Wang W, Zhong Q, Teng L, Bhatnagar N, Sharma B, Zhang X, Luther W, Haynes L P, Burgoyne R D, Vidal M, Volchenboum S, Hill D E, George R E
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
Oncogene. 2014 Jun 19;33(25):3316-24. doi: 10.1038/onc.2013.290. Epub 2013 Jul 22.
Heterozygous germline mutations in PHOX2B, a transcriptional regulator of sympathetic neuronal differentiation, predispose to diseases of the sympathetic nervous system, including neuroblastoma and congenital central hypoventilation syndrome (CCHS). Although the PHOX2B variants in CCHS largely involve expansions of the second polyalanine repeat within the C-terminus of the protein, those associated with neuroblastic tumors are nearly always frameshift and truncation mutations. To test the hypothesis that the neuroblastoma-associated variants exert their effects through loss or gain of protein-protein interactions, we performed a large-scale yeast two-hybrid screen using both wild-type (WT) and six different mutant PHOX2B proteins against over 10 000 human genes. The neuronal calcium sensor protein HPCAL1 (VILIP-3) exhibited strong binding to WT PHOX2B and a CCHS-associated polyalanine expansion mutant but only weakly or not at all to neuroblastoma-associated frameshift and truncation variants. We demonstrate that both WT PHOX2B and the neuroblastoma-associated R100L missense and the CCHS-associated alanine expansion variants induce nuclear translocation of HPCAL1 in a Ca(2+)-independent manner, while the neuroblastoma-associated 676delG frameshift and K155X truncation mutants impair subcellular localization of HPCAL1, causing it to remain in the cytoplasm. HPCAL1 did not appreciably influence the ability of WT PHOX2B to transactivate the DBH promoter, nor did it alter the decreased transactivation potential of PHOX2B variants in 293T cells. Abrogation of the PHOX2B-HPCAL1 interaction by shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B led to impaired neurite outgrowth with transcriptional profiles indicative of inhibited sympathetic neuronal differentiation. Our results suggest that certain PHOX2B variants associated with neuroblastoma pathogenesis, because of their inability to bind to key interacting proteins such as HPCAL1, may predispose to this malignancy by impeding the differentiation of immature sympathetic neurons.
PH0X2B是交感神经元分化的转录调节因子，其杂合种系突变易导致交感神经系统疾病，包括神经母细胞瘤和先天性中枢性低通气综合征（CCHS）。虽然CCHS中的PH0X2B变体主要涉及该蛋白质C末端第二个聚丙氨酸重复序列的扩增，但与成神经细胞瘤相关的变体几乎总是移码突变和截短突变。为了验证成神经细胞瘤相关变体通过蛋白质-蛋白质相互作用的丧失或获得发挥作用这一假设，我们使用野生型（WT）和六种不同的突变型PH0X2B蛋白对超过10000个人类基因进行了大规模酵母双杂交筛选。神经元钙传感器蛋白HPCAL1（VILIP-3）与WT PH0X2B和一个与CCHS相关的聚丙氨酸扩增突变体表现出强烈结合，但与成神经细胞瘤相关的移码突变和截短变体结合较弱或根本不结合。我们证明，WT PH0X2B以及与成神经细胞瘤相关的R100L错义突变体和与CCHS相关的丙氨酸扩增变体均以不依赖Ca(2+)的方式诱导HPCAL1的核转位，而成神经细胞瘤相关的676delG移码突变体和K155X截短突变体损害HPCAL1的亚细胞定位，使其保留在细胞质中。HPCAL1对WT PH0X2B反式激活DBH启动子的能力没有明显影响，也没有改变293T细胞中PH0X2B变体降低的反式激活潜能。在表达PH0X2B的成神经细胞瘤细胞中，通过shRNA敲低HPCAL1消除PH0X2B-HPCAL1相互作用，导致神经突生长受损，转录谱表明交感神经元分化受到抑制。我们的结果表明，某些与成神经细胞瘤发病机制相关的PH0X2B变体，由于它们无法与关键相互作用蛋白如HPCAL1结合，可能通过阻碍未成熟交感神经元的分化而导致这种恶性肿瘤。
