Mechanism of SPARC peptide FSEC inhibition of angiogenesis in neuroblastoma

描述（由申请人提供）：尽管在儿科癌症的治疗方面取得了很大进展，但高危神经母细胞瘤儿童的死亡率仍然很高，近60%的患者出现了现有疗法无法治愈的复发性疾病。传统 癌症治疗使用靶向肿瘤细胞的药物。然而，现在已经确定，肿瘤的非肿瘤成分（即，肿瘤基质/微环境）可以促进或抑制肿瘤生长，这取决于其细胞组成和细胞外基质的组织。基质细胞蛋白是癌细胞与其微环境之间相互作用的天然调节剂。我们和其他人已经表明，基质细胞蛋白分泌的蛋白质酸性和富含半胱氨酸（α）调节组装和细胞外基质的组成，阻断血管生成，并抑制神经母细胞瘤和其他肿瘤的生长。我们已经通过测试在3个主要结构域中具有缺失的cDNA 3构建体和许多cDNA 3肽的抗血管生成活性进行了结构-功能研究。我们进一步证明了对应于卵泡抑素结构域的EGF模块的肽FS-E（22个氨基酸）具有抗肿瘤活性。最近，我们设计了一种更小的FSEC肽（11个氨基酸），FSEC，对应于FS-E的C-末端部分，并且已经证明它也具有有效的抗血管生成活性，并在临床前模型中抑制神经母细胞瘤的生长。此外，我们的初步研究表明，FSEC抑制胰腺癌，肺癌和乳腺癌异种移植物的生长，表明FSEC可能在广泛的肿瘤类型中具有治疗活性。本研究的具体目的是通过鉴定FSEC结合配偶体来阐明肽FSEC抑制血管生成和神经母细胞瘤肿瘤生长的机制，所述FSEC结合配偶体通过：1）将用二氮丙啶基序工程化的FSEC与人神经母细胞瘤肿瘤组织蛋白交联; 2）通过亲和层析纯化结合配偶体;以及3）鉴定结合配偶体。 蛋白质与质谱分析法结合。我们将使用系统的蛋白质组学方法分离与FSEC相互作用的蛋白质。为了设计FSEC探针，我们将使用新技术，将高反应性的二氮丙啶基团并入合成的肽探针中。将这些探针与人神经母细胞瘤肿瘤组织一起孵育，并在UV照射下进行交联。结合配偶体将通过亲和色谱法纯化并通过质谱法鉴定。与FSEC相互作用的蛋白质的鉴定将导致更好地理解FSEC如何抑制血管生成和肿瘤生长，并且将为通过创建非允许的微环境来靶向肿瘤中的非肿瘤细胞的新型治疗剂的开发提供见解。我们研究的长期目标是通过开发针对微环境的有效治疗策略来改善高危神经母细胞瘤儿童的预后。

DESCRIPTION (provided by applicant): Despite great progress in the treatment of pediatric cancers, mortality among children with high-risk neuroblastoma remains high, with nearly 60% of the patients developing recurrent disease that is incurable with existing therapies. Traditional cancer treatment use drugs that target the neoplastic cells. However, it is now established that the non-neoplastic components of the tumor (i.e., the tumor stroma/microenvironment) can either promote or inhibit tumor growth depending upon its cellular composition and the organization of the extracellular matrix. Matricellular proteins are natural regulators of the interactions between cancer cells and their microenvironment. We and others have shown that the matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC) regulates the assembly and composition of the extracellular matrix, blocks angiogenesis, and inhibits the growth of neuroblastoma and other neoplastic tumors. We have conducted structure-functional studies by testing the anti-angiogenic activity of SPARC constructs with deletions in the 3 major domains, and a number of SPARC peptides. We have further demonstrated that peptide FS-E (22 amino acids), which corresponds to an EGF module of a follistatin domain, has anti-tumor activity. Most recently, we have designed a smaller SPARC peptide (11 amino acids), FSEC, corresponding to the C-terminal part of FS- E, and have demonstrated that it also has potent anti-angiogenic activity and inhibits the growth of neuroblastoma in preclinical models. Further, our preliminary studies show that FSEC inhibits the growth of pancreatic, lung, and breast cancer xenografts, indicating that FSEC may have therapeutic activity in a broad range of tumor types. The specific aim of this study is to elucidate the mechanisms by which peptide FSEC inhibits angiogenesis and neuroblastoma tumor growth by identifying FSEC-binding partners through: 1) cross-linking FSEC engineered with a diazirine motif with human neuroblastoma tumor tissue proteins; 2) purifying binding partners by affinity chromatography, and 3) identifying protein partners with mass-spectrometry. We will use a systemic proteomic approach to isolate the proteins that interact with FSEC. To engineer FSEC probes, we will use new technology that incorporates a highly reactive diazirine group into synthesized peptide probes. These probes will be incubated with human neuroblastoma tumor tissue, and cross-linking will be performed under UV-irradiation. Binding partners will be purified by affinity chromatography and identified by mass-spectrometry. Identification of the proteins that interact with FSEC will lead to a better understanding of how FSEC inhibits angiogenesis and tumor growth, and will provide insight for the development of novel therapeutics that target non-neoplastic cells in the tumor by creating a non- permissive microenvironment. The long-term goal of our research is to improve the outcome of children with high-risk neuroblastoma by developing effective therapeutic strategies that target the microenvironment.