1660P - Epigenetic Silencing of Arginino-Succinate Synthase (ASS1) Defines Arginine Depletion Therapy as a Novel Treatment Strategy for Breast Cancer

Abstract

ABSTRACT Background Loss of argininosuccinate synthetase (ASS1) or lyase (ASL) expression, critical for the biosynthesis of arginine in normal tissues, due to methylation-dependent silencing sensitises ovarian or glioblastoma cells to arginine depletion therapy (ADT). Cells not expressing sufficient levels of ASS1 or ASL become auxotrophic for arginine and require exogenous supply. Arginine deiminase (ADI), a novel arginine-degrading enzyme, can eliminate arginine from the circulation and has shown activity in cancers with low ASS1 and/or ASL activity. This study was performed to establish whether ASS1 or ASL are subject to methylation-dependent silencing in breast cancer to validate ADT as a novel therapeutic strategy for breast cancer. Methods Methylation of ASS1 and/or ASL was analysed by pyrosequencing and methylation-specific PCR (MSP) using primer sets validated by bisulphite sequencing. We used a panel of 19 breast cancer cell lines and two independent series of stage I-III primary breast cancers with linked mature clinical outcome that were randomly selected from the Tayside (n = 224) and Cuneo (n = 150) Tissue Banks. Tissue samples were subject to histopathological review to ensure adequate representation of cancer cells. Results Although ASS1 or ASL methylation could not be verified in breast cancer cell lines, ASS1 methylation was evident in a significant proportion of primary or metastatic breast cancer samples. In two independent series, aberrant methylation of ASS1 was detected in 4.5% and 16% of primary breast cancers. The incidence was even higher in metastatic breast cancer, with 47% of resected brain metastases (n = 19) showing methylation of ASS1 and 26% showing additional methylation of ASL, a combination that has shown to be highly sensitive to ADT. ASS1 methylation status as called by MSP agreed with that determined by pyrosequencing in all cases assessed by both methods. No significant association with a specific tumour type or outcome was found. Conclusions A significant proportion of primary and metastatic breast cancers show methylation-dependent transcriptional silencing of ASS1 or ASL and might be candidates for arginine depletion therapy. Disclosure All authors have declared no conflicts of interest.