Dissecting chromatin and cytoskeletal tumour suppressor functions of SETD2 in ccRCC

剖析 ccRCC 中 SETD2 的染色质和细胞骨架肿瘤抑制功能

• 批准号: 419592238
• 负责人: Professor Dr. Ian Frew
• 金额: --
• 依托单位: Zentrum für Translationale Zellforschung ZTZ
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/419592238?language=en
• 关键词: Dissecting; chromatin; cytoskeletal; tumour; suppressor

This project aims to understand how loss of function of the SETD2 tumour suppressor protein induces the development and progression of clear cell renal cell carcinoma (ccRCC). We propose that SETD2 not only acts as a tumour suppressor through its established chromatin regulatory function to control gene transcription, but additionally suppresses tumour invasion and metastasis through enzymatic modification and regulation of the actin and/or tubulin cytoskeletons. SETD2 writes the tubulin K40me3 mark and this enzymatic modification has been shown to be important for correct functioning of the mitotic spindle. While this tubulin modification is also present in interphase cells, a potential function of SETD2 in the regulation of cytoplasmic microtubules has yet to be uncovered. Similarly, while SETD2 trimethylates K68 of actin, the cytoplasmic functions of this modification remain unclear. We have identified that SETD2 knockout ccRCC cells exhibit elevated motility in scratch wound healing assays, increased invasion in transwell assays when stimulated with Matrigel or ECM components that activate Integrin signalling or when cultured on endothelial cell layers. In vivo, SETD2 knockout increased metastasis of xenograft tumours. Given the known roles of actin and tubulin in mediating cellular invasive processes, and of SETD2 in regulating actin and tubulin via Lysine trimethylation, we will characterise the signalling events by which SETD2 regulates the cytoplasmic cytoskeleton in response to Integrin-mediated extracellular signals. We will conduct quantitative assays of the actin and tubulin cytoskeletons as well as live-cell imaging to assess the effects of SETD2 mutation on dynamic turnover of actin and tubulin filaments. We will characterise the effects of rescue of different aspects of SETD2 function (chromatin versus cytoskeletal) on invasive phenotypes. We seek to gain mechanistic insight into how SETD2 regulates cellular migration. We also aim to generate a new autochthonous mouse model of ccRCC. We will recreate the mutational genotype of triple biallelic inactivation of VHL, PBRM1 and SETD2 that arises frequently in human ccRCC by generating renal epithelial-specific inducible Vhl/Pbrm1/Setd2 deletion mice to allow assessment of SETD2 functions in the most relevant physiological context. We envisage that this combination of gene deletions will yield a new model of mouse ccRCC that we will study using established workflows involving live-animal renal imaging by ultrasound, histology, immunohistochemistry/fluourescence and RNA seq. We will also use primary renal epithelial cell cultures and ccRCC tumour cell lines derived from these mice to dissect the role of SETD2 and PBRM1 in regulating the cytoskeleton, mitotic chromosome segregation, cellular migratory phenotypes and epigenetic control of transcription.

该项目旨在了解 SETD2 肿瘤抑制蛋白功能的丧失如何诱导透明细胞肾细胞癌 (ccRCC) 的发生和进展。我们认为SETD2不仅通过其已建立的染色质调节功能来控制基因转录而充当肿瘤抑制因子，而且还通过肌动蛋白和/或微管蛋白细胞骨架的酶促修饰和调节来抑制肿瘤侵袭和转移。 SETD2 写入微管蛋白 K40me3 标记，这种酶促修饰已被证明对于有丝分裂纺锤体的正确功能非常重要。虽然这种微管蛋白修饰也存在于间期细胞中，但 SETD2 在细胞质微管调节中的潜在功能尚未被发现。同样，虽然 SETD2 将肌动蛋白 K68 三甲基化，但这种修饰的细胞质功能仍不清楚。我们发现，SETD2 敲除的 ccRCC 细胞在划痕伤口愈合试验中表现出较高的运动性，在用激活整合素信号传导的基质胶或 ECM 成分刺激或在内皮细胞层上培养时，在 Transwell 试验中表现出较高的侵袭性。在体内，SETD2敲除增加了异种移植肿瘤的转移。鉴于肌动蛋白和微管蛋白在介导细胞侵袭过程中的已知作用，以及 SETD2 通过赖氨酸三甲基化调节肌动蛋白和微管蛋白的已知作用，我们将描述 SETD2 响应整合素介导的细胞外信号而调节细胞质细胞骨架的信号传导事件。我们将对肌动蛋白和微管蛋白细胞骨架进行定量分析以及活细胞成像，以评估 SETD2 突变对肌动蛋白和微管蛋白丝动态周转的影响。我们将描述 SETD2 功能不同方面（染色质与细胞骨架）的挽救对侵袭表型的影响。我们寻求深入了解 SETD2 如何调节细胞迁移。我们还旨在生成一种新的 ccRCC 本土小鼠模型。我们将通过生成肾上皮特异性诱导型 Vhl/Pbrm1/Setd2 缺失小鼠来重建 VHL、PBRM1 和 SETD2 三重双等位基因失活的突变基因型，这种突变基因型在人类 ccRCC 中经常出现，以便在最相关的生理背景下评估 SETD2 功能。我们设想这种基因删除组合将产生一种新的小鼠 ccRCC 模型，我们将使用已建立的工作流程进行研究，包括通过超声、组织学、免疫组织化学/荧光和 RNA seq 进行活体动物肾脏成像。我们还将使用来自这些小鼠的原代肾上皮细胞培养物和 ccRCC 肿瘤细胞系来剖析 SETD2 和 PBRM1 在调节细胞骨架、有丝分裂染色体分离、细胞迁移表型和转录的表观遗传控制中的作用。