SDHA isoform expression modulates reverse electron flow during hypoxia

Succinate dehydrogenase (SDH, also known as complex II) is a mitochondrial enzyme complex that directly connects the TCA cycle to the electron transport chain (ETC) via reductions of succinate to fumarate, leading to the reduction of coenzyme Q to high energy UQH2. The SDH complex is composed of four different subunits. SDHA and SDHB are the enzymatic subunits of SDH that forms the mitochondrial matrix-facing hydrophilic head whereas SDHC and SDHD are the hydrophobic mitochondrial inner membrane anchor subunits. When oxygen is present, electrons flow through the ETC, generating ATP, with oxygen serving as the terminal electron acceptor. However, hypoxic niches exist during a variety of physiological states including cancer where fumarate acts as an additional electron acceptor to regenerate CoQ for additional biological processes including pyrimidine biosynthesis. When SDH is utilized in reverse to recapture electrons onto fumarate, succinate is regenerated, leading to succinate accumulation in cancer cells in hypoxic conditions. It has been previously discovered that some tissues favor forward SDH activity (heart, lung) whereas other tissues (brain, kidney) favor the reverse flow. We hypothesized that different isoforms of SDHA, which contains the active site for the succinate to fumarate enzymatic reaction, could favor or disfavor forward and reverse flow of electrons through SDH and serve as a mechanistic basis for the previously observed experimental findings. We analyzed human tissue datasets and identified 3 major SDHA isoforms. We found tissue-specific differences in SDHA isoform expression with high expression of SDHA isoforms in kidney tissue, and low expression of SDHA isoforms in heart tissue, consistent with our hypothesis. We overexpressed SDHA isoforms in cancer cell lines and analyzed 13C15N-glutamine tracing to provide experimental support of our hypothesis that specific SDHA isoforms prioritize reverse electron flow under hypoxic conditions. We are currently assessing how CRISPR-Cas9 engineered cells to expression only one SDHA isoform (rather than combination of wild-type and variant transcript) affect cell responses to cancer therapy under normal and hypoxic conditions including epigenetic regulation of gene expression via the fumarate and succinate oncometabolites. This study will help us in identifying how SDHA isoforms contribute to cancer initiation and treatment strategies for hypoxic tumors.