Association ofSOD3 promoter DNA methylation with its down-regulation in breast carcinomas

ABSTRACT

Superoxide dismutase 3 (SOD3) is a secreted antioxidant enzyme that regulates reactive oxygen species in the microenvironment. It is also a potential tumour suppressor gene that is significantly downregulated in breast cancer. We have previously shown that its mRNA expression is inversely correlated with relapse free survival in breast cancer patients. This study aimed to investigate the correlation of SOD3 promoter DNA methylation with its expression in different molecular subtypes of breast carcinoma. We found that SOD3 expression was significantly reduced in breast carcinoma samples compared to normal tissues with the lowest levels observed in Luminal B subtype. Pyrosequencing analysis showed significant increase in methylation levels in the SOD3 promoter region (−108 and −19 from the TSS) in tumours vs normal tissues (53.6% vs 25.2%). The highest degree of correlation between methylation and SOD3 expression levels was observed in Luminal B subtype (Spearman’s R = −0.540, P < 0.00093). In this subtype, the −78 CpG position is the most significantly methylated site. The Spearman’s coefficient analysis also indicated the most significant correlation of DNA methylation at this site with SOD3 gene expression levels in tumours vs. normal tissues (R = −0.5816, P < 6.9E-12). Moreover, copy number variation analysis of TCGA database revealed that the more aggressive Triple Negative and Her2+ subtypes had higher levels of SOD3 gene deletion. The predominantly down-regulated expression pattern of SOD3 and the various genetic and epigenetic deregulations of its expression suggest that loss of this antioxidant promotes an advantageous tumour-promoting microenvironment in breast cancer.

KEYWORDS:

• SOD3
• extracellular superoxide dismutase
• breast cancer
• DNA methylation
• pyrosequencing

Acknowledgments

We thank Oleg Shats (Assistant Director for Cancer Informatics at UNMC), Dr. David Kelly (Director, Molecular Biology Core Facility, Eppley Cancer Institute, UNMC) for clinical data verification. We also acknowledge the contribution by Dr. Peng Xiao (Director, Bioinformatics and Systems Biology Core, UNMC) for analyzing the Agendia microarray data and providing bioinformatics supports. We thank the Epigenomic Core Facility and the director, Dr. David Klinkebiel at UNMC for performing the pyrosequencing analysis. In addition, we thank Agendia Inc. USA for performing the gene array expression profiling.

Authors’ contributions

BG and MTF analyzed most of the data, assembled results, generated majority of the figures, and wrote the manuscript. DK designed the pyrosequencing primers, performed the pyrosequencing analysis and the TCGA database queries, as well as generated the methylation heatmap figure. MF contributed in identifying the CpG sites for the pyrosequencing studies and generated the figure illustrating these six CpG sites in the SOD3 promoter region. MD, AK, and KC coordinated sample acquisitions and clinical data verification. All authors read and approved the final manuscript.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the TCGA network (https://www.cancer.gov/tcga), Oncomine platform (https://www.oncomine.org/resource/main.html) or from the corresponding author on reasonable request.

Disclosure statement

The authors declare no conflict.

Ethics approval and consent to participate

The Institutional Review Board at the University of Nebraska Medical Center approved the use of anonymized human DNA samples (IRB #030-17-EP).

Additional information

Funding

The Northern Great Plains Personalized Breast Cancer Program (NGPPBCP) was funded by The Leona M. and Harry B. Helmsley Charitable Trust. This work was financially supported by grants from the NIH R0I-CA182086A (Teoh-Fitzgerald). Brandon Griess was supported by the Eppley Institute in Cancer Biology Training Grant (NCI T32CA009476).