Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer.

Prostate cancer (PC) is still the second cause of cancer-related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone-sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non-raft into the raft compartment where it colocalized with the γ-secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.

Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression.

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-ß1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-ß1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-ß1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.

Notch signaling modulates hypoxia-induced neuroendocrine differentiation of human prostate cancer cells.

UNLABELLED: Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. RESULTS: Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and ß3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. CONCLUSIONS: Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription.

Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae.

Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

Gonadotropin-releasing hormone modulates cholesterol synthesis and steroidogenesis in SH-SY5Y cells.

Neurosteroids are involved in Central Nervous System development, brain functionality and neuroprotection but little is known about regulators of their biosynthesis. Recently gonadotropins, Gonadotropin-releasing Hormone (GnRH) and their receptors have been localized in different brain regions, such as hippocampus and cortex. Using human neuronal-like cells we found that GnRH up-regulates the expression of key genes of cholesterol and steroid synthesis when used in a narrow range around 1.0 nM. The expression of Hydroxysterol D24-reductase (seladin-1/DHCR24), that catalyzes the last step of cholesterol biosynthesis, is increased by 50% after 90 min of incubation with GnRH. StAR protein and P450 side chain cleavage (P450scc) are up-regulated by 3.3 times after 90 min and by 3.5 times after 3 h, respectively. GnRH action is mediated by LH and 1.0 nM GnRH enhances the expression of LHß as well. A two fold increase of cell cholesterol is induced after 90 min of GnRH incubation and 17ß-estradiol (E2) production is increased after 24, 48 and 72 h. These data indicate for the first time that GnRH regulates both cholesterol and steroid biosynthesis in human neuronal-like cells and suggest a new physiological role for GnRH in the brain.

Seladin-1 and testicular germ cell tumours: new insights into cisplatin responsiveness.

The molecular basis for the exquisite sensitivity of testicular germ cell tumours of adolescents and adults (TGCTs), ie seminomas and non-seminomatous germ cell tumours, to chemo/radiotherapy has not been fully clarified so far. It has been suggested that it may be dependent on factors involved in the regulation of apoptosis. Seladin-1 is a multi-functional protein involved in various biological processes, including apoptosis. The aim of our study was to assess the expression of seladin-1 in different histological types of TGCTs, known to have varying treatment sensitivity, in order to establish whether this protein may influence cisplatin responsiveness in vitro. Seladin-1 expression levels, both at the mRNA and at the protein level, were higher in the adjacent normal parenchyma than in the pathological counterparts. In tumoural tissues, the level of expression differed among TGCT histological types. The highest tumour-expression level was found in teratoma, whereas the lowest was detected in seminoma, corresponding to the different chemo/and radiosensitivities of these tumour types. In common with other cancers, in TGCT-derived cell lines seladin-1 showed anti-apoptotic properties through inhibition of caspase-3 activation. We confirmed our results using a non-seminomatous cell line model (NT2) before and after differentiation with retinoic acid. Significantly higher seladin-1 expression was observed in the differentiated derivatives (teratoma) and an inverse relationship was found between seladin-1 expression and the amount of cleaved caspase-3. Seladin-1 silencing or overexpression in this cell line supports involvement of seladin-1 in cisplatin responsiveness. Seladin-1 silencing was associated with greater cisplatin responsiveness demonstrated by decreased cell viability and increased expression of apoptotic markers. In contrast, overexpression of seladin-1 was associated with a higher survival rate and a clear anti-apoptotic effect. In conclusion, we have demonstrated for the first time an important role for seladin-1 in the biology of TGCTs and provided new insights into cisplatin responsiveness of these tumours.

New insights on the neuroprotective role of sterols and sex steroids: the seladin-1/DHCR24 paradigm.

In 2000 a new gene, i.e. seladin-1 (for selective Alzheimer's disease indicator-1) was identified and found to be down regulated in vulnerable brain regions in Alzheimer's disease. Seladin-1 was considered a novel neuroprotective factor, because of its anti-apoptotic properties. Subsequently, it has been demonstrated that seladin-1 corresponds to the gene that encodes 3-beta-hydroxysterol delta-24-reductase (DHCR24), that catalyzes the synthesis of cholesterol from desmosterol. There is evidence that cholesterol plays a fundamental role in maintaining brain homeostasis. Because of its enzymatic activity, seladin-1/DHCR24 has been considered the human homolog of the plant protein DIMINUTO/DWARF1, that is involved in the synthesis of sterol plant hormones. We have recently demonstrated that seladin-1/DHCR24 is a fundamental mediator of the protective effects of estrogens in the brain. This review describes how this protein interacts with cholesterol and estrogens, thus generating a neuroprotective network, that might open new possibilities in the prevention/treatment of neurodegenerative diseases.

Androgen receptor regulation of the seladin-1/DHCR24 gene: altered expression in prostate cancer.

Prostate cancer (CaP) represents a major leading cause of morbidity and mortality in the Western world. Elevated cholesterol levels, resulting from altered cholesterol metabolism, have been found in CaP cells. Seladin-1 (SELective Alzheimer Disease INdicator-1)/DHCR24 is a recently described gene involved in cholesterol biosynthesis. Here, we demonstrated the androgen regulation of seladin-1/DHCR24 expression, due to the presence of androgen responsive element sequences in its promoter region. In metastatic androgen receptor-negative CaP cells seladin-1/DHCR24 expression and cholesterol amount were reduced compared to androgen receptor-positive cells. In tumor samples from 61 patients who underwent radical prostatectomy the expression of seladin-1/DHCR24 was significantly higher with respect to normal tissues. In addition, in cancer tissues mRNA levels were positively related to T stage. In tumor specimens from 23 patients who received androgen ablation treatment for 3 months before surgery seladin-1/DHCR24 expression was significantly lower with respect to patients treated by surgery only. In conclusion, our study demonstrated for the first time the androgen regulation of the seladin-1/DHCR24 gene and the presence of a higher level of expression in CaP tissues, compared to the normal prostate. These findings, together with the results previously obtained in metastatic disease, suggest an involvement of this gene in CaP.

Seladin-1 is a fundamental mediator of the neuroprotective effects of estrogen in human neuroblast long-term cell cultures.

Estrogen exerts neuroprotective effects and reduces beta-amyloid accumulation in models of Alzheimer's disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3beta-hydroxysterol Delta24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17beta-estradiol (17betaE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17betaE2 against beta-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor alpha in Chinese hamster ovarian cells. The exposure to 17betaE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1.

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.

5alpha-Reductase activity in Lycopersicon esculentum: cloning and functional characterization of LeDET2 and evidence of the presence of two isoenzymes.

The full-length cDNA (LeDET2) encoding a 257 amino acid protein homolog of Arabidopsis DET2 (AtDET2) was isolated in tomato (Lycopersicon esculentum). LeDET2 has 76% similarity with AtDET2 and structural characteristics conserved among plant and mammalian steroid 5alpha-reductases (5alphaRs). LeDET2 is ubiquitously expressed in tomato tissues with higher levels in leaf than in stem, root, seed and callus. When expressed in mammalian cells (COS-7), recombinant LeDET2 was active on substrates typical of mammalian 5alphaRs (progesterone, testosterone, androstenedione), but reduced at very low levels campestenone, the substrate described for AtDET2. Similar results were obtained with the expression in COS-7 of recombinant AtDET2 that showed 5alphaR activity for progesterone and not for campestenone. Recombinant LeDET2 was inhibited by several inhibitors of the human 5alphaRs and the application of an active inhibitor to tomato seedlings induced dwarfism and morphological changes similar to BR-deficient mutants. In tomato tissues, campestenone was 5alpha-reduced in leaf, stem and root homogenates, like progesterone and testosterone, while androstenedione was converted to testosterone, evidencing for the first time a 17beta-hydroxysteroid dehydrogenase activity in plants. Moreover, two separate 5alphaR activities with different kinetic characteristic and response to inhibitors were characterized in tomato tissues. The presence of two 5alphaR isoenzymes was demonstrated also in Arabidopsis using the det2-1 mutant, in which a residual 5alphaR activity for campestenone and progesterone was evidenced and characterized. Therefore, the existence of two isoenzymes of 5alphaR is probably characteristic of the whole plant kingdom highlighting the similarities between the animal and plant steroid biosynthetic pathways.

New evidence of similarity between human and plant steroid metabolism: 5alpha-reductase activity in Solanum malacoxylon.

The physiological role of steroid hormones in humans is well known, and the metabolic pathway and mechanisms of action are almost completely elucidated. The role of plant steroid hormones, brassinosteroids, is less known, but an increasing amount of data on brassinosteroid biosynthesis is showing unexpected similarities between human and plant steroid metabolic pathways. Here we focus our attention on the enzyme 5alpha-reductase (5alphaR) for which a plant ortholog of the mammalian system, DET2, was recently described in Arabidopsis thaliana. We demonstrate that campestenone, the natural substrate of DET2, is reduced to 5alpha-campestanone by both human 5alphaR isozymes but with different affinities. Solanum malacoxylon, which is a calcinogenic plant very active in the biosynthesis of vitamin D-like molecules and sterols, was used to study 5alphaR activity. Leaves and calli were chosen as examples of differentiated and undifferentiated tissues, respectively. Two separate 5alphaR activities were found in calli and leaves of Solanum using campestenone as substrate. The use of progesterone allowed the detection of both activities in calli. Support for the existence of two 5alphaR isozymes in S. malacoxylon was provided by the differential actions of inhibitors of the human 5alphaR in calli and leaves. The evidence for the presence of two isozymes in different plant tissues extends the analogies between plant and mammalian steroid metabolic pathways.