Novel Roles for Manganese Superoxide Dismutase Polymorphisms and Epithelial Mesenchymal Transition in Prostate Cancer

Epithelial Mesenchymal Transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration. Reactive Oxygen Species (ROS), chemically reactive molecules of cellular metabolism, induce oxidative stress; increasing SNAI1 (Snail) transcription factor and promoting EMT. In Phase II clinical trials, prostate cancer patients with SOD2 Ala/Ala single nucleotide polymorphism (SNP) who were treated with natural product, Muscadine Grape Skin Extract (MSKE), responded better than those with Val/Val SNP. We hypothesize that increased SOD2 activity of Ala/Ala SNP is associated with enhanced EMT, potentially antagonized by MSKE in prostate cancer cells. Site-directed mutagenesis was conducted, creating Ala/Ala and Val/Val polymorphisms which were transiently and stably transfected into LNCaP prostate cancer cells. Baseline intracellular ROS and EMT marker expression were evaluated via ROS and immunoblot assays. Cell proliferation was measured using MTS assay. Prostate cancer cell line pyrosequencing determined that most prostate cancer cell lines were heterozygous for SOD2 (Ala/Val) SNPs, although several had extra copies, suggesting multiple copy numbers. Only metastatic MDA PCa-2a and -2b contained Ala/Ala homozygous SNPs. LNCaP cells stably transfected with SOD2 Ala/Ala were associated with higher SOD2 expression and more induction of EMT, indicated by higher levels of Snail and Vimentin compared to those overexpressing SOD2 Val/Val or Neo control. LNCaP Val/Val cells displayed high H₂O₂ levels and additional treatment with H₂O₂ decreased cell proliferation, more in comparison to Ala/Ala. However, LNCaP Ala/Ala cells contained higher Total ROS and Superoxide, and were more sensitive to co-treatment with H₂O₂ + MSKE. We believe that there is differential response to MSKE and Ala/Ala SNP may respond better due to MSKE targeting Superoxide. These studies may uncover differential biological behaviors of different SOD2 SNPs and how this may explain differential patient response to MSKE.

Acknowledgements: NIH/NIMHD/RCI Grant 5G12MD007590-31, NIH/NIGMS/RISE Grant 5R25GM060414, https://www.thecommunityfoundation.org/