PO.ET09.01 · 实验与分子治疗
将VHL与SETD2联系于一条汇聚于有丝分裂纺锤体的共同致癌通路
Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitotic spindle
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：3号染色体短臂（3p）缺失是透明细胞肾细胞癌（ccRCC）的标志性事件，导致VHL（von Hippel-Lindau）和SETD2（含Set结构域2）（以及位于3p上的其他肿瘤抑制因子）的单等位基因缺失。VHL的二次打击使这一关键肿瘤抑制因子失活，启动肿瘤进展。SETD2是一种组蛋白甲基转移酶，此前已被证明具有双重功能，可甲基化组蛋白和微管，从而参与组蛋白密码和微管蛋白密码。SETD2对微管的甲基化发生在有丝分裂纺锤体处，对于正常的有丝分裂和胞质分裂至关重要；SETD2的缺失是凋亡的强驱动因素。这引出一个难题：癌细胞如何在SETD2的早期单等位基因缺失中存活并逃避细胞死亡。
方法：使用生化激酶实验和质谱分析，我们鉴定出SETD2是AURKA的底物。此外，我们采用免疫印迹和免疫荧光实验来研究SETD2的磷酸化及其对染色质和细胞骨架靶点的影响。
结果：我们鉴定出有丝分裂激酶Aurora激酶A（AURKA）是SETD2的调控因子。我们的数据揭示SETD2是AURKA磷酸化的独特底物，质谱分析确定丝氨酸2080（S2080）为SETD2上的磷酸化位点。我们发现AURKA在S2080位点对SETD2的磷酸化有助于其对微管的甲基转移酶（即酶促）活性，但不影响H3K36处的染色质甲基化，后者保持不变。我们证明VHL通过AURKA调控SETD2，而SETD2磷酸化的丧失导致有丝分裂缺陷和基因组不稳定性。重要的是，我们证明在VHL和SETD2缺陷的情况下，抑制AURKA具有合成致死性。
结论：AURKA表达水平在VHL缺失细胞中很高，这是由于VHL无法将AURKA靶向降解所致，我们的数据现在凸显了VHL与SETD2之间的直接联系，这两个此前被认为独立驱动RCC发病机制的肿瘤抑制因子。总之，我们的数据揭示了一种与有丝分裂脆弱性相关的肿瘤特异性易感性，可被精准靶向以最终驱动有丝分裂灾难。
*报告者及主要作者。
查看英文原文 English abstract
Background: Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL ( von Hippel-Lindau ) and SETD2 ( Set-domain containing 2 ) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor, initiating tumor progression. SETD2, a histone methyltransferase, has been previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis; the loss of SETD2 acts as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive the early mono-allelic loss of SETD2 , escaping cell death.
Methods: Using biochemical kinase assays and mass spectrometry, we have identified SETD2 as a substrate for AURKA. Additionally, we have employed immunoblotting and immunofluorescence assays to investigate the phosphorylation of SETD2 and its effects on both chromatin and cytoskeleton targets.
Results: We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. We found that phosphorylation of SETD2 by AURKA at S2080 contributes to its methyltransferase (i.e., enzymatic) activity on microtubules but does not impact chromatin methylation at H3K36, which remains unchanged. We demonstrate that VHL regulates SETD2 through AURKA, and the loss of phosphorylation on SETD2 leads to mitotic defects and genomic instability. Importantly, we demonstrate that inhibition of AURKA is synthetic lethal in the setting of VHL and SETD2 deficiency.
Conclusions: AURKA expression levels are high in VHL -null cells, resulting from an inability of VHL to target AURKA for degradation, and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to drive RCC pathogenesis independently. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to drive mitotic catastrophe ultimately.
*Presenting and primary author.
利益披露 Disclosure
M. Motrapu, None.