Renal cell carcinoma (RCC), following prostate and bladder tumours, is the third most prevalent genitourinary malignancy. Clear cell renal cell carcinoma makes up the bulk of RCC cases (ccRCC). Despite the fact that ccRCC is the most aggressive type of RCC, our understanding of its pathophysiology is limited. Previous research in our laboratory revealed important oncogenic roles of contactin 1 (CNTN1), a neuronal cell adhesion protein, in prostate cancer. CNTN1 is involved in a number of signalling pathways that are often changed in cancer, including the VEGFC-VEGF receptor 3 (VEFGR3)/fms-related tyrosine kinase 4 (Flt4) axis, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) axis, and the Notch signalling system. Collectively, evidence suggests that CNTN1 facilitates ccRCC. To examine this possibility, I have established stable ccRCC 786-O and A498 cell lines expressing either empty vector (EV) or CNTN1. In comparison to the respective EV lines, ectopic expression of CNTN1 enhances colony formation and cell proliferation. In comparison to A498 EV cells, A498 CNTN1 cells seems to possess enhanced migration ability based on wound healing assay. Taken together, my research provides in vitro evidence supporting CNTN1 in facilitating ccRCC pathogenesis. Future research will be required to investigate this concept using in vivo systems and primary ccRCC tumor tissues. / Thesis / Master of Science (MSc)

Renal cell carcinoma is a high risk and high mortality cancer. While the VHL pathway is frequently altered in renal cell carcinoma, emerging evidences points towards involvement of multiple complex pathways in the progression of renal cell carcinoma. In this present work, we aimed to investigate the role of newly identified protein, Major Intrinsically disordered NOTCH2-Associated Receptor 1 (MINAR1), in renal cell carcinoma. We used immunohistochemistry and demonstrated that MINAR1 is highly expressed in normal kidney epithelium. Furthermore, MINAR1 was expressed at variable levels in human renal cell carcinoma cell lines. More importantly, we found that MINAR1 was significantly downregulated in human samples of renal cell carcinoma. Although further studies are needed, our data suggests a potential role for MINAR1 in renal biology. Given that MINAR1 expression appears to be downregulated in renal cancer patients, it is suggestive that MINAR1 could function as a tumor suppressor. / 2020-06-17T00:00:00Z

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

The Wilms’ tumor gene 1 (WT1) was first reported as a tumor suppressor gene in Wilms’ tumor. However, later studies have shown the oncogenic properties of WT1 in a variety of tumors. It was recently proposed that WT1 was a chameleon gene, due to its dual functions in tumorigenesis. We aimed to investigate the clinical significance of WT1 as biomarker in acute myeloid leukemia (AML) and clear cell renal cell carcinoma (ccRCC) and to elucidate the function of WT1 as an oncogene in squamous cell carcinoma of head and neck (SCCHN). In AML, it was suggested that WT1 expression was an applicable marker of minimal residual disease (MRD). In adult patients with AML, we found a good correlation between WT1 expression levels normalized to two control genes, β-actin and ABL. Outcome could be predicted by a reduction in WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis, when β-actin was used as control. Also, irrespective of the control gene used, outcome could be predicted by a reduction in WT1 expression in peripheral blood (≥ 2-log) detected between 1 and 6 months after treatment initiation. Previous studies in RCC demonstrated that WT1 acted as a tumor suppressor. Thus, we tested whether single nucleotide polymorphisms (SNPs) or mutations in WT1 might be associated with WT1 expression and clinical outcome in patients with ccRCC. We performed sequencing analysis on 10 exons of the WT1 gene in a total of 182 patient samples, and we identified six different SNPs in the WT1 gene. We found that at least one or two copies of the minor allele were present in 61% of ccRCC tumor samples. However, no correlation was observed between WT1 SNP genotypes and RNA expression levels. Moreover, none of the previously reported WT1 mutations were found in ccRCC. Nevertheless, we found that a favorable outcome was associated the homozygous minor allele for WT1 SNP. We then further investigated whether WT1 methylation was related to WT1 expression and its clinical significance. Methylation array and pyrosequencing analyses showed that the WT1 promoter region CpG site, cg22975913, was the most frequently hypermethylated CpG site. We found a trend that showed nearly significant correlation between WT1 mRNA levels and hypermethylation in the 5’-untranslated region. Hypermethylation in the WT1 CpG site, cg22975913, was found to be associated with patient age and a worse prognosis. One previous study reported that WT1 was overexpressed in SCCHN. That finding suggested that WT1 might play a role in oncogenesis. We found that both WT1 and p63 could promote cell proliferation. A positive correlation between WT1 and p63 expression was observed, and we identified p63 as a WT1 target gene. Furthermore, several known WT1 and p63 target genes were affected by knocking down WT1. Also, co-immunoprecipitation analyses demonstrated a protein interaction between WT1 and p53. In summary, WT1 gene expression can provide useful information for MRD detection during treatment of patients with AML. In RCC, our results suggested that the prognostic impact of WT1 SNPs was limited to the subgroup of patients that were homozygous for the minor allele, and that WT1 promoter hypermethylation could be used as a prognostic biomarker. In SCCHN, WT1 and p63 acted as oncogenes by affecting multiple genes involved in cancer cell growth.

<p>Despite clear cell renal cell carcinoma (ccRCC) being the most common and one of the most lethal forms of RCC, our understanding of ccRCC tumourigenesis remains limited. To identify factors contributing to ccRCC formation, we have found that raf kinase inhibitor protein (RKIP) is a candidate tumour suppressor of ccRCC. This is consistent with publications implicating that RKIP possesses tumour suppression functions in several human cancers. However, whether RKIP suppresses ccRCC tumorigenesis has yet to be determined. This thesis was therefore undertaken to examine the role of RKIP in the tumourigenesis of ccRCC as well as to gain knowledge about this understudied area of kidney cancer research. Upon examination of more than 600 patients with ccRCC in several independent patient cohorts, levels of RKIP protein are significantly reduced in more than 80% of ccRCC tumours in comparison to the adjacent non-tumour kidney or normal kidney tissues. This observed magnitude of RKIP reduction strongly suggests that RKIP is an important tumour suppressor of ccRCC. Supporting this concept is observations that ectopic expression of RKIP inhibited A498 and 786-O ccRCC cell invasion. Conversely, knockdown of RKIP in A498 and 786-O cells enhanced the invasion ability of those cells. Additionally, the progression of ccRCC into advanced tumour stages and tumour grades is correlated with a reduction in RKIP protein. This reduction of RKIP protein levels is more apparent in metastasized ccRCC tumours in which all tumours were negative for RKIP protein expression in comparison to the matched organ-confined tumours. To date there have been no reports investigating the role of RKIP in renal cell carcinoma, our results demonstrate a strong relationship between RKIP reduction and progression of this often fatal disease.</p> / Master of Science (MSc)

<p>The majority (75%) of renal cell carcinoma (RCC) is comprised of clear cell renal cell carcinoma (ccRCC). Despite ccRCC being the most aggressive form of RCC, our knowledge regarding the pathogenesis of the disease remains limited. We have identified upregulation of the extracellular protein periostin (POSTN) in ccRCC. Western blot analysis of ccRCC tumours from 27 patients demonstrated high POSTN protein expression in 26 tumours compared to their respective adjacent normal kidney tissue (ANK). Immunohistochemistry (IHC) analysis revealed high levels of stromal POSTN protein in the ccRCC primary tumours and 16 metastasized ccRCCs. Intriguingly, abundant stromal POSTN in the tumour and non tumour boundary were observed in local and metastaized ccRCC, and in A498 ccRCC cell-derived xenograft tumours. Collectively, these results suggest that the ccRCC-associated POSTN was derived from the stroma. This notion was supported by the co-existence of POSTN with α-smooth muscle actin (αSMA) in both local and metastasized ccRCC tumours. αSMA is a marker of activated stromal fibroblasts (myofibroblasts). Furthermore, co-culture of NIH3T3 murine fibroblasts with human A498 or 786-0 ccRCC cells dramatically enhanced POSTN transcription and secretion from NIH3T3 cells. Extracellular POSTN significantly enhanced A498 cell attachment. Upregulation of POSTN in NIH3T3 cells enhanced their proliferation. Taken together, my research demonstrates that 1) ccRCC induces fibroblast-mediated accumulation of extracellular POSTN, 2) stromal POSTN enhances ccRCC attachment, and 3) high levels of POSTN promotes fibroblasts' proliferation. These observations suggest a critical role for POSTN in mediating the co-evolving process between ccRCC and its stroma during ccRCC pathogenesis.</p> / Master of Science (MSc)

L’ensemble de ce travail vise à étudier les mécanismes de résistance aux thérapies anti-angiogéniques dans le traitement du cancer du rein à cellules claires (ccRCC). Nous avons mis en évidence que le bévacizumab (BVZ), un anticorps monoclonal humanisé anti-VEGF utilisé en clinique, accélère la croissance de ccRCC humain chez la souris nude. Ce modèle mime la phase d’échappement souvent observée chez les patients. Le traitement BVZ induit de la lymphangiogenèse associée à une surexpression du VEGF-C. Les cellules tumorales après traitement possèdent également des capacités d’invasion accrues. Ainsi, le traitement BVZ pourrait faciliter la progression tumorale et la formation de métastases dans les ccRCC. Le traitement entraine également une diminution de l’expression d’une phosphatase membranaire, la phospho-tyrosine phosphatase récepteur kappa (PTPR)impliquée dans le contrôle de l’activité de récepteurs à activité tyrosine kinase, comme le récepteur de l’EGF, du PDGF et de l’HGF. Ces récepteurs régulent la prolifération et la migration cellulaire. Le traitement BVZ faciliterait donc la croissance tumorale indépendante du VEGF. Enfin, le traitement induit une augmentation de la sécrétion de cytokines angiogéniques redondantes qui prennent le relais du VEGF, comme les cytokines CXCL7 et CXCL8, et facilitent le développement tumoral sous traitement BVZ. En particuliers, la cytokine CXCL7 et ses récepteurs CXCR1-2 ont un rôle central dans le développement des ccRCC. Cibler l’axe CXCL7/CXCR1-2 réduit efficacement la croissance tumorale. Les récepteurs cibles de PTPR, pour lesquels des inhibiteurs sont actuellement utilisés pour le traitement d’autres cancers, le VEGF-C et la cytokine CXCL7, pourraient donc constituer de nouveaux marqueurs prédictifs d’efficacité du BVZ et de nouvelles cibles thérapeutiques dans le traitement des ccRCC. La résistance au BVZ pourrait également s’expliquer par l’existence de formes "bénéfiques" anti-angiogéniques du VEGF qui sont reconnues par le BVZ avec la même affinité que les formes pro-angiogéniques. Nous avons mis en évidence qu’une immunisation prophylactique à l’aide d’un peptide spécifique du VEGF pro-angiogénique limite la croissance tumorale de ccRCC syngéniques de souris. De la même façon, en traitement curatif, l’utilisation d’anticorps spécifiques du VEGF pro-angiogénique bloque la croissance de ccRCC chez la souris nude sans induire les différents mécanismes d’échappement observés avec le BVZ. Ces résultats suggèrent la pertinence du ciblage spécifique des formes pro-angiogéniques de VEGF dans le traitement des ccRCC. / The aim of my work is to study resistance mechanisms to anti-angiogenic treatments of Clear Cell Renal Carcinoma (ccRCC). We observed that bevacizumab (BVZ) -a humanized monoclonal antibody targeting VEGF and currently used in the clinic- promotes the growth of human ccRCC xenografts in nude mice. This model mimics the “escape phase” widely observed in patients. BVZ treatment induces lymphangiogenesis and over-expression of VEGF-C. Tumor cells exposed to the treatment acquire an increased spreading capacity. Hence, BVZ might promote tumor progression and metastasis formation of ccRCC. Furthermore, this treatment decreases the expression of the receptor phosphor tyrosine phosphatase kappa (PTRP). This phosphatase is involved in the regulation of tyrosine kinase receptors controlling growth and migration, among others EGF, PDGF and HGF receptors. Thus, BVZ might promote tumor growth independently of VEGF. Moreover, the treatment increases secretion of redundant cytokines like CXCL7 and CXCL8. By their ability to exert similar effect as VEGF, these cytokines promote tumor development under BVZ treatment. In particular, CXCL7 and its receptors CXCR1 and CXCR2, play a central role in the development of ccRCC. Targeting this pathway efficiently reduces tumor growth. Target receptors of PTRP for which inhibitors are currently used for other cancers, VEGF-C and CXCL7 could therefore be regarded as new predictive markers for BVZ efficiency and may be considered as potential therapeutic targets. Resistance to BVZ could also be explained by the presence of "beneficial" forms of anti-angiogenic VEGF recognized by the BVZ with the same affinity as the pro-angiogenic forms. We have demonstrated that prophylactic immunization with a pro-angiogenic VEGF-specific peptide limits tumor growth of murine syngeneic ccRCC. Similarly, in curative therapy, antibodies specific for pro-angiogenic VEGF block growth of ccRCC in nude mice without inducing the escape mechanisms observed with BVZ. These results highlight the relevance of targeting such pro-angiogenic forms of VEGF for the treatment of ccRCC.

Le carcinome à cellules rénales (RCC) exprimant le facteur inductible de l'hypoxie (HIF) en raison de l'inactivation du gène de von Hippel Lindau (vhl), représente un modèle d'hypoxie chronique. Le devenir des patients dépend du stade de dissémination des cellules tumorales. Par conséquent, déchiffrer les mécanismes de métastase est une préoccupation majeure. Le développement dépendant du VEGF-C (Vascular Endothelial Growth Factor C) d'un réseau lymphatique est en première ligne de propagation métastatique. Pour étudier le rôle de VEGFC dans la dissémination du RCC, nous avons étudié son expression dans l'hypoxie et nous avons invalidé son gène dans des lignées cellulaires humaines et murines. L'hypoxie régule négativement l'ARNm de VEGFC par une diminution de la transcription et de la stabilité de l'ARNm mais l'expression de la protéine VEGF-C est induite par l’hypoxie. Des capacités accrues de prolifération et de migration, et une meilleure expression des marqueurs mésenchymateux et des marqueurs souches caractérisent les cellules vegf-c -/-. Alors que les cellules vegfc -/- ne forment pas de tumeurs chez les souris immunodéficientes, elles développent des tumeurs agressives chez les souris immunocompétentes. La surexpression de VEGFC, est liée à une augmentation de la survie sans progression et globale chez les patients atteints de tumeurs non métastatiques alors qu'une diminution de la survie sans progression et globale est observée chez les patients métastatiques. Nos expériences décrivent une régulation subtile du VEGF-C par hypoxie et mettent en évidence son rôle bénéfique ou péjoratif. Par conséquent, le ciblage VEGF-C pour la thérapie doit être considéré avec prudence. / Hypoxic zones are common features of metastatic tumors. Renal cell carcinoma (RCC) expressing the Hypoxia Inducible Factor (HIF) because of inactivation of the von Hippel Lindau gene (vhl), represent models of chronic hypoxia. Their outcome depends on the extent of their dissemination at diagnosis. Therefore, deciphering the mechanisms of metastasis is a major concern. The Vascular Endothelial Growth Factor C (VEGFC)-dependent development of a lymphatic network is in front line of metastatic spreading. To address the role of VEGFC in RCC dissemination, we studied its expression in hypoxia and we invalidated its gene in human and mouse model cell lines of RCC. Hypoxia down-regulates VEGFC mRNA through a decrease in transcription and mRNA stability but concomitantly induced VEGFC protein expression. Increased proliferation and migration abilities, over-activation of the AKT signaling pathway and enhanced expression of mesenchymal and stem cell markers characterized vegfc-/- cells. Whereas vegfc-/- cells do not form tumors in immuno-deficient mice, they develop aggressive tumors in immuno-competent mice. Moreover, mouse RCC cells generate fast-growing tumors in mice invalidated for six1 or eya2, two major regulators of VEGFC expression. Lymphangiogenic markers overexpression including VEGFC is linked to increased disease-free and overall survival in patients with non-metastatic tumors whereas decreased progression-free and overall survival is observed for metastatic patients. Our experiments describe a subtle regulation of VEGFC by hypoxia and highlight its beneficial or pejorative role. Therefore, targeting VEGFC for therapy must be considered with caution.

L'ensemble de ce travail vise à étudier les mécanismes de résistance aux thérapies anti-angiogéniques dans le traitement du cancer du rein à cellules claires (ccRCC). Nous avons mis en évidence que le bévacizumab (BVZ), un anticorps monoclonal humanisé anti-VEGF utilisé en clinique, accélère la croissance de ccRCC humain chez la souris nude. Ce modèle mime la phase d'échappement souvent observée chez les patients. Le traitement BVZ induit de la lymphangiogenèse associée à une surexpression du VEGF-C. Les cellules tumorales après traitement possèdent également des capacités d'invasion accrues. Ainsi, le traitement BVZ pourrait faciliter la progression tumorale et la formation de métastases dans les ccRCC. Le traitement entraine également une diminution de l'expression d'une phosphatase membranaire, la phospho-tyrosine phosphatase récepteur kappa (PTPR)impliquée dans le contrôle de l'activité de récepteurs à activité tyrosine kinase, comme le récepteur de l'EGF, du PDGF et de l'HGF. Ces récepteurs régulent la prolifération et la migration cellulaire. Le traitement BVZ faciliterait donc la croissance tumorale indépendante du VEGF. Enfin, le traitement induit une augmentation de la sécrétion de cytokines angiogéniques redondantes qui prennent le relais du VEGF, comme les cytokines CXCL7 et CXCL8, et facilitent le développement tumoral sous traitement BVZ. En particuliers, la cytokine CXCL7 et ses récepteurs CXCR1-2 ont un rôle central dans le développement des ccRCC. Cibler l'axe CXCL7/CXCR1-2 réduit efficacement la croissance tumorale. Les récepteurs cibles de PTPR, pour lesquels des inhibiteurs sont actuellement utilisés pour le traitement d'autres cancers, le VEGF-C et la cytokine CXCL7, pourraient donc constituer de nouveaux marqueurs prédictifs d'efficacité du BVZ et de nouvelles cibles thérapeutiques dans le traitement des ccRCC. La résistance au BVZ pourrait également s'expliquer par l'existence de formes "bénéfiques" anti-angiogéniques du VEGF qui sont reconnues par le BVZ avec la même affinité que les formes pro-angiogéniques. Nous avons mis en évidence qu'une immunisation prophylactique à l'aide d'un peptide spécifique du VEGF pro-angiogénique limite la croissance tumorale de ccRCC syngéniques de souris. De la même façon, en traitement curatif, l'utilisation d'anticorps spécifiques du VEGF pro-angiogénique bloque la croissance de ccRCC chez la souris nude sans induire les différents mécanismes d'échappement observés avec le BVZ. Ces résultats suggèrent la pertinence du ciblage spécifique des formes pro-angiogéniques de VEGF dans le traitement des ccRCC.