Abstract
RATIONALE AND OBJECTIVES: Remarkable intratumor heterogeneity of mitochondrial redox state was found in malignant tumors by optical redox imaging (ORI) of reduced nicotinamide adenine dinucleotide (NADH), oxidized flavoproteins (Fp) containing flavin adenine dinucleotide, and the optical redox ratio (ORR = Fp/(NADH + Fp)), with higher and lower ORR corresponding to more oxidative and more reductive redox status, respectively. Our previous reports suggested that ORR can be a biomarker for cancer aggressiveness or risk of progression. Our goal here is to explore the molecular basis of the ORR's biomarker value for breast cancer by investigating the expression and activity of PGC1α, a master regulator of mitochondrial metabolism and cancer progression. MATERIALS AND METHODS: Intratumor redox subpopulations were isolated from triple-negative breast cancer (TNBC) MDA-MB-231 mouse xenografts and grouped according to high, medium, and low levels of ORI indices (ORR, Fp, or NADH). Gene expression and associated gene networks were obtained by RNA sequencing and bioinformatics analysis, respectively. PGC1α gene expression was validated by RT-PCR. The role of PGC1α in TNBC progression was further investigated by knocking down PGC1α (validated by western blot and RT-qPCR) in MAD-MB-231 cells and measuring the changes in ORI indices and invasiveness in vitro. RESULTS: PGC1α was upregulated in the subpopulation with a high ORR compared to that with a medium ORR. A PGC1α associated gene network with 21 differentially expressed genes (DEGs) was also identified, implicating regulation of redox signaling, metabolism, and cancer progression. Important signaling regulating genes SIRT1 and FOXO1 were upregulated, whose activities influence the NAD(+)/NADH ratio or are influenced by the NAD(+)/NADH ratio. Decreased ORR and invasiveness were observed in vitro in PGC1α knockdown MDA-MB-231 cells, supporting the association of higher PGC1α expression with more oxidative redox status. CONCLUSION: ORI-based redox subpopulations in TNBC tumors exhibited differential expression of PGC1α gene that was associated with a gene network, providing a possible molecular basis underlying the potential value of ORR as a prognostic biomarker.