Preclinical characterization of EMT/CSC-specific small molecule inhibitors for TNBCs

三阴性（ER-、PR-、HER 2-）乳腺癌（TNBC）缺乏靶向治疗，导致高死亡率。 死亡率化疗耐药肿瘤的复发以及远处转移的形成是 这两种疾病都是头3-5年内死亡率的原因。我们和其他人已经证明， 具有癌症干细胞（CSC）和间充质特征，并且上皮间充质的诱导 转化（EMT）促进耐药CSC的从头产生/扩增。事实上， 通过诱导EMT产生的表达特征在低密蛋白乳腺癌中富集，这是一个特别的 内源性亚型占所有TNBC的近一半。 我们最近报道，表达的叉头盒转录因子FOXC 2是独特的诱导 在EMT后和CSC中，FOXC 2足以用于EMT、肿瘤起始和TNBC的转移， 细胞系和化疗抗性。值得注意的是，shRNA介导的FOXC 2敲低足以 阻断诱导EMT、转移以及肿瘤起始和转移的CSC相关特性， 化疗耐药性鉴于这些结果，我们推断， 选择性靶向表达FOXC 2的CSC将产生重要的化学试剂， 改善CSC富集TNBC的治疗。 我们已经对大约100，000种药物进行了高通量小分子筛选， 鉴定了7种对表达FOXC 2的CSC具有优先活性的候选分子。在这 建议，我们打算充分表征和验证这些分子的体外活性在广泛的 乳腺癌细胞系对CSC的选择性，其阻断诱导和逆转的能力 EMT相关肿瘤细胞特性（包括迁移和侵袭）及其改变CSC性状的能力 （包括乳腺球形成、标志物表达和集落形成）。此外，我们将阐明 通过使用光亲和试剂，随后进行质谱分析，确定这些试剂的相关蛋白质靶点，以及 还确定了这些靶蛋白对EMT/CSC特性的功能作用。最后，我们将优化 用于体内递送和可接受的药代动力学的候选分子，并进行广泛的临床前研究。 使用多个TNBC患者来源的异种移植物在小鼠中进行测试。 这项研究的结果将直接允许识别用于治疗复发和转移的新药 此外，还将阐明FOXC 2在TNBCs重编程过程中调控的分子网络。 肿瘤细胞向CSC/转移状态转移。马尼博士和莱尔森博士之间的密切合作， 我们大量的初步数据表明，成功的可能性很大。此外，扩展 该提案的范围，从使用高通量筛选的药物发现，到在患者来源的TNBC中的测试 异种移植，表明我们致力于迅速将新的癌症治疗方法投入临床试验。

Triple negative (ER-, PR-, HER2-) breast cancers (TNBCs) lack targeted treatments, resulting in a high mortality rate. Recurrence of chemotherapy-resistant tumors as well as formation of distant metastases are both contributors to mortality within the first 3-5 years. We and others have shown that TNBCs are enriched with cancer stem-cell (CSC)- and mesenchymal features, and that the induction of an epithelial-mesenchymal- transition (EMT) promotes the de novo generation/expansion of drug-resistant CSCs. Indeed, the gene expression signature generated by induction of EMT is enriched in claudin-low breast cancers, a particular intrinsic subtype comprising nearly half of all TNBCs. We recently reported that expression of the forkhead-box transcription factor FOXC2 is uniquely induced following EMT and in CSCs, and that FOXC2 is sufficient for EMT, tumor initiation, and metastasis of TNBC cell lines and chemotherapy-resistance. Remarkably, shRNA-mediated knockdown of FOXC2 is sufficient to block induction of EMT, metastasis as well as the CSC-associated properties of tumor initiation and chemotherapy-resistance. Given these results, we reasoned that a drug discovery screen for small molecules that selectively target FOXC2-expressing CSCs will yield important chemical agents that will dramatically improve treatment of CSC-enriched TNBCs. We have performed a high-throughput small molecule screen of approximately 100,000 agents, and have identified seven candidate molecules with preferential activity against FOXC2-expressing CSCs. In this proposal, we intend to fully characterize and validate the in vitro activity of these molecules across a wide spectrum of breast cancer cell lines for their selectivity for CSCs, their ability to block induction and reverse EMT-associated tumor cell properties (including migration and invasion), and their capacity to alter CSC traits (including mammosphere formation, marker expression and colony formation). In addition, we will elucidate the relevant protein targets for these agents, by use of photo-affinity reagents followed by mass spectrometry, and also determine the functional role of these target proteins for EMT/CSC properties. Finally, we will optimize candidate molecules for in vivo delivery and acceptable pharmacokinetics, and conduct extensive preclinical testing in mice using multiple TNBC patient-derived xenografts. The results of this study will directly permit the identification of novel drugs for treating recurrent and metastatic TNBCs, and will also shed light on the molecular networks regulated by FOXC2 during its reprogramming of tumor cells towards the CSC/metastatic state. The strong collaboration between Dr. Mani and Dr. Lairson, evidenced by our extensive preliminary data, indicates a high likelihood of success. Furthermore, the extended scope of this proposal, from drug discovery using high-throughput screens, to testing in patient-derived TNBC xenografts, indicates our commitment to rapidly moving novel cancer therapeutics into clinical trials.