Dissecting chromatin and cytoskeletal tumour suppressor functions of SETD2 in ccRCC

• Applicant: Professor Dr. Ian Frew
• Subject Area: Reproductive Medicine, Urology ; Nephrology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 419592238

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.

• DFG Programme: Research Grants