Functional characterization of brain-colonizing breast cancer CTC subsets

 描述(申请人提供)：乳腺癌脑转移(BCBM)是一种毁灭性的疾病，其机制还没有得到充分的研究，并在很大程度上仍不清楚。例如，虽然人类表皮生长因子受体1和2(EGFR和HER2)是BCBM的高危预测因子，但HER2/EGFR靶向治疗已被证明对BCBM大多无效。尽管循环肿瘤细胞(CTCs)是难治性转移的“种子”的概念已经确立，但对乳腺癌脑内定植CTCs的特性和生物标志物几乎一无所知。因此，迫切需要新的CTC靶向生物标记物来对抗BCBM。我们已经发现了不表达上皮细胞黏附分子的CTCs(EpCAM阴性CTCs)，并且在异种移植中具有高的BCBM能力。CellSearchTM无法检测到这些CTCs，CellSearchTM是FDA批准的仅评估EpCAM阳性CTC的预后测试。我们在EpCAM阴性的CTCs中发现了两个有希望的标记物来预测临床上的BCBM：NOTCH1，一个重要的肿瘤启动细胞生物标记物，以及肝素酶(HPSE)，一个强大的致瘤、血管生成和转移分子。我们的结果表明，BCBM诱导的CTCs具有脑转移选择标志物(BMSM)或HER2+/EGFR+/Notch1+/HPSE+CTCs。因此，NOTCH1和HPSE可以代表新的和特异的CTC生物标志物。此外，我们检测到在表达Notch1/HPSE的CTCs中，肿瘤调节因子Merlin(神经纤维瘤病2型)的表达最高。PAK1激酶对Merlin(P-S518 Merlin)的磷酸化导致了Merlin的致癌功能，并与BMSM CTC通路及其调控有关。基于这些发现，我们的假设是Notch1和HPSE是新的CTC生物标志物，可以预测原发乳腺癌脑转移的存在；它们可以成为预防继发性BCBM的潜在治疗靶点。这项建议的目的是进行临床前转化研究和靶点验证，以提名HPSE和Notch1作为乳腺癌脑归巢CTCs的关键生物标志物。我们将研究HER2+患者的CTCs，因为这种乳腺癌亚型发生BCBM的风险远远高于平均水平。具体地说，我们将：1)确定Notch1和HPSE CTC标记物在BCBM发病中的治疗抑制和调节作用；2)将Notch1/HPSE CTC亚群与临床BCBM联系起来；3)确定Merlin在BMSM CTC通路和BCBM肿瘤发生中的作用。这一机构间项目由一个跨学科和综合良好的小组开展，将研究和验证新的和具体的四氯化碳标记物，这些标记物负责四氯化碳引起的生物化学物质。该项目是一种范式转换，具有很高的治疗效果。我们处于进行这项研究的独特地位，这不仅是因为我们可以获得来自确诊为或不患有BCBM的患者的大量血液样本和数量，而且还因为我们实验室首次开发的人类CTC系的可用性，以及与该国其他群体不同的结合多个和互补的CTC平台和方法的广泛专业知识。

 DESCRIPTION (provided by applicant): Breast cancer brain metastasis (BCBM) is a devastating disease whose mechanisms are understudied and remain largely unknown. For example, while human epidermal growth factor receptor1 and 2 (EGFR and HER2) are high-risk predictors of BCBM, HER2/EGFR-targeted therapies have proven to be mostly ineffective against BCBM. Although notions that circulating tumor cells (CTCs) acting as "seeds" of intractable metastasis are established, virtually nothing is known about the properties and biomarkers of breast cancer brain-colonizing CTCs. Therefore, there is an urgent need for new CTC target biomarkers to combat BCBM. We have discovered CTCs that do not express the epithelial cell adhesion molecule (EpCAM-negative CTCs) and possess high BCBM competency in xenografts. These CTCs could not be detected by CellSearchTM, a FDA-cleared prognostic test which evaluates only EpCAM-positive CTCs. We found two promising markers in EpCAM-negative CTCs to predict BCBM in the clinic: Notch1, an important tumor-initiating cell biomarker, and heparanase (HPSE), a potent tumorigenic, angiogenic and pro-metastatic molecule. Our results indicate that BCBM-inducing CTCs have a "Brain Metastasis-Selected Marker (BMSM)" profile or HER2+/EGFR+/Notch1+/HPSE+ CTCs. Notch1 and HPSE can thus represent novel and specific CTC biomarkers. Furthermore, we detected highest expression of the tumor modulator Merlin (Neurofibromatosis type2) in Notch1/HPSE-expressing CTCs. The phosphorylation of Merlin (P-S518 Merlin) by PAK1 kinase resulted in oncogenic functions of Merlin which associate with BMSM CTC pathways and their regulation. Based on these findings, our hypothesis is that Notch1 and HPSE are novel CTC biomarkers that can predict the presence of primary breast cancer brain metastasis; and they can be potential therapeutic targets to prevent secondary BCBM. Objective of this proposal is to perform preclinical translational research and target validation to nominate HPSE and Notch1 as critical biomarkers of breast cancer brain-homing CTCs. We will study CTCs from HER2+ patients since this breast cancer subtype has a much higher than average risk of developing BCBM. Specifically, we will: 1) determine effects of therapeutic inhibition and regulation of Notch1 and HPSE CTC markers on BCBM onset; 2) link combinatorial Notch1/HPSE CTC subsets to clinical BCBM; 3) define roles of Merlin affecting BMSM CTC pathways and BCBM oncogenesis. This inter-institutional project by an inter-disciplinary and well-integrated team will study and validate new and specific CTC markers responsible for CTC-induced BCBM. This project is paradigm-shifting and has high therapeutic impact. We are uniquely positioned to perform this study not only for having access to an extensive cohort of blood samples and volume from patients diagnosed with or without BCBM but also for the availability of human CTC lines our laboratory was first to develop, and the extensive expertise combining multiple and complementary CTC platforms and approaches unlike other groups in the country.