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

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

Publication . Rocha, Sandra Catarina Moreira da; Socorro, Sílvia Cristina da Cruz Marques; Passarinha, Luís António Paulino

Prostate cancer (PCa) is one of the most common cancers in the male population and continues to be a considerable source of morbidity and mortality for men worldwide. In an early stage of the disease, the cancer is confined to the prostate gland depending on the stimulating action of androgens. This dependence is the characteristic that allows the use of androgen-deprivation therapy (ADT), which is based on reducing circulating levels of androgens or blocking their actions. The continuous administration of this therapy makes the PCa cells resistant to treatment, being able to survive, extravasate the prostate gland and cause metastases. This stage of the disease is the so-called castrate-resistant PCa (CRPC) and involves the use of chemotherapeutic agents. The limitations of the existent therapeutic options have been stimulating the use of combined therapies targeting survival regulators and proteins involved in the regulation of prostate cell growth and its emergence as a promising option for the treatment of PCa. The development and progression of PCa results from several genetic and metabolic changes, which give tumor cells treatment resistance, exacerbated proliferation capacity and invasive features. Interestingly, many cancer-associated proteins have been reported to have a multifunctional role in controlling the biological changes acquired by PCa cells. Among them is STEAP1 (“Six-Transmembrane Epithelial Antigen of the Prostate”). STEAP1 is a transmembrane protein identified as overexpressed in PCa and several other types of cancer. In non-tumor tissues, STEAP1 expression is low and only restricted to the prostate gland, suggesting that the increase in its expression may favor and accompany tumor development. STEAP1 has been suggested to act as an oncogene, as this protein appears to have the ability to regulate cell proliferation, apoptosis, invasion and metastasis leading to PCa progression. Furthermore, several studies have shown that STEAP1 is involved in intercellular communication and can function as an ion channel or transporter protein. Thus, taking into account the various biological processes that may be controlled by STEAP1, its location and overexpression at the cell surface, and its low expression in non-tumor tissues, it is predictable that this protein plays a crucial role in the maintenance of cell homeostasis and might be used as a possible therapeutic target for PCa. However, the clinical significance of STEAP1 expression in PCa and its usefulness as a biomarker still needs clarification. Furthermore, there are few studies on the regulation of STEAP1 expression in PCa and on cellular mechanisms underlying biological functions in PCa. The present thesis aimed to first establish the clinical significance of the STEAP1 protein in PCa and explore its usefulness as a biomarker. Its relationship with the other proteins of the STEAP family (STEAP2, STEAP3 and STEAP4) was also investigated. Using databases and bioinformatics tools, it was found that the STEAP1 gene is differentially expressed in PCa, from benign lesions to metastatic adenocarcinoma, suggesting that deregulation of this gene may be involved in the early stages of the development of the disease. Concerning to the other STEAP family proteins, our results indicate that STEAP2 and STEAP4 are also overexpressed in PCa, but STEAP3 is underexpressed. However, only the high expression of STEAP1 was associated with a poor prognosis of patients with PCa, compared to patients without alterations in the expression of STEAP family proteins. Furthermore, comparing only patients with alterations in the expression of these proteins, we found that the overexpression of STEAP1 continues to be associated with a worse prognosis, while the overexpression of STEAP4 indicates a good prognosis for patients with PCa, meaning a longer survival time. The dysregulation of STEAP1 can have a high impact on the development of PCa, and the work developed in this thesis demonstrated that STEAP1 can be regulated by epigenetic mechanisms. In addition to confirming the high expression of STEAP1 in neoplastic prostate cell lines (LNCaP) and the low expression of this gene in non-neoplastic prostate cell lines (PNT1A), it was found that the CpG island located at near the transcription start site is methylated in PNT1A cells and demethylated in LNCaP cells. This result suggested that demethylation of the STEAP1 gene promoter may contribute to its overexpression in PCa. The recruitment of enzymes, such as DNA methyltransferases and histone deacetylases, are essential for epigenetic modifications to occur. The treatment of PNT1A cells with inhibitors of these enzymes increased the expression of the STEAP1 gene, suggesting once again that the hypomethylation of the STEAP1 gene promoter may be associated with its overexpression in PCa. The global characterization of the proteome of LNCaP cells silenced for STEAP1 in order to unravel new molecular and cellular functions associated with STEAP1 was another focus of this dissertation. For this, LNCaP cells silenced, or not, for STEAP1 were subjected to protein identification and quantification techniques. Comparing the two experimental groups (STEAP1 siRNA LNCaP cells versus scramble siRNA-LNCaP cells), 526 proteins were found to be differentially expressed. The cellular processes of endocytosis, RNA transport, apoptosis, amino acid biosynthesis and metabolic pathways were the most significant, suggesting that STEAP1 can modulate these biological processes. In fact, STEAP1 silencing promotes apoptosis of PCa cells, attenuating cell proliferation and metabolism, stimulates endocytosis and diminishes intercellular communication by vesicles. These results emphasized the oncogenic role of STEAP1 in PCa. Finally, it was found that the action of antiandrogenic drugs on LNCaP cells, namely bicalutamide, enzalutamide and apalutamide, appears to be independent of STEAP1 expression levels. On the other hand, treatment of LNCaP cells with taxane-based chemotherapeutic drugs, paclitaxel, docetaxel and cabazitaxel, appears to depend on STEAP1 levels. High STEAP1 expression levels seem to promote a more efficient action of chemotherapeutic drugs based on taxanes, with stimulation of apoptosis and a decrease in uncontrolled proliferation of prostate cells. In conclusion, the main results of this thesis confirmed the crucial action of STEAP1 in development and progression of PCa. This dissertation showed that STEAP1 overexpression may be used as a prognostic biomarker, indicating that patients with PCa overexpressing this gene have a shorter survival time; also it showed that the reduction of STEAP1 expression might be a therapeutic strategy to decrease the development and progression of PCa; and yet, in an innovative way, this thesis demonstrated that the action of chemotherapeutic drugs based on taxanes are more effective in the treatment of PCa overexpressing STEAP1. Additional investigation into the relationship between STEAP1 and PCa will be a fundamental basis for the development of new therapeutic approaches for PCa, as well as its usefulness as biomarker.

2023-06Doctoral thesis

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