Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations in mismatch repair genes in methotrexate-resistant HT-29 cells

Ahreum Kim,Jong-Yeon Shin,Jeong-Sun Seo

doi:10.1038/s12276-021-00668-x

Abstract

DHFR gene amplification is commonly present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. In this study, we proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real-time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). We identified an amplification unit spanning 11 genes, from the DHFR gene to the ATP6AP1L gene position, with high adjusted interaction frequencies on chromosome 5 (~2.2 Mbp) and a twenty-fold tandemly amplified region, and novel inversions at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These mutations might stimulate chromosomal breakage and cause the dysregulation of mismatch repair. Characterizing the tandem gene-amplified unit may be critical for identifying the mechanisms that trigger genomic rearrangements. These findings may provide new insight into the mechanisms underlying the amplification process and the evolution of drug resistance.

Highlights

Gene amplification, the triggering of an abnormal copy number increase in a specific region of the genome of cells growing under a selective condition, is associated with the overexpression of oncogenes such as MYC, MYCN, and ERBB, which engender abnormal cell proliferation and replication[1,2,3]
Analysis of MTX-resistant HT-29 cells While generating MTX-resistant HT-29 cells from single-cell selection and MTX sensitization as previously described[30], dramatic morphological changes in the cells themselves were observed; rounded and circular cell shapes were observed during the first cycle of sensitization, and rod and irregular shapes were seen during the second cycle (Supplementary Fig. 2)
The original shapes were again observed during the third cycle of sensitization, which might indicate that HT-29 cells became resistant to MTX and grew rapidly under high MTX concentrations

Summary

Introduction

The triggering of an abnormal copy number increase in a specific region of the genome of cells growing under a selective condition, is associated with the overexpression of oncogenes such as MYC, MYCN, and ERBB, which engender abnormal cell proliferation and replication[1,2,3]. Gene amplification is an indicator of a drug-resistant sample in cancer and healthy cells[8], so it will be important to identify the genetic features or pathways that promote amplification in tumors These mechanisms might serve as therapeutic targets that can prevent drug resistance and arrest or eradicate tumor cells[9].

Methods

Results

Conclusion