Screening of inhibitors of SIRT1 and SIRT2 for the prevention of neuroblastoma

描述（由申请人提供）： 背景：神经母细胞瘤是儿童早期最常见的实体瘤。N-Myc癌基因扩增引起的神经母细胞瘤是一种侵袭性更强的亚型，临床结局更差。当通过细胞外信号调节蛋白激酶（ERK）在丝氨酸62处磷酸化时，N-Myc蛋白是稳定的。 III类组蛋白去乙酰化酶SIRT 1诱导组蛋白去乙酰化、启动子CpG岛甲基化、转录抑制和肿瘤抑制蛋白的去乙酰化。通过阻断细胞凋亡和促进细胞生长，SIRT 1可能在肿瘤的发生和发展中发挥关键作用。关于SIRT 2在癌症中的作用知之甚少。作为SIRT 1和SIRT 2的小分子抑制剂的阿替福林、cambinol和tenovin-6在各种癌症动物模型中显示出有希望的抗癌作用。 我们发现N-Myc诱导SIRT 1和SIRT 2在神经母细胞瘤细胞中过表达，SIRT 1和SIRT 2阻断蛋白酶体介导的N-Myc蛋白降解，并且SIRT 1或SIRT 2的抑制诱导细胞生长停滞。SIRT 1/SIRT 2抑制剂cambinol促进N-Myc蛋白降解并诱导剂量依赖性细胞生长停滞。此外，SIRT 1和SIRT 2抑制丝裂原活化蛋白激酶磷酸酶3（MKP 3）的基因转录，MKP 3使ERK去磷酸化和失活。目的：（1）证明SIRT 1和SIRT 2通过抑制MKP 3表达和调节N-Myc蛋白磷酸化来稳定N-Myc蛋白;（2）比较SIRT 1/SIRT 2抑制剂在促进N-Myc蛋白降解和阻断神经母细胞瘤和癌前神经母细胞瘤细胞的增殖和/或存活方面的功效;（3）鉴定预防神经母细胞瘤的最佳选择的SIRT 1/SIRT 2抑制剂。研究设计和方法：（i）SIRT 1和SIRT 2是否通过阻断N-Myc癌蛋白泛素化来阻断N-Myc癌蛋白降解？(ii)SIRT 1和SIRT 2是否通过抑制MKP 3基因转录，增强ERK磷酸化，从而诱导N-Myc蛋白在丝氨酸62位磷酸化来稳定N-Myc癌蛋白？(iii)SIRT 1/SIRT 2抑制剂是否能重新激活MKP 3基因表达，使ERK去磷酸化，促进N-Myc蛋白降解，并诱导神经母细胞瘤细胞生长停滞和/或凋亡？(iv)SIRT 1/SIRT 2抑制剂是否能重新激活N-Myc转基因小鼠癌前成神经细胞中MKP 3基因表达、ERK去磷酸化、促进N-Myc蛋白降解并诱导生长停滞和/或凋亡？(v)研究和比较SIRT 1/SIRT 2抑制剂对N-Myc转基因小鼠神经母细胞瘤的预防作用。

DESCRIPTION (provided by applicant): Background: Neuroblastoma is the commonest solid tumour in early childhood. Neuroblastoma due to N-Myc oncogene amplification represents a more aggressive subtype with a worse clinical outcome. N-Myc protein is stabilized when phosphorylated at Serine 62 by extracellular signal-regulated protein kinase (ERK). The class III histone deacetylase SIRT1 induces histone deacetylation, promoter CpG island methylation, transcriptional repression, and deacetylation of tumour suppressor proteins. By blocking apoptosis and promoting cell growth, SIRT1 may play a critical role in tumour initiation and progression. Much less is known about the role of SIRT2 in cancer. Aristoforin, cambinol and tenovin-6, which are small molecule inhibitors of both SIRT1 and SIRT2, have shown promising anti-cancer effects in various animal models of cancer. We have found that N-Myc induces SIRT1 and SIRT2 over-expression in neuroblastoma cells, that SIRT1 and SIRT2 block proteasome-mediated N-Myc protein degradation, and that repression of SIRT1 or SIRT2 induces cell growth arrest. The SIRT1/SIRT2 inhibitor cambinol promotes N-Myc protein degradation and induces dose-dependant cell growth arrest. Moreover, SIRT1 and SIRT2 repress gene transcription of mitogen-activated protein kinase phosphotase 3 (MKP3), which dephosphorylates and inactivates ERK. Aims: (1) To demonstrate that SIRT1 and SIRT2 stabilize N-Myc protein by repressing MKP3 expression and modulating N-Myc protein phosphorylation; (2) To compare the efficacy of SIRT1/SIRT2 inhibitors in promoting N-Myc protein degradation and blocking proliferation and/or survival of neuroblastoma and pre-cancer neuroblast cells; (3) To identify the SIRT1/SIRT2 inhibitor of the best choice for prevention of neuroblastoma. Research design and methods: (i) Do SIRT1 and SIRT2 block N-Myc oncoprotein degradation by blocking N-Myc oncoprotein ubiquitination? (ii) Do SIRT1 and SIRT2 stabilize N-Myc oncoprotein by repressing MKP3 gene transcription, enhancing ERK phosphorylation, and consequently inducing N-Myc protein phosphorylation at Serine 62? (iii) Do SIRT1/SIRT2 inhibitors reactivate MKP3 gene expression, dephosphorylate ERK, promote N-Myc protein degradation, and induce growth arrest and/or apoptosis in neuroblastoma cells? (iv) Do SIRT1/SIRT2 inhibitors reactivate MKP3 gene expression, dephosphorylate ERK, promote N-Myc protein degradation, and induce growth arrest and/or apoptosis in pre-cancer neuroblasts from N-Myc transgenic mice? (v) To examine and compare the efficacy of SIRT1/SIRT2 inhibitors in the prevention of neuroblastoma in N- Myc transgenic mice.