The effect of chronic mild hypoxia on genomic instability in HER2-overexpression breast cancer cell line SK-BR-3

Introduction Genomic instability (GIS) is a major tumorigenesis driving factor. Both - amplification of ERBB2 in breast cancer, and acute or cycling tumour hypoxia have been shown to result in increased GIS. Data concerning effects of chronic and mild hypoxia on GIS in cancer cells are conflicting and scarce. Therefore the aim of this study was to explore effects of chronic mild hypoxia (cmH) on GIS in HER2-positive breast cancer cell line SK-BR-3. Material and methods State of GIS was characterised by proportion of micronuclei containing cells (immunostaining with anti-tubulin and DAPI), chromosomal breakpoints, copy number alterations (Illumina CytoSNP12 v2-1 Bead Chip), expression of genome stability related genes (qPCR); relative telomere length (RTL) (qPCR) after prolonged cultivation of SK-BR-3 (three passages) in hypoxia (2% O2) or normoxia (control). Results and discussions Prolonged exposure to cmH resulted in significant 3.3-fold increase of micronuclei containing cells (hypoxia vs normoxia: 25.38 and 5.86 micronuclei per 1000 cells). Initial adaptation to cmH manifested as contraction of genome and decrease of chromosomal breakpoints (normoxia vs hypoxia: DNA index 2.06 and 1.90; breakpoint count: 4573 and 2678). Further cultivation in cmH resulted in additional reduction of DNA index (1.89) and increase in number of breakpoints (3037). CmH increased expression of ATM dependent DDR genes, significantly decreased expression of dsDNA-break reparation genes (H2AFX, BRCA1, FANCD2) and had no significant effect on aneuploidy-related gene expression. Initial exposure to cmH resulted in major increase of RTL (from 1.1 to 7.8), but further culture in hypoxia showed gradual decrease of RTL (down to 1.3). Low expression levels of telomerase (TERT) through all passages did not change significantly. Conclusion Initial adaptation to hypoxia was characterised by increased RTL, contraction of genome and decrease of genomic heterogeneity. Initial selection of hypoxia-fit SK-BR-3 subpopulations was followed by increased formation of chromosomal rearrangements. CmH in SK-BR-3 activated ATMCHEK2 branch of DDR and decreased expression of genes involved in reparation of dsDNA breaks. Low expression levels of TERT through all passages did not change significantly and did not correlate with the RTL.