Targeting the circC16orf62/miR-1299/PHF8 axis to suppress metastasis and overcome immune evasion in triple-negative breast cancer

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive malignancy with high metastatic potential and poor prognosis. Lymph node metastasis is a pivotal event and a key negative prognostic indicator. However, the molecular drivers of this process in TNBC remain unclear. Circular RNAs (circRNAs) have emerged as important cancer regulators, but their specific roles in TNBC metastasis remain largely unexplored, highlighting a critical knowledge gap. METHODS: Through comprehensive circRNA sequencing of lymph node-positive (LN+) versus lymph node-negative (LN-) TNBC tissues, we revealed that circC16orf62 is significantly upregulated in LN+ tumors. Functional studies were conducted in vitro and in vivo to assess the effects of circC16orf62 and related components on metastasis, epithelialmesenchymal transition (EMT), and PD-L1 expression. Mechanistic investigations were included identifying miRNA interactions and downstream targets. RESULTS: We demonstrated that elevated circC16orf62 expression promotes tumor metastasis by driving EMT and upregulating PD-L1 expression in TNBC cells. Mechanistically, circC16orf62 functions as an efficient miRNA sponge, sequestering miR-1299 to relieve its suppressive activity on its direct target, PHF8, a histone demethylase. Consequently, circC16orf62-mediated inhibition of miR-1299 increases PHF8 expression, triggering EMT and PD-L1 induction. Depletion of circC16orf62 or restoration of miR-1299 expression significantly attenuated EMT, reduced PD-L1 levels, and impaired metastatic capacity. Conversely, PHF8 overexpression phenocopied the prometastatic effects of circC16orf62. Pharmacological inhibition of PHF8 (iPHF8) potently suppressed EMT and PD-L1 expression. Given the dual role of this axis in metastasis and immune evasion, a combinatorial therapeutic strategy was evaluated. iPHF8 synergized with anti-PD-1 immunotherapy to profoundly inhibit EMT, reduce metastasis, and increase antitumor immunity in preclinical TNBC models. CONCLUSION: This study revealed that the circC16orf62/miR-1299/PHF8 axis is a critical regulator that couples EMT-driven metastasis and PD-L1-mediated immunosuppression in TNBC. Targeting PHF8 combined with immune checkpoint blockade presents a promising novel therapeutic paradigm for aggressive, metastatic TNBC.