Non-Canonical Role of Singleminded 2 in Energy Metabolism

Abstract

Dysregulation of cellular metabolism is a defining hallmark of breast cancer progression and is associated with metastasis and therapeutic resistance. The mechanisms by which cells undergo the switch from respiration to glycolysis and the subsequent impact on cancer progression, however, are not fully understood. We show the short splice variant of the bHLH/PAS transcription factor Singleminded 2 (SIM2s) promotes oxidative phosphorylation (OXPHOS) using breast cancer cell line models. Mechanistically, we found that SIM2s functions in a non-canonical manner directly interacting with the mitochondrial respiratory chain (MRC) to facilitate supercomplex (SC) formation. Using breast cancer cell lines with gain and loss of SIM2s, we discovered SIM2s is required for and promotes mitochondrial respiration and metabolic homeostasis. MRC complex expression was inconsistent in gain and loss of SIM2s cells. We then examined the formation of MRC complexes and SCs via blue-native polyacrylamide electrophoresis (BN-PAGE) and discovered SC formation was impaired with loss of SIM2s and stabilized with overexpression of SIM2s. Interestingly, SIM2s was shown to be localized to the mitochondria as well as the nucleus. Immunoblotting for SIM2 after BNPAGE revealed an association with SCs. Mass spectrophotometry analysis indicated an interaction between SIM2 and NADH dehydrogenase 1 beta subcomplex subunit 10 (NDUFB10), which was confirmed by co-immunoprecipitation and proximity ligation assays. Metabolomic analysis showed that knock-out of SIM2 led to compensatory increases in ATP production through glycolysis and increased glutamine anaplerosis to produce NAD+ as well, creating a favorable environment for high proliferation. To determine if SIM2 promoted OXPHOS universally, we used a Sim2+/- mouse model and a skeletal muscle differentiation model. We determined Sim2+/- mice had impaired fatty acid metabolism and dysfunctional skeletal muscle mitochondria. In vitro loss of Sim2 impaired skeletal muscle differentiation through dysregulated mitochondrial turnover. Moreover, Sim2 promoted OXPHOS in differentiated myofibrils. Our findings indicate that SIM2s is a novel stabilizing factor required for SC assembly, providing insight into the impact of the MRC in metabolic adaptation and breast cancer progression. Interrogating these novel mechanisms by which cells undergo metabolic transitions will provide crucial therapeutic targets to treat a variety of cancers and other pathologies.