Unrevealing the effect of STEAP1 knockdown in prostate cancer cells: from protein expression profile to clinical applications

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Enhanced stability of detergent-free human native STEAP1 protein from neoplastic prostate cancer cells upon an innovative isolation procedure

Publication . Barroca-Ferreira, Jorge; Cruz-Vicente, Pedro; Santos, Marino F. A.; Rocha, Sandra M.; Santos-Silva, Teresa; Maia, Cláudio J.; Passarinha, Luís A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.

2021-09Artigo científico

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Promoter demethylation upregulates STEAP1 gene expression in human prostate cancer

Publication . Rocha, Sandra M.; Sousa, Inês; Gomes, Inês M.; Arinto, Patrícia; Costa-Pinheiro, Pedro; Coutinho, Eduarda; Santos, Cecília R. A.; Jerónimo, Carmen; Lemos, Manuel C.; Passarinha, Luís A.; Socorro, Sílvia; Maia, Cláudio J.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute

The Six Transmembrane Epithelial Antigen of the Prostate (STEAP1) is an oncogene overexpressed in several human tumors, particularly in prostate cancer (PCa). However, the mechanisms involved in its overexpression remain unknown. It is well known that epigenetic modifications may result in abnormal gene expression patterns, contributing to tumor initiation and progression. Therefore, this study aimed to analyze the methylation pattern of the STEAP1 gene in PCa versus non-neoplastic cells. Bisulfite amplicon sequencing of the CpG island at the STEAP1 gene promoter showed a higher methylation level in non-neoplastic PNT1A prostate cells than in human PCa samples. Bioinformatic analysis of the GEO datasets also showed the STEAP1 gene promoter as being demethylated in human PCa, and a negative association with STEAP1 mRNA expression was observed. These results are supported by the treatment of non-neoplastic PNT1A cells with DNMT and HDAC inhibitors, which induced a significant increase in STEAP1 mRNA expression. In addition, the involvement of HDAC in the regulation of STEAP1 mRNA expression was corroborated by a negative association between STEAP1 mRNA expression and HDAC4,5,7 and 9 in human PCa. In conclusion, our work indicates that STEAP1 overexpression in PCa can be driven by the hypomethylation of STEAP1 gene promoter.

2021-11-17Artigo científico

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Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/115693/2016