Heparanase Mechanisms in Brain-metastatic Breast Cancer

描述(申请人提供)：我的实验室的长期目标是确定乙酰肝素酶在脑转移瘤中的作用机制，并针对这种毁灭性的疾病进行肝素酶的靶向治疗。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HER2/EGFR通路的下游靶点。我们现在的目标是确定肝素酶是如何调节BMBC的，并利用这一知识开发基于肝素酶的新疗法。为了强调靶向乙酰肝素酶的重要性，我们有四个关键发现，这些发现为这种分子与侵袭性BMBC表型的相关性提供了新的线索。首先，我们鉴定了microRNA-1258是一种抑制乙酰肝素酶和抑制BMBC的microRNA。其次，我们发现肝素酶在拉帕替尼(一种双重的HER2/EGFR激酶抑制剂)靶点以外的位置调节EGFR的磷酸化，提示肝素酶在拉帕替尼耐药机制中发挥作用。第三，我们发现，HPSE调节Rac和Rho，这是细胞骨架动力学的关键介质，是肿瘤细胞和微环境相互作用的基本过程。第四，通过对BMBC患者血液中循环肿瘤细胞(CTCs)的研究，我们发现CTCs中有乙酰肝素酶的表达，并且其存在、EGFR基因扩增与已知的肿瘤干细胞标志物ALDH1之间存在显著的相关性。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，例如非抗凝剂、乙二醇裂解型肝素。其中之一，SST0001，已经成为一种有效的肝素酶小分子抑制剂，并可用于我们的治疗。根据我们的发现，我们假设乙酰肝素酶的表达和功能调节了BMBC和脑微环境细胞之间的串扰，并启动了对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，以下目的是为了确定这种分子的新机制，该分子密切参与BMBC的进展。目的1确定miR-1258对肝素酶的调节与抑制BMBC有关的机制。目的2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性的能力。目标3将描述乙酰肝素酶在BMBC发育的初始步骤、信号传递和脑归巢CTC模式中的作用。我们的方法将包括体外和体内模型，加上通过miR-1258靶向HPSE的慢病毒传递，新的HPSE抑制剂，以及尖端的CTC技术。这些研究强调了我们拟议工作的强大翻译部分，通过提供临床前数据，将肝素酶抑制剂引入更有效的治疗脑转移，特别是脑转移乳腺癌。

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.