Dissecting chromatin and cytoskeletal tumour suppressor functions of SETD2 in ccRCC

该项目旨在了解SETD 2肿瘤抑制蛋白的功能丧失如何诱导透明细胞肾细胞癌（ccRCC）的发展和进展。我们认为，SETD 2不仅通过其已建立的染色质调节功能来控制基因转录，而且还通过肌动蛋白和/或微管蛋白细胞骨架的酶修饰和调节来抑制肿瘤侵袭和转移。SETD 2写入微管蛋白K40 me 3标记，并且这种酶修饰已被证明对于有丝分裂纺锤体的正确功能是重要的。虽然这种微管蛋白修饰也存在于间期细胞中，但SETD 2在调节细胞质微管中的潜在功能尚未被发现。类似地，虽然SETD 2三甲基化肌动蛋白的K68，这种修饰的细胞质功能仍然不清楚。我们已经确定，当用激活整联蛋白信号传导的基质胶或ECM组分刺激时或当在内皮细胞层上培养时，SETD 2敲除的ccRCC细胞在划痕伤口愈合测定中表现出运动性升高，在transwell测定中表现出侵袭性增加。在体内，SETD 2敲除增加了异种移植肿瘤的转移。已知肌动蛋白和微管蛋白在介导细胞侵袭过程中的作用，以及SETD 2在通过赖氨酸三甲基化调节肌动蛋白和微管蛋白中的作用，我们将研究SETD 2调节细胞质细胞骨架响应整合素介导的细胞外信号的信号传导事件。我们将进行肌动蛋白和微管蛋白细胞骨架的定量分析以及活细胞成像，以评估SETD 2突变对肌动蛋白和微管蛋白丝动态周转的影响。我们将研究拯救SETD 2功能的不同方面（染色质与细胞骨架）对侵袭性表型的影响。我们试图获得机制洞察如何设置2调节细胞迁移。我们还旨在产生新的ccRCC的本地小鼠模型。我们将通过产生肾上皮特异性诱导型Vhl/Pbrm 1/Setd 2缺失小鼠来重建在人ccRCC中频繁出现的VHL、PBRM 1和SETD 2的三重双等位基因失活的突变基因型，以允许在最相关的生理环境中评估SETD 2功能。我们设想，这种基因缺失的组合将产生一种新的小鼠ccRCC模型，我们将使用现有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测序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SETD 2和PBRM 1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。

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.