PRDX6 Drives Breast Cancer Progression Through Mitochondrial Biosynthesis and Oxidative Phosphorylation

Abstract

Background: Peroxiredoxin 6 (PRDX6) scavenges reactive oxygen species (ROS) and plays a key role in antioxidant defense. Although PRDX6 is involved in various cancers, its role in breast cancer (BRCA) remains unclear.

Methods: Cell proliferation was assessed using CCK-8, EdU staining, and colony formation assays. Migration and invasion were evaluated via wound-healing and transwell assays. ROS levels and mitochondrial membrane potential were measured by fluorescence microscopy or flow cytometry. Oxidative phosphorylation (OXPHOS) activity was determined by ATP production and NAD⁺/NADH ratio. Mitochondria were visualized by TEM, and mitochondrial complex subunits were detected by quantitative real-time PCR and Western blotting. In vivo effects were evaluated using a xenograft tumor model.

Results: Although PRDX6 was downregulated in BRCA overall, it showed elevated expression in aggressive subtypes and advanced-stage tumors, correlating with poor prognosis. Overexpression of PRDX6 enhanced BRCA cell proliferation, migration, and invasion. PRDX6 reduced ROS levels, upregulated mitochondrial transcription factor A (TFAM) expression, and promoted mitochondrial complex subunit expression and OXPHOS. Inhibition of TFAM led to a decrease in the expression of some of the mitochondrial complex subunits, which reversed the pro-carcinogenic phenotype of the tumor. PRDX6 also promoted tumor growth in vivo.

Conclusion: PRDX6 maintains intracellular homeostasis by reducing ROS and promotes mitochondrial biogenesis and OXPHOS through TFAM-dependent and -independent pathways, driving BRCA progression.

Keywords: PRDX6; breast cancer; mitochondria; oxidative phosphorylation (OXPHOS); tumorigenesis.

FIGURE 1

High PRDX6 expression was associated with poor prognosis in BRCA patients. (a) The mRNA expression profiles of PRDX6 in BRCA tissues compared with normal tissues. (b) PRDX6 expression according to PAM50 typing. (c) PRDX6 expression based on BRCA cancer stage. (d) The OS, DFS, and DMFS were determined according to PRDX6 expression in BRCA patients by the Kaplan–Meier database. BRCA, breast cancer; OS, overall survival; DFS, disease‐free survival; DMFS, distant metastasis‐free survival.

FIGURE 2

PRDX6 promoted cell proliferation, migration, and invasion in vitro. (a–c) Validation of knockdown and overexpression effects of PRDX6 protein (a, b) and mRNA (c). (d–h) CCK8 assay (d), EdU staining (e, f), and colony formation assay (g, h) were used to evaluate the effect of PRDX6 on cell proliferation. The results showed that knockdown of RDDX6 inhibited the proliferative capacity of MCF‐7, and PRDX6 overexpression promoted the proliferative capacity of SUM159PT cells in vitro. (i, j) Overexpression of PRDX6 led to increased phosphorylation levels of mTOR and AKT at Ser473, and the opposite phenomenon was observed when PRDX6 was knocked down. (k, l) Knockdown of PRDX6 inhibited the invasive ability of MCF‐7 cells, while overexpression of PRDX6 enhanced the invasiveness of SUM159PT cells. (m, n) Overexpression of PRDX6 promotes migration of SUM159PT cells. **p < 0.01; ***p < 0.001.

FIGURE 3

PRDX6 promoted mitochondrial biosynthesis and OXPHOS. (a) Heatmap of PRDX6‐associated genes generated using the LinkedOmics database. (b) Genes associated with PRDX6 were enriched in the OXPHOS pathway (FDR < 0.05). (c–f) Fluorescence microscopy (c, d) and flow cytometry (e, f) were used to assess intracellular ROS levels. (g, h) The mitochondrial membrane potential levels in cells were evaluated using fluorescence microscopy. (i) In the vector group, mitochondria were fewer, swollen, with sparse matrix, dissolved contents, and reduced cristae. In the PRDX6 overexpression group, mitochondria increased, with clear double membranes, lamellar cristae, and parallel arrangement. Red arrows: Mitochondria; N: Nucleus. Top: 2500×; bottom: 10,000×. (j) PRDX6 promoted intracellular ATP production. (k) PRDX6 overexpression led to an elevated intracellular NAD+/NADH ratio. (l) Regulation of mitochondrial complex subunit mRNA expression levels by PRDX6 expression. (m, n) PRDX6 regulated protein expression of mitochondrial complex subunits NDUFB8, MTCO2 and ATP5A1. **p < 0.01; ***p < 0.001.

FIGURE 4

TFAM was a target of PRDX6. (a) PRDX6 expression was positively correlated with TFAM (r = 0.35, p < 0.0001) and TFB2M (r = 0.47, p < 0.0001) expression. (b) PRDX6 affected the mRNA expression level of TFAM. (c, d) PRDX6 affected the protein expression level of TFAM. (e) TFAM was highly expressed in BRCA. (f) TFAM was associated with worse OS, DFS, and DMFS in BRCA patients. (g, h) siRNA against TFAM knocked down the expression level of TFAM. (i–l) Knockdown of TFAM increased ROS production (i, j) and reduced the mitochondrial membrane potential (k, l). (m) Knockdown of TFAM reduced the expression of ATP5A1, MT‐CYB, and MTCO2 caused by PRDX6 overexpression. (n, o) Knockdown of TFAM reduced the synthesis of MTCO2 and ATP5A1 caused by PRDX6 overexpression. (p, r) Enhanced proliferation (p) and invasive capacity (q,r) Induced by PRDX6 was suppressed after knockdown of TFAM. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

PRDX6 promoted tumor growth in vivo. (a–c) Compared with the control group, the tumors in the PRDX6 overexpression group of xenograft model mice were larger in size (a, b) and heavier in mass (c); Top: 4×; bottom: 10×. (d) HE staining sections of tumor tissue. (e) Representative IHC staining of Ki67 in tumor tissues from the control and PRDX6 overexpression groups; Top: 4×; bottom: 10×. *p < 0.05; **p < 0.01. HE, Hematoxylin–eosin; IHC, immunohistochemical staining.