OTX2 drives medulloblastoma proliferation via direct regulation of cell cycle genes and inhibits differentiation

The transcription factor OTX2 has been implicated as an oncogene in medulloblastoma, which is the most common malignant brain tumor in children. It is highly expressed in most medulloblastomas and amplified in a subset of them. The role of OTX2 in medulloblastoma and its downstream targets are unclear. Therefore, we generated D425 medulloblastoma cells in which we can silence endogenous OTX2 by inducible shRNA. Silencing of OTX2 strongly inhibited cell proliferation and resulted in a neuronal-like differentiation. Expression profiling of time courses after silencing showed a progressive change in gene expression for many cellular processes. Down regulated genes were highly enriched for cell cycle and visual perception genes, while up regulated genes were enriched for genes involved in development and differentiation. This shift in expression profiles is reminiscent to changes described to occur during normal cerebellum development. OTX2 is expressed in proliferating granular progenitor cells, but the expression diminishes when these cells exit the cell cycle and start differentiating. ChIP-on-chip analyses of OTX2 in D425 cells showed that cell cycle and perception genes were direct OTX2 targets, while regulation of most differentiation genes appears to be indirect. These analyses provide the first insight in the molecular network of OTX2, demonstrating that OTX2 is essential in medulloblastoma and directly drives proliferation by regulating the expression of cell cycle genes. Since many of these genes also correlate in expression with OTX2 in primary tumors, they might be potential targets for therapy in medulloblastoma patients. Keywords: OTX2, medulloblastoma, mRNA profiling *** This Series represents the gene expression component of the study. Three independent time course experiments of OTX2 silencing, and 1 control experiment in D425 medulloblastoma cells.

转录因子OTX2（transcription factor OTX2）已被认定为髓母细胞瘤（medulloblastoma，儿童最常见的恶性脑肿瘤）中的致癌基因。该因子在多数髓母细胞瘤中呈高表达，并在部分病例中发生扩增。目前OTX2在髓母细胞瘤中的作用及其下游靶标仍不明确。
因此，我们构建了可通过诱导型短发夹RNA（inducible shRNA）沉默内源性OTX2的D425髓母细胞瘤细胞系。OTX2的沉默可显著抑制细胞增殖，并诱导神经元样分化。
对沉默后的时间进程进行基因表达谱分析显示，众多细胞过程相关基因的表达呈进行性改变。下调基因显著富集于细胞周期与视觉感知相关基因，而上调基因则富集于发育与分化相关基因。这种表达谱变化与正常小脑发育过程中的报道改变高度相似。
OTX2在增殖的颗粒层祖细胞中表达，但当这些细胞退出细胞周期并启动分化时，其表达会逐渐减弱。
对D425细胞中OTX2的染色质免疫沉淀-芯片（ChIP-on-chip）分析显示，细胞周期与感知相关基因为OTX2的直接靶标，而多数分化相关基因的调控则为间接调控。
这些分析首次揭示了OTX2的分子调控网络，证实OTX2在髓母细胞瘤中发挥必需作用，并通过调控细胞周期基因的表达直接驱动细胞增殖。由于这些基因中的多数在原发性髓母细胞瘤中也与OTX2的表达存在相关性，它们或可成为髓母细胞瘤患者的潜在治疗靶点。
关键词：OTX2、髓母细胞瘤、mRNA表达谱
*** 本系列代表本研究的基因表达组学分析部分：包含3次独立的OTX2沉默时间进程实验，以及1次D425髓母细胞瘤细胞对照实验。