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Engineering Targeted Chromosomal Amplifications in Human Breast Epithelial Cells

2015-06-23 00:01:35

Breast Cancer Research and Treatment; 23 June 2015: DOI:10.1007/s10549-015-3468-2



Simeon Springer, Kyung H. Yi, Jeenah Park, Anandita Rajpurohit, Amanda J. Price and Josh Lauring



Abstract



Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.



INTRODUCTION



Chromosomal amplifications are a major type of genetic aberration found in human cancers of many histologies. The importance of chromosomal amplifications to tumor biology is exemplified by the ERBB2/HER-2 amplification in 15–20 % of breast cancers. Identification of this amplification was initially shown to be a poor prognostic feature. Subsequently molecular genetic analysis identified the HER-2 gene as a major oncogenic driver in the amplicon, which has been subsequently targeted for therapy with great success in the clinic. Chromosomal amplification of the dihydrofolate reductase (DHFR) gene was initially described as a mechanism of resistance to the chemotherapeutic drug methotrexate, and there are recent examples of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors for lung cancer via MET amplification. Thus, chromosomal amplification plays a key role in cancer origination and therapeutic response.



Much of our knowledge of chromosomal amplification derives from studies in model organisms such as yeast or from experimental amplification of the endogenous DHFR locus. Stepwise increases in methotrexate concentration in culture can lead to amplification of the endogenous DHFR locus or a DHFR-linked transgene in rodent and some human cell lines. Random insertion of a mutant DHFR transgene in HCT-116 + chr3 colon cancer cells was used with methotrexate selection to amplify insertion sites and surrounding genomic loci.



Some recurrent amplifications in human cancers occur near the so-called DNA fragile sites, which are prone to spontaneous DNA breakage under conditions of replication stress, but the mechanisms underlying recurrent amplification at many loci remain poorly understood. We hypothesize that there are likely locus and cell-type-specific differences in terms of susceptibility to chromosomal amplification and that it would be useful to be able to study amplification of a greater variety of loci beyond DHFR. While genetic engineering has enabled modeling of specific mutations, deletions, and chromosomal translocations found in human cancer, we are not aware of a method for engineering specific, rather than random, chromosomal amplification events. We have developed such a method and as a proof of principle have engineered a recurrent amplification found in human breast cancers in a human breast cancer cell line.



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Key Words



Chromosomal amplifications | Gene targeting | ZNF703 | Breast cancer | 8p11-12