Cecília R. A. Santos

STEAP proteins: from structure to applications in cancer therapy.

The 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. Mol Cancer Res; 10(5); 573–87. ©2012 AACR.

Regucalcin Is Under―Expressed in Human Breast and Prostate Cancers: Effect of Sex Steroid Hormones

Regucalcin plays an important role in maintenance of intracellular Ca2+ homeostasis, suppresses cell proliferation, inhibits expression of oncogenes, and increases the expression of tumour suppressor genes. This suggests that regucalcin functions may be altered in cancer tissues. In this study the regucalcin expression in breast and prostate cancer cases was analysed by RT‐PCR and immunohistochemistry showing that the mRNA and/or protein are under‐expressed in these tumors. The effect of sex steroid hormones on regucalcin expression in breast and prostate cancer cells was determined by real‐time PCR. MCF‐7 and LNCaP cells were stimulated with 0, 1, and 10 nM of 17β‐estradiol (E2) or 5α‐dihydrotestosterone (DHT), respectively, for 0, 6, 12, 24, and 48 h. MCF‐7 cells were also stimulated with E2 conjugated to BSA (E2‐BSA). To explore the mechanisms underlying the sex steroid regulation of regucalcin expression, control treatments with ICI 182,780, flutamide and cyclohexamide were carried out. E2 effects regulating regucalcin expression were not abrogated in the presence of ICI 182,780, and were similar to those observed with E2‐BSA, which suggests the involvement of a membrane‐bound estrogen receptor. In LNCaP cells, DHT down‐regulated regucalcin expression, an effect inhibited by the presence of both flutamide and cyclohexamide, suggesting the involvement of androgen receptor and de novo protein synthesis. The loss of regucalcin expression in breast and prostate cancer cases and the regulation of its expression by sex steroid hormones suggest that it may be associated with development and progression of these human tumors. J. Cell. Biochem. 107: 667–676, 2009.

Neuroprotective and neuroregenerative properties of metallothioneins

Metallothioneins (MTs) are low‐molecular weight cysteine‐ and metal‐rich proteins with unquestionable metal binding capacity, antioxidant and anti‐inflammatory properties, and a clear involvement in diverse physiological actions as inhibition of proapoptotic mechanisms, enhancement of cell survival, and tissue regeneration. Concurrent with this wide array of functions, MT‐1/2 have been implicated in neuroprotection and neuroregeneration. The zinc binding capacity and antioxidant properties of MTs may account for most of their physiological features in the brain. However, some receptor‐mediated actions of MT‐1/2 have also been reported recently, a subject to be fully elucidated. This review analyses and updates the current knowledge on the actions of MTs related to neuroprotection and neuroregeneration in an effort to distinguish receptor‐mediated actions of MTs from those arising from its zinc binding capacity and its antioxidant properties.

Transthyretin interacts with metallothionein 2.

Transthyretin (TTR) is a 55 kDa homotetrameric protein known for the transport of thyroxine and the indirect transportation of retinol. Within the central nervous system, TTR is primary synthesized and secreted into the cerebral spinal fluid by the choroid plexus (CP), whereas most TTR in the systemic circulation is produced and secreted by the liver. TTR is involved in two types of amyloid disease, the senile systemic amyloidosis and the familial amyloidotic polyneuropathy. TTR has also been implicated in the sequestration of amyloid beta peptide (Abeta), preventing its deposition. To explore other biological roles for TTR, we searched for protein-protein interactions using the yeast two-hybrid system with the full-length human TTR cDNA as bait. We found a novel interaction between TTR and metallothionein 2 (MT2) in human liver. This interaction was confirmed by competition binding assays, co-immunoprecipitation, cross-linking, and Western blotting experiments. Binding studies using MT1 showed a saturable specific interaction with TTR with a Kd of 244.8 +/- 44.1 nM. Western blotting experiments revealed a TTR-MT1/2 protein complex present in rat CP and kidney tissue extracts. Immunofluorescence experiments, in CP primary cell cultures and in CP paraffin sections, showed co-localization of TTR and MT1/2 in the cytoplasm of epithelial CP cells and localization of MT1/2 in the endoplasmic reticulum. Moreover, dot blot immunoassays of rat CSF provided the first evidence, to our knowledge, of circulating metallothionein in CSF. Taken together, we suggest that TTR-MT1/2 complexes may be functionally significant not only in healthy conditions but also in Abeta deposition in Alzheimer disease, thereby providing a novel potential therapeutic target.

5α-dihydrotestosterone up-regulates transthyretin levels in mice and rat choroid plexus via an androgen receptor independent pathway

Transthyretin (TTR) is a 55 kDa plasma homotetrameric protein mainly synthesized in the liver and choroid plexuses (CPs) of the brain that, functions as a carrier for thyroxin and retinol binding protein. It sequesters amyloid beta (Abeta) peptide, and TTR levels in the cerebrospinal fluid (CSF) appear to be inversely correlated with Alzheimers disease (AD) onset and progression. Androgen deprivation increases plasma Abeta levels, which indicate that androgens may reduce the levels of soluble Abeta, the peptide widely implicated in the initiation of AD pathogenesis; however, the underlying mechanisms are still poorly understood. In this study we examined the effects of 5alpha-dihydrotestosterone (DHT) on TTR protein and mRNA levels, in primary cultures of rat CPs epithelial cells (CPEC) by Western blot, and real time PCR, respectively. Moreover, TTR concentrations were measured in the CSF of castrated wild-type, and transgenic mice expressing human TTR subjected to DHT treatment, by radioimmunoassay and ELISA, respectively. TTR mRNA expression was also compared in the CPs, of the animals from each experimental group by real time PCR. DHT treatment increased TTR protein levels in CPEC, and induced TTR transcription in these cells. The combination of flutamide with DHT in the treatment of CPEC did not abrogate DHT-induced TTR levels, suggesting that TTR is up-regulated via an androgen receptor independent pathway. In the CPs of both mice strains, DHT also increased TTR mRNA levels, but no significant differences in TTR protein levels were detected in the CSF of these animals. These findings open a wide range of possibilities for future studies on Abeta deposition and cognitive function, in response to androgen induction of TTR in animal models of AD.

17β-Estradiol Induces Transthyretin Expression in Murine Choroid Plexus via an Oestrogen Receptor Dependent Pathway

Oestrogen protects against AD by multiple mechanisms, including the enhancement of Aβ clearance. Transthyretin (TTR) is a homotetrameric protein mainly synthesized by the liver and choroid plexus (CP) of the brain that sequesters the amyloid beta (Aβ) peptide. In this study we examined the effects of 17β-estradiol (E2) on TTR protein and mRNA levels, in primary cultures of rat CP epithelial cells (CPEC) by Western blot and Real Time PCR, respectively. Moreover, the localization of oestrogen receptors alpha (ERα) and beta (ERβ) in response to E2 treatment was analysed by confocal microscopy in these cells. The expression of TTR, ERα and ERβ was also compared in the CP of castrated female mice treated with E2 to vehicle-treated animals by Real Time PCR. TTR concentration in the CSF of all these animals was measured by radioimmunoassay. E2 treatment induced TTR transcription and increased TTR protein content in CPEC. Pre-treatment with ICI 182,780 (ICI) abrogated E2-induced TTR expression suggesting that, TTR is up-regulated via an ER-dependent pathway. Confocal microscopy demonstrated extranuclear ERα and ERβ localization in untreated CPEC. Upon E2 treatment, translocation of ERα to the nucleus occurred, while ERβ remained in the cytosol. These data was concurrent with the up-regulation of TTR expression detected in the CP of castrated female mice subjected to E2 treatment. Our results highlight the importance of E2 on the regulation of TTR, which may participate in the oestrogen-induced decrease in Aβ levels and deposition described in the literature.

STEAP1 is over-expressed in breast cancer and down-regulated by 17β-estradiol in MCF-7 cells and in the rat mammary gland

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) was identified as a prostate-specific cell-surface antigen over-expressed in prostate cancer, and in human cancer cell lines obtained from several other tissues. Its cell surface location in all tumor types analyzed so far, and its absence in most vital organs in humans, turned STEAP1 into a potential target for anti-tumor immunotherapy. This study provides experimental evidence that STEAP1 is also over-expressed in human breast cancer cases, and in normal breast tissue adjacent to breast tumors, where it is localized in the cell membrane of epithelial cells. It is also demonstrated that STEAP1 transcription correlates negatively with estrogen receptor (ER) immunoreactivity, and positively with tumor grading in breast cancer cases. As estrogens are involved in breast cancer onset and progression, the response of STEAP1 to 17β-estradiol (E2) was investigated in the mammary gland of rats, and in the human breast cancer cell line, MCF-7. These experiments demonstrated that STEAP1 is down-regulated by E2 in both models. The mechanisms underlying the STEAP1 response to E2 in vitro were further investigated in MCF-7 cells, and the results obtained suggest an effect mediated by the membrane-bound ERα (mbERα).

Human metallothioneins 2 and 3 differentially affect amyloid-beta binding by transthyretin.

Transthyretin (TTR), an amyloid‐beta (Aβ) scavenger protein, and metallothioneins 2 and 3 (MT2 and MT3), low molecular weight metal‐binding proteins, have recognized impacts in Aβ metabolism. Because TTR binds MT2, an ubiquitous isoform of the MTs, we investigated whether it also interacts with MT3, an isoform of the MTs predominantly expressed in the brain, and studied the role of MT2 and MT3 in human TTR–Aβ binding. The TTR–MT3 interaction was characterized by yeast two‐hybrid assays, saturation‐binding assays, co‐immunolocalization and co‐immunoprecipitation. The effect of MT2 and MT3 on TTR–Aβ binding was assessed by competition‐binding assays. The results obtained clearly demonstrate that TTR interacts with MT3 with a Kd of 373.7 ± 60.2 nm. Competition‐binding assays demonstrated that MT2 diminishes TTR–Aβ binding, whereas MT3 has the opposite effect. In addition to identifying a novel ligand for TTR that improves human TTR–Aβ binding, the present study highlights the need to clarify whether the effects of MT2 and MT3 in human TTR–Aβ binding observed in vitro have a relevant impact on Aβ deposition in animal models of Alzheimer’s disease.

Stress and Glucocorticoids Increase Transthyretin Expression in Rat Choroid Plexus via Mineralocorticoid and Glucocorticoid Receptors

Transthyretin (TTR) is a carrier for thyroid hormones and retinol binding protein. Several mutated forms of TTR cause familial amyloidotic polyneuropathy, an inheritable lethal disease. On the other hand, wild-type TTR has a protective role against Alzheimer’s disease. Despite its overall importance in normal animal physiology and in disease, few studies have focused on its regulation. An in silico analysis of the rat TTR gene revealed a glucocorticoid responsive element in the 3′ region of the first intron. Thus, we hypothesised that TTR could be regulated by glucocorticoid hormones and investigated the regulation of TTR expression in response to hydrocortisone in a rat choroid plexus cell line (RCP) and in primary cultures of choroid plexus epithelial cells (CPEC). In addition, the effect of psychosocial stress on TTR expression was analysed in rat liver, choroid plexus (CP) and cerebrospinal fluid (CSF). In RCP and CPEC cultures hydrocortisone upregulated TTR expression, an effect suppressed by glucocorticoid receptor and mineralocorticoid receptor antagonists. Moreover, induction of psychosocial stress increased TTR expression in liver, CP and CSF of animals subjected to acute and chronic stress conditions. Overall, we conclude that stress upregulates TTR expression in CP.

STEAP1 is overexpressed in prostate cancer and prostatic intraepithelial neoplasia lesions, and it is positively associated with Gleason score

BACKGROUND Six 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. MATERIALS AND METHODS Human tissue microarrays were constructed from tissue biopsies of prostate adenocarcinoma (n = 63), including nonneoplastic adjacent tissue (n = 41), prostatic intraepithelial neoplasia (PIN) lesions (n = 7), and 41 prostate samples from patients diagnosed with benign prostatic hyperplasia (BPH). The histologic features of tumor specimens were classified and clinical and pathologic data were retrieved. STEAP1 expression was evaluated by immunohistochemical analysis, and a semiquantitative quantification was performed using a grade score system based on the intensity and percentage of stained cells. RESULTS Overexpression of STEAP1 protein was found in both plasma membrane and cytoplasm of prostate cancer and PIN lesions when compared with nonneoplastic adjacent tissue and BPH samples. Furthermore, its expression associates positively with higher Gleason scores, but not with other clinical data, such as age, prostate-specific antigen levels, pathologic stage, and metastasis. Regarding its role as a biomarker, STEAP1 is highly liable for distinguishing malignant prostate stages from BPH. On the contrary, it lacks specificity in distinguishing PIN lesions from prostate cancer. CONCLUSIONS STEAP1 is consistently overexpressed in malignant prostate tissue, namely adenocarcinoma and PIN lesions. Its overexpression in PIN lesions and positive association with higher Gleason scores suggest that STEAP1 could be involved in tumor initiation and progression. The high sensitivity and specificity for detection of malignant lesions suggests that STEAP1 may be of clinical usefulness in early disease diagnosis.