A detailed investigation of clear cell renal cell carcinoma cell and organoid cultures to find putative therapeutic targets and novel therapy approaches

Abstract

Therapy options for metastasized renal cell carcinoma are limited. While immune checkpoint inhibitors and therapies targeting the tumor microenvironment have drastically changed treatment options, response rates are hardly predictable. Despite the development of novel therapies, clear cell renal cell carcinomas account for the most deaths of renal cancer, namely 180,000 in 2020. The scarcity of effective therapies highlights the importance of novel therapeutic targets and alternative therapy approaches for this cancer entity.<br /> Therefore, in the first part of this thesis I investigated PARK2, which codes for the protein Parkin. It was first described in autosomal recessive juvenile Parkinson’s disease, however, evidence is growing that it can also function as a tumor suppressor gene. Thus, I aimed to thoroughly investigate Parkin’s function in clear cell renal cell carcinoma cell lines.<br /> I found that overexpression of PARK2 in clear cell renal cell carcinoma cells leads to reduced aggressiveness in vitro indicated by decreased migration and invasion. Moreover, lack of Parkin caused accumulation of CKS2, which was shown by mass spectrometry. CKS2 has been described to be highly expressed in many cancers, therefore, it was intriguing to examine the link between Parkin and the expression levels of CKS2. When mutating the catalytic site of PARK2, which results in E3 ligase deficiency, the migratory capacities of clear cell renal cell carcinoma cells were abolished leading to a similar migratory phenotype as wild-type cells, which lack Parkin. Next, I found that aggressiveness can be reduced by silencing of CKS2 resulting in decreased migration, which was comparable to cells expressing Parkin. Furthermore, analysis of a patient cohort showed that high CKS2 levels are associated with high tumor grading and poor survival.<br /> In conclusion, this first part of the thesis suggests a link between Parkin and CKS2, which affects tumor aggressiveness. Parkin may influence CKS2 levels via its E3 ubiquitin ligase which causes reduced migration and invasion. Altogether, this provides valuable information that CKS2 may be a potential biomarker in clear cell renal cell carcinoma and may serve as a novel therapeutic target in the future.<br /> Since therapy responses in patients are hardly predictable, I was interested in researching a novel tool, which assists present platforms, for its suitability in clear cell renal cell carcinoma therapy testing and its potential for developing more personalized therapy approaches.<br /> For that, I used fresh tissue from 42 patients, which underwent complete or partial nephrectomy, to generate patient-derived organoids based on an air-liquid interface culture system. The generated air-liquid interface patient-derived organoids were thoroughly characterized to validate them as a novel tool for therapy testing. For that, the obtained tissue was partly minced and cultured, and partly used for isolating RNA to conduct RNA sequencing. Further, immunohistochemistry was performed to verify the similarity of the cultured air-liquid interface patient-derived organoids and the tumor of origin. Common pathological markers and cells of the tumor microenvironment could be identified. Lastly, to test it as a tool for therapy testing, ten air-liquid interface patient-derived organoids were treated with the targeted therapy agent cabozantinib or the immune checkpoint inhibitor nivolumab and responses were obtained. In agreement with observations in the clinics, the response rates varied drastically. This indicates that this model system may give valuable information about the response rates in patients and possible therapy options.<br /> In conclusion, these findings show that the established patient-derived organoids are a promising tool for therapy testing under a controlled setting. Moreover, they resemble the tumor of origin and therapy responses can be quickly obtained.