Análise do perfil proteômico tecidual de tumores uroteliais de bexiga não músculo-invasivo
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Data
2024-05-24
Autores
Silva, Tiago Aparecido [UNIFESP]
Orientadores
Azevedo, Hatylas Felype Zaneti de [UNIFESP]
Tipo
Tese de doutorado
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Objetivo: Caracterizar o perfil proteômico tecidual do carcinoma urotelial de bexiga não músculo-invasivo. Métodos: Estudo transversal comparando o perfil proteômico tecidual de tumores de bexiga com tecido vesical adjacente macroscopicamente normal submetidos à ressecção transuretral de bexiga. As amostras teciduais tiveram confirmação histopatológica e foram submetidas a protocolos de extração proteica, purificação e análise proteômica tipo shotgun, com a utilização de cromatografia líquida acoplada à espectrometria de massa e recursos de bioinformática. As proteínas encontradas tiveram sua expressão comparadas e aquelas que apresentaram fold-change ≥2 ou ≤0,5 foram consideradas diferencialmente expressas. Resultados: Quarenta e cinco pacientes preencheram os critérios de inclusão e tiveram tecido tumoral e adjacente submetidos à análise proteômica. Os dados clínicos e epidemiológicos revelaram que: 34 (75,6%) pacientes eram do sexo masculino; a idade média foi de 66 anos; 75,6% das lesões eram de novo; e o tamanho médio da lesão foi de 2,8 cm. O resultado anatomopatológico revelou 26 (57,8%) lesões pTa baixo grau, 6 (13,3%) lesões de pTa alto grau, 1 (2,2%) lesão PUNLMP e 12 (26,7%) lesões pT1 alto grau. Cento e oitenta e oito proteínas apresentaram-se diferentemente expressas entre tecido tumoral e adjacente. Além disso, 11 proteínas foram encontradas exclusivamente no tecido tumoral (SPINT1, TXNDC12, GTF2F1, COPZ1, RS25, PTK2, LSR, SNRNP40, NCOA5, SEC63, CD2AP). Após construção de rede de interação proteína-proteína, as proteínas AGR2, FLNA, CALD1, TPM1, VCL e TPM3 destacaram-se por apresentar maior número de interações. Além disso, notam-se principalmente dois conjuntos de proteínas enriquecidas formando módulos (clusters): proteínas ribossomais (RPL7, RPL 24, RPL 19, RPL 29, RPL 15, RPL 35) e queratinas (KRT5, KRT7 KRT8, KRT13, KRT 15, KRT17, KRT19, KRT20). O enriquecimento funcional das proteínas evidenciou importância nas funções de aderência focal, proteoglicanos ligados ao câncer, e proteínas ligadas à interação da matriz extracelular, além dos processos biológicos de organização de fibras supramoleculares e organização de actina. As proteínas foram analisadas conforme variáveis clínicas, e CNDP relacionou-se à recorrência tumoral, e a EPS8L ao estadiamento clínico. Conclusão: A comparação proteômica entre o carcinoma urotelial não músculo-invasivo e o tecido adjacente evidenciou 188 proteínas diferentemente expressas e 11 proteínas exclusivas (SPINT1, TXNDC12, GTF2F1, COPZ1, RS25, PTK2, LSR, SNRNP40, NCOA5, SEC63, CD2AP). As proteínas AGR2, FLNA, TPM1, CALD1, foram diferencialmente expressas no tecido tumoral e possuem papel de destaque pelo número elevado de interações proteína-proteína. A expressão da proteína CNDP relacionou-se à recorrência tumoral, e a EPS8L ao estadiamento tumoral. Todas essas proteínas representam potenciais biomarcadores tumorais no câncer de bexiga não músculo-invasivo e merecem estudos de validação futuros.
Objective: characterization of the tissue proteomic profile in nonmuscleinvasive bladder cancer by comparing paired tumor and normal samples. Methods: crosssectional study comparing the tissue proteomic profile of bladder tumors with macroscopically normal adjacent bladder tissue undergoing transurethral resection. The tissue samples were submitted to histopathological confirmation and subjected to protein extraction, purification, and shotgun proteomic analysis, using liquid chromatography coupled to mass spectrometry and bioinformatics analyses. Differentially expressed and exclusive proteins were identified using statistical analysis and selecting the proteins with at least an absolute fold change ≥2. Results: fortyfive patients met the inclusion criteria and had their tumor and adjacent tissue subjected to proteomic analysis. Clinical and epidemiological data revealed: 34 (75.6%) patients were male; the mean age was 66 years; 75.6% of the tumors were de novo; and the average lesion size was 2.8 cm. The anatomopathological analysis revealed that 26 (57.8%) samples were lowgrade pTa lesions, 6 (13.3%) were highgrade pTa lesions, 1 (2.2%) was a PUNLMP lesion, and 12 (26.7%) were highgrade pT1 lesions. One hundred and eightyeight proteins were differentially expressed between tumor and adjacent tissue. Furthermore, 11 proteins were exclusively found in tumor tissue (SPINT1, TXNDC12, GTF2F1, COPZ1, RPS25, PTK2, LSR, SNRNP40, NCOA5, SEC63, CD2AP). The network analysis of the protein interactions revealed that proteins AGR2, FLNA, CALD1, TPM1, VCL, and TPM3 exhibited the greatest number of interactions. Furthermore, two sets of enriched proteins formed clusters: ribosomal proteins (RPL7, RPL24, RPL19, RPL29, RPL15, RPL35) and keratins (KRT5, KRT7, KRT8, KRT13, KRT15, KRT17, KRT19, KRT20). The functional enrichment analysis of the differentially expressed proteins disclosed the importance of focal adhesion, proteoglycans linked to cancer, and proteins linked to the interaction of the extracelular matrix, supramolecular fiber organization, and actin organization. Proteins were analyzed according to clinical variables, and CNDP2 correlated with tumor recurrence, whereas EPS8L2 was associated with clinical staging. Conclusions: the proteomic comparison between nonmuscleinvasive urothelial carcinoma and adjacent tissue revealed 188 differentially expressed proteins and 11 unique proteins (SPINT1, TXNDC12, GTF2F1, COPZ1, RPS25, PTK2, LSR, SNRNP40, NCOA5, SEC63, CD2AP). The proteins AGR2, FLNA, TPM1, and CALD1 were differentially expressed in the tissue and play a prominent role due to their high number of proteinprotein interactions. CNDP2 and EPS8L2 expression were associated with tumor recurrence and tumor staging, respectively. All these proteins represent potential tumor biomarkers in nonmuscleinvasive bladder cancer and deserve future validation studies.
Objective: characterization of the tissue proteomic profile in nonmuscleinvasive bladder cancer by comparing paired tumor and normal samples. Methods: crosssectional study comparing the tissue proteomic profile of bladder tumors with macroscopically normal adjacent bladder tissue undergoing transurethral resection. The tissue samples were submitted to histopathological confirmation and subjected to protein extraction, purification, and shotgun proteomic analysis, using liquid chromatography coupled to mass spectrometry and bioinformatics analyses. Differentially expressed and exclusive proteins were identified using statistical analysis and selecting the proteins with at least an absolute fold change ≥2. Results: fortyfive patients met the inclusion criteria and had their tumor and adjacent tissue subjected to proteomic analysis. Clinical and epidemiological data revealed: 34 (75.6%) patients were male; the mean age was 66 years; 75.6% of the tumors were de novo; and the average lesion size was 2.8 cm. The anatomopathological analysis revealed that 26 (57.8%) samples were lowgrade pTa lesions, 6 (13.3%) were highgrade pTa lesions, 1 (2.2%) was a PUNLMP lesion, and 12 (26.7%) were highgrade pT1 lesions. One hundred and eightyeight proteins were differentially expressed between tumor and adjacent tissue. Furthermore, 11 proteins were exclusively found in tumor tissue (SPINT1, TXNDC12, GTF2F1, COPZ1, RPS25, PTK2, LSR, SNRNP40, NCOA5, SEC63, CD2AP). The network analysis of the protein interactions revealed that proteins AGR2, FLNA, CALD1, TPM1, VCL, and TPM3 exhibited the greatest number of interactions. Furthermore, two sets of enriched proteins formed clusters: ribosomal proteins (RPL7, RPL24, RPL19, RPL29, RPL15, RPL35) and keratins (KRT5, KRT7, KRT8, KRT13, KRT15, KRT17, KRT19, KRT20). The functional enrichment analysis of the differentially expressed proteins disclosed the importance of focal adhesion, proteoglycans linked to cancer, and proteins linked to the interaction of the extracelular matrix, supramolecular fiber organization, and actin organization. Proteins were analyzed according to clinical variables, and CNDP2 correlated with tumor recurrence, whereas EPS8L2 was associated with clinical staging. Conclusions: the proteomic comparison between nonmuscleinvasive urothelial carcinoma and adjacent tissue revealed 188 differentially expressed proteins and 11 unique proteins (SPINT1, TXNDC12, GTF2F1, COPZ1, RPS25, PTK2, LSR, SNRNP40, NCOA5, SEC63, CD2AP). The proteins AGR2, FLNA, TPM1, and CALD1 were differentially expressed in the tissue and play a prominent role due to their high number of proteinprotein interactions. CNDP2 and EPS8L2 expression were associated with tumor recurrence and tumor staging, respectively. All these proteins represent potential tumor biomarkers in nonmuscleinvasive bladder cancer and deserve future validation studies.
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SILVA, Tiago Aparecido. Análise do perfil proteômico tecidual de tumores uroteliais de bexiga não músculo-invasivo. 2024. 93 f. Tese (Doutorado em Urologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.