The TOX2/MT1F Axis Mediates Breast Cancer Cell Proliferation and Xenograft Growth

Abstract

Background: Aberrant DNA methylation is a key epigenetic mechanism involved in breast cancer (BC) progression. Thymocyte selection-associated high mobility group box 2 (TOX2) has been reported to undergo hypermethylation in BC; however, its functional role and downstream regulatory network remain unclear. This study aimed to explore the biological role of TOX2 and its regulatory interaction with metallothionein-1F (MT1F) in BC progression.Methods: TOX2 and MT1F expression were modulated in MDA-MB-231 cells using plasmid overexpression and small interfering RNA (siRNA) transfection, and TOX2 and MT1F expression were modulated in MCF-7 cells using plasmid overexpression. Methylation-specific PCR (MSP) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to assess TOX2 promoter methylation and gene expression levels in BC (MDA-MB-231 and MCF-7) and normal breast cells (MCF-10A). 5-ethynyl-2ʹ-deoxyuridine incorporation and Cell Counting Kit-8 assays were used to calculate the proliferation and viability of MDA-MB-231 and MCF-7 cells. Tumorigenicity in vivo was assessed in a nude mouse xenograft model by tumor volume and weight measurements. MT1F expression in both cells and tumor tissues was determined using qRT-PCR.Results: MDA-MB-231 and MCF-7 cells showed TOX2 promoter methylation and downregulated TOX2 versus MCF-10A cells (p < 0.001). TOX2 overexpression markedly inhibited MDA-MB-231 and MCF-7 cell viability and proliferation, accompanied by reduced MT1F expression, whereas TOX2 knockdown exerted opposite effects in MDA-MB-231 cells (p < 0.001). In contrast, MT1F overexpression promoted MCF-7 cell proliferation, while its silencing suppressed MDA-MB-231 cell proliferation (p < 0.01). Rescue experiments demonstrated that TOX2 and MT1F could reciprocally reverse each other’s regulatory effects on BC cell proliferation (p < 0.05). In vivo, TOX2 knockdown significantly increased xenograft tumor burden, reflected by larger tumor volume and weight, along with elevated MT1F mRNA expression in tumor tissues (p < 0.001).Conclusion: TOX2 downregulation promotes BC cell proliferation and increases xenograft tumor burden, in association with upregulation of MT1F. The TOX2-MT1F-associated regulatory axis may represent a potential therapeutic target in BC. However, further validation in clinical specimens is warranted.