Ovarian cancer (OC) accounts for 4% of all cancer cases and 4.2% of all cancer deaths worldwide. OC is the most lethal gynecological cancer because it lacks early disease symptoms and does not have a specific diagnostic marker. As a result, more than 70% of OC patients are diagnosed in later stages when the disease has already metastasized and the 5-year survival rate has decreased to less than 20% compared with approximately 90% survival for women diagnosed with early stage disease. Therefore, I initiated my studies with a computational analysis of the 27 most commonly reported literature-derived ovarian cancer (LDOC) protein biomarkers. I found that LDOC protein biomarkers share many biochemical features including a preponderance for a stable protein structure, the ability to be secreted, and functionality related to extracellular matrix (ECM) modification, immune response and/or energy production. Subsequently, I analyzed the human proteome to identify proteins that also share these biochemical features. Of the 70,616 proteins in the human proteome, 683 proteins were found to have similar biochemical features to the 27 LDOC proteins. I also identified a subset of 21 potential additional protein regulators of ovarian cancer (APROC) that interact with LDOCs. Three of the APROCs identified were amylase proteins AMY1A, AMY2A, and AMY2B which cleaves alpha 1, 4-glycosidic bonds in polysaccharides. Amylase is reportedly overexpressed in and secreted by ovarian tumors but its functional contribution to OC remains unknown[1]. In this thesis, I posit that amylase contributes to OC invasion. I initiated my studies by computational characterizing the different amylase isozymes to predict which amylase isozyme(s) is most likely overexpressed in and contributory to OC invasion. I found that AMY1 and AMY2B have unique regions of disorder and unique phosphorylation sites indicating that AMY1 and AMY2B would be more likely to interact with other proteins, and to be easily secreted. Using OC patient serum samples, I was able to validate AMY1 and AMY2B overexpression by western immunoblotting. I then developed an in vitro model system to study the molecular contribution of amylase to OC invasion using normal ovarian surface epithelial (IOSE) and OC cell lines. I showed that OC cells generally overexpress and secrete metabolically active amylase isozymes AMY1 and AMY2B. Abrogating amylase activity using siRNA silencing technology decreased the capacity of OC cells to invade collagen coated Boyden chambers and increased sulfated glycosaminoglycans (sGAG) production. Since a survey of OC cell lines indicated that cancer cells have a bulkier glycocalyx compared to IOSE cells and immunogold labeling studies indicated the presence of amylase within the immediate OC microenvironment, my data suggest that, by cleaving alpha 1, 4-glycosidic bonds in glycoconjugates present within ECM, amylase may remodel the ECM to promote an invasive cancer phenotype. Amylase is therefore a target for therapeutic intervention in OC patients with hyperamylasemia. I established Spirulina, a dietary supplement, as a novel transcriptional inhibitor of amylase. Spirulina inhibited amylase expression in OC cell lines at both the message and protein levels. Spirulina reduced OC cell invasion and migration in vitro, putatively by decreasing amylase expression.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause the emerging tick-transmitted disease, human granulocytic anaplasmosis (HGA). Following entry, the pathogen replicates within a host cell-derived vacuole that fails to mature along the endocytic pathway, does not acidify, and does not fuse with lysosomes. Selective fusogenicity is prototypical of many vacuole-adapted pathogens and has been attributed, at least in part, to pathogen modification of the vacuolar inclusion membrane and/or to selective recruitment or exclusion of host trafficking regulators. As a result, the A. phagocytophilum-occupied vacuolar membrane (AVM) provides a unique interface to study the host-pathogen interactions critical to A. phagocytophilum intracellular survival. Diverse vacuole-adapted pathogens; including Chlamydia, Legionella, and Salmonella; selectively recruit host Rab GTPases to their vacuolar membranes to establish replicative permissive niches within their host cells. Rab GTPases coordinate many aspects of endocytic and exocytic cargo delivery. We determined that the A. phagocytophilum-occupied vacuole (ApV) selectively recruits a subset of fluorescently-tagged Rabs that are predominantly associated with recycling endosomes. Another emerging theme among vacuole-adapted pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. We show that monoubiquitinated proteins decorate the AVM during infection of promyelocytic HL-60 cells, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Importantly, tetracycline treatment concomitantly promotes loss of the recycling endosome-associated GFP-Rabs and ubiquitinated proteins and acquisition of the late endosomal marker, Rab7, and lysosomal marker, LAMP-1, implicating bacterial-derived proteins in the ApV's altered fusogenicity. Therefore, we rationalized that A. phagocytophilum-encoded proteins that associate with the AVM may establish interactions with the host cell that are important for intracellular survival. By focusing on A. phagocytophilum proteins that are induced during host infection, we identified the first two bacterial-encoded proteins -- APH_1387 and APH_0032 -- that modify the AVM. Although functional studies are hindered by the lack of a system to genetically manipulate Anaplasma, the pathobiological roles of APH_1387 and APH_0032 are likely unique, as both proteins exhibit very little or no homology with any previously described protein. APH_1387 and APH_0032 are present at the cytoplasmic face of the AVM, therefore they likely interact with host proteins. We demonstrate that ectopic expression of APH_1387 and APH_0032 inhibits the ApV development in A. phagocytophilum infected cells. The results presented in this dissertation contribute to our understanding of how A. phagocytophilum modifies the vacuolar membrane in which it resides to establish a safe haven and evade lysosomal degradation.

The Staphylococcus aureus two component system (TCS) sae governs expression of numerous virulence factors, including Eap (extracellular adherence protein), which in turn among other functions also mediates invasion of host cells. The sae TCS is encoded by the saePQRS operon, with saeS coding for the sensor histidine kinase (SaeS) and saeR encoding the response regulator (SaeR). The saeRS system is preceded by two additional open reading frames (ORFs), saeP and saeQ, which are predicted to encode a lipoprotein (SaeP) and a membrane protein (SaeQ), respectively. Earlier, we have shown that SDS-containing subinhibitory concentrations of biocides (Perform®) and SDS alone activate sae transcription and increase cellular invasiveness in S. aureus strain Newman. The effect is associated with an amino acid exchange in the N-terminus of SaeS (L18P), specific to strain Newman. In this work, the role of whether the two additional genes, saePQ coding for the accessory proteins SaeP and SaeQ, respectively, are involved in SDS-mediated saeRS was investigated. It could demonstrated that the lack of the SaeP protein resulted in an increased saeRS transcription without SDS stress in both SaeSL/P variants, while the SDS effect was less pronounced on sae and eap expression compared to the Newman wildtype, suggesting that the SaeP protein represses the sae system. Also, SDS-mediated inductions of sae and eap transcription along with enhanced invasion were found to be dependent on presence of the SaeSP variant in Newman wildtype. On the other hand, the study also shows that the saePQ region of the sae operon is required for fully functional two-component system saeRS under normal growth conditions, but it is not involved in SDS-mediated activation of the saeS signaling and sae-target class I gene, eap. In the second approach, the study investigates whether SDS-induced sae expression and host cell invasion is common among S. aureus strains not carrying the (L18P) point mutation. To demonstrate this strain Newman, its isogenic saeS mutants, and various S. aureus isolates were analysed for sae, eap expression and cellular invasiveness. Among the strains tested, SDS exposure resulted only in an increase of sae transcription, Eap production and cellular invasiveness in strain Newman wild type and MRSA strain ST239-635/93R, the latter without an increase in Eap. Interestingly, the epidemic community-associated MRSA strain, USA300 LAC showed a biphasic response in sae transcription at different growth stages, which, however, was not accompanied by increased invasiveness. All other clinical isolates investigated displayed a decrease of the parameters tested. While in strain Newman the SDS effect was due to the saeSP allele, this was not the case in strain ST239-635/93R and the biphasic USA300 strains. Also, increased invasiveness of ST239-635/93R was found to be independent of Eap production. Furthermore, to investigate the global effect of SDS on sae target gene expression, strain Newman wild-type and Newman ∆sae were treated with SDS and analyzed for their transcription profiles of sae target genes using microarray assays. We could show that subinhibitory concentrations of SDS upregulate and downregulate gene expression of several signaling pathways involved in biosynthetic, metabolic pathways as well as virulence, host cell adherence, stress reponse and many hypothetical proteins. In summary, the study sheds light on the role of the upstream region saePQ in SDS-mediated saeRS and eap expression during S. aureus SDS stress. Most importantly, the study also shows that subinhibitory SDS concentrations have pronounced strain-dependent effects on sae transcription and subsequent host cell invasion in S. aureus, with the latter likely to be mediated in some strains by other factors than the known invasin Eap and FnBP proteins. Moreover, there seems to exist more than the saeSP-mediated mechanism for SDS-induced sae transcription in clinical S. aureus isolates. These results help to further understand and clarify virulence and pathogenesis mechanisms and their regulation in S. aureus. / Das Zwei Komponenten-Systems (TCS) Sae in S. aureus reguliert die Expression einer Vielzahl von Virulenzfaktoren, dazu gehört unter anderem das extrazelluläre Adhärenzprotein Eap, welches neben weiteren Funktionen, die Invasion in eukaryotische Wirtszellen vermittelt. Die Gene des sae TCS sind in einem Operon organisiert (saePQRS), wobei saeS für die sensorische Histidinkinase (SaeS) und saeR für den „Response Regulator“ (SaeR) kodieren. Diesen Genen sind zwei weitere Genabschnitte, saeP und saeQ, vorangestellt, wobei saeP vermutlich für ein Lipoprotein (SaeP) und saeQ für ein Membranprotein (RelQ) kodieren. In einer früheren Arbeit konnten wir zeigen, dass SDS-haltige Biozide (Perform©) unter sub- inhibitorischen Konzentrationen, sowie reines SDS, die sae Transkription aktiviert und dadurch zu einer erhöhten Invasion des S. aureus Stamms Newman in Wirtszellen führt. Dieser Effekt ist assoziiert mit einem spezifischen Aminosäureaustausch im N-terminus von SaeS (L18P) des Stamm Newman. In dieser Arbeit soll nun die Beteiligung der zwei zusätzlichen Gene, saeP und saeQ, an der SDS vermittelten transkriptionellen Induktion von saeR/S untersucht werden. Es konnte gezeigt werden, dass ohne SaeP, die saeR/S Transkription in beiden SaeL/P Varianten erhöht war, wobei eine zusätzliche SDS Behandlung hierfür nicht notwendig war. Im Gegenteil, es zeigte sich, dass der SDS Effekt auf die sae und eap Expression in der saeP Mutante deutlich weniger ausgeprägt ist als im Wildtyp Stamm. Das läßt vermuten, dass das Lipoprotein SaeP repremierend auf das sae System einwirkt. Des Weiteren wurde festgestellt, dass die SDS vermittelte transkriptionelle Induktion von sae und eap, zusammen mit der erhöhten Invasion, abhängig vom vorhanden sein der SaeSP Variante im Newman Wildtyp Stamm ist. Die Arbeit zeigt, dass die saePQ Region wichtig ist für die vollständige Funktion des Zwei Komponenten Systems SaeRS unter normalen Wachstumsbedingungen. Jedoch ist diese Region nicht involviert in der Aktivierung von SaeS, mit SDS als Signalgeber, sowie der darauffolgenden Aktivierung des sae Zielgens eap. In einem zweiten Ansatz wurde untersucht, ob die SDS induzierte sae Expression und Wirtszellinvasion auch häufig in S. aureus Stämmen auftritt, welche keine (L18P) Punktmutation besitzen. Dafür wurde Stamm Newman, die isogene saeS Mutante und verschiedene S. aureus Klinikisolate auf ihre sae, eap Expression, sowie zelluläre Invasionsfähigkeit hin analysiert. Von den getesteten Stämmen reagiert nur Wildtyp Stamm Newman und ein MRSA Stamm ST239-635/93R mit gesteigerter sae Transkription, Eap Produktion und zellulärer Invasion. Der MRSA Stamm jedoch ohne erhöhte Eap Produktion. Interessanterweise zeigt der „community- associated“ MRSA Stamm USA300 LAC eine biphasische sae Transkription in verschiedenen Wachstumsphasen, welche jedoch nicht einhergeht mit erhöhter Invasion. Alle anderen Klinikisolate zeigten abnehmende Tendenzen in den getesteten Parametern. Während im Stamm Newman der SDS Effekt auf das saeSP Allel zurückzuführen ist, gilt dies nicht für den Stamm ST239-635/93R, sowie den biphasischen Stamm USA300. Außerdem konnte gezeigt werden, dass die erhöhte Invasion des Stamms ST239-635/93R unabhängig von seiner Eap Produktion ist. Des Weiteren zeigten wir den globalen Effekt von SDS auf die sae Zielgenexpression. Dafür behandelten wir Wildtyp Stamm Newman mit SDS und analysierten die Transkription der sae Zielgene mittels Microarray Analyse. Wir konnten zeigen, dass subinhibitorische SDS Konzentrationen, induzierende als auch repremierende Auswirkungen auf die Genexpression haben. Dabei sind Gene betroffen, die involviert sind in verschiedene Signalwege, Biosynthese/Metabolismus als auch in Virulenz, Wirtzelladhärenz und Stressantwort. Zusammenfassend gibt die Arbeit Aufschluss über die Rolle der „upstream“ Region saePQ hinsichtlich der SDS-abhängigen saeRS und eap Expression in S. aureus. Am wichtigsten ist hierbei die Erkenntnis, das subinhibitorische SDS Konzentrationen einen deutlichen stammabhängigen Effekt auf die sae Transkription und daraus folgernd auf die Wirtszellinvasion von S. aureus haben. Letzteres wird vermutlich in manchen Stämmen durch andere Faktoren als die bekannten Invasinproteine Eap und FnBP vermittelt. Außerdem scheint es in den klinischen S. aureus Isolaten mehr als nur den saeSP abhängigen Mechanismus der sae Induktion durch SDS zu geben. Diese Ergebnisse helfen uns die Virulenz und pathogenen Mechanismen als auch deren Regulation in S. aureus zu verstehen. Die Beobachtungen tragen zu unserem Verständnis bei, wie das sae System Signale der Umgebung detektieren kann. Dies ist bis jetzt eine Fragestellung mit vielen Unbekannten.

Understanding cancer biomarkers for diagnosis and prognosis leads to improved patient treatments and care. This thesis addresses the relevance of thymidine kinase 1 (TK1) as a cancer biomarker and the role of TK1 in cancer progression. Worldwide, cancer leads to more than 12 million deaths annually. In the United States alone, each year over 1.5 million cases will be diagnosed and over half a million persons will die. The most prevalent cancer types include skin, lung, breast, prostate, and colon. TK1 is known to be present in the serum of patients with multiple cancer types, including lung, breast, colon and prostate. In fact, it is shown to be detectable in cancer patients even before they manifest clinical symptoms. Additionally, the levels of TK1 increase progressively with increasing tumor grade; meaning that levels of TK1 can indicate tumor grade. Cellular proliferation markers such as p53 and Ki-67 have been compared to TK1 in cancer diagnosis and prognosis. TK1 has potential as both a prognostic and diagnostic biomarker in various cancer types including breast. Breast cancer is one of the most aggressive cancer types with 20-30% of diagnosed tumors becoming metastatic. Recent findings have identified additional involvement of TK1 downstream of cellular proliferation in cancer progression, including cellular invasion which is a part of cancer metastasis. These findings while efficacious, fail to identify the individual contribution of TK1 in downstream processes that aid in cancer progression. As mentioned previously, TK1 is upregulated in several different cancer types. We propose that there is an advantage to upregulated levels of TK1 in cancer progression and seek to explore its role specifically in cell invasion and survival. Based on our current understanding of TK1, we first wanted to review the history of TK1 and show the importance of understanding this crucial enzyme. Finally, we report our results from experiments exploring the influence of TK1 in vitro on breast cancer cell invasion and survival.

Fusobacterium nucleatum is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacterium species exhibit genetic recalcitrance, which has led to hindrance in the understanding of their biology and molecular pathogenesis. Though the association of Fusobacterium in diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum ATCC 23726 because it is genetically tractable. Here, in this dissertation, we show that we are able to enhance our existing molecular tools for genome editing to introduce the first mutants in a clinically relevant strain, F. nucleatum ATCC 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cellular invasion and survival. In this work, we identified a novel small adhesin, FadA2, that played a significant role in the invasive ability of F. nucleatum ATCC 25586 to colorectal cancer cells. This dissertation also sheds the first insight into the roles of the type 5a autotransporters. Using a deletion library of genes encoding for the type 5a autotransporter proteins in F. nucleatum ATCC 23726, we systemically characterized altogether 12 type 5a proteins with a focus on the invasion of colorectal cancer cells. Most notably, we found that a wide assortment of type 5a proteins contributing to binding and invasion of F. nucleatum to HCT116 cancer cells. Furthermore, we identified that RadD was not directly involved in inducing secretions of the cytokines IL-8 and CXCL1 while confirmed the specific association of Fap2 in bacterial-induced cytokine secretion. Thus, our findings provided the first comparative and functional analysis of Fusobacterium type 5a autotransporter proteins in colorectal cancer cells which will be crucial to the understanding of Fusobacterium involvement in cancer progression. Finally, this dissertation reported on the first ever observation on the survival strategy of Fusobacterium inside the host cells. We uncovered a novel protein that contributed to enhanced survival of Fusobacterium residing in colorectal cancer cells. This work undoubtedly helps expand the current Fusobacterium genetic toolkit to study proteins and mechanisms relevant to Fusobacterium-accelerated diseases. By identifying and characterizing novel virulence strategies that Fusobacterium can take advantage of, we can increase our comprehension on this opportunistic microbe while devising innovative therapeutic treatments. / Doctor of Philosophy / Fusobacterium, a member of the microbial community in our mouth, has been a captivating study target due to its association with human health and diseases. By nature, Fusobacterium lives in oxygen-free pockets between our teeth and gumline in which this organism has been correlated with a multitude of complications and diseases including periodontitis, inflammatory bowel disease, preterm birth, and most importantly colorectal cancer. Though the connection to human health is established, we still have to learn more about the mechanisms utilized by Fusobacterium to exacerbate diseases. This challenge is mainly hindered by the lack of efficient tools and resources to systematically investigate the relationship between the bacterium and its human host. Therefore, the work in this dissertation focuses on expanding the existing molecular toolkit to study clinically relevant Fusobacterium strain, which provides the power and convenience to discover novel mechanisms that Fusobacterium can take advantage of to be a successful pathogen. Accordingly, we first enhanced our ability to work with a wider range of Fusobacterium species. We successfully introduced exogenous genetic materials into a clinical strain of Fusobacterium, Fusobacterium nucleatum ATCC 25586. This breakthrough was built on the success of our current toolkit to make genetic modifications to a sister strain, Fusobacterium nucleatum ATCC 23726. With this newfound capacity to modify F. nucleatum ATCC 25586, we have described the importance of a novel protein aiding in the invasion of Fusobacterium to colorectal cancer. Furthermore, we have determined that certain proteins within the fusobacterial type 5a protein family can play a key role in governing binding and invasion of colorectal cancer cells in this study. Concurrently, for the first time, we provided the snapshot of a small protein and its role in fusobacterial long-term survival inside its targeted host cells. Altogether, the findings in this dissertation will bring forth an innovative framework to better the comprehension of current Fusobacterium-induced disease implications, while exploring alternative treatments for enhanced patient health.

Nous étudions la migration et la protéolyse de la matrice extracellulaire dans le cancer du sein. Pour cela, nous avons mis en place deux systèmes modèles. Le premier, se base sur une lame basale reconstituée et permet d'évaluer le potentiel invasif de lignées cellulaires tumorales. Nous montrons que les cellules cancéreuses migrent différemment à travers un gel pour former des amas de taille variable directement corrélé à leur pouvoir invasif. Dans notre système, seule la migration de type mésenchymateuse est utilisée par les cellules. Ce type de mouvement est directement dépendant de protéases sécrétées par les cellules. Nous avons, donc mesurée la synthèse au niveau transcriptionnel de la classe d'enzyme majoritairement impliquée dans la dissémination tumorale, les matrice métalloprotéases (MMPs). Nous avons ainsi pu montrer que l'expression de 3 MMPs est corrélée aux capacités migratoires des cellules donc à leur potentiel invasif. Le processus physique par lequel les enzymes dégradent les matrices est très peu étudié au niveau expérimental. Le second système que nous utilisons se base sur un modèle de matrice conjonctive majoritairement composer de collagène de type I. Nous utilisons la gélatine, pour étudier la protéolyse de gels protéiques par différentes classes de protéases. A partirdes études sur la solubilisation enzymatique des gels par l'-chymotrypsine, la protéinase K et la papaïne, nous montrons qu'il existe des mécanismes de dégradation distincts. Le premier est un mécanisme anormal dont la cinétique est limitée par la diffusion de l'enzyme, le second est brownien et la cinétique est limitée par la réaction. Ce second mécanisme dépend directement d'interactions eléctrostatiques entre l'enzyme et le gel. Nous observons pour deux des enzymes que l'évolution des temps de dégradation mais également la cinétique dépendent de la concentration en protéine dans les gels. / We study the migration and proteolysis of the extracellular matrix in breast cancer. For this, we set up two model systems. The first is based on a reconstituted basement membrane and allows the evaluation of invasive potential tumor cell lines. We show that cancer cells migrate differently across the gel to form clusters of variable size directly correlates with their invasiveness. In our system, only the migration of mesenchymal type is used by the cells. This type of movement is directly dependent proteases secreted by the cells. We therefore measured the synthesis at the transcriptional level of the enzyme class mainly involved in tumor dissemination, the matrix metalloproteases (MMPs). We were able to show that the expression of 3 MMPs is correlated with migratory capacity of cells, therefore their invasive potential. The physical process by which enzymes degrade the matrix is very little studied at the experimental level. The second system we use is based on a model of connective matrix mainly composed of collagen type I. We use gelatin for the study of protein gels proteolysis by different classes of proteases. Based on the study of gels enzymatic solubilization by a- chymotrypsin, proteinase K and papain, we show that there are distinct mechanisms of degradation. The first mechanism is abnormal whose kinetic is limited by enzyme diffusion, and the second is Brownian and the kinetic is reaction limited. The second mechanism depends directly on electrostatic interactions between enzyme and gel. We observe for two enzymes that the evolution of degradation time but also the degradation kinetics depend on the concentration of protein in gels.

Ovarian cancer (OC) has the highest mortality among gynecological cancers. The high mortality is associated with the lack of an accurate screening tool to detect disease in early stage. As a result the majority of OCs are diagnosed in late stage. Further, the molecular events responsible for malignant transformation in the ovary remain poorly understood. Consequently, delineating key molecular players driving OC could help elucidate potential diagnostic, prognostic and therapeutic targets. Receptor for hyaluronan-mediated motility (RHAMM) belongs to a group of hyaladherins, which share a common ability to bind to hyaluronan (HA). Intracellularly, RHAMM is involved in microtubule spindle assembly contributing to cell cycle progression. On the cell surface, loosely tethered RHAMM forms a complex with cluster differentiation 44 and HA to activate cell signaling pathways that promote cellular migration, invasion and proliferation. Since RHAMM is overexpressed in a number of cancer types and it is often associated with an aggressive cancer phenotype, I sought to determine if RHAMM similarly contributes to OC. I found that RHAMM is overexpressed in clinical specimens of OC by immuno-histochemistry and although both primary and metastatic OCs stain equally for RHAMM, RHAMM staining was most intense among clinically aggressive OC histologic subtypes. Further, using an in vitro model system, I was able to show that OC cells express and secrete RHAMM. Abrogation of RHAMM using silencing RNA technology inhibited OC cell migration and invasion suggesting that RHAMM may contribute, at least in part, to the metastatic propensity of OC. Since RHAMM lacks an export signal peptide sequence and has not been reported to employ alternate mechanisms for extracellular secretion, I utilized computational analyses to predict post-translational glycosylation events as a novel mode for RHAMM secretion. N- glycosylation inhibitors abrogated RHAMM secretion by OC cells in vitro validating my prediction and identify a novel and potentially unconventional mode for RHAMM secretion. Lastly, since RHAMM is secreted by OC cells, I sought to determine whether RHAMM could be detected in bodily fluids. In a pilot study, I found that urinary levels of RHAMM are elevated in OC patients as measured by enzyme-linked immunosorbant assays. Decreased urinary RHAMM levels noted following cytoreductive surgery support OC as the source of elevated urinary RHAMM levels. Finally, while obesity was associated with high urinary RHAMM levels in OC patients, combined measurements of urinary RHAMM and serum CA125 improved prediction of OC. Taken together, the studies described herein suggest that RHAMM contributes to OC and that further studies are warranted to further elucidate the clinical role of RHAMM in OC.

A causa de morte da maioria das pacientes com câncer de mama se deve à doença metastática desenvolvida a partir do tumor primário. A degradação dos componentes da matriz extracelular (MEC), um dos principais eventos do processo metastático, é regulada pelo balanço entre as atividades das metaloproteinases de matriz (MMPs) e dos seus inibidores, tanto os inibidores teciduais (TIMPs) como o inibidor associado à membrana (RECK). Contudo, ainda existe pouca informação sobre os mecanismos moleculares responsáveis pela manutenção deste balanço. No presente trabalho, foi investigado o envolvimento de TGF-β1 (Transforming Growth Factor-β1), uma citocina multifuncional é capaz tanto de inibir o crescimento celular, quanto de promover invasão e metástase, dependendo do estadiamento e do tipo de tumor, na regulação da expressão de MMPs, TIMPs e RECK, em modelo de câncer de mama. Primeiramente, examinou-se os níveis de expressão de mRNA das isoformas e receptores de TGF-β, em um painel de cinco linhagens de carcinoma mamário humano, com diferentes potenciais invasivos e metastáticos, por qRT-PCR. Os resultados obtidos demonstraram uma correlação positiva entre a expressão dessas moléculas, e a progressão do caráter invasivo e metastático celular. Em seguida, a linhagem altamente invasiva, MDA-MB-231, foi tratada com diferentes concentrações de TGF-β1 recombinante. Esta citocina foi capaz de modular a expressão gênica de MMPs (MMP-2 e MMP-9) e de seus inibidores (TIMP- 2 e RECK). Tanto ERK½, quanto p38MAPK mostraram-se envolvidas neste mecanismo. Foi demonstrado que a inibição da atividade de ERK½ alterou a expressão das proteínas MMP-9, TIMP-2 e RECK, enquanto o bloqueio de p38 MAPK afetou os níveis protéicos de MMP-2 e TIMP-2. O aumento do potencial migratório e invasivo da linhagem MDA-MB-231, induzido por TGF-β1, mostrou-se também dependente da atividade de MMPs, ERK½ e p38MAPK. Dada a ausência de informações sobre o papel de RECK em modelo mamário, a função deste inibidor de MMPs também foi investigada. Primeiramente, analisou-se a expressão de RECK ao longo do desenvolvimento da mama e, posteriormente, em 1040 amostras tumorais de mama humana, através da metodologia de Tissue Microarray, tendo sido possível demonstrar que a alta expressão de RECK associa-se a menor tempo de sobrevida global e livre de doença em 10 anos. Os resultados obtidos indicaram que a expressão da proteína RECK, em oposição ao verificado em outros tipos de tumores, está relacionada ao fenótipo mais agressivo de tumores de mama. Entretanto, a análise funcional de RECK, realizada por meio da utilização de vetores shRNA específicos para a inibição desta proteína, demonstrou que RECK também atua como um inibidor de invasão celular e da expressão de MMP-9, na linhagem MDA-MB-231. Em conjunto, os resultados obtidos neste trabalho contribuíram para a elucidação dos mecanismos moleculares de regulação de RECK, por clássicas moléculas associadas ao processo de tumorigênese (TGF-β1 e MAPKs), bem como para o esclarecimento de suas funções em modelo mamário, sugerindo-o como mais um promissor candidato a marcador prognóstico e alvo molecular para a terapia do câncer de mama. / The metastatic disease is the main mortality cause of breast cancer patients. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix (ECM) compounds. The degradation of ECM is tightly regulated by the balance between the activities of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors (TIMPs) and the membrane-associated inhibitor (RECK). Among the several molecules released and activated by ECM remodeling, TGF-β1 (Transforming Growth Factor-β1) is a multifunctional cytokine able to regulate both cell growth inhibition and invasion and metastasis promotion, depending on the tumor stage and type. Since the molecular mechanisms involved in the ECM remodeling control are still not completed understood, in this study, we investigated the involvement of TGF-β1 in regulating of MMPs, TIMPs and RECK expression, in the breast cancer model. By qRT-PCR, we first examined the gene expression levels of TGF-β isoforms and receptors, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. Our results suggest a positive correlation between the mRNA expression of these molecules and the breast cancer progression. Moreover, the highly invasive breast cancer cell line MDA-MB-231 was treated with different concentrations of recombinant TGF-β1. We described that this cytokine was able to modulate the gene expression of MMPs (MMP-2 and MMP-9) and MMPs inhibitors (TIMP-2 and RECK) at both the mRNA and protein levels, with ERK½ and p38 MAPK being involved in this molecular mechanism. However, while ERK½ activity inhibition altered MMP-9, TIMP-2 and RECK expression, the p38 MAPK blockage affected the protein levels of MMP-2 and TIMP-2. Finally, we reposted that the TGF-β1-enhanced migration and invasion capacities of MDA-MB- 231 cells were blocked by MMPs, ERK½ and p38 MAPK inhibitors. Analysis of the RECK function in the breast model was also an objective of this study. We analyzed RECK expression during mammary gland development. We evaluated the RECK protein profile in 1040 breast tumor tissue samples using Tissue Microarray assays. We demonstrated that high expression levels of RECK were associated with shorter overall and disease-free survival in 10 years. Moreover, we verified that RECK is a biomarker of poor prognosis mainly for patients diagnosed with less aggressive breast tumor. Therefore, in contrast to other tumor types, our results indicate that high protein expression levels of RECK are related to a more aggressive phenotype. In fact, the RECK functional analysis, performed by using of shRNA vectors, showed that RECK function remains as an inhibitor of cellular invasion and MMP-9 expression, in MDA-MB-231 cells. Taken together, our results contribute to better understanding of the molecular mechanisms associated to RECK regulation by TGF-β1 and MAPK as well as to clarify its role in breast model. Thus, we suggests RECK as a new and promising prognostic marker and molecular target candidate for breast cancer therapy.

A causa de morte da maioria das pacientes com câncer de mama se deve à doença metastática desenvolvida a partir do tumor primário. A degradação dos componentes da matriz extracelular (MEC), um dos principais eventos do processo metastático, é regulada pelo balanço entre as atividades das metaloproteinases de matriz (MMPs) e dos seus inibidores, tanto os inibidores teciduais (TIMPs) como o inibidor associado à membrana (RECK). Contudo, ainda existe pouca informação sobre os mecanismos moleculares responsáveis pela manutenção deste balanço. No presente trabalho, foi investigado o envolvimento de TGF-β1 (Transforming Growth Factor-β1), uma citocina multifuncional é capaz tanto de inibir o crescimento celular, quanto de promover invasão e metástase, dependendo do estadiamento e do tipo de tumor, na regulação da expressão de MMPs, TIMPs e RECK, em modelo de câncer de mama. Primeiramente, examinou-se os níveis de expressão de mRNA das isoformas e receptores de TGF-β, em um painel de cinco linhagens de carcinoma mamário humano, com diferentes potenciais invasivos e metastáticos, por qRT-PCR. Os resultados obtidos demonstraram uma correlação positiva entre a expressão dessas moléculas, e a progressão do caráter invasivo e metastático celular. Em seguida, a linhagem altamente invasiva, MDA-MB-231, foi tratada com diferentes concentrações de TGF-β1 recombinante. Esta citocina foi capaz de modular a expressão gênica de MMPs (MMP-2 e MMP-9) e de seus inibidores (TIMP- 2 e RECK). Tanto ERK½, quanto p38MAPK mostraram-se envolvidas neste mecanismo. Foi demonstrado que a inibição da atividade de ERK½ alterou a expressão das proteínas MMP-9, TIMP-2 e RECK, enquanto o bloqueio de p38 MAPK afetou os níveis protéicos de MMP-2 e TIMP-2. O aumento do potencial migratório e invasivo da linhagem MDA-MB-231, induzido por TGF-β1, mostrou-se também dependente da atividade de MMPs, ERK½ e p38MAPK. Dada a ausência de informações sobre o papel de RECK em modelo mamário, a função deste inibidor de MMPs também foi investigada. Primeiramente, analisou-se a expressão de RECK ao longo do desenvolvimento da mama e, posteriormente, em 1040 amostras tumorais de mama humana, através da metodologia de Tissue Microarray, tendo sido possível demonstrar que a alta expressão de RECK associa-se a menor tempo de sobrevida global e livre de doença em 10 anos. Os resultados obtidos indicaram que a expressão da proteína RECK, em oposição ao verificado em outros tipos de tumores, está relacionada ao fenótipo mais agressivo de tumores de mama. Entretanto, a análise funcional de RECK, realizada por meio da utilização de vetores shRNA específicos para a inibição desta proteína, demonstrou que RECK também atua como um inibidor de invasão celular e da expressão de MMP-9, na linhagem MDA-MB-231. Em conjunto, os resultados obtidos neste trabalho contribuíram para a elucidação dos mecanismos moleculares de regulação de RECK, por clássicas moléculas associadas ao processo de tumorigênese (TGF-β1 e MAPKs), bem como para o esclarecimento de suas funções em modelo mamário, sugerindo-o como mais um promissor candidato a marcador prognóstico e alvo molecular para a terapia do câncer de mama. / The metastatic disease is the main mortality cause of breast cancer patients. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix (ECM) compounds. The degradation of ECM is tightly regulated by the balance between the activities of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors (TIMPs) and the membrane-associated inhibitor (RECK). Among the several molecules released and activated by ECM remodeling, TGF-β1 (Transforming Growth Factor-β1) is a multifunctional cytokine able to regulate both cell growth inhibition and invasion and metastasis promotion, depending on the tumor stage and type. Since the molecular mechanisms involved in the ECM remodeling control are still not completed understood, in this study, we investigated the involvement of TGF-β1 in regulating of MMPs, TIMPs and RECK expression, in the breast cancer model. By qRT-PCR, we first examined the gene expression levels of TGF-β isoforms and receptors, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. Our results suggest a positive correlation between the mRNA expression of these molecules and the breast cancer progression. Moreover, the highly invasive breast cancer cell line MDA-MB-231 was treated with different concentrations of recombinant TGF-β1. We described that this cytokine was able to modulate the gene expression of MMPs (MMP-2 and MMP-9) and MMPs inhibitors (TIMP-2 and RECK) at both the mRNA and protein levels, with ERK½ and p38 MAPK being involved in this molecular mechanism. However, while ERK½ activity inhibition altered MMP-9, TIMP-2 and RECK expression, the p38 MAPK blockage affected the protein levels of MMP-2 and TIMP-2. Finally, we reposted that the TGF-β1-enhanced migration and invasion capacities of MDA-MB- 231 cells were blocked by MMPs, ERK½ and p38 MAPK inhibitors. Analysis of the RECK function in the breast model was also an objective of this study. We analyzed RECK expression during mammary gland development. We evaluated the RECK protein profile in 1040 breast tumor tissue samples using Tissue Microarray assays. We demonstrated that high expression levels of RECK were associated with shorter overall and disease-free survival in 10 years. Moreover, we verified that RECK is a biomarker of poor prognosis mainly for patients diagnosed with less aggressive breast tumor. Therefore, in contrast to other tumor types, our results indicate that high protein expression levels of RECK are related to a more aggressive phenotype. In fact, the RECK functional analysis, performed by using of shRNA vectors, showed that RECK function remains as an inhibitor of cellular invasion and MMP-9 expression, in MDA-MB-231 cells. Taken together, our results contribute to better understanding of the molecular mechanisms associated to RECK regulation by TGF-β1 and MAPK as well as to clarify its role in breast model. Thus, we suggests RECK as a new and promising prognostic marker and molecular target candidate for breast cancer therapy.