Investigation of genetic and translational effects of tumor suppressor proteins in two hereditary forms of renal cell carcinoma
von Katja Dinkelborg
Datum der mündl. Prüfung:2021-06-09
Betreuer:Prof. Dr. Matthias Dobbelstein
Gutachter:Prof. Dr. Matthias Dobbelstein
Gutachter:Prof. Dr. Dörthe Katschinski
EnglischSome familial cancer syndromes are known to cause renal cell carcinoma (RCC) in afflicted subjects. I studied the role of tumor suppressor genes in two syndromes: Birt-Hogg-Dubé (BHD) and von Hippel-Lindau (VHL). BHD is caused by a germline mutation of the tumor suppressor gene folliculin (FLCN). Patients are at risk of developing RCC throughout their lifetime. In order to functionally classify the FLCN germ line mutations encountered during genetic testing, we established an in vivo assay, based on a mouse xenograft model. In this model, reintroduction of wildtype FLCN into FLCN-/- cells, when implanted into immunocompromised mice, showed a significant reduction of tumor growth compared to a comparable transfection of a non-functional exon 5 deletion. This particular FLCN deletion (FLCN p.Gly84_Glu132del) has been detected in a 14 year old patient with RCC of the left kidney. Germ line mutations in the tumor suppressor von Hippel-Lindau (VHL) gene cause VHL disease, which can lead to the development of clear cell RCC. VHL is also mutated autosomal in the majority of sporadic clear cell RCC. Binding of the VHL protein to hypoxia inducible factors (HIF) leads to their degradation and subsequent suppression of tumor growth. Downregulation of the HIF2a subunit is necessary and sufficient for tumor growth suppression and thus a promising target for cancer therapy. Small molecules inhibiting translation of HIF2a as chemical biology probes were used to identify proteins that regulate HIF expression and therefore may be involved in initiation and/or progression of RCC. We chemically modified the compounds to track their interaction with cellular protein-targets by incorporating an alkyne group in its structure. This compound (83), because of the alkyne group, can be isolated or marked with azide containing probes via click chemistry. Compound 83 can be biotinylated with biotin-azide through a click reaction after cell lysis of treated VHL-/- cells. Proteins in these cell lysates bound in a strong way to compound 83 can be western blotted and made visible using streptavidin. This method showed a specific reproducible pattern of protein bands and ensured specificity. Using click chemistry, we performed two different isolation methods to later identify candidate proteins through mass spectrometry analysis. Among others, iron regulatory protein 1, peflin and Tumor susceptibility gene 101 were indicated as the most promising candidate target proteins by these methods. However, further experiments will have to be performed in order to evaluate these results. Both FLCN and VHL play an important role in the development of renal cell carcinoma. The results presented here offer an assay to test FLCN mutations seen in BHD patients. Also, they give new insights into investigation of hypoxia signaling and its role in RCC.
Keywords: Renal Cell Carcinoma; von Hippel Lindau; Birt-Hogg-Dubé; Folliculin