Background: Oral cancer, the largest subset of head and neck cancer, has become one of the most lethal malignancies during the last two decades. Although several diagnostic tools have been applied for the early detection of oral malignancies, it is still urgent to identify novel tumor markers. In this study, we explored the cell surface N-glycomes of primary cultured human oral keratinocytes (HOK), immortalized human gingival keratinocytes (SG cells), and oral squamous cell carcinoma (OC2). Methods: Enzymatically hydrolyzed cell surface N-glycans were analyzed by MALDI-TOF mass spectrometry. Results: High levels of fucosylated N-glycans, especially core-fucosylated N-glycans, were observed on the OC2 cell surface whereas the major N-glycans on SG and HOK cells were high mannose type. In addition, the mRNA expression level of fucosyltransferase 8 was elevated significantly in OC2 cells than in SG and HOK cells. Core-fucosylated glycoproteins of OC2 cells were then purified with lectin affinity chromatography and a key adhesion molecule in cancer cells, CD147, was identified. Finally, overexpression of cell surface CD147 was confirmed on OC2 cells and oral cancer tissues (tissue array). Conclusions: CD147 was discovered by glycoproteomic approaches and suggested to be a potential novel tumor marker for oral cancer diagnosis.

Background: Oral cancer, the largest subset of head and neck cancer, has become one of the most lethal malignancies during the last two decades. Although several diagnostic tools have been applied for the early detection of oral malignancies, it is still urgent to identify novel tumor markers. In this study, we explored the cell surface N-glycomes of primary cultured human oral keratinocytes (HOK), immortalized human gingival keratinocytes (SG cells), and oral squamous cell carcinoma (OC2). Methods: Enzymatically hydrolyzed cell surface N-glycans were analyzed by MALDI-TOF mass spectrometry. Results: High levels of fucosylated N-glycans, especially core-fucosylated N-glycans, were observed on the OC2 cell surface whereas the major N-glycans on SG and HOK cells were high mannose type. In addition, the mRNA expression level of fucosyltransferase 8 was elevated significantly in OC2 cells than in SG and HOK cells. Core-fucosylated glycoproteins of OC2 cells were then purified with lectin affinity chromatography and a key adhesion molecule in cancer cells, CD147, was identified. Finally, overexpression of cell surface CD147 was confirmed on OC2 cells and oral cancer tissues (tissue array). Conclusions: CD147 was discovered by glycoproteomic approaches and suggested to be a potential novel tumor marker for oral cancer diagnosis.

Les fibroblastes activés qui composent les stromas tumoraux sont des acteurs majeurs des interactions tumeur-stroma impliquées dans la croissance et la dissémination des cellules tumorales. Ce processus d'activation des fibroblastes est caractérisé par l'expression de marqueurs protéiques spécifiques parmi lesquels figure l'alphaSMA et FAPalpha;. Le TGFbeta;, cytokine secrétée massivement par les cellules tumorales, est un des éléments impliqués dans l'activation des fibroblastes et la formation du stroma tumoral qui en résulte. L'EMMPRIN, glycoprotéine transmembranaire surexprimée dans les cellules tumorales est également un médiateur des interactions tumeur-stroma puisqu'il a la capacité d'induire la synthèse des MMPs par les fibroblastes péri-tumoraux accroissant ainsi la propagation des cellules tumorales à travers l'organisme. Nos travaux identifièrent que le TGFbeta secrété par les cellules tumorales induisait la synthèse du marqueur FAPalpha par les fibroblastes. L'EMMPRIN stromal apparaît comme récepteur de ces signaux tumoraux et est nécessaire à la synthèse du marqueur FAPalpha; par les fibroblastes. L'EMMPRIN participe donc à l'activation TGFbeta; dépendante des fibroblastes. Son inhibition dans ces cellules conduit à un dysfonctionnement de la signalisation médiée par les protéines Smad2/Smad3 aboutissant à une diminution de la synthèse du marqueur alphaSMA ainsi que de certaines protéines matricielles induites par le TGFbeta. L'étude du mécanisme d'action de l'EMMPRIN dans ce processus a permis d'identifier l'EMMPRIN comme nouvelle protéine chaperonne du récepteur de type I au TGFbeta;. / Tumor stroma activated fibroblasts are major actors of tumor stroma interactions taking to tumor growth and spreading. Activated fibroblasts are characterized by the expression of specific markers including alphaSMA and FAPalpha;. The TGFbeta;, a cytokine highly secreted by tumor cells, is one of the key factors involved in fibroblast activation and tumor stroma formation. EMMPRIN, a transmembrane glycoprotein overexpressed in tumor cells, is also a mediator of tumor-stroma interactions by its ability to induce the synthesis of MMPs by peri-tumor fibroblasts enhancing then tumor cells dissemination across the organism.Here, we demonstrate that TGFbeta; secreted by tumor cells is the tumor factor involved in the synthesis of FAPalpha; by fibroblasts. Stromal EMMPRIN appeared to be the receptor of these tumor-stroma interactions and is required for the synthesis of FAPalpha; by fibroblasts. EMMPRIN was also evidenced to take part in TGFbeta;-dependent fibroblast activation. Its inhibition in these cells correlate to a dysfunction in Smad2/Smad3 signaling leading to a decrease in the expression of alphaSMA and matrix proteins induced by TGFbeta;. The study of the mechanism used by EMMPRIN in this process evidenced this protein as a new chaperone for the type I TGFbeta; receptor.

Bladder cancer is a potentially curable malignancy; however in regards to the state of current therapy regimens, a plateau has been reached in both the non-muscle and muscle invasive types. To obtain effective treatment, and consequently a decreased mortality, it has become imperative to test and understand aspects affecting therapy response. The aim of this thesis is to illustrate a better understanding of clinical factors affecting therapy response using new drug combinations and new tumor markers alongside established risk criteria. In Paper I we reported the 5 year follow up from a multicenter, prospectively randomized study and we evaluated the 5-year outcomes of BCG alone compared to a combination of epirubicin and interferon-a2b in the treatment of patients with T1 bladder cancer. Treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant Cis was not predictive of failure of BCG therapy. Independent factor for treatment failure was remaining T1 stage at second resection. In Paper II &III we investigated the validity of emmprin, survivin and CCTα proteins as biomarkers for response and survival before neoadjuvant cisplatin chemotherapy. Bladder tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry (IHC). Patients in the chemotherapy cohort with negative emmprin and CCTα expression had significantly better overall survival (OS) than those with positive expression. In Paper IV primary end point was examining STMN1 as prognostic factor in bladder cancer.  Analysis was performed on three bladder cancer patient cohorts using IHC, western blot and a bladder cancer cell line. High levels of STMN1, expression correlated to shorter disease-specific survival and the growth and migration of the cells were significantly reduced when transfecting the cells with STMN1 siRNA. Conclusion Risk assessment and predictors of outcomes could help in individualized treatment and follow up.  Biomarkers will become more important for treatment choices in bladder cancer management.

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Made available in DSpace on 2015-03-03T15:38:43Z (GMT). No. of bitstreams: 1 PedroPAS_TESE_1-70.pdf: 4719637 bytes, checksum: 8f16cb0e2326a80cfc947b1ea2b89641 (MD5) Previous issue date: 2012-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Myofibroblasts are cells that exhibit a hybrid phenotype, sharing the morphological characteristics of fibroblasts and smooth muscle cells, which is acquired during a process called differentiation. These cells then start to express -SMA, a marker that can be used for their identification. Studies suggest that myofibroblasts are related to the aggressiveness of different tumors and that TGF-1 and IFN- play a role in myofibroblast differentiation, stimulating or inhibiting this differentiation, respectively. The objective of this study was to investigate the role of myofibroblasts in epithelial odontogenic tumors, correlating the presence of these cells with the aggressiveness of the tumor. Immunohistochemistry was used to evaluate the expression of TGF-1 and IFN- in myofibroblast differentiation, as well as the expression of MMP-13, which is activated by myofibroblasts, and of EMMPRIN (extracellular matrix metalloproteinase inducer) as a precursor of this MMP. The sample consisted of 20 solid ameloblastomas, 10 unicystic ameloblastomas, 20 odontogenic keratocysts, and 20 adenomatoid odontogenic tumors. For evaluation of myofibroblasts, anti- -SMA-immunoreactive cells were quantified in connective tissue close to the epithelium. Immunoexpression of TGF-1, IFN-, MMP-13 and EMMPRIN was evaluated in the epithelial and connective tissue components, attributing scores of 0 to 4. The results showed a higher concentration of myofibroblasts in solid ameloblastomas (mean of 30.55), followed by odontogenic keratocysts (22.50), unicystic ameloblastomas (20.80), and adenomatoid odontogenic tumors (19.15) (p=0.001). No significant correlation between TGF-1 and IFN- was observed during the process of myofibroblast differentiation. There was also no correlation between the quantity of myofibroblasts and MMP-13 expression. Significant correlations were found between MMP-13 and TGF-1 (r=0.087; p=0.011), between MMP- 13 and IFN- (r=0.348; p=0.003), as well as between EMMPRIN and MMP-13 (r=0.474; p<0.001) and between EMMPRIN and IFN- (r=0.393; p=0.001). The higher quantity of myofibroblasts observed in solid ameloblastomas, odontogenic keratocysts and unicystic ameloblastomas suggests that these cells are one of the factors responsible for the more aggressive biological behavior of these tumors, although the myofibroblast population was not correlated with TGF-1, IFN-, MMP-13 or EMMPRIN. The correlation between MMP- 13 and TGF-1 suggests that the latter induces the expression of this metalloproteinase. The present results also support the well-established role of EMMPRIN as an inducer of MMP-13. Furthermore, the relationship between EMMPRIN and IFN- and between MMP-13 and IFN- suggests synergism in the antifibrotic effect of these markers / Os miofibroblastos s?o c?lulas que apresentam um fen?tipo h?brido exibindo caracter?sticas morfol?gicas de fibroblastos e de c?lulas musculares lisas, sendo a aquisi??o de tal fen?tipo denominada diferencia??o, passando ent?o a expressar a -SMA, a qual ? importante na identifica??o dessas c?lulas. Estudos t?m sugerido que os miofibroblastos apresentam rela??o com a agressividade de diversas les?es e que o seu processo de diferencia??o estaria relacionado ? express?o do TGF- 1 e do IFN- atuando, respectivamente, no est?mulo e na inibi??o dessa diferencia??o. O objetivo deste trabalho foi investigar o papel dos miofibroblastos em les?es odontog?nicas epiteliais, relacionando-os ? agressividade das les?es e analisar por meio da imuno-histoqu?mica, a express?o do TGF- 1 e IFN- no processo de diferencia??o, al?m da an?lise da MMP-13 que ? ativada por miofibroblastos e do indutor de metaloproteinases de matriz (EMMPRIN) como precursor desta MMP. A amostra foi constitu?da por 20 ameloblastomas s?lidos, 10 ameloblastomas unic?sticos, 20 ceratocistos odontog?nicos e 20 tumores odontog?nicos adenomat?ides. Para a avalia??o dos miofibroblastos, foram quantificadas as c?lulas imunorreativas ao anticorpo - SMA presentes no tecido conjuntivo, pr?ximo ao tecido epitelial. As express?es de TGF- 1, IFN- , MMP-13 e EMMPRIN, foram avaliadas no componente epitelial e no conjuntivo, estabelecendo-se o percentual de imunorreatividade e atribuindo-se escores de 0 a 4. A an?lise dos miofibroblastos evidenciou maior concentra??o nos ameloblastomas s?lidos (m?dia de 30,55), seguido pelos ceratocistos odontog?nicos (22,50), ameloblastomas unic?sticos (20,80) e tumores odontog?nicos adenomat?ides (19,15) com valor de p= 0,001. N?o foi encontrada correla??o significativa entre TGF- 1 e IFN- no processo de diferencia??o dos miofibroblastos, bem como na rela??o entre a quantidade de miofibroblastos e a express?o da MMP-13. Constatou-se, correla??o estat?stica entre MMP-13 e TGF- 1 (r= 0,087; p= 0,011) al?m de significante correla??o entre MMP-13 e IFN- (r=0,348; p=0,003). Entre EMMPRIN e MMP-13 verificou-se signific?ncia (r= 0,474; p<0,001) assim como entre EMMPRIN e IFN- (r=0,393; p=0,001). A maior quantidade de miofibroblastos evidenciada nos ameloblastomas s?lidos, ceratocistos odontog?nicos e ameloblastomas unic?sticos sugere que estas c?lulas podem ser um dos fatores respons?veis para um comportamento biol?gico mais agressivo destas les?es, embora a popula??o de miofibroblastos n?o tenha apresentado correla??o com TGF- - 1, IFN- ,MMP-13 e EMMPRIN. Quanto a correla??o evidenciada entre MMP-13 e TGF- 1, isto pode sugerir um papel indutor do TGF- 1 para a express?o da MMP-13, assim como os resultados deste estudo refor?am a rela??o bem estabelecida do EMMPRIN como indutor da MMP-13. Constatou-se tamb?m rela??o entre EMMPRIN e IFN- assim como entre MMP-13 e IFN- sugerindo, dessa forma, um sinergismo na a??o anti-fibr?tica desses marcadores