Transfected poly(I:C) activates different dsRNA receptors, leading to apoptosis or immunoadjuvant response in androgen-independent prostate cancer cells.

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-ß, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-ß expression.

TLR3 engagement induces IRF-3-dependent apoptosis in androgen-sensitive prostate cancer cells and inhibits tumour growth in vivo.

Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.

Mouse Sertoli cells sustain de novo generation of regulatory T cells by triggering the notch pathway through soluble JAGGED1.

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.

Autophagy in prostate cancer and androgen suppression therapy.

The role of autophagy is known to be highly complex and context-dependent, leading to both cancer suppression and progression in several tumors including melanoma, breast and prostate cancer. In the present review, recent advances in an understanding of the involvement of autophagy in prostate cancer treatment are described. The regulatory effects of androgens on prostate cancer cell autophagy are particularly discussed in order to highlight the effects of autophagy modulation during androgen deprivation. A critical evaluation of the studies examined in the present review suggests the attractive possibility of autophagy inhibition combined with hormonal therapy as a promising approach for prostate cancer treatment.

TLR stimulation of prostate tumor cells induces chemokine-mediated recruitment of specific immune cell types.

TLRs boost antimicrobial response mechanisms by epithelial cells and represent the first line of defense at mucosal sites. In view of these immunomodulatory properties, TLR stimulation may represent a novel means to activate anticancer immune responses. In the present study, the ability of TLR ligands to affect the recruitment of different immune cell populations by human prostate cancer cell lines and the underlying mechanisms were investigated. We showed that LNCaP and DU-145 cells express functionally active TLR3 and TLR5. Treatment with their respective agonists, polyinosinic:polycytidylic acid and flagellin, rapidly triggered NF-kappaB-dependent upregulation of different inflammatory molecules, as assayed by microarray and ELISA. Furthermore, we demonstrated that conditioned media from polyinosinic:polycytidylic acid- and flagellin-treated LNCaP and DU-145 cells induced the recruitment of different leukocyte subpopulations, suggesting that TLR stimulation is able to activate the earliest step of immune response mediated by soluble factors. Interestingly, the more aggressive cancer cell line PC3 expressed TLR3 and TLR5 but failed to respond to TLR agonists in terms of NF-kappaB activation and the ability to attract immune effectors. Overall, these data show for the first time that TLR3 and TLR5 stimulation of human prostate cancer cells triggers the production of chemokines, which, in turn, favor the attraction of immune effectors, thereby representing a tool to enhance the efficacy of conventional therapies by stimulating anticancer immune responses.

TNF-α inhibits GDNF levels in Sertoli cells, through a NF-κB-dependent, HES1-dependent mechanism.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is a soluble molecule crucial for the regulation of the spermatogonial stem cells (SSC) of the testis. The effects of GDNF on target cells have been extensively described, but mechanisms underlying GDNF regulation are currently under investigation. In the nervous system, GDNF expression is regulated by pro-inflammatory cytokines including lipopolysaccharide (LPS), interleukin 1 beta (IL-1ß), and tumor necrosis factor alpha (TNF-α) but the effect of these cytokines on GDNF expression in the testis is unclear. OBJECTIVES: The aim of the present study was to investigate the impact of TNF-α on GDNF expression levels using primary murine Sertoli cells as experimental model. MATERIAL AND METHODS: The expression of TNF-α-regulated genes including Gdnf in different culture conditions was determined by real-time PCR. GDNF protein levels were determined by ELISA. The activation of the NF-κb pathway and HES1 levels were assessed by Western Blot analysis and immunofluorescence. HES1 expression was downregulated by RNAi. RESULTS: In primary Sertoli cells, TNF-α downregulates GDNF levels through a nuclear factor-κB (NF-κB)-dependent mechanism. Mechanistically, TNF-α induces the transcriptional repressor HES1 by a NF-Κb-dependent mechanism, which in turn downregulates GDNF. DISCUSSION: Under physiological conditions, TNF-α is secreted by germ cells suggesting that this cytokine plays a role in the paracrine control of SSC niche by modulating GDNF levels. HES1, a well-known target of the Notch pathway, is implicated in the regulation of GDNF expression. In Sertoli cells, TNF-α and Notch signaling may converge at molecular level, to regulate the expression of HES1 and HES1- target genes, including GDNF. CONCLUSIONS: Because of the importance of GDNF for spermatogonial stem cell self-renewal and proliferation, this data may give important insights on how cytokine signals in the testis modulate the expression of niche-derived factors.

Altered Tregs Differentiation and Impaired Autophagy Correlate to Atherosclerotic Disease.

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-ß (TGFß). TGFß is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.

Toll-like receptor 3 triggers apoptosis of human prostate cancer cells through a PKC-alpha-dependent mechanism.

Toll-like receptors (TLRs) are known to play a key role in the innate immune system particularly in inflammatory response against invading pathogens. Recent reports strongly indicate that they play important roles in cancer cells. Prostate cancer represents one of the most common cancer for which no cure is available once metastatic and androgen refractory. Since TLR3 has been recently suggested as a possible therapeutic target in some cancer cell lines, we studied TLR3 expression and functionality in two human prostate cancer cell lines, LNCaP and PC3. We report that both cell lines express TLR3 and that the TLR3 agonist poly (I:C) activates mitogen-activated protein kinases and induces inhibition of proliferation as well as caspase-dependent apoptosis. By using pharmacological and genetic approaches, we demonstrate the involvement of TLR3 in poly (I:C)-induced effects. We also show that a novel interferon-independent pathway involving protein kinase C (PKC)-alpha activation, upstream of p38 and c-jun N-terminal kinase, is responsible for poly (I:C) pro-apoptotic effects on LNCaP cells. To our knowledge, this is the first report describing a role of PKC-alpha in poly (I:C)-mediated apoptosis. The comprehension of the mechanisms underlying TLR3-mediated apoptosis can contribute tools to develop new agonists useful for the treatment of prostate cancer.

Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells.

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.

Characterization of signaling pathways leading to Fas expression induced by TNF-alpha: pivotal role of NF-kappaB.

TNF-alpha is known to induce a strong up-regulation of Fas expression in mouse Sertoli cell cultures, leading to their apoptosis triggered by effector FasL-bearing cells. These data suggest that increased Fas expression on the cell surface might be a key event in the pathogenesis of autoimmune orchitis, by inducing a leakage of the blood-tubular barrier as a consequence of Sertoli cell apoptosis. In the present paper, we have investigated the signal transduction mechanisms involved in the regulation of Fas expression induced by TNF-alpha in mouse Sertoli cells. We studied the role of the transcription factor NF-kappaB and of MAPKs in regulating Fas expression. By using Sertoli cells transfected with a NF-kappaB Luc reporter gene, we proved that TNF-alpha activates the IkappaB/NF-kappaB system. Moreover, the use of the proteasome inhibitor lactacystin led us to demonstrate that NF-kappaB is required for TNF-alpha mediated Fas expression. By using specific inhibitors for each MAPK, we confirmed the pivotal role of the IkappaB/NF-kappaB system by demonstrating that ERKs, p38, and JNK are not involved in Fas up-regulation by TNF-alpha. The comprehension of these pathways could be relevant to the knowledge of the pathogenesis of autoimmune disorders in immune privileged districts of the body.

Germ cell apoptosis control during spermatogenesis.

The aim of the present study was to investigate the expression and role of c-Flip long isoform (c-FlipL), a known anti-apoptotic protein. No data are currently available on c-FlipL in male gonad before puberty; therefore, this study was carried out in prepuberal mouse testis. We investigated pachytene spermatocytes and spermatogonia by immunostaining of testis sections and found a strong and specific expression of c-FlipL in pachytene spermatocytes, while spermatogonia expressed very low levels of c-FlipL. This finding inversely correlated with the caspases activity, which was higher in spermatogonia as compared to pachytene spermatocytes. Other experiments carried out in an organ-culture model revealed that Fas-induced apoptosis was higher in spermatogonia as compared to pachytene spermatocytes. These data suggest that c-FlipL may play a role as an anti-apoptotic molecule in the prepuberal mouse testis and open new perspectives in the comprehension of the mechanisms controlling germ cells apoptosis.

Necroptosis: molecular signalling and translational implications.

Necroptosis is a form of programmed necrosis whose molecular players are partially shared with apoptotic cell death. Here we summarize what is known about molecular signalling of necroptosis, particularly focusing on fine tuning of FLIP and IAP proteins in the apoptosis/necroptosis balance. We also emphasize necroptosis involvement in physiological and pathological conditions, particularly in the regulation of immune homeostasis.

Toll-like receptor 3 regulates angiogenesis and apoptosis in prostate cancer cell lines through hypoxia-inducible factor 1 alpha.

Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-inducible factor 1 (HIF-1) regulates several cellular processes, including apoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific I.3 isoform of HIF-1 alpha and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1 alpha. However, the transfection of I.3 isoform of hif-1 alpha in LNCaP cells allows poly(I:C)-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1 alpha expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.

Peculiar subcellular localization of Fas antigen in human and mouse spermatozoa.

The highly polarized structure and function of mammalian spermatozoa dictate that these cells compartmentalize specific metabolic and signaling pathways to regions where they are needed. Fas was initially identified as membrane receptor for pro-apoptotic signals, has been recently recognized as a molecule with pleiotropic functions. In this article, we provide evidence of a peculiar Fas localization: it is closely associated to the perinucleus, mainly at the level of the inner acrosomal membrane, as well as in the inner compartment of mitochondria. Immunoelectron microscopy and Western blot analysis indicated that intracellular Fas was associated with mitochondria in mouse epididymal spermatozoa. Accordingly, also in human ejaculated sperm, immunofluorescence analysis showed Fas localized in the middle piece of sperm flagellum where mitochondria are grouped. The potential functional implications of these findings are discussed.

Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.

Sertoli cells initiate testicular innate immune responses through TLR activation.

TLRs play a crucial role in early host defense against invading pathogens. In the seminiferous epithelium, Sertoli cells are the somatic nurse cells that mechanically segregate germ cell autoantigens by means of the blood-tubular barrier and create a microenvironment that protects germ cells from both interstitial and ascending invading pathogens. The objective of this study was to examine TLR expression and their functional responses to specific agonists in mouse Sertoli cells. We measured the expression of TLR2, TLR4, TLR5, and TLR6 mRNAs and confirmed by FACS analysis the presence of proteins TLR2 and TLR5 on which we focused our study. Stimulation of Sertoli cells with macrophage-activating lipopeptide-2, agonist of TLR2/TLR6, and with flagellin, agonist of TLR5, induces augmented secretion of the chemokine MCP-1. To assess the functional significance of MCP-1 production following TLR stimulation, conditioned medium from either macrophage-activating lipopeptide-2 or flagellin-treated Sertoli cells was tested for in vitro chemotaxis assay, and a significant increase of macrophage migration was observed in comparison with unstimulated conditioned medium. Moreover, we studied the role of NF-kappaB and of MAPKs in regulating TLR-mediated MCP-1 secretion by using inhibitors specific for each transduction pathway and we demonstrated a pivotal role of the IkappaB/NF-kappaB and JNK systems. In addition, TLR2/TLR6 and TLR5 stimulation induces increased ICAM-1 expression in Sertoli cells. Collectively, this study demonstrates the novel ability of Sertoli cells to potentially respond to a wide variety of bacteria through TLR stimulation.