A single kinase modulates the diverse extracellular phosphoproteome of glioblastoma multiforme

“癌症病理方面的最佳研究工作现在是由那些研究种子性质的人完成的......但是他对土壤性质的观察也可能是有用的。斯蒂芬·佩吉特博士，《柳叶刀》，1889年。佩吉特博士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”与周围组织或基质或“土壤”之间复杂的串扰在促进癌症中起着至关重要的作用。在这种背景下，我的建议计划建立一个主要的参与者来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，只有十分之一的患者能存活5年以上。当低度脑肿瘤发展为IV级GBM时，会发生包括染色体增殖在内的多种变化。事实上，表现出7号染色体增殖的GBM肿瘤患者的预后是所有患者中最差的。世界各地的许多实验室都在努力了解如何利用7号染色体特定基因作为脑癌分级进展的标记，然而，所有这些研究都需要首先进行高风险的神经外科手术来切除肿瘤。研究切除的GBM肿瘤或GBM患者血液的各个小组都报道了各种分泌蛋白，酶，生长因子在高级别脑肿瘤患者中升高。这些分泌的蛋白质要么直接促进GBM肿瘤生长，要么从周围组织或“土壤”（也称为“基质支持”）为肿瘤生长提供外部支持。尽管许多实验室都在研究这些分泌蛋白，但很少有人意识到所有这些分泌蛋白都是由磷酸盐分子（称为磷酸化）通过一种分泌酶修饰的。有趣的是，该酶存在于7号染色体上，在IV期GBM中显著升高。升高后，酶的活性增加了20倍，它把多个磷酸盐放在分泌的蛋白质上，然后“磷酸化”的蛋白质在肿瘤内执行它们的功能，然后经常从肿瘤进入血液。因此，如果我们能够研究这种酶如何催化分泌蛋白的磷酸化在直接肿瘤生长或基质支持的情况下影响蛋白质功能的机制，我们将能够开发出新的治疗方法来干预这些机制。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问题：这种酶在肿瘤中是否更活跃？如果是这样，7号染色体很可能是倍增的，所以肿瘤可能是IV级。为了确定这一点，我提出以下步骤：研究磷酸化分泌蛋白如何促进GBM肿瘤或“种子”生长。我们将利用原代患者来源的GBM细胞和Crispr/cas9基因组编辑技术来研究小鼠大脑中GBM细胞的生长和侵袭特性。2. 确定细胞外磷酸化蛋白如何调节细胞内生长信号并促进GBM肿瘤。将采用各种生化、生物物理和质谱方法来实现这一目标。建立首个预测脑癌的磷酸化蛋白检测血液试验。阐明该酶如何为GBM肿瘤生长提供细胞外支持或“土壤”。我们将利用各种脑癌小鼠模型来研究大脑中的酶如何为肿瘤生长提供支持。成功完成这些目标将建立多种预测、预后和治疗GBM的生物标志物。建立一种血液检测方法来检测低级别到高级别脑肿瘤的进展，可能是本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.