The study of underlying mechanism of EGFR-Ras signaling in glioblastoma

准确的基因工程小鼠（GEM）疾病模型可能是疾病机制研究和药物测试的有力工具。我们使用这个工具来研究胶质母细胞瘤（GBM），这是一种临床上非常具有挑战性的疾病。在之前的一项研究中，我们通过将通路畸变[视网膜母细胞瘤蛋白（RB）， K-Ras和磷酸酶和紧张素同源物（PTEN）]靶向人类星形细胞瘤中经常失调的星形细胞，建立了可诱导的基因工程小鼠模型。每一个基因工程小鼠系都是一个有条件的柔停或柔外显子系，其中突变的激活依赖于活性Cre酶的存在。我们将这些小鼠与GFAP- creer小鼠杂交，发现突变仅限于表达GFAP的星形细胞细胞。这些突变在任意时间点都是可诱导的，因为它们是潜伏的，直到小鼠腹腔注射4-羟基他莫昔芬（4-OHT）来激活Cre活性。这种方法导致了GBM的逐步发展。简而言之，单独具有异常视网膜母细胞瘤蛋白通路的基因工程小鼠发展为低级别星形细胞瘤（II级）。组构性K-Ras激活和PTEN失活都不能单独产生可检测的脑病理。含有灭活的视网膜母细胞瘤蛋白和组成性活性K-Ras的基因工程小鼠发生高级别星形细胞瘤（III级）；随后的PTEN失活产生具有胶质母细胞组织病理学特征的肿瘤（IV级）。为了确定表皮生长因子受体（EGFR）在星形细胞瘤/胶质母细胞瘤发生中的作用，我们首先在我们的模型中研究了表皮生长因子受体（EGFR）是否在胶质母细胞瘤的发展中被激活。特别是，我们检查了同时具有K-Ras突变和Rb失活的模型。我们在诱导后的不同时间点收获了小鼠的大脑。通过对这些样本的分析，我们发现EGFR信号被多种方法激活，包括免疫组织化学、western blot和实时逆转录酶PCR。通过实时逆转录酶PCR，我们发现除了EGFR本身，肿瘤中EGFR配体的水平也增加了。我们证实，在K-Ras突变和Rb失活的小鼠模型中，EGFR信号被激活。同时，我们通过基因敲除EGFR基因来检测该模型的肿瘤发生是否需要EGFR信号。我们将上述基因工程小鼠模型与EGFR条件敲除小鼠系杂交。如果肿瘤发生需要EGFR，那么肿瘤的发展有望在一定程度上受到抑制。然而，我们的研究结果表明，在EGFR丢失时，肿瘤实际上进展到更晚期，这表明在EGFR丢失时，某种代偿机制促进了肿瘤的发生。为了探索其潜在的机制，我们比较了有和没有EGFR的肿瘤中EGFR信号和相关受体酪氨酸激酶信号。为此，我们使用了脑组织和肿瘤细胞培养物。我们在EGFR野生型、EGFR异质缺失和EGFR均质缺失的肿瘤上产生了多种肿瘤细胞系。我们对培养的肿瘤细胞做了一些测试。所有的结果都支持EGFR丢失后肿瘤发生的代偿机制的可能性。我们还收集了一组星形细胞瘤/胶质母细胞瘤样本进行无偏差微阵列实验，这将有助于我们了解其潜在机制。总预算的47%用于建立新实验室。

Accurate genetically engineered mouse (GEM) disease models are potentially very powerful tools for disease mechanistic studies and drug tests. We used this tool to study glioblastoma (GBM), a very challenging disease clinically. In a previous study, we generated inducible genetically engineered mouse models by targeting pathway aberrations [retinoblastoma protein (RB), K-Ras and phosphatase and tensin homolog (PTEN)] to astrocytes that are frequently dysregulated in human astrocytomas. Each genetically engineered mouse line is a conditional floxed-stop or floxed-exon line, in which the activation of mutation is dependent on the presence of active Cre enzyme. We crossed those mice to GFAP-CreER mouse line, so that the mutations are restricted in the glial fibrillary acidic protein (GFAP) expressing astrocytic cells. The mutations are inducible at arbitrary time points because they are latent until mice are intraperitoneally injected with 4-hydroxytamoxifen (4-OHT) to activate Cre activity. This approach has resulted in stepwise progression of GBM. In brief, genetically engineered mice with aberrant retinoblastoma protein pathway alone developed low-grade astrocytomas (grade II). Neither constitutive K-Ras activation nor PTEN inactivation alone produced detectable brain pathology. Genetically engineered mice harboring both inactivated retinoblastoma protein and constitutively active K-Ras developed high-grade astrocytomas (grade III); subsequent PTEN inactivation produced tumors with histopathological features of glioblastom (grade IV). To determine the role in the astrocytoma/glioblastoma genesis of epidermal growth factor receptor (EGFR), a frequent mutation and a hot therapeutic target in glioblastoma treatments, we firstly investigated whether EGFR is activated in glioblastoma development in our models. Particularly, we examined the model that has both K-Ras mutation and Rb inactivation. We have harvested the brains of mice at different time points after induction. After analyses of those samples, we have found that EGFR signal is activated by multiple methods, including immunohistochemistry, western blot and real time reverse transcriptase PCR. We found that, other than EGFR itself, the level of EGFR ligands were also increased in the tumors by using real time reverse transcriptase PCR. We confirmed that EGFR signal was activated in the mouse model that has both K-Ras mutation and Rb inactivation. In the meanwhile, we tested if EGFR signal was required for the tumorigenesis in this model by genetically knocking out the EGFR gene. We have crossed the above genetically engineered mouse model to EGFR conditional knockout mouse line. If EGFR is required for the tumorigenesis, then tumor development was expected to be inhibited to a certain degree. However, our results showed that tumors actually progressed to a more advanced stage upon the loss of EGFR, suggesting some compensatory mechanism contributing to the tumorigenesis upon EGFR loss. To explore the underlying mechanism, we have compared the EGFR signal and related receptor tyrosine kinase signals between the tumors with and without EGFR. To do this, we used both brain tissues and tumor cell cultures. We have generated multiple tumor cell lines on tumors with EGFR wild type, EGFR heterogeneous deletion and EGFR homogeneous deletion. We have done some testing on the culture tumor cells. All of the results support the possibility of a compensatory mechanism of tumorigenesis upon EGFR loss. We also have collected a bank of astrocytoma/glioblastoma samples for the non-bias micro array experiment, which will help us understand the underlying mechanisms. 47% of the overall budget was spent on establishing the new laboratory.