A single kinase modulates the diverse extracellular phosphoproteome of glioblastoma multiforme

“现在对癌症病理学最好的研究是那些研究种子性质的人……但他对土壤性质的观察也可能是有用的。”斯蒂芬·佩杰博士，《柳叶刀》，1889年。佩杰博士1889年提出的“种子和土壤假说”在过去130多年里一直是一个核心概念。我们已经知道，癌细胞或“种子”与周围组织或间质或“土壤”之间复杂的相互作用在促进癌症方面起着至关重要的作用。在这种背景下，我的提议计划建立一个主要参与者，同时调节多形性胶质母细胞瘤(GBM)的“种子”和“土壤”。IV期脑癌或多形性胶质母细胞瘤(GBM)是一个重大挑战，只有4种FDA批准的化疗药物可用于治疗。自最初诊断以来，每10名患者中只有1名能存活5年以上。当低级别脑瘤进展到IV级GBM时，会发生多种变化，包括其染色体7的倍增。事实上，在所有患者中，具有7号染色体倍增的GBM肿瘤的患者预后最差。世界各地的许多实验室都在努力了解7号染色体特异基因如何被用作脑癌分级进展的标志，然而，所有此类研究都需要首先切除肿瘤的高风险神经外科手术。研究切除的GBM肿瘤或GBM患者血液的不同小组报告称，在较高级别的脑肿瘤患者中，各种分泌的蛋白质、酶和生长因子升高。这些分泌的蛋白质或者直接促进基底膜肿瘤的生长，或者从周围的组织或‘土壤’为肿瘤生长提供外部支持，也被称为‘基质支持’。尽管许多实验室都在研究这些分泌的蛋白质，但很少有人意识到所有这些分泌的蛋白质都是通过一种分泌的酶被磷酸分子(称为磷酸化)修饰的。有趣的是，该酶存在于7号染色体上，并在IV期GBM中显著升高。一旦升高，酶的活性就会增加20倍，它会在分泌的蛋白质上放置多种磷酸盐，然后“磷酸化”的蛋白质在肿瘤内发挥它们的功能，然后经常从肿瘤进入血液。因此，如果我们能够研究酶催化的分泌蛋白的磷酸化如何在直接的肿瘤生长或间质支持的背景下影响蛋白质的功能的机制，我们将能够开发新的疗法来干预这些机制。此外，捕获患者血液中分泌蛋白的磷酸化形式将使我们能够预测以下情况：这种酶在肿瘤中是否更活跃？如果是这样的话，7号染色体很可能是倍增的，所以肿瘤可能是IV级的。为了确定这一点，我建议采取以下步骤：1.研究磷酸化的分泌蛋白如何促进GBM肿瘤或种子的生长。我们将利用原代患者来源的GBM细胞和Crispr/Cas9基因组编辑来研究小鼠脑内GBM细胞的生长和侵袭特性。2.确定细胞外的磷酸化蛋白如何调节细胞内的生长信号，促进GBM肿瘤的发生。将采用各种生化、生物物理和质谱学方法来解决这一目标。建立有史以来第一个预测脑癌的磷酸蛋白检测血液试验。阐明这种酶如何为GBM肿瘤的生长提供细胞外支持或“土壤”。我们将利用不同的脑癌小鼠模型来研究大脑中的酶如何支持肿瘤的生长。成功完成这些目标将建立多种预测、预后和治疗基底膜的生物标志物。建立一种血液测试来检测低级别到高级别脑瘤的进展可能是这十年的一项重大突破。

'The best work on the pathology of cancer is now done by those who are studying the nature of the seed......but his observation of the properties of the soil may also be useful'. Dr. Stephen Paget, The Lancet, 1889. Dr. Paget's 'seed and soil hypothesis' of 1889 remains a central concept for the last 130+ years. We have come to know that an intricate cross-talk between cancer cells or 'seed' and surrounding tissue or stroma or 'soil' plays a vital role in promoting cancer. In that context, my proposal plans to establish a major player modulating both the 'seed' and the 'soil' of glioblastoma multiforme (GBM). Stage IV brain cancer or glioblastoma multiforme (GBM) poses a major challenge with only 4 FDA approved chemotherapeutic drugs available for treatment. Since initial diagnosis, only 1 out of 10 patients will survive beyond 5 years. When a low- grade brain tumour progresses to grade IV GBM, multiple changes take place including multiplication of its chromosome7. In fact, patients with GBM tumours that exhibit chromosome7 multiplication have the poorest outcome of all patients. Efforts are underway across many laboratories worldwide to understand how chromosome 7 specific genes could be used as markers for grade progression of brain cancer, however, all such studies require risky neurosurgery for removal of the tumour first. Various groups studying the excised GBM tumour or blood of GBM patients have reported various secreted proteins, enzymes, growth factors to be elevated in higher grade brain tumour patients. These secreted proteins either directly promote the GBM tumour growth or provide support externally for tumour growth from the surrounding tissue or 'soil' also referred to as 'stromal support'. Although many laboratories study these secreted proteins, very few realize that all of these secreted proteins are modified by phosphate molecules (called phosphorylation) by a single secreted enzyme. Interestingly, the enzyme is present on chromosome7 and remarkably elevated in stage IV GBM. Upon elevation, the enzyme activity is increased by >20-fold and it puts multiple phosphates on the secreted proteins and then the 'phosphorylated' proteins carry out their functions within the tumour and then often travel from the tumour to blood. Hence if we can study the mechanism of how the enzyme catalysed phosphorylation of the secreted protein affect the function of the proteins either in the context of direct tumour growth or stromal support, we would be able to develop novel therapies to intervene in those mechanisms. Furthermore, capturing the phosphorylated form of the secreted proteins in patients' blood will allow us to predict the following: Is the enzyme more active in the tumour? If so, chromosome7 is probably multiplied, so the tumour might be grade IV. To establish this, I propose the following steps:1. Study how does phosphorylated secreted proteins promote GBM tumour or 'seed' growth. We will utilise primary patient derived GBM cells and Crispr/cas9 genome editing to study GBM growth and invasion properties of cells in mouse brains. 2. Identify how the extracellular phosphorylated proteins could modulate the intracellular growth signals and promote GBM tumours. Various biochemical, biophysical and mass-spectrometry approaches will be carried out to address this aim.3. Establish the first ever phospho-protein detection blood-test to predict brain cancer.4. Elucidate how the enzyme could provide extracellular support or 'soil' for GBM tumour growth. We will utilise various brain cancer mouse models to study how the enzyme in the brain could provide support to tumour growth.Successful completion of these aims will establish multiple predictive, prognostic and therapeutic biomarkers of GBM. Establishing a blood-test to detect progression of low to high-grade brain tumour could be a major breakthrough of the decade.