetastatic brain tumors (MBTs) are the most common type of malignant brain tumors. Due to the deviation of MBTs from the parental tumors, the effective therapies for primary tumors often are not working in brain metastases. Even more new intracranial lesions were developed though the primary lesion was controlled. The occurrence of brain metastasis-initiating cells (BMICs) suggested the possibility of its spread intracranially. Here we aimed to explore the biological behavior in cell motility of BMICs and understand the potential mechanisms.MethodsIn vitro and in vivo phage display biopanning strategies were used to isolate dodecapeptides that specifically target BMICs by selecting against primary lung cancer cells and normal brain cells. In silico analysis was used to derive specific protein targets in BMICs. Potential targets were narrowed down through analysis in patient databases and verified for their presence in BMIC through RT-PCR. Cell migration and adhesion in BMICs were analyzed using Transwell, scratch, and adhesion assays. Protein expression and cell morphology were detected by immunofluorescence. Immune blot was performed to detect the epithelial-mesenchymal related molecules and explore protein-protein interactions.ResultsIn silico analysis of BMICs specific peptides revealed Angiomotin (Amot) as a potential target in BMICs. Amot was found to be overexpressed in BMICs compared to primary lung cancer cells. Kaplan-Meier analysis demonstrated Amot was negatively correlated with overall survival among lung adenocarcinoma patients. Knockdown of AMOT in BMICs decreased the capability of cell migration and adhesion, through the downregulation of E-Cadherin. Amot was found to maintain the E-Cadherin in BMICs through reducing ubiquitination of E-Cadherin. Furthermore, the knockdown of E-Cadherin decreased cell migration and adhesion due to the decrease in cdc42 activity.ConclusionsAmot plays a role in promoting migration and adhesion in BMICs through preservation of E-Cadherin.