Alokda Abdulrahman M, Soffar Ahmed Abdelmagied, Yousef Amany I, Ibrahim Fawziya A R, El-Sewedy Tarek, Elmetwalli Alaa
Department of Applied Medical Chemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt.
Department of Zoology, Faculty of Sciences, Alexandria University, Alexandria, Egypt.
Naunyn Schmiedebergs Arch Pharmacol. 2025 Mar 10. doi: 10.1007/s00210-025-03975-6.
Prefoldin1 (PFDN1), a molecular chaperone, is essential for stabilizing cytoskeletal proteins like actin and tubulin, supporting cellular processes such as survival, migration, and cell cycling. Recent evidence suggests that PFDN1 also influences key cancer-related signaling pathways. However, the complete mechanisms involved and the downstream genes implicated in such action remain relatively undiscovered. This study investigated the effects of PFDN1 silencing on cellular processes and gene expression in triple-negative breast cancer (TNBC) cells, focusing on its potential as a therapeutic target. MDA-MB-231 cells, a TNBC model, were transfected with PFDN1-targeting siRNA to knock down PFDN1 expression. The effects of PFDN1 silencing were assessed through various assays, including phase contrast and scanning electron microscopy (SEM) for morphological changes, colony formation and wound healing assays for proliferation and migration, and flow cytometry for cell cycle and apoptosis analysis. Gene expression changes were evaluated using a qRT-PCR array targeting 84 genes involved in cancer progression. PFDN1 silencing resulted in a 54.8% reduction in PFDN1 protein levels (p < 0.0001). Morphological analysis revealed cytoplasmic shrinkage, chromatin condensation, roughened membranes, and microvilli loss, consistent with apoptotic changes. Colony formation assays showed a 10.33% reduction in colony number and size (p < 0.05) in PFDN1-silenced cells. Migration was significantly impaired, with reduced wound closure observed in wound healing assays (p < 0.01). Flow cytometry revealed a G2/M phase arrest (p < 0.05) and increased early apoptotic populations (20.93% vs. 5.42% in controls, p < 0.01). Gene expression analysis showed downregulation of genes associated with angiogenesis (KDR, TEK), EMT (FOXC2, SNAI1), and hypoxia signaling (CA9, EPO), while proapoptotic genes, such as FASLG, were upregulated. This study highlights the critical role of PFDN1 in TNBC progression, demonstrating that its silencing disrupts survival, migration, cell cycling, and apoptosis pathways. PFDN1 knockdown also significantly alters the expression of key cancer-related genes, further impairing angiogenesis, EMT, and hypoxia adaptation. These findings suggest that targeting PFDN1 could be a promising therapeutic strategy for TNBC, warranting further investigation in preclinical models.
前折叠蛋白1（PFDN1）是一种分子伴侣，对于稳定肌动蛋白和微管蛋白等细胞骨架蛋白至关重要，支持诸如存活、迁移和细胞周期等细胞过程。最近的证据表明，PFDN1还影响关键的癌症相关信号通路。然而，其中涉及的完整机制以及这种作用中涉及的下游基因仍相对未被发现。本研究调查了PFDN1沉默对三阴性乳腺癌（TNBC）细胞中细胞过程和基因表达的影响，重点关注其作为治疗靶点的潜力。使用靶向PFDN1的小干扰RNA（siRNA）转染TNBC模型MDA-MB-231细胞，以敲低PFDN1表达。通过各种检测评估PFDN1沉默的影响，包括相差显微镜和扫描电子显微镜（SEM）观察形态变化、集落形成和伤口愈合检测评估增殖和迁移，以及流式细胞术分析细胞周期和凋亡。使用针对84个参与癌症进展的基因的qRT-PCR阵列评估基因表达变化。PFDN1沉默导致PFDN1蛋白水平降低54.8%（p < 0.0001）。形态学分析显示细胞质收缩、染色质浓缩、细胞膜粗糙和微绒毛丧失，与凋亡变化一致。集落形成检测显示，PFDN1沉默细胞中的集落数量和大小减少10.33%（p < 0.05）。迁移明显受损，伤口愈合检测中观察到伤口闭合减少（p < 0.01）。流式细胞术显示G2/M期阻滞（p < 0.05）和早期凋亡细胞群体增加（对照组为5.42%，而PFDN1沉默组为20.93%，p < 0.01）。基因表达分析显示，与血管生成（KDR、TEK）、上皮-间质转化（EMT）（FOXC2、SNAI1）和缺氧信号传导（CA9、EPO）相关的基因下调，而促凋亡基因如FASLG上调。本研究强调了PFDN1在TNBC进展中的关键作用，表明其沉默会破坏存活、迁移、细胞周期和凋亡途径。PFDN1敲低还显著改变关键癌症相关基因的表达，进一步损害血管生成、EMT和缺氧适应。这些发现表明，靶向PFDN1可能是TNBC一种有前景的治疗策略，值得在临床前模型中进一步研究。
