Deletion of the long arm of chromosome 6 is one of the most common genetic alterations in human malignant melanoma. Recently, a reciprocal translocation between chromosomes 6q and 17p was detected in a melanoma cell line, UACC-930, using arm painting probes of 6p and 6q. Reciprocal translocation is seldom observed in solid tumors. Upon further characterization of the translocation marker using techniques such as Southern blotting, genomic library screening and DNA sequencing, a complex rearrangement including two inversions of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q21q22)(q22q27)t(6;17)(q27;p13)], was detected. An NCBI blast search revealed 3 genes being interrupted by the breakpoints: prenyl diphosphate synthase subunit 2 (PDSS2) at 6q21, Parkin at 6q27 and p53 at 17p13. Down-regulation of PDSS2 was commonly observed in 59/87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7/66, 10.6%, p<0.001), indicating the tumor-suppressive potential of PDSS2 in melanoma development. To characterize the function of PDSS2 in tumorigenesis, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was demonstrated by both in vitro and in vivo assays. The results showed that PDSS2 could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. PDSS2 is the first enzyme involved in the CoQ10 biosynthesis pathway. Other studies have demonstrated PDSS2 mutations can cause severe CoQ10 deficiency and markedly reduced ATP production because of respiratory chain dysfunction. Interestingly, proteomics analysis revealed 7 out of 11 identified proteins (HSPA8, GAPDHS, TPI1, HSPA5, PGK1, ENO1, and ATP5B) differentially expressed in PDSS2-overexpressing cells were related to energy metabolism. Further studies are required to determine how PDSS2 could alter the energy supply in tumor cells. Taken together, these results support the proposal that PDSS2 is a novel tumor suppressor gene which may play an important role in the development of malignant melanoma via altering tumor metabolism.

No description available.

Mitofusin 2 (MFN2), an outer mitochondrial membrane protein expressed in virtually all human tissues, is a multi-faceted protein known to affect mitochondrial morphology, metabolism, tethering, and movement as well as overall cell cycle progression. Most intriguing among its characteristics is its ability to bind to Ras and Raf, upstream effectors in the MAPK/ERK pathway. Conditional knockout (cKO) of renal proximal tubule MFN2 in vivo showed a post-ischemic protective effect. While the two day survival of control mice was only 28%, an unexpected 86% of the MFN2 cKO mice were alive at two days post-ischemia. This is likely explained by MFN2's ability to bind and sequester Ras at baseline. Because the MFN2 deficient mice did not sequester as much Ras, renal proximal tubule cells were able to proliferate at a greater rate and restore organ function more quickly. Immunoprecipitation studies confirm a strong interaction between Ras and MFN2 in resting cells but a weaker one immediately following ischemic insult, even in cells replete with MFN2. These results suggest that blocking the MFN2-Ras interaction may be a novel method to treat acute kidney injury. A small peptide mimicking Ras to block MFN2 could be feasible. This should grant ischemic tissue an increased propensity to regenerate healthy cells while leaving non-ischemic tissue completely unaffected. Such a therapeutic agent would be novel in the treatment of acute kidney injury and may have uses in other tissues as well due to MFN2's widespread expression profile.

Philosophiae Doctor - PhD / Cancer is a group of diseases that arises from irreversible genomic and epigenomic alterations that result in unrestrained proliferation of abnormal cells. Detailed understanding of the molecular mechanisms underlying a cancer would aid the identification of most, if not all, genes responsible for its progression and the development of molecularly targeted chemotherapy. The challenge of recurrence after treatment shows that our understanding of cancer mechanisms is still poor. As a contribution to overcoming this challenge, we provide an integrative multi-omic analysis on glioblastoma multiforme (GBM) for which large data sets on di erent classes of genomic and epigenomic alterations have been made available in the Cancer Genome Atlas data portal. The rst part of this study involves protein network analysis for the elucidation of GBM tumourigenic molecular mechanisms, identification of driver genes, prioritization of genes in chromosomal regions with copy number alteration, and co-expression and transcriptional analysis. Functional modules were obtained by edge-betweenness clustering of a protein network constructed from genes with predicted functional impact mutations and differentially expressed genes. Pathway enrichment analysis was performed on each module to identify statistical overrepresentation of signaling pathways. Known and novel candidate cancer driver genes were identi ed in the modules, and functionally relevant genes in chromosomal regions altered by homologous deletion or high-level amplication were prioritized with the protein network. Co-expressed modules enriched in cancer biological processes and transcription factor targets were identified using network genes that demonstrated high expression variance. Our findings show that GBM's molecular mechanisms are much more complex than those reported in previous studies. We next identified differentially expressed miRNAs for which target genes associated with the protein network were also differentially expressed. MiRNAs and target genes were prioritized based on the number of targeted genes and targeting miRNAs, respectively. MiRNAs that correlated with time to progression were selected by an elastic net-penalized Cox regression model for survival analysis. These miRNA were combined into a signature that independently predicted adjuvant therapy-linked progression-free survival in GBM and its subtypes and overall survival in GBM. The results show that miRNAs play significant roles in GBM progression and patients' survival finally, a prognostic mRNA signature that independently predicted progression-free and overall survival was identified. Pathway enrichment analysis was carried on genes with high expression variance across a cohort to identify those in chemoradioresistance associated pathways. A support vector machine-based method was then used to identify a set of genes that discriminated between rapidly- and slowly-progressing GBM patients, with minimal 5 % cross-validation error rate. The prognostic value of the gene set was demonstrated by its ability to predict adjuvant therapy-linked progression-free and overall survival in GBM and its subtypes and was validated in an independent data set. We have identified a set of genes involved in tumourigenic mechanisms that could potentially be exploited as targets in drug development for the treatment of primary and recurrent GBM. Furthermore, given their demonstrated accuracy in this study, the identified miRNA and mRNA signatures have strong potential to be combined and developed into a robust clinical test for predicting prognosis and treatment response.

Metastasis suppressors regulate multiple steps during the process of dissemination of tumor cells from primary sites to distant organs, while they do not affect the growth of the primary tumor. Previously, we identified NDRG1 (N-myc downstream regulated gene 1) as a tumor metastasis suppressor gene and found that it is negatively involved in metastatic progression of prostate and breast cancers. To elucidate the molecular mechanism of NDRG1 function, we used the yeast two-hybrid system to identify proteins interacting with NDRG1. In the first part of this project, we demonstrate that NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signaling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumor cells in vitro and in animal model. We also found that restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumor cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a negative master regulator of Wnt signaling during the metastatic progression, and therefore revealed a novel control mechanism of Wnt signaling in tumor progression. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of KAI1 gene. In the second part of this project, we examine a possible link between two metastasis suppressor genes, NDRG1 and KAI1, through ATF3. We demonstrated that ectopic expression of NDRG1 was able to augment endogenous KAI1gene expression in prostate cancer cell lines, while silencing NDRG1 accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFêB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional down-stream target of NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through intrinsic transcriptional cascade.

Des mutations acquises du gène TET2 ont été décrites dans les hémopathies malignes humaines. La fréquence de ces anomalies dans les hémopathies myéloïdes est de 10 à 20%, atteignant 50% dans les échantillons de leucémies myélo-monocytaires chroniques (LMMC). Les mutations observées sont inactivatrices, ce qui suggère que TET2 est un gène de type suppresseur de tumeur et que les mutations retrouvées conduisent à une perte de fonction de la protéine. Ce gène code pour une enzyme capable de modifier les cytosines méthylées. Il participerait ainsi au contrôle de la méthylation de l'ADN et donc à la régulation épigénétique de l’expression génique. Afin de mieux comprendre son rôle au cours de l’hématopoïèse, deux modèles murins d'inactivation du gène Tet2 ont été développés. Des expériences de greffe de cellules médullaires dans des souris syngéniques montrent que les cellules déficientes pour ce gène présentent un avantage compétitif par rapport aux cellules sauvages. L’analyse des souris invalidées pour ce gène montre une amplification des populations hématopoïétiques immatures, ainsi que des anomalies de la différenciation des lignages myéloïdes et également des lignages lymphoïdes. Une fraction des souris invalidées pour Tet2 âgées de plus de six mois développe des hémopathies malignes ressemblant à la LMMC humaine. Des anomalies équivalentes sont retrouvées dans les souris hémizygotes pour Tet2 et dans des souris portant un allèle hypomorphe du gène. L’ensemble de ces résultats montre qu’une dérégulation de l'activité de Tet2 conduit à des anomalies précoces de l'hématopoïèse, mais n'entraine pas directement la transformation des cellules progénitrices immatures. La latence du développement de ces tumeurs suggère la nécessité d'une coopération avec d'autres évènements oncogéniques, comme des anomalies d’autres acteurs épigénétiques / Acquired loss-of-function mutations of TET2 gene are frequently observed in patients with myeloid malignancies, including acute myeloblastic leukemia, myeloproliferative neoplasm, myelodysplastic syndrome, and chronic myelomonocytic leukemia (CMML). The Ten-Eleven-Translocation (TET) family proteins are 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze the conversion of 5-methyl-cytosine into 5-hydroxymethyl-cytosine, which is proposed to constitute a first step toward cytosine demethylation. To study the function of Tet2 in murine hematopoiesis, we developed two mouse models in which the catalytic domain of the protein is disrupted. In both models, Tet2 deficiency leads to the progressive expansion of the immature hematopoietic compartment that includes stem cell and multipotent progenitors. In addition, both Tet2-deficient animals display abnormalities of erythroid, megakaryocytic, myelo-monocytic and lymphoid lineages, recapitulated in competitive transplantation assays. With age, Tet2-deficient mice develop bona fide myeloid tumors. All these properties were shown to be cell-autonomous by bone marrow cells transplantation and in vitro assays. Together these data suggest that TET2 activity is essential for normal homeostasis of the hematopoietic system. Its inactivation results in the development of hematologic disorders resembling human CMML myeloid disorders. TET2 deficiency endows the cells with a competitive advantage over wild type cells, induces hematopoietic differentiation abnormalities but is not responsible for full cellular transformation. The latency observed for CMML development in mouse models of Tet2 deficiency suggests a requirement for cooperating mutations, such other epigenetic regulator alterations.

Le mélanome est un cancer de la peau agressif et au mauvais pronostic. Si de nouvelles solutions thérapeutiques efficaces ont été développées, les taux de réponses sont variables et transitoires. Découvrir de nouveaux mécanismes oncogéniques dans cette pathologie reste donc nécessaire. Durant mes travaux, j’ai pu démontrer que la protéine tyrosine-kinase FES est exprimée dans les mélanocytes normaux. Cette expression est largement perdue dans un panel de lignées cellulaires de mélanome, au niveau protéique et transcriptionnel ainsi que dans des cultures primaires d’échantillons de patients. La perte de FES est due à une hyper-méthylation de son promoteur et est réversible. En ré-exprimant FES de manière stable dans deux lignées cellulaires de mélanomes, j’ai montré que cette réexpression entraînait une diminution des capacités oncogéniques des cellules. De plus, en analysant les données d’une cohorte de mélanomes (TCGA), j’ai pu établir qu’une diminution importante ou une perte d’expression de FES se retrouve dans près de 40% des patients, et qu’elle est corrélée à une hyper-méthylation du gène FES. Les patients ayant une faible expression de FES présentent un moins bon pronostic soulignant l’importance de ce phénomène. Enfin, en croisant un modèle murin déficient pour le gène Fes avec un modèle de mélanome, nous observons que les tumeurs sous fond Fes KO sont plus prolifératives et plus volumineuses.Ainsi, par des analyses in vitro, sur des données de patients ou en croisant des modèles murins, j’ai pu démontrer que FES est exprimée au niveau des mélanocytes normaux et y exerce un rôle de suppresseur de tumeur. / Among skin cancers, melanoma is the most aggressive and has the worst prognosis. In the last years, new therapeutic tools have been developed but responses differ between patients and are often transient due to resistance mechanisms. This highlights the need to improve understanding of molecular mechanisms of the disease. During my thesis, I have shown for the first time that FES tyrosine kinase is expressed in normal melanocytes, and that its expression is lost at the protein and RNA levels in most melanoma cell lines. The same result is observed in a panel of 12 patients’ short-term cultures. The lack of expression is due to FES promoter hyper-methylation and can be reverted using a hypomethylating agent. By restoring FES expression in two melanoma cell lines, I observe a decrease of oncogenic properties of the cells. Moreover, the analysis of the TCGA data on melanoma indicate that FES expression is strongly decreased or lost in about 40% of patients, and that this loss of expression is correlated with FES promoter methylation. Importantly, patients with low level of FES mRNA have poor prognosis compared to FES expressing patients. Finally, Fes knock-out mice crossed with an inducible melanoma mouse model indicate that tumors proliferation and size are more important under a Fes KO background.In conclusion, by using melanoma cells in vitro, data from melanoma patients and mouse models, I have demonstrated that FES is expressed in normal melanocytes and clearly plays a tumor suppressor role.in melanoma.

IFN-α has been applied in medical treatment of various neuroendocrine (NE) tumors, either alone or combination with somatostatin analogues. They can improve clinical symptoms in 50-70% of patients but a significant tumor reduction is only observed in 5-15% patients. Vasostatin (vaso) is believed to be an angiogenic inhibitor. The aim of this study is to evaluate the feasibility to use IFN-α and vasostatin gene therapy in NE tumors. We constructed plasmid vectors carrying human IFN-α2 (hIFN-α2) gene and human vasostatin gene, which were transfected into BON I cell to obtain stable gene-expressing cell lines. We found that in animal tumor model and cell experiments gene transfer of vasostatin caused a faster cell growth and tumor development via down-regulation of the tumor suppressor gene and p27. Cell adhesion, spreading, migration and invasion ability were increased in vaso-expressing BON I cells. Transfecting chicken vinculin could reverse the malignant behavior and restored expression of tumor suppressor genes. Moreover, vinculin knockdown could result in a faster cell growth and an increased colony formation. Condition medium taken from hIFN-α2-expressing BON I cells showed significant antiproliferative effects both on the NE tumor cells, BON I and LCC18, and the endothelial cells, PAE. It also suppressed cell adhesion and cell invasion and inhibited angiogenesis on CAM assay. Mice implanted with a mixture of WT BON cells and hIFN-α2-expressing BON cells (1:1) demonstrated significantly lower tumor incidence and longer tumor doubling time. Furthermore, long-acting IFN-α2b (PEGIntron®) demonstrated a better anti-tumor effect in contrast with IFN-α2b (IntronA®). Intratumoral injection of hIFN-α2 plasmids significantly inhibited NE tumor growth and caused tumor regression. We concluded that gene transfer of vasostatin into BON I cells might cause an enhanced malignant tumor behavior. Therefore, vasostatin therapy can not be recommended for patients with NE tumors. Vinculin might play an important role in NE tumor development and growth regulation. Gene therapy by using plasmid DNA carrying hIFN-α2 gene is feasible and promising in NE tumors.

WW domain-containing oxidoreductase (WWOX) is a novel tumor suppressor gene discovered few years ago. Many researches indicate that expression of WWOX is reduced in a variety of cancers including heptocellular carcinoma (HCC). A recent report suggests that WWOX is implicated in Wnt/£]-catenin pathway which is frequently affected in HCC. In this study, we used immunohistochemical (IHC) staining to analyze the expression of WWOX and Wnt/£]-catenin pathway components in HCC and adjacent non-tumor tissues. Our result showed that WWOX was significantly downregulated in poor differentiated HCC. In addition, downregulation of WWOX was significantly correlated with cytoplasmic £]-catenin expression. We also found that TCF4 was strongly expressed in HCC tissues and the expression was associated with tumor grade and stage. Consequently, our result implied that downregulation of WWOX in HCC might lead to accumulation of £]-catenin in the cytoplasm and the subsequent activation of Wnt/£]-catenin signaling pathway.

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.