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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.
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Fundação para a Ciência e a Tecnologia
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Funding Award Number
COVID/BD/151732/2021