Renal cell carcinoma (RCC) is a cancer that originates from renal tubular epithelial cells in the kidneys. Studies focusing on RCC are vital as it accounts for around 3% of all human cancers as of 2014 and accounts for over 90% of cancers affecting the kidneys. Being a heterogenous disease, RCC consists of multiple subtypes including papillary RCC (pRCC) with a 10% incidence rate. The heterogenous nature of RCC makes it difficult to determine biomarkers and therapeutic treatments that can provide adequate treatment targeting RCC in different patients, which stresses the need to discover as many therapeutic targets as possible. OIP5, also known as Mis18β, is a 25-kDa protein that is accumulated in the telophase-G1 centromere during mitosis and plays a role in centromere/kinetochore structure formation and functionality. As Mis18β, it interacts with Mis18α in a heterotetramer complex to bind and localize centromeric protein A (CENP-A) to the proper centromere region to allow for formation for centromere/kinetochore formation and function. OIP5 is normally highly expressed in the testes but recent studies have suggested that OIP5 overexpression is linked to the progression of some types of human cancers, including promoting pRCC cell proliferation and tumorigenesis. Furthermore, past studies have shown that other genes may impact RCC progression through regulation of cell growth and substrate activation extending past just the cell cycle. PCSK5 is another target of interest of focus in this study. Known also as PC6, it is a proprotein convertase which acts on precursor proteins to turn them into their active forms through post-translational modification and plays a role in cell growth and bone remodeling. This study aims to investigate the role of OIP5 during RCC progression both in vitro and in vivo with a focus on pRCC as well as investigate the expression of PCSK5 in conjunction with OIP5 and pRCC. In vitro studies confirmed previous results that OIP5 promotes colony formation in ACHN pRCC cells. Using a bioluminescent reporter alongside ACHN OIP5 and ACHN empty vector (EV) cell lines, mice were injected through renal subcapsule transplantation. It was seen that, while OIP5 produced larger tumors over a wider area after 12 weeks, metastasis did not appear to occur as largely as hoped though this may be due to limitations of the model. Analysis of the ACHN xenografts showed increased expression of PCSK5 and PLK1 in the OIP5 tumors. For PLK1, this is likely due to the close nature of its function with OIP5 during mitosis while PCSK5 may be regulated by OIP5 through an unknown mechanism or through microRNA miR-101-5p. Despite this, mRNA levels of PCSK5 were insignificant between OIP5 and EV groups. pRCC patient data showing PCSK5 expression levels showed a minor correlation with OIP5 expression, as well as higher expression at later pRCC stages and leading to reduced survival probability. Overall, this thesis analyzes the relationship of OIP5 in RCC, specifically pRCC, and introduces PCSK5 as another intriguing target for further study in treating pRCC. / Thesis / Master of Science (MSc) / This project focuses on renal cell carcinoma (RCC) which is a cancer that originates from renal tubular epithelial cells in the kidneys. The elusive and vast nature of RCC leads to many subtypes that differ histologically and biologically and thus require different diagnoses and treatments. By investigating the papillary RCC (pRCC) subtype, the goal is to identify potential biomarkers for pRCC that may aid in future clinical diagnosis and treatment. OIP5 is a protein scarcely investigated and past studies have shown its overexpression linked to pRCC progression and tumorigenesis, but further metastatic investigation is warranted. This study aimed to confirm OIP5 overexpression plays a role in cancer as well as use a luciferase firefly reporter gene to monitor OIP5’s role in tumorigenesis and metastatic potential in vivo using a live mouse model. Results suggest that OIP5 overexpression plays a role in enhanced tumor growth in vivo. PCSK5 is another fairly novel gene of interest in this study, and while research involving PCSK5 and RCC is severely limited, recent studies imply it may be affected by different oncogenes such as OIP5 and specific microRNA during ccRCC and pRCC progression. Therefore, this study investigated the presence of PCSK5 in OIP5 pRCC tissue and tumors. The results suggest that PCSK5 has a higher presence in OIP5 overexpressed cells and tissue as well as a higher mRNA presence. Figures created using public genomic datasets show a correlation between PCSK5 and OIP5 expression during the later stages of pRCC progression and with lower survival probabilities. This indicates the possibility of interaction between OIP5 and PCSK5 and if true, provides a potential link for finding more biomarkers for potential future therapeutic targeting.

Cotutelle - Mélanie Dieudé / L’ischémie-reperfusion inhérente à toute transplantation d’organe solide induit l’apoptose des cellules endothéliales. Les cellules endothéliales apoptotiques sécrètent des vésicules extracellulaires apoptotiques de type exosome (ApoExo). L’internalisation des ApoExo par les cellules endothéliales (CE) adjacentes conduit à des changements fonctionnels importants dont le dysfonctionnement endothélial. Cependant, les mécanismes d’internalisation des ApoExo par les CE sont méconnus. Des marqueurs fluorescents spécifiques aux protéines et à l’ARN ont été utilisés afin de marquer spécifiquement les ApoExo et étudier leur internalisation par microscopie confocale et cytométrie de flux. Les ApoExo ont été internalisés par les CE en fonction du temps et de la concentration. L’inhibition des voies classiques d’endocytose à l’aide d’inhibiteurs pharmacologiques et d’interférence par ARN n’a pas réduit les niveaux d’internalisation des ApoExo. Le blocage de la phosphatidylsérine des ApoExo avec l’annexine-V a réduit leur internalisation. L’analyse ultrastructurelle par microscopie électronique des CE a révélé la présence de structures lamellipodes importantes pour la macropinocytose dont l’inhibition a diminué le transfert d’ARN et de protéines dans les CE. L’analyse par RT-qPCR a révélé que l’ARNm PCSK5, le plus enrichi dans les ApoExo, est augmenté dans les CE traitées aux ApoExo. Cette augmentation est abolie avec ApoExo exempts d’ARNm PCSK5. Ces résultats démontrent que les ApoExo sont activement internalisés par macropinocytose dépendante de la phosphatidylsérine, favorisant leur internalisation en augmentant l’activité macropinocytique des CE. Les ApoExo transfèrent ainsi des ARN fonctionnels capables de moduler le protéome des CE. Ces résultats ouvrent de nouvelles portes pour la prévention de l’internalisation des ApoExo, et donc de la dysfonction endothéliale. / Ischemia-reperfusion injury inherent to solid organ transplantation induces endothelial apoptosis, releasing apoptotic exosome-like vesicles (ApoExo) which in turn induce endothelial dysfunction. We showed that ApoExo modulates gene expression, functions, and morphology of endothelial cells (EC) towards endothelial dysfunction. However, the mechanism by which EC internalize ApoExo remains unclear. Fluorescent probes specifically targeting proteins and RNA were used to track ApoExo uptake in EC by flow cytometry and confocal microscopy. Pharmacological inhibitors and gene silencing were used to probe uptake mechanisms. RNA and protein expression were quantified using Taqman RT-qPCR and immunoblot, respectively. Uptake of ApoExo by EC was observed in a time- and concentration-dependent manner. Inhibition of clathrin- and caveolae-dependent endocytosis did not decrease ApoExo internalization by EC. Blocking phosphatidylserine on ApoExo surface with annexin-V decreased ApoExo uptake. Ultrastructural analysis of serum-starved EC via electron microscopy revealed lamellipodia-like structures, hallmark of macropinocytosis, whose number increased following ApoExo exposure. Inhibition of macropinocytosis abrogated both RNA and protein transfers from ApoExo to EC. The most enriched mRNA in ApoExo, coding for PCSK5, showed enhanced levels in ApoExo-treated EC along with increased PCSK5 protein levels. This was abrogated by both macropinocytosis inhibition and depletion of PCSK5 mRNA in ApoExo. These results demonstrate that EC actively internalize ApoExo through phosphatidylserine-dependent macropinocytosis, and moreover, that ApoExo further increase macropinocytosis. These findings also show that functional RNAs can be delivered to EC through ApoExo. These results open new avenues for preventing ApoExo internalization and counteracting the development of endothelial dysfunction.