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Breast cancer is a heterogeneous disease, and can be classified according to histological subtypes based on cellular morphology. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most common histological subtypes, accounting for approximately 80% and 12% of cases respectively. ILC exhibits a number of distinct clinico-pathological features in comparison with IDC, and is understudied as a breast cancer subtype. ILC tumours are typically oestrogen receptor positive, HER2 negative, and frequently demonstrate early loss of Ecadherin expression, which is a hallmark of the lobular phenotype. ILC is presently treated in a similar manner to IDC, with treatment generally directed against hormone receptors. Upon acquisition of hormone resistance, limited secondary options are available; patients are rarely candidates for agents targeting HER2, and are recognised to be poorly responsive to chemotherapeutics. We therefore need to advance our understanding of lobular tumour biology, in order to identify suitable biomarkers that will guide the development of targeted therapies for ILC patients. As protein expression levels determine cellular phenotype, a protein-based approach has the potential to provide biologically relevant insight into the mechanisms driving ILC. A range of protein analysis platforms, including reverse phase protein array, label-free mass spectrometry and immunohistochemistry, were therefore used to elucidate biological mechanisms active in the ILC subtype. Such experiments led to the identification of activated PI3K-Akt signalling in mouse and human ILC, suggesting that inhibition of this pathway may be an effective treatment strategy in lobular breast cancer. Preliminary evidence of differences in cytoskeletal and extracellular matrix (ECM) proteins was also acquired, providing an interesting basis for future research. A further major strand of this project was the development of in vitro and in vivo tools, to facilitate further interrogation of lobular biology. This included determination of a representative mouse model of ILC, and generation of primary cancer cells and cancer-associated fibroblasts (CAFs) from patient-derived material. Analysis of CAFS showed differential expression of ECM-associated genes, consistent with proteomic analyses. In addition, a tissue micro-array (TMA) comprising primary ILC and IDC tumours, with associated clinical data, was developed. Immunohistochemical staining of the TMA identified a potential role for IGF-1 pathway signalling in ILC, with increased expression of IGF-1 ligand associating with increased tumour size and metastasis in ILC patients. Taken together, the generation and validation of a range of useful tools in the course of this work has provided useful insight into the unique biology of ILC.

The response of cells to mechanical inputs is a key determinant of cell behavior. In response to changes in the mechanical environment of epithelial cells, E-cadherin initiates signal transduction cascades that allow the cells to modulate their contractility to withstand the force. Much attention has focused on identifying the E-cadherin signaling pathways that promote contractility, but the negative regulators remain undefined. In this thesis, we identify SHP2 as a force-activated phosphatase that negatively regulates E-cadherin force transmission by dephosphorylating vinculin Y822. To specifically probe a role for SHP2 in E-cadherin mechanotransduction, we innovatively mutated vinculin so that it retains its phosphorylation but cannot be dephosphorylated. Cells expressing the mutant vinculins have increased contractility. This work provides the first mechanism for inactivating E-cadherin mechanotransduction and provides a new method for specifically targeting the action of phosphatases in cells.

Epithelial adhesion molecule, E-cadherin plays important role in maintaining structural integrity of epithelial tissue. Altered expression of E-cadherin might result in the loss of contact inhibition growth property, abnormal cell growth and differentiation, hence the oncogenic transformation. To explore the role of E-cadherin in the tumor progression, we have investigate the expression pattern of E-cadherin in invasive cervical carcinomas. 77 paraffin embedded specimen with FIGO staging Ia¡]N=15¡^, Ib¡]N=19¡^, Ib meta¡]N=13¡^ IIa¡]N=17¡^, IIb¡]N=13¡^were included in immunohistochemical study, 18 surgical removed tumor tissues of Ib stage and its normal counter parts were used for semi-quantitative RT-PCR analysis. The results indicated that no significant correlation between E-cadherin expression level and tumor metasis, patient prognosis. However, E-cadherin immunostainimg intensity was significanty inverse correlated with tumor size¡]p < 0.001¡^. Notably, loss of membranes E-cadherin expression might be a significant factor that correlated with tumor metasis¡]p = 0.001,< 0.005¡^.

Signal Transducer and Activator of Transcription 3, Stat3, has been associated with cytokine-induced proliferation, anti-apoptosis and neoplastic transformation, while constitutively active Stat3 has been found in many human tumors. Stat3 activation by the Src oncoprotein leads to specific gene regulation and the Stat3 mediated signaling pathway is one of the critical signaling pathways involved in Src oncogenesis. Our laboratory demonstrated that engagement of cadherins, which are a class of cell-cell adhesion molecules, can activate Stat3, even in the absence of direct cell to cell contact. Interestingly, a significant increase in total Rac1 and Cdc42 protein levels triggered by cadherin engagement, and an increase in Rac1 and Cdc42 activity, which led to Stat3 activation by a mechanism involving gp130, a common subunit of the IL-6 family of cytokines, was also observed. To investigate the role of gp130 in vSrc transformation, we knocked down gp130 in vSrc transformed cells and found a decrease in the levels of phosphorylated Stat3, the rate of cell migration, rate of cell proliferation and the anchorage-independent growth. It was also previously demonstrated that vSrc had a negative effect on the function of cadherins. Surprisingly, however, despite the fact that vSrc may reduce cadherin adhesion, previous results in our lab showed that cell density still caused a significant increase in Stat3 activity in vSrc transformed cells. Moreover, Stat3 downregulation induced apoptosis in transformed cells which was more pronounced at high cell densities. This may reflect an increased need for Stat3 activity at high densities, possibly to overcome apoptosis, which raised the question of the actual role of cadherins in the density-mediated activation of Stat3 in such cells. The expression of cadherin-11, a type II cell adhesion molecule, is associated with invasive breast cancer and many studies suggest that it may play a significant role in facilitating tumor cell invasion and the formation of metastatic tumors. Since Src and its family members participate in many aspects of tumor progression and metastasis, it was interesting to see if Src needed cadherin-11 for neoplastic transformation. To this effect, when we knocked down cadherin-11 in Balb/c3T3 cells, we observed a significant reduction in levels of phosphorylated Stat3-ptyr705 which was also observed when vSrc was expressed in them. Moreover, expressing vSrc in cells in which cadherin-11 was knocked down also decreased the anchorage –independent growth and increased apoptosis indicating that cadherin-11 is needed for transformation and survival respectively, in vSrc transformed cells. Our results thus demonstrate that cadherin-11 may be a good target for the selective elimination of cells expressing Src and presumably other oncogenes as well. Stat3 activation by cadherins is so potent and important that tumor cell death can be enhanced by cadherin inhibition. In our experiments, the inhibition of cadherin-11 induced apoptosis in Src expressing cells, while the normal cells were not affected. Elucidation of the functions and regulation of cadherin-11 may enhance our understanding of the roles of cadherins in invasive cancer and may provide future targets for therapy. Through our work, the cadherin/IL-6 pathway may emerge as a crucial Stat3 activator in vSrc expressing cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-11-27 18:54:05.777

Student No. 9404705H MDent Research Report School of Oral Sciences / Expression of the E-cadherin/β-catenin complex in oral squamous cell carcinoma and its correlation with histomorphology and metastasis. The immunohistochemical expression of E-cadherin and β-catenin was examined in 30 primary oral squamous cell carcinomas in patients with (n=19) and without (n=11) nodal metastasis, as confirmed on histopathological examination of the resected regional lymph nodes. The corresponding primary and nodal metastases tissue samples were available for 17 patients. The 30 primary carcinomas were histologically graded according to the invasive tumour front grading system and by conventional Broders’ criteria. None of the 30 primary carcinomas showed homogenous, membranous E-cadherin and β-catenin expression when compared to the normal oral squamous epithelium. Staining was heterogeneous in 73% (22/30) and in 77% (23/30) of the primary carcinomas stained for E-cadherin and β-catenin respectively. There was a highly significant reduction (P<0,001) of both E-cadherin and β- catenin expression with progression from the well-differentiated areas to the less differentiated tumour cells at the invasive tumour front. At the invasive tumour front, however, irrespective of the nodal status and invasive tumour front grading score, 28/30 (93%) tumours showed loss of E-cadherin expression. Loss of β-catenin expression was recorded in 22/30 (73%) cases. These findings indicate that E-cadherin and β-catenin play a key role in the loss of differentiation of tumour cells in oral squamous cell carcinoma and while they may be permissive for metastasis, in isolation, E-cadherin and β-catenin are probably not predictive of metastatic potential in oral squamous cell carcinoma.

Cells experience force throughout their lifetimes. Cells sense force via adhesion receptors, such as the cadherins, which anchor cells to neighboring cells, and integrins, which tether cells to the underlying matrix. Both adhesion receptors respond to force by activating signaling pathways inside the cell. These pathways trigger growth of adhesion complexes and reinforcement of the cytoskeleton in order to resist the force. These activities are energetically costly. Thus, mechanisms are needed to couple force transmission and energy production. In this thesis, I demonstrated force on cadherins activates a master regulator of energy homeostasis known as AMP-activated kinase (AMPK). In response to force, AMPK was recruited to the cadherins. AMPK promoted growth of the adhesion complex and cytoskeletal reinforcement by stimulating energy production in the cell. Additionally, AMPK formed a complex with vinculin—a protein that is recruited to both cadherins and integrins. I observed AMPK activation of vinculin dictates whether vinculin joins the cadherin complex. Conversely, AMPK activation has no bearing on vinculin recruitment to integrins. This work provides three novel contributions: (1) the first link between energy production and force transmission, (2) a molecular mechanism for how AMPK increases adhesion complex growth, and (3) an explanation for how vinculin discriminates between cadherins and integrins.

Les infections génitales par les virus du papillome humains (HPV) sont les infections virales sexuellement transmises, les plus communes chez les femmes en âge de procréer. Il est désormais bien établi que l’infection persistante par les HPV classés «à haut risque» est l’un des facteurs indispensables au développement de lésions précancéreuses et cancéreuses du col de l’utérus. Ces HPV semblent aussi être impliqués dans le développement d’autres cancers de la région ano-génitale et pourraient être également impliqués dans les cancers de la tête et du cou. Durant cette dernière décennie, des études croissantes tendent à établir un rôle étiologique des HPV dans les dysfonctionnements gestationnels. La détection des ADN HPV dans les placentas issus d’avortements spontanés et leur capacité exceptionnelle à se répliquer in vitro dans les cellules trophoblastiques cultivées en monocouche, ont apporté de nouvelles perspectives quant à la possibilité que le placenta pourrait constituer aussi un tropisme naturel des infections par HPV. Six jours après la fécondation et suite à l’accolement du blastocyste à l’épithélium utérin, le trophoblaste s’engage dans des processus actifs de prolifération, d’invasion et de différenciation complexe pour la construction de l’interface physiologique indispensable aux échanges essentiels entre la mère et l’enfant ; le placenta. De façon intéressante, ses propriétés sont similaires à celles de la cellule tumorale maligne. Néanmoins, ses mécanismes sont étroitement régulés dans le trophoblaste, à la fois dans l’espace et le temps, assurant un développement normal à chaque étape de la grossesse. Devant toutes ces données, nous avions émis l’hypothèse que l’expression des protéines précoces E5, E6 et E7 d’HPV de type 16 (de haut risque), pourraient modifier le développement des trophoblastes infectés. Les résultats obtenus durant ce travail de doctorat démontrent que la protéine virale E5, hautement hydrophobe, est cytotoxique et affecte la viabilité du trophoblaste. Cette cytotoxicité est neutralisée, et la viabilité est améliorée, lorsque les oncoprotéines majeures E6 et E7 sont exprimées en présence de la protéine E5. Lorsque toutes les protéines précoces sont exprimées sous le contrôle de leur propre promoteur (LCR), la viabilité est favorisée. Ces observations ont été confirmées dans les cellules cervicales également. Il a été précédemment rapporté que les oncoprotéines E6 et E7 affectaient l’adhésion du trophoblaste aux cellules endométriales. Dans le présent travail, il a été retrouvé que la protéine E5 diminuait elle aussi l’adhésion, non seulement aux cellules endométriales, mais aussi au support de culture cellulaire. Les capacités de migration et d’invasion de la matrice extracellulaire sont augmentées par l’expression de E5 et dans une plus large proportion par l’expression de E6 et E7. Des résultats similaires ont été obtenus lorsque toutes les protéines de la région précoces sont exprimées sous le contrôle de leur propre promoteur (LCR). La diminution de l’expression de la E-cadhérine est considérée comme un marqueur de malignité et de mauvais pronostic pour les cancers. Nous avons démontré que l’expression de E5, E6 ou de E7, inhibait l’expression de la E-cadhérine, reflétant l’impact des oncoprotéines du virus HPV-16 sur la diminution de l’adhésion et l’augmentation du pouvoir invasif des cellules trophoblastiques. L’investigation d’autres marqueurs de malignité et de tolérance immunitaire, l’étude de l’impact du virus HPV-6 (de bas risque) sur la migration et l’invasion des cellules trophoblastiques, et l’étude de la capacité des protéines précoces d’HPV-16 à influencer l’entrée des particules virales, ont fait l’objet de résultats préliminaires, ouvrant de larges perspectives. Genital Human Papillomavirus (HPV) infections are the most common sexually transmitted infections amongst women on the age of reproduction. It is well established that persistent infection with high-risk HPVs is the necessary factor in the causation of precancerous and cancerous cervical lesions. High-risk HPVs have also been reported to be involved in the causation of head and neck cancers and other anogenital cancers. On this last decade, growing data are attempting to study the potential etiological association of HPV with gestational dysfunctions. The detection of HPV DNA in placentas resulting from spontaneous abortions and the unique ability of multiple HPV types to replicate in vitro in trophoblastic cells cultured in a monolayer system, rise new questions over the HPV tropism. Six days following fertilization and once the apposition of the blastocyst on the uterine wall takes place, the trophoblast, in a very active and complex process, starts to proliferate, invade and to differentiate in order to build a physiological interface; the placenta, from where multiple mother/foetus exchanges occur. Interestingly, the way that the trophoblast behaves is very similar to malignant tumoural cells. However, the trophoblast obeys to strict spatial-temporal regulatory confines, insuring a proper development all along the pregnancy. In regard to these data, we hypothesised that the expression of the high-risk HPV type 16 oncoproteins E5, E6 and E7, might modify the development of the infected trophoblast. During my Ph.D study, I demonstrated that the highly hydrophobic protein E5 is localized in many interne membranes compartments of the transfected trophoblast. E5 affects the viability of transiently and stably transfected trophoblastic cells. E6 and E7, favouring cell growth, neutralised the E5 cytotoxic effect. All HPV-16 early proteins, when expressed under the control of their endogenous promoter (LCR), favoured trophoblastic growth. These observations were also observed in cervical cell lines. In addition, E5 decreased the adhesiveness of trophoblastic cells to the tissue culture plastic and to endometrial cells similarly as previously described for E6 and E7. Cells expressing E6, E7 and in less extend E5 favoured chemotaxic migration and matrigel invasion compared to the cells expressing the LacZ control. These effects were also observed when early proteins were expressed under the control of their own viral promoter (LCR). Interestingly, the E-cadherin was down regulated in trophoblastic cells expressing E5, E6 and E7. In conclusion, HPV-16 early proteins enhanced trophoblastic growth and intensify the malignant phenotype by impairing cell adhesion leading to increased cellular motile and invasive properties. HPV-16 E5 participated, with E6 and E7, in these changes by impairing E-cadherin expression, a hallmark of malignant progression. Additional preliminary results consisting on the investigation of other markers of malignancy and immune tolerance, on studying the impact of the low-risk HPV type 6 early proteins on the migratory and invasive properties of trophoblastic cells and on the study of the ability of HPV-16 to influence the entry of virus particules, allowed to open wide perspectives.

Durant la transició Epiteli-Mesènquima (TEM), el factor de transcripció Snail reprimeix la transcripció de l'E-cadherina unint-se a les caixes E presents en el promotor d'aquest gen.Les cèl·lules que expressen Snail presenten un fenotip fibroblastoide amb pocs contactes cel·lulars: els gens epitelials són reprimits (E-cadherina, MUC1 i VDR) i els gens mesenquimals són induïts (Zeb1).La sobre-expressió de Snail en varies línies cel·lulars donarà lloc a un augment en els nivells de RNA i de l'activitat del promotor de Zeb1. A més, Zeb1 reprimeix l'E-cadherina i MUC1.Estàvem interessats en el mecanisme de repressió i inducció de gens per Snail. Snail necessita de HDAC per a realitzar el seu mecanisme de repressió. A més, es va estudiar el mecanisme de inducció del promotor de Zeb1. El promotor de Zeb1 era activat en línies cel·lulars que responien a estímuls de TEM, com la sobre-expressió de ILK, de l' oncogen Ha-Ras o de la cPK-Ca. Finalment es va descriure que el promotor de Zeb1 responia a NF-kB, b-catenina/TCF4 i Twist. / During Epithelial mesenchymal transition (EMT), the transcriptional factor Snail represses E-cadherin transcription by binding to E-box sequence of the E-cadherin promoter. Cells expressing Snail presented a scattered flattened phenotype with low intercellular contacts: epithelial gene are repressed (E-cadherin, VDR and Muc-1) and mesenchymal genes are induced (Zeb1). Snail overexpression in several lines raised ZEB1 RNA levels and increased the activity of ZEB1 promoter. ZEB1 repressed E-cadherin and MUC1.We were interested in Snail repression and induction mechanism. We analysed whether Snail needs other proteins for its repression function and we found HDAC such as partners in Snail repression. In addition, we are studying its activation activity upon Zeb1 promoter. The human Zeb1 promoter was activated in cell lines that respond to agents that induce mesenchymal phenotype, as overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or cPK-Ca. Moreover, Zeb1 promoter was activated by different proteins implicated in EMT, such as, NF-kB, b-catenin/TCF4 and Twist.

In the Xenopus embryo, cell rearrangements during early development require the dynamic modulation of adhesion. Cells primarily use the integrin family of transmembrane receptors for attachment to and interpretation of the extracellular environment. While acting as adhesion receptors, integrins also have bidirectional signalling properties essential for driving cellular movements. The regulation of integrin activity is thought to stem from cytoplasmic assemblies of constitutively expressed molecules. PINCH (Particularly Interesting New cysteine-histidine rich protein), an adapter protein, is part of an IPP complex that has emerged as a key signalling scaffold indispensable for integrin function in vitro. As such, I tested the hypothesis that PINCH regulates integrin function in the Xenopus embryo. Xenopus PINCH was successfully cloned using RT-PCR. The predicted amino acid sequence of PINCH shares a 98% similarity with mammalian orthologs, and comprises of five highly conserved LIM domains. PINCH mRNA and protein are ubiquitously expressed throughout embryogenesis. In situ hybridization indicates that PINCH mRNA is expressed in the blastocoel roof and the pre-involution mesoderm. The localization and temporal expression of PINCH suggests a role in mediating cell adhesive events during gastrulation. A functional approach was used to examine the role of PINCH during gastrulation. I used site-directed mutagenesis to generate non-functional LIM1 (LIM1mut) and LIM4 (LIM4mut) domains that have been proposed to bind ILK and Grb4 respectively. Over-expression of PINCH leads to a delay in blastopore closures, while the expression of both LIM1mut and LIM4mut relieve this inhibition at lower concentrations. Further analysis indicates that PINCH, LIM1mut, and LIM4mut inhibit FN matrix assembly independent of integrin adhesion. Contradictory to in vitro studies, co-immunoprecipitation analysis indicates that endogenous PINCH does not bind ILK, confirming an integrin-independent role during gastrulation. Furthermore, in the embryo PINCH is found at cell boundaries but does not appear to directly modulate cadherin adhesion. As such this thesis provides evidence that PINCH regulates cell intercalation movements independent of integrin and cadherin receptors and raises the possibility that the LIM4 domain is involved in PINCH regulation of cell adhesion during early development.