Department of Internal Medicine, College of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, 731014, USA.
Department of Internal Medical Oncology, Harbin Medical University Cancer Hospital, Harbin, 150040, Heilongjiang Province, People's Republic of China.
Mol Cancer. 2019 Nov 7;18(1):156. doi: 10.1186/s12943-019-1077-0.
Aspartate β-hydroxylase (ASPH) is silent in normal adult tissues only to re-emerge during oncogenesis where its function is required for generation and maintenance of malignant phenotypes. Exosomes enable prooncogenic secretome delivering and trafficking for long-distance cell-to-cell communication. This study aims to explore molecular mechanisms underlying how ASPH network regulates designated exosomes to program development and progression of breast cancer.
Stable cell lines overexpressing or knocking-out of ASPH were established using lentivirus transfection or CRISPR-CAS9 systems. Western blot, MTT, immunofluorescence, luciferase reporter, co-immunoprecipitation, 2D/3-D invasion, tube formation, mammosphere formation, immunohistochemistry and newly developed in vitro metastasis were applied.
Through physical interactions with Notch receptors, ligands (JAGs) and regulators (ADAM10/17), ASPH activates Notch cascade to provide raw materials (especially MMPs/ADAMs) for synthesis/release of pro-metastatic exosomes. Exosomes orchestrate EMT, 2-D/3-D invasion, stemness, angiogenesis, and premetastatic niche formation. Small molecule inhibitors (SMIs) of ASPH's β-hydroxylase specifically/efficiently abrogated in vitro metastasis, which mimics basement membrane invasion at primary site, intravasation/extravasation (transendothelial migration), and colonization/outgrowth at distant sites. Multiple organ-metastases in orthotopic and tail vein injection murine models were substantially blocked by a specific SMI. ASPH is silenced in normal adult breast, upregulated from in situ malignancies to highly expressed in invasive/advanced ductal carcinoma. Moderate-high expression of ASPH confers more aggressive molecular subtypes (TNBC or Her2 amplified), early recurrence/progression and devastating outcome (reduced overall/disease-free survival) of breast cancer. Expression profiling of Notch signaling components positively correlates with ASPH expression in breast cancer patients, confirming that ASPH-Notch axis acts functionally in breast tumorigenesis.
ASPH-Notch axis guides particularly selective exosomes to potentiate multifaceted metastasis. ASPH's pro-oncogenic/pro-metastatic properties are essential for breast cancer development/progression, revealing a potential target for therapy.
天冬氨酸 β-羟化酶（ASPH）在正常成年组织中是沉默的，只有在癌变过程中才重新出现，其功能是为恶性表型的产生和维持提供必要条件。外泌体能够实现前癌基因分泌组的传递和运输，从而实现长距离的细胞间通讯。本研究旨在探讨 ASPH 网络调节特定外泌体以编程乳腺癌发生和发展的分子机制。
使用慢病毒转染或 CRISPR-CAS9 系统建立稳定过表达或敲除 ASPH 的细胞系。应用 Western blot、MTT、免疫荧光、荧光素酶报告、共免疫沉淀、2D/3D 侵袭、管形成、类器官形成、免疫组化和新开发的体外转移实验。
通过与 Notch 受体、配体（JAGs）和调节剂（ADAM10/17）的物理相互作用，ASPH 激活 Notch 级联反应，为合成/释放促转移外泌体提供原料（特别是 MMPs/ADAMs）。外泌体协调 EMT、2D/3D 侵袭、干性、血管生成和前转移龛形成。ASPH 的β-羟化酶的小分子抑制剂（SMIs）特异性/有效地阻断了体外转移，该转移模拟了原发性部位的基底膜侵袭、血管内/血管外（跨内皮迁移）以及远处部位的定植/生长。在原位癌发展为浸润性/进展性导管癌的过程中，ASPH 逐渐上调，在原位癌和高度表达的浸润性/高级别导管癌中均上调。ASPH 的中高度表达赋予了更具侵袭性的分子亚型（三阴性乳腺癌或 Her2 扩增）、早期复发/进展和破坏性结局（降低总生存/无病生存）。乳腺癌患者 Notch 信号通路成分的表达谱与 ASPH 表达呈正相关，证实了 ASPH-Notch 轴在乳腺癌发生中具有功能性作用。
ASPH-Notch 轴指导特定的外泌体来增强多方面的转移。ASPH 的致癌/促转移特性对于乳腺癌的发展/进展是必不可少的，这为治疗提供了一个潜在的靶点。
