Les échanges entre les cellules tumorales et le microenvironnement jouent un rôle essentiel dans le développement des tumeurs. Dans ce contexte, la nouvelle famille de métalloprotéases, les protéines ADAM, constituent aujourd’hui des régulateurs majeurs de la progression tumorale en agissant sur la biodisponibilité des médiateurs de la communication cellulaire que sont les cytokines, chimiokines et facteurs de croissance. Au sein de cette famille, ADAM12 est la plus associée au cancer. Elle possède la particularité de jouer un rôle dans la signalisation cellulaire, de façon indépendante de son activité métalloprotéase, notamment dans les voies de signalisation du TGFβ. Notre étude montre pour la première fois un rôle pour la forme membranaire d'ADAM12 dans l'induction de la transition épithélio-mésenchymateuse (EMT), un processus essentiel à l'invasion tumorale dont le TGFβ est un inducteur majeur. Cet effet est médié par l'activation des voies de signalisation du TGFβ, impliquant les protéines SMAD3, AKT et ERK et requiert le domaine cytoplasmique d'ADAM12L mais pas son domaine catalytique. L'activation de ces voies de signalisation pourrait impliquer une relocalisation d'ADAM12L au sein de plates-formes de signalisation dans les radeaux lipidiques. Par ailleurs, nous avons montré qu'ADAM12L interagit avec les protéines ZO-1 et ZO-2, des protéines des jonctions serrées, et pourrait favoriser leur désassemblage au cours de l'EMT. Nos travaux ont permis de mettre en évidence une nouvelle fonction pour ADAM12L dans l'EMT, par un mécanisme impliquant une modulation des signaux régulant ce processus. Une meilleure compréhension de la dynamique de ces mécanismes moléculaires pourrait permettre de développer de nouvelles thérapies ciblées pour lutter contre la progression tumorale. / Communication between tumoral cells and the microenvironnement plays an essential role in the developpement of tumors. In that context, the new family of metalloproteases, the ADAM proteins, are major regulators of the tumoral progression by acting on the bioavaibility of importants mediators of cellular communication as cytokines and growth factors. Among this family, ADAM12 is the most associated with cancer. It has been shown to mediate signaling pathways by a process independant of its metalloproteasis activity, in particular for TGFβ signaling. This study show for the first time a role for the membrane form of ADAM12 in the induction of epithelial to mesenchymal transition (EMT), a essential process involved in tumor invasion, whom TGFβ is a main inducer. This effect is mediated by the activation of TGFβ signaling pathways, SMAD3, AKT and ERK and require the cytoplasmic tail of ADAM12L but not its catalytic activity. Activation of these pathways could involve a relocalisation of ADAM12L in special signaling platform in lipid rafts. Moreover, we have shown that ADAM12L interact with ZO-1 and ZO-2, two proteins of tight junctions, and could facilitate their desassembling during EMT. This work underscore for the first time a new function of ADAM12L in EMT, by a mecanism invovlving a modulation of signals regulating this process. A better understanding of the dynamic of these molecular mecanisms could allow the developpement of new targeted therapies to fight against tumoral progression.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / ADAMs (a disintegrin and metalloprotease) can influence multiple cellular processes involved in normal development and pathogenesis. ADAM12 expression levels are elevated in many pathological conditions including cancer, cardiovascular disease, and muscle regeneration. Recently, ADAM12 has emerged as a candidate cancer gene in a comprehensive genetic analysis of human breast cancers. The regulation of ADAM12 expression is poorly understood. Identification of new substrates for ADAM12 metalloprotease can expand our knowledge of processes in which ADAM12 is involved. Here, we show that ADAM12 expression is upregulated by transforming growth factor beta (TGF-beta), an essential signaling pathway for many cellular processes. This upregulation requires proteosomal degradation of a transcriptional repressor SnoN. Furthermore, breast cancer cell lines expressing high levels of SnoN have significantly impaired induction of ADAM12 by TGF-beta, suggesting an inverse correlation between SnoN and the extent of regulation of ADAM12 by TGF-beta. Additionally, we demonstrate that ADAM12 is one of the metalloproteases involved in shedding a Notch ligand, Delta like 1 (Dll1). The Notch signaling pathway plays a crucial role in cell fate decision during development and in adults. Cleavage of Dll1 by ADAMs occurs in cis and results in activation of Notch signaling in a cell-autonomous manner. Furthermore, the intracellular domain of Dll1 created after cleavage further enhances TGF-beta signaling in response to TGF-beta. Our analysis of breast cancer-associated mutations in the ADAM12 gene showed a lack of proper proteolytic processing of the ADAM12 protein and its mislocalization to the endoplasmic reticulum. Additionally, ADAM12 mutants show a dominant-negative effect on the processing of the wild-type ADAM12 and result in loss of the functional ADAM12 at the cell surface. Collectively, our results indicate a new mechanism of regulation of ADAM12 expression, expand the role of ADAM12 in the regulation of Notch signaling, and characterize cancer-associated mutations in the ADAM12 gene.

Glioblastoma is the most common malignant brain tumor, accounting for 17% of all primary brain tumors in the United States. Despite the available surgical, radiation, and chemical therapeutic options, the invasive and infiltrative nature of the tumor render current treatment options minimally effective. Recent reports have identified multiple regulators of glioblastoma progression and invasiveness. It has been demonstrated that ADAM12, A Disintegrin And Metalloproteinase encoded by ADAM12 gene, is over-expressed in glioblastoma and directly correlated with tumor proliferation. Additionally, dysregulation of the Notch signaling pathway has been implicated in the pathogenesis of many gliomas. Lastly, an evolving role of microRNAs, small noncoding RNAs, in carcinogenesis is progressively growing. A recent study has identified ADAM12 as a notch-related gene, and another demonstrated that inhibition of notch signaling decreased glioblastoma recurrence. However the mechanisms of regulation are still unknown. In this study, we hypothesize that direct downregulation of microRNA-29, downstream of over-expression of notch enhances glioblastoma malignancy through upregulation of ADAM12. Although our data demonstrate upregulation of Notch1, its downstream target HES1, and ADAM12 in U87MG glioblastoma cell line. Expression of the cleaved intracellular Notch1 was not detected. Furthermore, we were unable to demonstrate an inhibitory effect of ɣ-secretase inhibitor on Notch signaling, likely reflecting the requirement for modifying culturing conditions or detection in our assays. Furthermore, miR-29 was detected in glioblastoma cells. The expression of miR-29 was further elevated by ɣ-secretase inhibitor treatment, suggesting a role for Notch1 inhibition on miR-29 expression. Although no conclusive results are shown in our work, a role of Notch1 through miR-29 is implicated in the pathogenesis of glioblastoma pathogenesis warranting further investigation into the role downstream target genes in the Notch signaling pathway.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Anna Zolkiewska / The ADAM (A Disintegrin and Metalloprotease) family of multi-domain proteins modulates a number of cellular signaling pathways in both normal and cancerous cells. ADAM12 has been shown to be a candidate cancer gene for breast cancer and its expression is up-regulated in breast tumors. The human ADAM12 transcript is alternatively spliced. One of these splice variants encodes a transmembrane ADAM12 isoform, ADAM12-L, which has been demonstrated to release cell signaling molecules from the cell surface. Another variant encodes a secreted protease, ADAM12-S, which cleaves extracellular matrix proteins and other secreted proteins. Although these variants are expressed from the same promoter, their relative expression levels are highly discordant. Here, I demonstrate variant-specific regulation of ADAM12 transcripts by microRNAs. Members of the microRNA-29 and microRNA-200 families target the unique 3’UTR of the ADAM12-L transcript and cause transcript degradation. Additionally, I show the presence of a novel ADAM12 splicing event in which 9 additional nucleotides are inserted in the region encoding the autoinhibitory pro-domain. I demonstrate that this novel variant is expressed in breast epithelial cells and breast cancer cell lines. The resulting protein isoform does not undergo proteolytic processing to activate the protease. Additionally, trafficking of the novel isoform to the cell surface is impaired and this isoform is localized to the endoplasmic reticulum. Finally, I determined a role for ADAM12-L in the progression of triple negative breast cancers (TNBCs). These tumors are lacking expression of hormone receptors and the HER2 receptor. HER2 is a member of the epidermal growth factor receptor (EGFR) family and the loss of the HER2 receptor causes tumors to rely on EGFR for propagating pro-growth signals. I show here that, in TNBC tumors, ADAM12-L expression is strongly correlated with poor patient prognosis and increased activation of EGFR. These data suggest that in TNBCs, ADAM12-L enhances tumor growth via EGFR activation. Collectively, the data presented here demonstrate (a) transcript-specific regulation of ADAM12 in breast cancer, (b) the existence of a novel splice variant and protein isoform with impaired cellular trafficking, and (c) an important role of the ADAM12-L isoform in EGFR activation in TNBC.