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乙酰肝素酶在脑转移中的机制，并靶向乙酰肝素酶治疗这种毁灭性疾病。我们的工作表明乙酰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HER 2/EGFR途径的下游靶点发挥作用。我们的目标是确定乙酰肝素酶如何调节BMBC，并利用这些知识开发新的基于乙酰肝素酶的疗法。为了强调靶向乙酰肝素酶的重要性，我们已经取得了四项关键发现，这些发现揭示了这种分子与侵袭性BMBC表型的相关性。首先，我们鉴定了microRNA-1258作为抑制乙酰肝素酶和抑制BMBC的microRNA。其次，我们发现乙酰肝素酶调节EGFR磷酸化的位点不是拉帕替尼（一种双重HER 2/EGFR激酶抑制剂）的靶点，这表明乙酰肝素酶在拉帕替尼耐药机制中的作用。第三，我们发现HPSE调节Rac和Rho，细胞骨架动力学的关键介质，这是肿瘤细胞和微环境之间相互作用的基本过程。第四，通过研究来自BMBC患者血液的循环肿瘤细胞（CTC），我们发现了CTC中乙酰肝素酶的表达，以及其存在、EGFR基因扩增和已知癌症干细胞标志物ALDH 1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，无抗凝剂，乙二醇裂解肝素。其中之一，SST 0001，已成为一个有效的小分子乙酰肝素酶抑制剂，并提供给我们。基于我们的发现，我们假设乙酰肝素酶的表达和功能调节BMBC和脑微环境细胞之间的串扰，并启动对BMBC的发展和进展至关重要的多种效应。通过提出乙酰肝素酶更广泛的作用，涉及酶和非酶功能，以下目的旨在确定这种分子强烈参与BMBC进展的新机制。目的1将确定miR-1258调节乙酰肝素酶与BMBC抑制相关的机制。目的2将鉴定乙酰肝素酶抑制剂SST 0001和miR-1258的功能，以及它们克服BMBC细胞中拉帕替尼耐药性的能力。目的3将描述乙酰肝素酶在BMBC发展、信号传导和脑归巢CTC模式的初始步骤中的作用。我们的方法将包括体外和体内模型，再加上慢病毒递送靶向HPSE与miR-1258，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来强调我们所提出的工作的强有力的转化组成部分，以将乙酰肝素酶抑制剂引入更有效的治疗脑转移的疗法中，特别是脑转移性乳腺癌。 公共卫生相关性： 乙酰肝素酶是一种由癌细胞产生的蛋白质，在帮助癌细胞生长和扩散（转移）到全身方面起着重要作用。最高水平的乙酰肝素酶活性已被发现，特别是在细胞转移到脑。该项目旨在揭示乙酰肝素酶作为酶或蛋白质的功能。它还旨在测试新型抗乙酰肝素酶药物，以确定这些药物如何抑制乳腺癌脑转移。

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.