■癌症干细胞(CSC)驱动启动,维护,和乳腺肿瘤的治疗反应。CD49f在乳腺CSC中表达并在维持干性中起作用。因此,阻断CD49f是靶向乳腺CSC的潜在治疗方法。在本研究中,我们的目标是将药物重新用作CD49f拮抗剂。
■我们使用对异源二聚化至关重要的CD49f亚结构域和一组临床测试的药物化学物质进行了共有分子对接。采用分子动力学模拟来进一步表征药物-靶标结合。使用MDA-MB-231细胞,我们评估了潜在的CD49f拮抗剂对1)细胞与层粘连蛋白的粘附;2)乳腺球形成;和3)细胞活力的影响。我们通过蛋白质印迹(WB)和共免疫沉淀分析了具有更好CSC选择性的药物对CD49f下游信号激活的影响。通过流式细胞术和WB分析干细胞标志物CD44和SOX2的表达,分别。通过荧光素酶报告基因测定评价S0X2启动子的反式激活。通过有限稀释异种移植评估CSC数量的变化。
■Pranlukast,一种用于治疗哮喘的药物,与CD49f结合并抑制CD49fMDA-MB-231细胞与层粘连蛋白的粘附,表明它拮抗含CD49f的整联蛋白。分子动力学分析表明,pranlukast结合可诱导CD49f的构象变化,从而影响其与β1-整联蛋白亚基的相互作用,并限制了异源二聚体的构象动力学。普伦司特在乳腺球形成试验中降低了乳腺癌细胞的克隆形成性,但对大块肿瘤细胞的活力没有影响。MDA-MB-231细胞短暂暴露于pranlukast改变了CD49f依赖性信号,减少粘着斑激酶(FAK)和磷脂酰肌醇3-激酶(PI3K)激活。Further,pranlukast处理的细胞显示CD44和SOX2表达降低,SOX2启动子反式激活,和体内致瘤性,支持这种药物降低CSC的频率。
■我们的结果支持普朗司特作为CD49f拮抗剂的功能,可减少三阴性乳腺癌细胞中的CSC群体。该药物的药代动力学和毒理学已经建立,为乳腺癌患者提供潜在的辅助治疗。
UNASSIGNED: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors.
CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of
CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as
CD49f antagonists.
UNASSIGNED: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of
CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation.
UNASSIGNED: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of
CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes
CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC.
UNASSIGNED: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.