Heparanase Mechanisms in Brain-metastatic Breast Cancer

描述（由申请人提供）：我实验室的长期目标是确定肝素酶在脑转移中的作用机制，并针对这种毁灭性疾病进行肝素酶靶向治疗。我们的研究表明肝素酶是脑转移的促进剂。特别是，我们已经证明HPSE在脑转移性乳腺癌（BMBC）细胞和组织中表达，并作为影响BMBC细胞增殖的HER2/EGFR通路的下游靶点。我们现在的目标是确定肝素酶如何调节BMBC，并利用这些知识开发新的基于肝素酶的治疗方法。强调靶向肝素酶的重要性，我们已经取得了四个关键发现，揭示了该分子与侵袭性BMBC表型的相关性。首先，我们确定了microRNA-1258是抑制肝素酶和抑制BMBC的microRNA。其次，我们发现肝素酶在非拉帕替尼（一种HER2/EGFR激酶抑制剂）靶点处调节EGFR磷酸化，这表明肝素酶在拉帕替尼耐药机制中起作用。第三，我们发现HPSE调控Rac和Rho，这是细胞骨架动力学的关键介质，是肿瘤细胞与微环境相互作用的基本过程。第四，通过研究BMBC患者血液中的循环肿瘤细胞（CTCs），我们发现了肝素酶在CTCs中的表达，以及其存在、EGFR基因扩增和已知癌症干细胞标志物ALDH1之间的显著相关性。合乎逻辑的下一步是制定抑制HPSE和抑制BMBC的策略。这可以通过使用miR-1258以及新的HPSE抑制剂来实现，例如，非抗凝血，乙二醇分裂肝素。其中一种，SST0001，已经成为一种有效的小分子肝素酶抑制剂，并可供我们使用。根据我们的发现，我们假设肝素酶的表达和功能调节了BMBC与脑微环境细胞之间的串导，并启动了对BMBC的发展和进展至关重要的多种作用。通过提出肝素酶更广泛的作用，包括酶和非酶功能，以下目标旨在确定该分子在BMBC进展中的新机制。目的1将确定miR-1258调节肝素酶与BMBC抑制的机制。目的2将鉴定肝素酶抑制剂SST0001和miR-1258的功能，以及它们在BMBC细胞中克服拉帕替尼耐药的能力。目的3将描述肝素酶在BMBC发展、信号传导和脑归巢CTC模式的初始阶段的作用。我们的方法将包括体外和体内模型，结合使用miR-1258靶向HPSE的慢病毒递送，新的HPSE抑制剂和尖端的CTC技术。这些研究通过提供临床前数据，强调了我们提出的工作中强大的翻译成分，以引入肝素酶抑制剂更有效地治疗脑转移，特别是脑转移性乳腺癌。

DESCRIPTION (provided by applicant): The long-term goal of my laboratory is to determine mechanisms of heparanase in brain metastasis and to target heparanase therapeutically for this devastating disease. Our work has implicated heparanase as a promoter of brain metastasis. In particular, we have demonstrated that HPSE is expressed in brain metastatic breast cancer (BMBC) cells and tissues, and functions as a downstream target of HER2/EGFR pathways affecting BMBC cell proliferation. Our objective is now to determine how heparanase regulates BMBC and to use this knowledge to develop new heparanase-based therapies. Underscoring the importance of targeting heparanase, we have made four key discoveries that shed new light on the relevance of this molecule towards the aggressive BMBC phenotype. First, we identified microRNA-1258 as a microRNA that inhibits heparanase and suppresses BMBC. Second, we found that heparanase modulates EGFR phosphorylation at sites which are not targets of lapatinib, a dual HER2/EGFR kinase inhibitor, suggesting heparanase roles in mechanisms of lapatinib resistance. Third, we discovered that HPSE regulates Rac and Rho, critical mediators of cytoskeletal dynamics which is a fundamental process in the interplay between tumor cells and the microenvironment. Fourth, by investigating circulating tumor cells (CTCs) from the blood of BMBC patients, we discovered the expression of heparanase in CTCs, and a significant correlation between its presence, EGFR gene amplification, and ALDH1, a known cancer stem cell marker. A logical next step is to formulate strategies to inhibit HPSE and suppress BMBC. This can be achieved by using miR-1258 as well as new HPSE inhibitors, e.g., non-anticoagulant, glycol-split heparins. One of them, SST0001, has emerged as a potent small-molecule inhibitor of heparanase and is available to us. Based on our discoveries, we hypothesize that heparanase expression and function regulates the cross-talk between BMBC and cells of the brain microenvironment, and initiates multiple effects that are critical for the development and progression of BMBC. By proposing much broader roles for heparanase, which involve enzymatic and non-enzymatic functions, the following aims are designed to identify new mechanisms for this molecule keenly involved in BMBC progression. Aim 1 will determine the mechanisms of heparanase regulation by miR-1258 in relation to BMBC suppression. Aim 2 will identify functions of heparanase inhibitors, SST0001 and miR-1258, and their ability to overcome lapatinib resistance in BMBC cells. Aim 3 will delineate the roles of heparanase during the initial steps of BMBC development, signaling, and in brain-homing CTC modalities. Our approaches will include in vitro and in vivo models, coupled with lentiviral delivery targeting HPSE with miR-1258, new HPSE inhibitors, and cutting-edge CTC technologies. These studies emphasize the strong translational component of our proposed work by providing pre-clinical data to introduce heparanase inhibitors in more effective therapies to treat brain metastasis, in particular brain metastatic breast cancer. PUBLIC HEALTH RELEVANCE: Heparanase is a protein produced by cancer cells that plays a major role in helping them grow and spread (metastasize) throughout the body. Highest levels of heparanase activity have been found specifically in cells metastasizing to brain. This project is designed to reveal how heparanase functions, either as enzyme or protein. It is also designed to test new anti-heparanase drugs to determine how these drugs can suppress brain metastasis in breast cancer.