Department of Neurosurgery, Beth Israel Deaconess Medical Center, Boston, MA, USA.
Br J Cancer. 2011 Oct 11;105(8):1235-43. doi: 10.1038/bjc.2011.345. Epub 2011 Sep 20.
Epidermal growth factor receptor (EGFR) signalling is frequently altered during glioblastoma de novo pathogenesis. An important downstream modulator of this signal cascade is SHP2 (Src homology domain-containing phosphatase 2).
We examined the The Cancer Genome Atlas (TCGA) database for SHP2 mutations. We also examined the expression of a further 191 phosphatases in the TCGA database and used principal component and comparative marker analysis available from the Broad Institute to recapitulate the TCGA-defined subgroups and identify the specific phosphatases defining each subgroup. We identified five siRNAs from two independent commercial sources that were reported by the vendor to be pre-optimised in their specificity of SHP2 silencing. The specificity and physiological effects of these siRNAs were tested using an in vitro glioma model.
TCGA data demonstrate SHP2 to be mutated in 2% of the glioblastoma multiforme's studied. Both mutations identified in this study are likely to be activating mutations. We found that the four subgroups of GBM as defined by TCGA differ significantly with regard to the expression level of specific phosphatases as revealed by comparative marker analysis. Surprisingly, the four subgroups can be defined solely on the basis of phosphatase expression level by principal component analysis. This result suggests that critical phosphatases are responsible for the modulation of specific molecular pathways within each subgroup. Src homology domain-containing phosphatase 2 constitutes one of the 12 phosphatases that define the classical subgroup. We confirmed the biological significance by siRNA knockdown of SHP2. All five siRNAs tested reduced SHP2 expression by 70-100% and reduced glioblastoma cell line growth by up to 80%. Profiling the established molecular targets of SHP2 (ERK1/2 and STAT3) confirmed specificity of these siRNAs. The loss of cell viability induced by SHP2 silencing could not be explained by a significant increase in apoptosis alone as demonstrated by terminal deoxyribonucleotidyl transferase-mediated nick-end labelling and propidium iodide staining. Src homology domain-containing phosphatase 2 silencing, however, did induce an increase in β-galactosidase staining. Propidium iodide staining also showed that SHP2 silencing increases the population of glioblastoma cells in the G1 phase of the cell cycle and reduces the population of such cells in the G2/M- and S-phase.
Src homology domain-containing phosphatase 2 promotes the growth of glioblastoma cells by suppression of cellular senescence, a phenomenon not described previously. Selective inhibitors of SHP2 are commercially available and may be considered as a strategy for glioblastoma therapy.
表皮生长因子受体（EGFR）信号在胶质母细胞瘤新生发病机制中经常发生改变。该信号级联的一个重要下游调节剂是 SHP2（Src 同源结构域含磷酸酶 2）。
我们在癌症基因组图谱（TCGA）数据库中检查 SHP2 突变。我们还在 TCGA 数据库中检查了另外 191 种磷酸酶的表达，并使用 Broad 研究所提供的主成分和比较标记分析来重现 TCGA 定义的亚组，并确定定义每个亚组的特定磷酸酶。我们从两个独立的商业来源中确定了五个 siRNA，供应商报告说这些 siRNA 在 SHP2 沉默的特异性方面经过了预先优化。我们使用体外神经胶质瘤模型测试了这些 siRNA 的特异性和生理效应。
TCGA 数据表明，2%的多形性胶质母细胞瘤发生了 SHP2 突变。本研究中鉴定的两种突变均可能为激活突变。我们发现，TCGA 定义的四个 GBM 亚组在特定磷酸酶的表达水平上有显著差异，这可以通过比较标记分析来揭示。令人惊讶的是，四个亚组可以仅根据主成分分析的磷酸酶表达水平来定义。这一结果表明，关键的磷酸酶负责调节每个亚组内的特定分子途径。含 Src 同源结构域的磷酸酶 2 是定义经典亚组的 12 种磷酸酶之一。我们通过 SHP2 的 siRNA 敲低证实了这一生物学意义。测试的所有五个 siRNA 都将 SHP2 表达降低了 70-100%，并将神经胶质瘤细胞系的生长降低了高达 80%。对 SHP2 建立的分子靶点进行分析，证实了这些 siRNA 的特异性。如末端脱氧核苷酸转移酶介导的缺口末端标记和碘化丙啶染色所示，SHP2 沉默诱导的细胞活力丧失不能仅通过明显增加细胞凋亡来解释。然而，含 Src 同源结构域的磷酸酶 2 沉默确实会诱导β-半乳糖苷酶染色增加。碘化丙啶染色还表明，SHP2 沉默会增加神经胶质瘤细胞在细胞周期 G1 期的数量，并减少 G2/M 和 S 期的此类细胞数量。
含 Src 同源结构域的磷酸酶 2 通过抑制细胞衰老来促进神经胶质瘤细胞的生长，这是以前未描述过的现象。SHP2 的选择性抑制剂已上市，可考虑作为神经胶质瘤治疗的一种策略。
