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- STEAP1 is overexpressed in prostate cancer and prostatic intraepithelial neoplasia lesions, and it is positively associated with Gleason scorePublication . Gomes, Inês; Arinto, Patrícia; Lopes, Carlos; Santos, Cecilia; Baptista, CláudioSix transmembrane epithelial antigen of the prostate 1 (STEAP1) is a transmembrane protein of epithelial cells, mostly located at cell-cell junctions, and is overexpressed in several types of tumors, particularly prostate cancer. Several studies have pointed STEAP1 as a biomarker, but the clinical significance of its overexpression is not fully understood. Therefore, we aimed to establish the association of STEAP1 immunoreactivity with histologic diagnosis and clinical data of patients.
- Estrogens down-regulate the stem cell factor (SCF)/c-KIT system in prostate cells: Evidence of antiproliferative and proapoptotic effectsPublication . Figueira, Marília I; Correia, Sara; Vaz, Cátia; Cardoso, HJ; Gomes, Inês; Marques, Ricardo; Baptista, Cláudio; Socorro, SílviaThe development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17β-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.
- Six transmembrane epithelial antigen of the prostate 1 is down-regulated by sex hormones in prostate cellsPublication . Gomes, Inês; Santos, Cecilia; Socorro, Sílvia; Baptista, CláudioSTEAP1 is over-expressed in several types of tumors, especially prostate cancer, where it is localized in the plasma membrane of epithelial cells, at cell-cell junctions. Its role in prostate carcinogenesis and its regulation in prostate cells remain unknown. Therefore, we propose to study the effect of sex hormones in the regulation of STEAP1 expression in prostate cells in vitro and in vivo.
- Regulation of STEAP1 gene and its clinical significance in human prostate cancerPublication . Gomes, Inês Margarida Amaral Santos; Baptista, Cláudio Jorge Maia; Santos, Cecília Reis Alves dosThe prostate gland is an accessory gland of the male reproductive system and displays a critical role in male fertility. This gland is dependent of sex steroid hormones, namely androgens and estrogens, for the gland morphogenesis, development and cellular homeostasis. Prostate cancer (PCa) is a multifactorial disease, affected by both endogenous and exogenous risk factors, especially family history, age and sex hormones. PCa is the second most common type of cancer among men, and along with BPH, is found in the majority of men over 40 years old. Although PCa etiology is complex, inflammatory processes are thought to be behind the appearance of pre-cancerous lesions. At molecular level, PCa is often described as a two step disease. Initially it is responsive to androgens, but usually, the PCa becomes androgen-independent. In the end, the clinical outcome of PCa is defined by the potential of the tumor to grow, invade and metastasize. Regardless of all the knowledge gathered from prostate cancer pathophysiology and clinical management, identification of genes associated with the pathology, the role of sex steroid hormones in their regulation, and their potential to be used as biomarkers or immunotherapeutic targets is urgent. STEAP1 was firstly identified as being overexpressed in prostate cancer. However, it can also be found overexpressed in several other types of tumors. STEAP1 expression in normal tissues is almost restricted to the prostate, where it is primarily localized in the plasma membrane of epithelial cells, especially at cell-cell junctions. STEAP1 regulation and biological function are yet uncertain, but it is believed to act as an ion channel or transporter protein, regulating inter- and intra-cellular communications. Finally, although its clinical significance is not clear, STEAP1 features stress its potential as biomarker and immunotherapeutic target. This way, it was evaluated if STEAP1 expression is regulated by sex steroid hormones, using both in vitro and in vivo assays. It was found that STEAP1 is down-regulated by DHT and E2 in LNCaP prostate cancer cells and in rat prostate, suggesting that STEAP1 may have a role on prostate cancer progression from androgen-dependent to androgen-independent. Subsequently, it was also evaluated the expression pattern of a STEAP1 related gene (STEAP1B) in cancer cell lines, and whether STEAP1 expression is subjected to posttranscriptional or post-translational regulatory mechanisms. In silico analysis revealed that STEAP1 and STEAP1B1 share high homology, and STEAP1 is more stable in prostate cancer cells rather than in non-malignant ones. Furthermore, it was demonstrated by in silico analysis that STEAP1 expression may be regulated by several post-translational modifications. In order to clarify the role of STEAP1 in prostate cancer, it was used a specific siRNA to decrease the levels of STEAP1 in LNCaP cells. Then, it was evaluated the role of STEAP1 in cell proliferation and apoptosis, as well as the effect of dihydrotestosterone (DHT) in LNCaP cells with low levels of STEAP1. Using MTS assay, flow cytometry and TUNEL assay, it was evident that STEAP1 gene silencing appears to reduce cell viability and growth, and to increase apoptosis of LNCaP cells. On the other hand, the DHT action seems to be dependent of STEAP1 levels as it could not revert the effects induced by STEAP1 knockdown. Much effort has been done to clarify the potential of STEAP1 as biomarker and immunotherapeutic target. Here, it was investigated the association between STEAP1 expression with histologic and clinical data of patients. It was demonstrated that STEAP1 is overexpressed in PCa and PIN lesions, and it is positively associated with Gleason score. Taken together, the results here presented demonstrate that STEAP1 expression is negatively regulated by the two main sex steroid hormones presents on prostate. STEAP1 high stability in prostate tumor cells in comparison to normal ones lead us to believe that post-transcription and translational regulation mechanisms are dependent on the tumor stage. Moreover, STEAP1 gene silencing inhibits cell viability and proliferation, ate the same time that increases apoptosis in LNCaP cells, suggesting that STEAP1 may have an important role on PCa progression from androgen-dependent to hormone refractory. The use of STEAP1 per se may not be sufficient to be used as a biomarker in the daily clinical practice, but may open novel strategies for diagnosis and treatment of prostate cancer.
- Expression of STEAP1 and STEAP1B in prostate cell lines, and the putative regulation of STEAP1 by post-transcriptional and post-translational mechanismsPublication . Gomes, Inês; Santos, Cecilia; Baptista, CláudioSTEAP1 gene is overexpressed in several kinds of tumors, particularly in prostate cancer. Besides STEAP1, there is another related gene, STEAP1B, which may encode two different transcripts. Although several studies have been pointing STEAP1 as a putative immunotherapeutic target and biomarker, the mechanisms underlying its regulation are not fully understood. In silico analysis allowed us to show that STEAP1 and STEAP1B share high homology, but with slight differences at structural level. Experiments with prostate cells showed that STEAP1B2 is overexpressed in cancer cells. Regarding STEAP1 regulation, it is demonstrated that the stability of mRNA and protein is higher in LNCaP than in PNT1A cells. Of note, serum triggered opposite effects in LNCaP and PNT1A in relation to STEAP1 stability, e.g., increasing it in PNT1A and decreasing in LNCaP. These results suggest that STEAP1 may be regulated by post-transcriptional and post-translational modifications (PTM), which may differ between non-neoplastic and neoplastic cells. These PTM are supported through in silico analysis, where several modifications such as N-glycosylation, N-Glycation, Phosphorylation and O-linked β-N-acetylglucosamine, may occur in STEAP1 protein. In conclusion, these data indicate that STEAP1B2 is overexpressed in neoplastic cells, and PTM may be involved in regulation of STEAP1 expression in prostate cells.
- STEAP Proteins: From Structure to Applications in Cancer TherapyPublication . Gomes, Inês; Maia, C J; Santos, CeciliaThe human 6-transmembrane epithelial antigen of prostate (STEAP) family comprises STEAP1, STEAP2, STEAP3, and STEAP4. All of these proteins are unique to mammals and share an innate activity as metalloreductases, indicating their importance in metal metabolism. Overall, they participate in a wide range of biologic processes, such as molecular trafficking in the endocytic and exocytic pathways and control of cell proliferation and apoptosis. STEAP1 and STEAP2 are overexpressed in several types of human cancers, namely prostate, bladder, colon, pancreas, ovary, testis, breast, cervix, and Ewing sarcoma, but their clinical significance and role in cancer cells are not clear. Still, their localization in the cell membrane and differential expression in normal and cancer tissues make STEAP proteins potential candidates as biomarkers of several cancers, as well as potential targets for new immunotherapeutic strategies for disease attenuation or treatment. This review brings together the current knowledge about each STEAP protein, giving an overview of the roles of this family of proteins in human physiology and disease, and analyzes their potential as immunotherapeutic agents in cancer research.
- STEAP1 exepression in prostate cancer and its regulation by androgensPublication . Gomes, Inês Margarida Amaral Santos; Baptista, Cláudio Jorge Maia; Santos, Cecília Reis Alves dosSix transmembrane epithelial antigen of the prostate 1 (STEAP1) was identified as a gene overexpressed in human prostate cancer and spontaneous transgenic mouse model of prostate cancer. It is localized in cell junctions of epithelial cells, and its structure with six transmembrane domains, suggests that it may act as a membrane channel or transporter protein in tight junctions, gap junctions or in cell adhesion, helping in intercellular communication in a way that allows growth of cancer cells. Although STEAP1 expression seems to be up-regulated in all stages of prostate cancer, its clinical significance remains to be clarified. Moreover, STEAP1 is more expressed in LNCaP than in PC3, suggesting that androgens may regulate its expression. Therefore, the goals of this experimental work were: i) to evaluate if STEAP1 expression correlates with clinical reports from patients; ii) to analyze if STEAP1 is regulated by 5α- dihydrotestosterone (DHT) in vitro and in vivo, by Real-time PCR and Western blot. Immunohistochemical analysis revealed that STEAP1 expression is principally associated with epithelial cells, but it is also present in some stromal cells. Analysis of STEAP1 immunoreactivity in prostate cancer is underway. In vitro results demonstrated that both STEAP1 mRNA and protein expression are down-regulated in the presence of 1nM or 10nM DHT after 24h of stimulation. However, at least one more experimental assay is required to confirm these results. In vivo results demonstrated that castration visibly increases STEAP1 protein expression when compared to intact rats, and treatment with DHT abrogates the effect of castration in STEAP1 expression, suggesting that STEAP1 protein is down-regulated by DHT. However, these results do not correlate with STEAP1 mRNA expression, suggesting that mechanisms at the translation level may be involved
- Regucalcin is an androgen-target gene in the rat prostate modulating cell-cycle and apoptotic pathwaysPublication . Vaz, C V; Baptista, Cláudio; Marques, Ricardo; Gomes, Inês; Correia, Sara; Alves, Marco G; Cavaco, JE; Oliveira, Pedro F.; Socorro, SílviaRegucalcin (RGN) is a calcium (Ca(2+) )-binding protein underexpressed in prostate adenocarcinoma comparatively to non-neoplastic prostate or benign prostate hyperplasia cases. Moreover, RGN expression is negatively associated with the cellular differentiation of prostate adenocarcinoma, suggesting that loss of RGN may be associated with tumor onset and progression. However, the RGN actions over the control of prostate cell growth have not been investigated.
- 5α-Dihydrotestosterone regulates the expression of L-type calcium channels and calcium-binding protein regucalcin in human breast cancer cells with suppression of cell growthPublication . Marques, Ricardo; Peres, Carina; Vaz, Cátia; Gomes, Inês; Figueira, Marília I; Cairrão, Elisa; Verde, Ignacio; Baptista, Cláudio; Socorro, SílviaAndrogens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100 nM of 5α-dihydrotestosterone (DHT) for 24-72 h. The obtained results showed that 1 nM DHT up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.
- Promoter Demethylation Upregulates STEAP1 Gene Expression in Human Prostate Cancer: In Vitro and In Silico AnalysisPublication . Rocha, Sandra; Sousa, Inês; Gomes, Inês M.; Arinto, Patrícia; Pinheiro, Pedro Costa; Coutinho, Eduarda; Santos, Cecilia; Jerónimo, Carmen; Lemos, Manuel C.; Passarinha, L A; Socorro, Sílvia; Baptista, Cláudio MaiaThe 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.