Epigenetically Regulated DNA Rereplication as a Precursor to Inherited Gene Amplification

Abstract

Genome‐wide analysis of copy number variation (CNV) in cancers has identified chromosomal regions with higher frequencies of gain or amplification. However, we do not understand the origins of many of these CNVs or whether it is possible to prevent their development. We previously demonstrated that epigenetic enzymes can regulate CNVs during S phase by inducing Transient Site‐specific Gains (TSSGs) through DNA rereplication. Regions undergoing TSSG are frequently amplified in tumors suggesting that TSSG events could precede stable inherited amplifications. These previously undetected and unanticipated transient CNVs alter how we think about tumor heterogeneity and the development of inherited CNV.To understand the origins of environmentally induced gene amplifications, we used the classical gene amplification model of the metallothionein (MT) genes. The MT locu sis amplified in cells selected by exposure to copper, zinc or cadmium. Increased expression and amplification of the MT locus correlates with poor overall survival in breast cancer. However, the mechanisms that lead to MT amplification are poorly understood.Here we describe derivation of cadmium resistant breast cancer cells that exhibit progressive amplification of the MT locus. Surprisingly, early focal, transient amplification of the MT locus (MT TSSG) precedes larger inherited gene amplifications. Using novel sequencing technology for detecting DNA rereplication, we demonstrate that the focal transient MT locus amplifications correlate with DNA rereplication. Importantly, regions of DNA rereplication resemble MT amplifications observed in breast cancer patients.We demonstrate that TSSG of the MT locus is epigenetically regulated and long‐term inhibition of epigenetic silencing pathways allows transition from focal MT TSSG to inherited gene amplifications in the absence of selective pressure from cadmium. Furthermore, we demonstrate that transition from focal transient amplifications to stable inherited MT amplifications requires the homologous recombination pathway. Our results describe a new paradigm for the origin of targeted gene amplifications where epigenetically regulated DNA rereplication precedes stable inherited gene amplification.