背景:乳腺癌是女性中最致命的癌症之一。尽管在乳腺癌的诊断和治疗方面取得了重大进展,许多患者仍然屈服于这种疾病,因此,迫切需要新的有效治疗方法。天然产物香豆素已被广泛研究,因为它揭示了医药领域的各种生物学特性。越来越多的证据表明,组蛋白去乙酰化酶抑制剂(HDACIs)是有前途的新型抗乳腺癌药物。然而,目前的大多数HDACIs对实体肿瘤仅表现出中等效果,并伴有严重的副作用。因此,开发更有效的乳腺癌治疗HDACIs,HDACIs的异羟肟酸盐与香豆素核心有关,并设计合成了香豆素-异羟肟酸盐杂种。
方法:通过药效团融合策略将取代的香豆素部分掺入到经典的异羟肟酸盐HDACIs中。通过使用HDACI筛选试剂盒和细胞活力测定鉴定ZN444B。进行分子对接以探索ZN444B与HDAC1的结合模式。蛋白质印迹,免疫荧光染色,细胞活力,使用集落形成和细胞迁移以及流式细胞术测定来分析ZN444B的体外抗乳腺癌作用。将小鼠模型中的原位研究用于体内功效和毒性的临床前评估。蛋白质组学分析,双荧光素酶报告分析,染色质免疫沉淀,免疫共沉淀,免疫荧光染色分析和免疫组织化学(IHC)分析用于阐明ZN444B作用的分子基础。
结果:我们合成并鉴定了一种新的香豆素-异羟肟酸盐偶联物,ZN444B在体外和体内均具有有希望的抗乳腺癌活性。分子对接模型显示ZN444B以高亲和力结合HDAC1。进一步的机理研究表明,ZN444B通过抑制HDAC1在K703对Sp1的脱乙酰酶活性而特异性降低FOS样抗原2(FOSL2)mRNA水平,并消除Sp1与FOSL2启动子的结合能力。此外,FOSL2表达与乳腺癌进展和转移呈正相关。沉默FOSL2表达降低了乳腺癌细胞对ZN444B治疗的敏感性。此外,ZN444B在小鼠中没有显示全身毒性。
结论:我们的发现强调了FOSL2作为乳腺癌新的生物标志物和治疗靶点的潜力,并且用ZN444B靶向HDAC1-Sp1-FOSL2信号轴可能是一种有希望的乳腺癌治疗策略。
BACKGROUND: Breast cancer is one of the most lethal cancers in women. Despite significant advances in the diagnosis and treatment of breast cancer, many patients still succumb to this disease, and thus, novel effective treatments are urgently needed. Natural product coumarin has been broadly investigated since it reveals various biological properties in the medicinal field. Accumulating evidence indicates that histone deacetylase inhibitors (HDACIs) are promising novel anti-breast cancer agents. However, most current HDACIs exhibit only moderate effects against solid tumors and are associated with severe side effects. Thus, to develop more effective HDACIs for breast cancer therapy, hydroxamate of HDACIs was linked to coumarin core, and coumarin-hydroxamate hybrids were designed and synthesized.
METHODS: A substituted coumarin moiety was incorporated into the classic hydroxamate HDACIs by the pharmacophore fusion strategy. ZN444B was identified by using the HDACI screening kit and cell viability assay. Molecular docking was performed to explore the binding mode of ZN444B with HDAC1. Western blot, immunofluorescent staining, cell viability, colony formation and cell migration and flow cytometry assays were used to analyze the anti-breast cancer effects of ZN444B in vitro. Orthotopic studies in mouse models were applied for preclinical evaluation of efficacy and toxicity in vivo. Proteomic analysis, dual-luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation, immunofluorescent staining assays along with immunohistochemical (IHC) analysis were used to elucidate the molecular basis of the actions of ZN444B.
RESULTS: We synthesized and identified a novel coumarin-hydroxamate conjugate, ZN444B which possesses promising anti-breast cancer activity both in vitro and in vivo. A molecular docking model showed that ZN444B binds to HDAC1 with high affinity. Further mechanistic studies revealed that ZN444B specifically decreases FOS-like antigen 2 (
FOSL2) mRNA levels by inhibiting the deacetylase activity of HDAC1 on Sp1 at K703 and abrogates the binding ability of Sp1 to the FOSL2 promoter. Furthermore,
FOSL2 expression positively correlates with breast cancer progression and metastasis. Silencing
FOSL2 expression decreases the sensitivity of breast cancer cells to ZN444B treatment. In addition, ZN444B shows no systemic toxicity in mice.
CONCLUSIONS: Our findings highlight the potential of
FOSL2 as a new biomarker and therapeutic target for breast cancer and that targeting the HDAC1-Sp1-
FOSL2 signaling axis with ZN444B may be a promising therapeutic strategy for breast cancer.