Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, expression to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in several solid cancers including those from breast, prostate, cervix, and lung. To study the role of CD177 in breast cancer, we generated a total body Cd177 knockout mouse. These mice had no profound phenotype at 3 - months of age or younger. The only phenotype found at this age was reduced peripheral neutrophil counts, but no difference in their ability to clear infections. Upon further analysis these mice developed an age dependent hyperproliferative mammary gland phenotype at 10 - months of age that was lost in mice 15 - months and older. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancer. We found that CD177 suppresses breast cancer pathogenesis. To understand the mechanism behind CD177 mediated suppression of breast cancer, we performed mass spectrometry on the purified CD177 complex. Mass spectrometry and co-immunoprecipitation results revealed CD177 interacts with β-Catenin and glycolytic enzymes PFK, aldolase A, GAPDH and enolase-ɑ. Further studies revealed that mechanistically CD177 forms a complex with ECadherin and β-Catenin at adherens junctions. This physical interaction between CD177, E-Cadherin and β-Catenin prevents β-Catenin activation via the canonical WNT. We also found CD177 suppressed WNT/β-Catenin signaling independent of E-Cadherin with an unknown protein. Thus, we identified a novel protein complex involving CD177 and proteins from adherens junctions that can suppress cancer formation via inhibiting the WNT/β-Catenin signaling pathway, a key cellular biological process relevant to the oncogenesis of multiple cancer types and tissue development. The lack of WNT/β- Catenin signaling control explains how mice without CD177 develop hyperproliferation of mammary epithelium in the mouse mammary gland. Interestingly, this phenotype is lost with age, possibly due to a decrease in WNT/β-Catenin signaling resulting from a decrease in progesterone and estrogen. In addition to CD177’s role in the regulation of WNT/β-Catenin signaling we also identified that CD177 plays a role in cancer cell metabolism. Since metabolism plays a significant role in cancer and CD177 interacts with glycolytic enzymes, we sought to determine if CD177 plays a role in metabolism. CD177 appears to interact with glycolytic enzymes, PFK, aldolase A, GAPDH, and ɑ-enolase and ultimately suppresses their mRNA expression. Furthermore, we found novel localization of CD177 at the mitochondrion, thus providing a potential explanation as to how an extracellular membrane bound protein such as CD177 interacts with glycolytic enzymes. Metabolic analysis of CD177 expression on cancer cells revealed that CD177 leads to a decrease in glucose uptake and a slight decrease in basal glycolysis, but an increase in lactate concentration. Further metabolic profiling also revealed that CD177 expression results in a significant decrease in glycolytic capacity (ECAR). Expression of CD177 also resulted in a significant decrease in basal respiration, ATP production, maximal respiration, and spare capacity (OCR) as well as an increase in reactive oxygen species. These data reveal that CD177 plays a novel role in cancer cell metabolism.

Acute myeloid leukemia (AML) is a genetic disease whereby sequential genetic aberrations alter essential white blood cell development leading to differentiation arrest and hyperproliferation. Pertinent animal models serve as essential intermediaries between in vitro molecular studies and the use of new agents in clinical trials. We previously generated a transgenic zebrafish model expressing human NUP98-HOXA9 (NHA9), a fusion oncogene found in high-risk AML. This expression yields a pre-leukemic state in both embryos and adults. Using this model, we have identified the overexpression of dnmt1 and the Wnt/β-catenin pathway as downstream contributors to the myeloproliferative phenotype. Targeted dnmt1 morpholino knockdown and pharmacological inhibition with methyltransferase inhibitors rescues NHA9 embryos. Similarly, inhibition of β-catenin with COX inhibitors partially restores normal hematopoiesis. Interestingly, concurrent treatment with a histone deacetylase inhibitor and either a methyltransferase inhibitor or a COX inhibitor, synergistically inhibits the effects of NHA9 on embryonic hematopoiesis. Thus, we have identified potential pharmacological targets in NHA9-induced myeloid disease that may offer a highly efficient therapy with limited toxicity – addressing a major long-term goal of AML research.

La transició epiteli-mesènquima (TEM) és un procés en què cèl lules epitelials, immòbils i amb polaritat apico-basal transiten cap un fenotip mesenquimal o fibroblàstic. L'expressió del factor de transcripció snail1 és suficient per induir TEM en cèl lules en cultiu i és necessari per la majoria de les TEM fisiològiques descrites. Snail1 és un membre de la família de proteïnes amb dits de Zinc que reprimeix gens epitelials (com l'E-cadherina) a través de la unió directa a seqüències especifiques dels promotors anomenades caixes E i posterior reclutament de corepressors. La TEM també es caracteritza per l'activació de gens mesenquimals, però el mecanisme pel qual snail1 indueix l'expressió d'aquests és poc conegut. En aquest treball demostrem que snail1 actua a nivell transcripcional per incrementar els nivells dels marcadors mesenquimals FN1 (fibronectina) i LEF1 (de l'anglès, lymphoid enhancer-binding factor 1) a través d'un mecanisme nou per aquesta proteïna de dits de Zn que no requereix ni caixes E ni unió directa a l'ADN. A més a més, mostrem que, per a dur a terme l'activació, snail1 coopera amb dos factors de transcripció ja descrits en relació a la TEM: beta-catenina i NF-kappa-B. Els nostres resultats també proven que l'expressió forçada de la E-cadherina evita aquesta cooperació i conseqüent activació gènica. A banda d'aquest mecanisme, també hem descrit que el factor de transcripció TFCP2c, que no havia estat prèviament relacionat amb TEM, és necessari per l'activació del gen FN1 induïda per snail1. / Epithelial-mesenchymal transition (EMT) is a cellular process by which no motile epithelial, apico-basal-polarized cells transit towards a motile mesenchymal front-backpolarized phenotype. Expression of the transcription factor snail1 is sufficient to induce EMT in cultured cells and it is required for most of the physiological EMTs described. Snail1 is a member of the Zn finger protein family that represses epithelial genes (such as E-cadherin) by directly binding to specific promoter sequences called E-boxes and subsequent recruitment of corepressors. EMT is also accompanied by activation of mesenchymal genes, however, little is known of how snail1 induces their expression.In this work we provide evidence that snail1 acts at the transcriptional level to increase the levels of the mesenchymal FN1 (fibronectin) and LEF1 (lymphoid enhancer-binding factor 1) genes through a novel mechanism for this Zn finger protein that does not require neither E-boxes nor direct binding to DNA. Furthermore, we describe a cooperative action in such mechanism between snail1 and two transcription factors previously related to EMT: beta-catenin and NF-kappaB. Our results also show that restoration of E-cadherin levels prevents such cooperation and subsequent activation. In addition, we also demonstrate that TFCP2c, which had not been previously linked to EMT, is also required for snail1-induced transcriptional activation of the FN1 gene.

The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), and is a frequent event in sporadic thyroid carcinomas. Multiple endocrine neoplasia 2B (MEN 2B), a subtype of MEN 2, is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET), however the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10 fold increase in ATP binding affinity, and leads to a more stable receptor-ATP complex, relative to the wildtype receptor. We also show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease. In the second part of the study, we identified a novel β-catenin-RET kinase signaling pathway which is a critical contributor to the development and metastasis of human thyroid carcinoma. We show that RET binds to, and tyrosine phosphorylates, β-catenin and demonstrate that the interaction between RET and β-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, β-catenin escapes cytosolic downregulation by the APC/Axin/GSK3 complex and accumulates in the nucleus, where it can stimulate β-catenin-specific transcriptional programs in a RET-dependent fashion. We show that downregulation of β-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumour growth in nude mice. Finally, we used a structure guided approach to identify and characterize a novel, non-ATP competitive, RET inhibitor; SW-01. We show that SW-01 provides significant RET inhibition in an in vitro kinase assay using purified RET. Moreover, RET phosphorylation is blocked, or dramatically reduced, in vivo in cells overexpressing active RET. We observe a significant decrease in cell proliferation and colony formation in RET-expressing cells in the presence of SW-01. Together, our data suggest that SW-01 has potential as a novel RET kinase inhibitor with clinical utility. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-09-15 16:20:59.976

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Oct. 31, 2007). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

The canonical Wnt pathway is a vitally important signalling pathway that plays an important role in cell proliferation, differentiation and fate decisions in embryonic development and in the maintenance of adult tissues. The twelve Armadillo (ARM) repeat-containing protein beta-catenin acts as the signal transducer in this pathway and is continuously degraded in the cytosol by the beta-catenin destruction complex (BDC). Upon receiving the Wnt signal the BDC is inactivated, allowing beta-catenin to accumulate in the cytosol and be transported to the nucleus where it binds to the TCF/LEF family of transcription factors, inducing the expression of cell cycle promotor genes. In this Thesis I describe investigations into the roles of leucine-rich repeat kinase 2 (LRRK2) and the transcription factor TCF7L2 within this signalling pathway. LRRK2 is a large multi-domain protein with strong links to Parkinson’s disease and suggested to play a role in inactivating the BDC in response to the Wnt signal. A recent paper proposed that the previously uncharacterised regions of LRRK2 contain a series of tandem repeat sub-domains. I began an investigation into these sub-domains but I was unable to produce soluble protein constructs despite the use of a range of common techniques, and so I was forced to conclude this project early. The main body of this thesis focuses on the interaction between the intrinsically disordered TCF7L2 and the repeat protein beta-catenin, a very long interface of approximately 4800 Å2 that spans from the third to the eleventh ARM repeat of beta-catenin and residues 12 to 50 of TCF7L2, as determined by X-ray crystal structures. First, a fluorescence reporter system for the binding interaction was developed and used to determine the kinetic rate constants for the association and dissociation of the wild-type construct using stopped-flow fluorescence spectroscopy and time-dependent fluorescence spectroscopy. It was found that association of TCF7L2 and beta-catenin was rapid (7.3 ± 0.1 x107 M-1s-1) with only a single phase was observed, whereas dissociation was biphasic and slow (5.7 ± 0.4 x10-4 s-1, 15.2 ± 2.8 x10-4 s-1). Using either of these two dissociation rate constants the calculated Kd value obtained is much lower than the values previously reported in the literature (8 ± 1 / 20 ± 2 pM compared with 16 nM). This reporter system was then used to investigate the striking variability between three crystal structures previously obtained for the TCF7L2-beta-catenin complex, in which different regions of TCF7L2 show different elements of secondary structure. Mutational analysis revealed that the interface residues on TCF7L2 identified in these structures make little or no contribution to the overall binding affinity, pointing to a transient nature of these contact in solution and suggesting that the observed differences between the structures are due to differences in crystal packing. Further experiments into the effect of osmolarity on the binding equilibrium and kinetics supported this conclusion and suggest a change in the association/dissociation mechanism as a function of ionic strength. Lastly, further mutational analysis of TCF7L2 revealed two regions that contribute particularly strongly to the binding kinetics, suggesting that TCF7L2-beta-catenin assembly proceeds via a two-site avidity mechanism. Some of the most destabilising variants display two additional dissociation phases, indicating the presence of an alternative dissociation pathway that is inaccessible to the wild-type. In summary, the results presented here provide insights into the kinetics of molecular recognition of a long intrinsically disordered region with an extended repeat protein surface, a process shown to involve multiple routes with multiple steps in each.

Orientador: Rebeca de Souza Azevedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T01:17:16Z (GMT). No. of bitstreams: 1 Dutra_SabrinaNogueira_M.pdf: 2889311 bytes, checksum: 3530f20f2b5d10e200ba02ec5d6d58b2 (MD5) Previous issue date: 2012 / Resumo: O ameloblastoma (AME) é um dos tumores odontogênicos (TO) mais comuns e, apesar de ser uma lesão benigna, pode apresentar comportamento clínico agressivo, localmente destrutivo e invasivo, enquanto o tumor odontogênico cístico calcificante (TOCC) é um TO benigno cístico que apresenta similaridades microscópicas com o AME, mas, geralmente, exibe comportamento clínico de menor agressividade. A via de sinalização Wnt/ß-catenina está envolvida no desenvolvimento e progressão tumoral de neoplasias benignas e malignas, e a expressão alterada de suas proteínas já foi identificada em alguns TO, provavelmente contribuindo para suas biologias tumorais. Dessa forma, o objetivo deste estudo foi avaliar e comparar a expressão imunoistoquímica de marcadores associados a via de sinalização Wnt/ß-catenina - Wnt1, Wnt5a, ß--catenina e syndecan-1(SDC-1) - em 17 casos de AME sólido e de 6 casos de TOCC. Os resultados revelaram que nos AME, a maioria dos casos foram focalmente positivos para Wnt1 e Wnt5a, a ß--catenina foi positiva no citoplasma isoladamente ou em associação a positividade de membrana, e SDC-1 exibiu positividade epitelial em 100% dos casos e positividade estromal em 40% dos casos. Nos casos de TOCC, a positividade para Wnt1 e Wnt5a foi de 100% e incluiu também as células fantasmas. A ß--catenina foi positiva no citoplasma e no núcleo das células epiteliais da maioria dos casos, e SDC-1 exibiu positividade epitelial em 100% dos casos e positividade estromal em 16,7% dos casos. Em conclusão, este estudo evidenciou que a expressão de Wnt1 e Wnt5a é mais proeminente nos casos de TOCC, a expressão de ß--catenina em AME é citoplasmática e em TOCC é nuclear, e que a expressão estromal de SDC-1 é mais proeminente nos casos de AME, reforçando, assim, o papel de Wnt1 e Wnt5 no desenvolvimento do TOCC, de ß--catenina no desenvolvimento do TOCC e do AME, e de SDC-1 estromal no fenótipo invasivo do AME. Além disso, a expressão de Wnt1 e de Wnt5a nas células fantasmas do TOCC pode contribuir para esta histogênese. Por conseguinte, a via de sinalização Wnt/ß--catenina parece contribuir para o desenvolvimento das lesões de AME sólido e de TOCC / Abstract: Ameloblastoma (SMA) is one of the most common odontogenic tumors (OT), and although benign, it may have aggressive clinical behavior, be locally destructive and invasive, while calcifying cystic odontogenic tumor (CCOT) is a benign cystic OT that presents microscopic similarities with AME, but, in general, it has a less aggressive clinical behavior. Wnt/ß--catenin signaling pathway is involved in benign and malignant tumor development and progression, and changes in expression of its proteins have been identified in some OT, probably contributing to their tumoral biologies. Thus, the aim of this study was to evaluate and compare the immunohistochemical expression of markers associated with Wnt/ß--catenin signaling pathway - Wnt1, Wnt5a, ß--catenin and syndecan-1 (SDC-1) - in 17 cases of solid SMA and 6 cases of CCOT. The results showed that in AME, most fo the cases were focally positive for Wnt1 and Wnt5a, ß--catenin was positive in the cytoplasm only or in association with membrane positivity, and SDC-1 had epithelial positivity in 100% of the cases and stromal positivity in 40% of the cases. In CCOT cases, Wnt1 and Wnt5a was of 100% and also included ghost cells. ß--catenin was positive in the cytoplasm and in the nucleus of epithelial cells in most of the cases, and SDC-1 had epithelial positivity in 100% of the cases and stromal positivity in 16.7% of the cases. In conclusion, this study highlighted that Wnt1 and Wnt5a expression was most prominent in CCOT, ß--catenin expression was cytoplasmatic in AME and nuclear in CCOT, and that stromal expression of SDC-1 was most prominent in AME, so, reinforcing Wnt1 and Wnt5 role in CCOT development, ß--catenin role in CCOT and AME development, and stromal SDC-1 role in the AME invasive phenotype. In addition, Wnt1 and Wnt5a expression in ghost cells of CCOT may contribute to this histogenesis. Therefore, Wnt/ß--catenin signaling pathway seems to contribute to solid AME and CCOT development / Mestrado / Estomatologia / Mestre em Estomatopatologia

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