Triple-negative breast cancer (TNBC) remains the most challenging subtype of breast cancer and lacks definite treatment targets. Aerobic glycolysis is a hallmark of metabolic reprogramming that contributes to cancer progression. PFKP is a rate-limiting enzyme involved in aerobic glycolysis, which is overexpressed in various types of cancers. However, the underlying mechanisms and roles of the posttranslational modification of PFKP in TNBC remain unknown. To explore whether PFKP protein has a potential role in the progression of TNBC, protein levels of PFKP in TNBC and normal breast tissues were examined by CPTAC database analysis, immunohistochemistry staining (IHC), and western blotting assay. Further CCK-8 assay, colony formation assay, EDU incorporation assay, and tumor xenograft experiments were used to detect the effect of PFKP on TNBC progression. To clarify the role of the USP5-PFKP pathway in TNBC progression, ubiquitin assay, co-immunoprecipitation (Co-IP), mass spectrometry-based protein identification, western blotting assay, immunofluorescence microscopy, in vitro binding assay, and glycolysis assay were conducted. Herein, we showed that PFKP protein was highly expressed in TNBC, which was associated with TNBC progression and poor prognosis of patients. In addition, we demonstrated that PFKP depletion significantly inhibited the TNBC progression in vitro and in vivo. Importantly, we identified that PFKP was a bona fide target of deubiquitinase USP5, and the USP5-mediated deubiquitination and stabilization of PFKP were essential for cancer cell aerobic glycolysis and TNBC progression. Moreover, we found a strong positive correlation between the expression of USP5 and PFKP in TNBC samples. Notably, the high expression of USP5 and PFKP was significantly correlated with poor clinical outcomes. Our study established the USP5-PFKP axis as an important regulatory mechanism of TNBC progression and provided a rationale for future therapeutic interventions in the treatment of TNBC.

中文翻译：

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USP5 使 PFKP 去泛素化和稳定化，激活有氧糖酵解，促进三阴性乳腺癌进展

三阴性乳腺癌（TNBC）仍然是乳腺癌中最具挑战性的亚型，缺乏明确的治疗目标。有氧糖酵解是代谢重编程的标志，有助于癌症进展。PFKP 是一种参与有氧糖酵解的限速酶，在多种类型的癌症中过度表达。然而，PFKP 翻译后修饰在 TNBC 中的潜在机制和作用仍不清楚。为了探讨 PFKP 蛋白在 TNBC 进展中是否具有潜在作用，通过 CPTAC 数据库分析、免疫组织化学染色 (IHC) 和蛋白质印迹法检测 TNBC 和正常乳腺组织中 PFKP 的蛋白水平。进一步采用CCK-8测定、集落形成测定、EDU掺入测定和肿瘤异种移植实验来检测PFKP对TNBC进展的影响。为了阐明 USP5-PFKP 通路在 TNBC 进展中的作用，进行了泛素测定、免疫共沉淀 (Co-IP)、基于质谱的蛋白质鉴定、蛋白质印迹测定、免疫荧光显微镜、体外结合测定和糖酵解测定。在此，我们发现PFKP蛋白在TNBC中高表达，这与TNBC进展和患者不良预后相关。此外，我们还证明，PFKP 消耗可显着抑制体外和体内 TNBC 的进展。重要的是，我们发现 PFKP 是去泛素酶 USP5 的真正靶标，并且 USP5 介导的去泛素化和 PFKP 的稳定对于癌细胞有氧糖酵解和 TNBC 进展至关重要。此外，我们发现 TNBC 样本中 USP5 和 PFKP 的表达之间存在很强的正相关性。值得注意的是，USP5 和 PFKP 的高表达与不良临床结果显着相关。我们的研究确立了 USP5-PFKP 轴作为 TNBC 进展的重要调节机制，并为未来治疗 TNBC 的治疗干预提供了理论基础。