Division of Pediatric Oncology and Departments of Pathology and Pediatrics and Adolescent Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Clin Cancer Res. 2011 Jun 1;17(11):3590-9. doi: 10.1158/1078-0432.CCR-10-3349.
BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain.
The constitutively active BRAF(V600E) allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16(INK4a) levels in pilocytic astrocytoma.
BRAF(V600E) expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF(V600E)-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16(INK4a) whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16(INK4a) immunoreactivity in the majority of cases, but patients with tumors negative for p16(INK4a) had significantly shorter overall survival.
BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16(INK4a) expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.
BRAF 基因融合或点突变在毛细胞型星形细胞瘤中经常被激活，这是最常见的儿童脑肿瘤。我们研究了组成性 BRAF 激活对正常人类神经干细胞和祖细胞的功能影响，以确定其在脑肿瘤诱导中的作用。
将组成性激活的 BRAF(V600E)等位基因导入人神经球，并检测其对 MAPK（丝裂原活化蛋白激酶）信号转导、增殖、软琼脂集落形成、干细胞表型和诱导细胞衰老的影响。免疫组织化学检测毛细胞型星形细胞瘤中 p16(INK4a)的水平。
BRAF(V600E)表达最初强烈促进集落形成，但不会导致增殖显著增加。BRAF(V600E)表达的细胞随后停止增殖，并诱导致癌基因诱导的衰老标志物，包括酸性β-半乳糖苷酶、PAI-1 和 p16(INK4a)，而对照细胞则没有。衰老的发生与神经干细胞标志物包括 SOX2 的表达下调有关。原代毛细胞型星形细胞瘤培养也显示出酸性β-半乳糖苷酶活性的诱导。对 66 例毛细胞型星形细胞瘤的免疫组织化学检查显示，大多数病例存在 p16(INK4a)免疫反应性，但 p16(INK4a)阴性肿瘤患者的总生存期明显缩短。
BRAF 在人类神经干细胞和祖细胞中的激活最初促进软琼脂中的克隆生长，提示部分细胞转化，但致癌基因诱导的衰老随后限制了增殖。BRAF 诱导的衰老可能有助于解释毛细胞型星形细胞瘤的低级别病理生物学，而与缺乏 p16(INK4a)表达的肿瘤相关的更差的临床结果可能反映了衰老诱导的失败或致癌基因诱导的衰老的逃逸。
