The Role of HSPB8, a Component of the Chaperone-Assisted Selective Autophagy Machinery, in Cancer.

The cellular response to cancer-induced stress is one of the major aspects regulating cancer development and progression. The Heat Shock Protein B8 (HSPB8) is a small chaperone involved in chaperone-assisted selective autophagy (CASA). CASA promotes the selective degradation of proteins to counteract cell stress such as tumor-induced stress. HSPB8 is also involved in (i) the cell division machinery regulating chromosome segregation and cell cycle arrest in the G0/G1 phase and (ii) inflammation regulating dendritic cell maturation and cytokine production. HSPB8 expression and role are tumor-specific, showing a dual and opposite role. Interestingly, HSPB8 may be involved in the acquisition of chemoresistance to drugs. Despite the fact the mechanisms of HSPB8-mediated CASA activation in tumors need further studies, HSPB8 could represent an important factor in cancer induction and progression and it may be a potential target for anticancer treatment in specific types of cancer. In this review, we will discuss the molecular mechanism underlying HSPB8 roles in normal and cancer conditions. The basic mechanisms involved in anti- and pro-tumoral activities of HSPB8 are deeply discussed together with the pathways that modulate HSPB8 expression, in order to outline molecules with a beneficial effect for cancer cell growth, migration, and death.

Gonadotropin-Releasing Hormone Receptors in Prostate Cancer: Molecular Aspects and Biological Functions.

Pituitary Gonadotropin-Releasing Hormone receptors (GnRH-R) mediate the activity of the hypothalamic decapeptide GnRH, thus playing a key role in the regulation of the reproductive axis. Early-stage prostate cancer (PCa) is dependent on serum androgen levels, and androgen-deprivation therapy (ADT), based on GnRH agonists and antagonists, represents the standard therapeutic approach for PCa patients. Unfortunately, the tumor often progresses towards the more aggressive castration-resistant prostate cancer (CRPC) stage. GnRH receptors are also expressed in CRPC tissues, where their binding to both GnRH agonists and antagonists is associated with significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic effects, mediated by the Gαi/cAMP signaling cascade. GnRH agonists and antagonists are now considered as an effective therapeutic strategy for CRPC patients with many clinical trials demonstrating that the combined use of these drugs with standard therapies (i.e., docetaxel, enzalutamide, abiraterone) significantly improves disease-free survival. In this context, GnRH-based bioconjugates (cytotoxic drugs covalently linked to a GnRH-based decapeptide) have been recently developed. The rationale of this treatment is that the GnRH peptide selectively binds to its receptors, delivering the cytotoxic drug to CRPC cells while sparing nontumor cells. Some of these compounds have already entered clinical trials.

Cellular and molecular biology of cancer stem cells in melanoma: Possible therapeutic implications.

Malignant melanoma is a tumor characterized by a very high level of heterogeneity, responsible for its malignant behavior and ability to escape from standard therapies. In this review we highlight the molecular and biological features of the subpopulation of cancer stem cells (CSCs), well known to be characterized by self-renewal properties, deeply involved in triggering the processes of tumor generation, metastasis, progression and drug resistance. From the molecular point of view, melanoma CSCs are identified and characterized by the expression of stemness markers, such as surface markers, ATP-binding cassette (ABC) transporters, embryonic stem cells and intracellular markers. These cells are endowed with different functional features. In particular, they play pivotal roles in the processes of tumor dissemination, epithelial-to-mesenchymal transition (EMT) and angiogenesis, mediated by specific intracellular signaling pathways; moreover, they are characterized by a unique metabolic reprogramming. As reported for other types of tumors, the CSCs subpopulation in melanoma is also characterized by a low immunogenic profile as well as by the ability to escape the immune system, through the expression of a negative modulation of T cell functions and the secretion of immunosuppressive factors. These biological features allow melanoma CSCs to escape standard treatments, thus being deeply involved in tumor relapse. Targeting the CSCs subpopulation is now considered an attractive treatment strategy; in particular, combination treatments, based on both CSCs-targeting and standard drugs, will likely increase the therapeutic options for melanoma patients. The characterization of CSCs in liquid biopsies from single patients will pave the way towards precision medicine.

Unraveling the molecular mechanisms and the potential chemopreventive/therapeutic properties of natural compounds in melanoma.

Melanoma is the most fatal form of skin cancer. Current therapeutic approaches include surgical resection, chemotherapy, targeted therapy and immunotherapy. However, these treatment strategies are associated with development of drug resistance and severe side effects. In recent years, natural compounds have also been extensively studied for their anti-melanoma effects, including tumor growth inhibition, apoptosis induction, angiogenesis and metastasis suppression and cancer stem cell elimination. Moreover, a considerable number of studies reported the synergistic activity of phytochemicals and standard anti-melanoma agents, as well as the enhanced effectiveness of their synthetic derivatives and novel formulations. However, clinical data confirming these promising effects in patients are still scanty. This review emphasizes the anti-tumor mechanisms and potential application of the most studied natural products for melanoma prevention and treatment.

Role of Endoplasmic Reticulum Stress in the Anticancer Activity of Natural Compounds.

Cancer represents a serious global health problem, and its incidence and mortality are rapidly growing worldwide. One of the main causes of the failure of an anticancer treatment is the development of drug resistance by cancer cells. Therefore, it is necessary to develop new drugs characterized by better pharmacological and toxicological profiles. Natural compounds can represent an optimal collection of bioactive molecules. Many natural compounds have been proven to possess anticancer effects in different types of tumors, but often the molecular mechanisms associated with their cytotoxicity are not completely understood. The endoplasmic reticulum (ER) is an organelle involved in multiple cellular processes. Alteration of ER homeostasis and its appropriate functioning originates a cascade of signaling events known as ER stress response or unfolded protein response (UPR). The UPR pathways involve three different sensors (protein kinase RNA(PKR)-like ER kinase (PERK), inositol requiring enzyme1α (IRE1) and activating transcription factor 6 (ATF6)) residing on the ER membranes. Although the main purpose of UPR is to restore this organelle's homeostasis, a persistent UPR can trigger cell death pathways such as apoptosis. There is a growing body of evidence showing that ER stress may play a role in the cytotoxicity of many natural compounds. In this review we present an overview of different plant-derived natural compounds, such as curcumin, resveratrol, green tea polyphenols, tocotrienols, and garcinia derivates, that exert their anticancer activity via ER stress modulation in different human cancers.

Tocotrienols and Cancer: From the State of the Art to Promising Novel Patents.

BACKGROUND: Tocotrienols (TTs) are vitamin E derivatives naturally occurring in several plants and vegetable oils. Like Tocopherols (TPs), they comprise four isoforms, α, ß, γ and δ, but unlike TPs, they present an unsaturated isoprenoid chain. Recent studies indicate that TTs provide important health benefits, including neuroprotective, anti-inflammatory, anti-oxidant, cholesterol lowering and immunomodulatory effects. Moreover, they have been found to possess unique anti-cancer properties. OBJECTIVE: The purpose of this review is to present an overview of the state of the art of TTs role in cancer prevention and treatment, as well as to describe recent patents proposing new methods for TTs isolation, chemical modification and use in cancer prevention and/or therapy. METHODS: Recent literature and patents focusing on TTs anti-cancer applications have been identified and reviewed, with special regard to their scientific impact and novelty. RESULTS: TTs have demonstrated significant anti-cancer activity in multiple tumor types, both in vitro and in vivo. Furthermore, they have shown synergistic effects when given in combination with standard anti-cancer agents or other anti-tumor natural compounds. Finally, new purification processes and transgenic sources have been designed in order to improve TTs production, and novel TTs formulations and synthetic derivatives have been developed to enhance their solubility and bioavailability. CONCLUSION: The promising anti-cancer effects shown by TTs in several preclinical studies may open new opportunities for therapeutic interventions in different tumors. Thus, clinical trials aimed at confirming TTs chemopreventive and tumor-suppressing activity, particularly in combination with standard therapies, are urgently needed.

Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells.

Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma.

Estrogen Receptor ß Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

BACKGROUND: Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor ß (ERß) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERß activation on melanoma cell growth. METHODS AND RESULTS: Protein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERß transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERß isoforms were identified by qRT-PCR. We demonstrated that ERß is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ERß agonists significantly and specifically inhibited cell proliferation. ERß activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERß agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERß activation. ERß agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells. Finally, we could observe that ERß isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERß agonists. CONCLUSIONS: Our results demonstrate that ERß is expressed in melanoma cell lines and that ERß agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a heterogeneous tumor and that genetic profiling is mandatory for the development of effective personalized therapeutic approaches for melanoma patients.

Oxime bond-linked daunorubicin-GnRH-III bioconjugates exert antitumor activity in castration-resistant prostate cancer cells via the type I GnRH receptor.

It is well established that gonadotropin-releasing hormone receptors (GnRH-R) are expressed in different types of cancers, including castration-resistant prostate cancer (CRPC) and mediate the antiproliferative effect of GnRH analogs. Thus, these compounds are employed as targeting moieties to selectively deliver chemotherapeutic agents to cancer cells. GnRH-III, the decapeptide isolated from the sea lamprey brain, has lower potency than GnRH in stimulating gonadotropin secretion, but it exerts antiproliferative effects on many tumors expressing the GnRH-R. GnRH-III-based peptides are considered promising targeting moieties for the preparation of anticancer drug delivery systems. These studies were aimed at i) evaluating the antitumor activity of two cytotoxic oxime bond-linked daunorubicin (Dau)-GnRH-III derivative bioconjugates (Dau-GnRH-III, in which daunorubicin was coupled to the 8Lys in the native form of GnRH-III, and Dau-[4Lys(Ac)]-GnRH-III, in which daunorubicin was attached to the 8Lys of a GnRH-III derivative where 4Ser was replaced by an acetylated lysine) on CRPC cells; and ii) to elucidate the involvement of the classical GnRH-R (type I GnRH-R) in this antitumor activity. Our results demonstrated that both Dau-GnRH-III and Dau-[4Lys(Ac)]-GnRH-III were rapidly internalized into DU145 prostate cancer cells and exerted a significant cytostatic effect. Both bioconjugates increased the levels of the active form of caspase-3, indicating the involvement of apoptosis in their antitumor activity. The antiproliferative effect of both Dau-GnRH-III and Dau-[4Lys(Ac)]-GnRH-III was counteracted by the simultaneous treatment of the cells with Antide, an antagonist of the GnRH-R. Moreover, after silencing the type I GnRH-R the antitumor activity of both bioconjugates was completely abolished. These data demonstrate that in CRPC cells, daunorubicin-GnRH-III derivative bioconjugates: i) inhibit tumor cell proliferation, by triggering the apoptosis process; ii) exert their antitumor effect through the activation of the type I GnRH-R expressed on these cells. Cytotoxic-GnRH-III derivative may represent promising targeted chemotherapeutics for the treatment of CRPC patients.

Targeting hormonal signaling pathways in castration resistant prostate cancer.

It is now well established that hormonal pathways are involved in the development of prostate cancer towards the castration resistant (CRPC) stage and can be effective molecular targets for novel treatment strategies. Most CRPC are sensitive to androgens and this can be due to the intratumoral production of androgens, androgen receptor (AR) amplification/ mutations and epigenetic modifications of AR expression/signaling. Based on these observations, potent agents targeting the AR axis were developed: 1) inhibitors of CYP17 (a key enzyme in the production of androgens), such as abiraterone and orteronel; 2) AR antagonists that bind to AR and impair AR activation, such as enzalutamide and ARN-509. Moreover, gonadotropin-releasing hormone receptors (GnRH-R), associated with a strong antitumor activity, are expressed in CRPC cells, indicating that they might represent an important target for GnRH analog-based therapeutic strategies. In addition to GnRH agonists and antagonists (i.e., degarelix), cytotoxic GnRH-based bioconjugates, delivering chemotherapeutic drugs to cancer cells expressing the GnRH-R, were developed and reported to exert antitumor effects on CRPC cells; some of them (i.e., AN-152) have already entered clinical trials. This review discusses the most relevant patents and recent observations on the anti-cancer efficacy of novel drugs targeting the AR and the GnRH-R pathways in CRPC.

Gonadotropin-releasing hormone agonists sensitize, and resensitize, prostate cancer cells to docetaxel in a p53-dependent manner.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. We previously showed that GnRH agonists exert an antitumor activity in CRPC cells; however, a link between GnRH receptors and the apoptotic machinery remains to be defined. Aim of this study was to evaluate whether, in CRPC cells, GnRH agonists might affect the expression/activity of apoptosis-related proteins and might sensitize, or resensitize, cancer cells to chemotherapeutics. We demonstrated that, in p53-positive DU145 cells, GnRH agonists: a) increase the expression of the proapoptotic protein Bax; this effect is mediated by the phosphorylation (activation) of p53, triggered by the p38 MAPK; b) potentiate the antiproliferative/proapoptotic activity of docetaxel; c) resensitize docetaxel-resistant cells to the antitumor activity of the cytotoxic drug. These data indicate that GnRH agonists sensitize and, more importantly, resensitize DU145 CRPC cells to chemotherapy in a p53-dependent manner. To confirm the crucial role of p53 in the activity of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein.

GnRH receptors in cancer: from cell biology to novel targeted therapeutic strategies.

The crucial role of pituitary GnRH receptors (GnRH-R) in the control of reproductive functions is well established. These receptors are the target of GnRH agonists (through receptor desensitization) and antagonists (through receptor blockade) for the treatment of steroid-dependent pathologies, including hormone-dependent tumors. It has also become increasingly clear that GnRH-R are expressed in cancer tissues, either related (i.e. prostate, breast, endometrial, and ovarian cancers) or unrelated (i.e. melanoma, glioblastoma, lung, and pancreatic cancers) to the reproductive system. In hormone-related tumors, GnRH-R appear to be expressed even when the tumor has escaped steroid dependence (such as castration-resistant prostate cancer). These receptors are coupled to a G(αi)-mediated intracellular signaling pathway. Activation of tumor GnRH-R by means of GnRH agonists elicits a strong antiproliferative, antimetastatic, and antiangiogenic (more recently demonstrated) activity. Interestingly, GnRH antagonists have also been shown to elicit a direct antitumor effect; thus, these compounds behave as antagonists of GnRH-R at the pituitary level and as agonists of the same receptors expressed in tumors. According to the ligand-induced selective-signaling theory, GnRH-R might assume various conformations, endowed with different activities for GnRH analogs and with different intracellular signaling pathways, according to the cell context. Based on these consistent experimental observations, tumor GnRH-R are now considered a very interesting candidate for novel molecular, GnRH analog-based, targeted strategies for the treatment of tumors expressing these receptors. These agents include GnRH agonists and antagonists, GnRH analog-based cytotoxic (i.e. doxorubicin) or nutraceutic (i.e. curcumin) hybrids, and GnRH-R-targeted nanoparticles delivering anticancer compounds.

Molecular mechanisms of the antimetastatic activity of nuclear clusterin in prostate cancer cells.

The proapoptotic activity of nuclear clusterin (nCLU) in cancer cells is now well established. We previously showed that nCLU decreases the motility of prostate cancer cells by triggering a dramatic dismantling of the actin cytoskeleton. Here, we sought to unravel the molecular mechanisms of the antimetastatic activity of nCLU. We found that nCLU: i) decreases LIMK1 expression, thus increasing the levels of the active (unphosphorylated) form of cofilin, the well known actin depolymerizing factor; ii) binds to vimentin, sequestering the protein from its adhesion sites at the cell periphery, thus interfering with its role in cell motility and adhesion; iii) affects the intracellular distribution of E-cadherin (the major component of epithelial adherens junctions) which appears to be diffusely distributed in the cells. Through these mechanisms nCLU reduces the migratory/invasive behavior of PC3 cells; this effect is further demonstrated by a decreased secretion of active MMP-2 from the cells. Thus, in addition to its proapoptotic function, nCLU also exerts a strong anti-migratory/anti-invasive activity in prostate cancer cells, by interfering with the cytoskeletal components and by decreasing MMP-2 activity.

Dual targeting of tumor and endothelial cells by gonadotropin-releasing hormone agonists to reduce melanoma angiogenesis.

We showed previously that GnRH receptors are expressed in melanoma cells; their activation reduces cell growth and metastatic behavior. Here, we investigated whether GnRH agonists might affect the expression of genes involved in melanoma progression. By genome-wide transcriptomic and real-time PCR analysis, we first observed that GnRH agonists decrease the expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) (all isoforms) in BLM melanoma cells. Then, we demonstrated that GnRH agonists specifically decrease the expression of the VEGF165 isoform as well as its secretion from BLM cells. These data suggested that activation of GnRH receptors might reduce the pro-angiogenic behavior of melanoma cells. To verify this hypothesis, we treated BLM cells with a GnRH agonist; the conditioned medium from these cells was tested to assess its capability to stimulate human umbilical vein endothelial cell (HUVEC) motility. The migration of HUVECs towards the conditioned medium of GnRH agonist-treated BLM cells was significantly lower than the migration of HUVECs toward the conditioned medium of untreated cells. Thus, GnRH agonists reduce the pro-angiogenic behavior of melanoma cells through a decreased production of bioactive VEGF. We then found that GnRH receptors are also expressed on HUVECs and that GnRH agonists reduce their ability to proliferate and to form capillary-like tubes when stimulated by VEGF. These findings suggest that GnRH agonists exert an anti-angiogenic activity indirectly by decreasing VEGF secretion from tumor cells and directly by counteracting the pro-angiogenic activity of the growth factor. These data might lead to the development of novel targeted approaches for melanoma.

Novel insights into GnRH receptor activity: role in the control of human glioblastoma cell proliferation.

Malignant glioblastoma is one of the highest proliferative and invasive tumors within the central nervous system (CNS); the therapeutical options for this disease are still very poor. Receptors for gonadotropin-releasing hormone (GnRH) have been reported to be present in glioblastoma tissues. This study aimed to determine the role of these receptors in the control of glioma growth. In two human glioblastoma cell lines, U87MG and U373, we demonstrated the expression of GnRH receptors, both at mRNA and protein levels. We also found that GnRH receptor is expressed in glioblastoma tissues from tumor patients as shown by Western blotting. In U87MG and U373 cell lines, we found the expression of mRNA for GnRH, indicating the presence of an autocrine GnRH-based system in these cell lines. Treatment of the two cell lines with a GnRH agonist resulted in a significant decrease of cell proliferation. Moreover, the GnRH agonist significantly counteracted the forskolin-induced increase of intracellular cAMP levels in these cells. These findings suggest that the GnRH receptor might represent a molecular target for an endocrine therapeutical approach against gliomas.

Type I gonadotropin-releasing hormone receptor mediates the antiproliferative effects of GnRH-II on prostate cancer cells.

BACKGROUND: GnRH-II has been shown to exert a strong antiproliferative action on tumors of the female reproductive system. The data so far reported on the effects of GnRH-II on prostate cancer growth are controversial. Moreover, it is still unclear through which receptor [type I or type II GnRH-receptor (GnRH-R)] GnRH-II might modulate cancer cell proliferation. OBJECTIVE: The objective of this work was to investigate whether GnRH-II might affect the proliferation of prostate cancer cells and to identify the GnRH-R through which the peptide might exert its activity. DESIGN: We investigated the effects of GnRH-II on prostate cancer cell proliferation. We then transfected PC3 cells with a small interfering RNA targeted to type I GnRH-R. After receptor silencing we evaluated the effects of GnRH-II on cell proliferation and on forskolin-induced intracellular cAMP accumulation. Similar experiments were performed by silencing type II GnRH-R. RESULTS: GnRH-II exerted an antiproliferative activity on prostate cancer cells. Transfection of PC3 cells with a type I GnRH-R small interfering RNA resulted in a significant decrease of the expression of this receptor. After type I GnRH-R silencing: 1) the antiproliferative effect of GnRH-II was completely abrogated; and 2) GnRH-II lost its capacity to counteract the forskolin-induced cAMP accumulation. On the contrary, type II GnRH-R silencing did not counteract the antiproliferative effect of GnRH-II. CONCLUSIONS: GnRH-II exerts a specific and significant antiproliferative action on prostate cancer cells. This antitumor effect is mediated by the activation of type I (but not of type II) GnRH-R and by its coupled cAMP intracellular signaling pathway.

Gonadotropin-releasing hormone agonists suppress melanoma cell motility and invasiveness through the inhibition of alpha3 integrin and MMP-2 expression and activity.

Cutaneous melanoma represents the leading cause of skin cancer deaths. The prognosis of highly aggressive, metastatic melanoma is still very poor, due to the resistance of the disseminated tumor to existing therapies. The clarification of the molecular mechanisms regulating melanoma growth and progression might help identify novel molecular targets for the development of new therapeutic interventions. We previously showed that gonadotropin-releasing hormone (GnRH) receptors are expressed in melanoma cells; activation of these receptors by means of GnRH agonists significantly reduces cell proliferation. In the current study, we first showed that GnRH agonists significantly reduced the metastatic behavior of melanoma cells in terms of both cell motility (haptotactic assay using laminin as the chemoattractant) and invasiveness (cell invasion assay evaluating the capacity of the cells to invade a reconstituted extracellular matrix barrier). On the basis of this observation, we then investigated the molecular mechanisms underlying the antimetastatic activity of GnRH agonists. We found that, in melanoma cells, a) the activity of the alpha3 integrin subunit is crucial for the migratory behavior of the cells; b) GnRH agonists significantly reduced alpha3 integrin expression (Western blotting and immunofluorescence studies); c) GnRH agonists significantly reduced MMP-2 expression (comparative RT-PCR) and activity (zymographic analysis performed on cell culture media). These data indicate that GnRH agonists, in addition to the previously reported antiproliferative effect, elicit a strong inhibitory activity on the migratory/invasive behavior of melanoma cells expressing GnRH receptors. These compounds reduce the metastatic potential of melanoma cells by interfering with the expression/activity of cell adhesion molecules (alpha3 integrin) and matrix metalloproteinase (MMP-2).

Clusterin isoforms differentially affect growth and motility of prostate cells: possible implications in prostate tumorigenesis.

Besides a fully processed, secreted form of clusterin (sCLU), an alternative proapoptotic form of the protein targeting the nucleus (nCLU) was recently described. The possible differential roles played by the two clusterin forms in growth and motility of nonmalignant and malignant prostate cells are investigated here. sCLU or nCLU was transiently transfected in both androgen-independent prostate cancer cells (PC3 and DU 145) and immortalized prostate epithelial cells (PNT1A, a nontumoral control). Then, cell growth, motility, and cytoskeleton organization were studied. We found that (a) in PNT1A cells, both sCLU and nCLU significantly decreased cell proliferation and motility; (b) in PC3 and DU 145 cancer cells, only nCLU inhibited cell growth and migration, with sCLU being ineffective; and (c) the antimotility effect of nCLU was accompanied by a dramatic dismantling of the actin cytoskeleton. Moreover, transfection with "full-length" CLU cDNA produced both sCLU and nCLU in nonmalignant PNT1A cells, whereas only sCLU was found in cancer cells. Thus, CLU gene expression might play a crucial role in prostate tumorigenesis by exerting differential biological effects on normal versus tumor cells through differential processing of CLU isoforms in the two cell systems. We also found that nCLU binds to alpha-actinin, a key protein for the regulation of actin cytoskeleton, and that nCLU and alpha-actinin colocalize in the cytoplasm. Thus, the antimotility activity of nCLU and its ability to cause dismantling of the actin cytoskeleton seem to be mediated by its binding to alpha-actinin.

Gonadotropin-releasing hormone agonists reduce the migratory and the invasive behavior of androgen-independent prostate cancer cells by interfering with the activity of IGF-I.

Androgen-independent prostate carcinoma is characterized by a high proliferation rate and by a strong metastatic behavior. We have previously shown that GnRH agonists exert a direct and specific inhibitory action on the proliferation of androgen-independent prostate cancer cells (DU 145). These compounds mainly act by interfering with the mitogenic activity of growth factors, such as the insulin-like growth factor-I (IGF-I). The present experiments were performed to clarify whether GnRH agonists might also affect the migratory and the invasive behavior of androgen-independent prostate cancer cells and to define their mechanism of action. First we showed that the GnRH agonist Leuprolide reduces the migration of DU 145 cells towards a chemoattractant and their ability to invade a reconstituted basement membrane. Experiments were then performed to clarify whether the GnRH agonist might act by interfering with the pro-metastatic activity of IGF-I. We found that, in androgen-independent prostate cancer cells, Leuprolide: a) interferes with the IGF-I system (receptor protein expression and tyrosine-phosphorylation); b) abrogates the IGF-I-induced phosphorylation of Akt (a kinase previously shown by us to mediate the pro-metastatic activity of IGF-I in prostate cancer cells); c) counteracts the migration and the invasive activity of the cells stimulated by IGF-I; d) abolishes the effects of IGF-I on cell morphology, on actin cytoskeleton organization and on alphavbeta3 integrin expression/cellular localization. These data indicate that GnRH agonists, in addition to their well known antiproliferative effect, can also exert a significant inhibitory activity on the migratory and invasive behavior of androgen-independent prostate cancer cells, expressing the GnRH receptor. GnRH agonists act by interfering with the pro-metastatic activity of the growth factor IGF-I.

Insulin-like growth factor-I promotes migration in human androgen-independent prostate cancer cells via the alphavbeta3 integrin and PI3-K/Akt signaling.

In its phase of androgen-independence, prostate carcinoma is characterized by a high proliferation rate and by a strong ability to give rise to metastases. IGF-I has been shown to exert a potent mitogenic action on prostate cancer. We investigated whether IGF-I might also affect the motility of prostate cancer cells and defined the mechanism of action. We found that IGF-I promotes the migratory capacity of androgen-independent prostate cancer cells through the activation of its specific receptor, IGF-IR. This effect was accompanied by a change in cell morphology (as revealed by scanning electron microscopy), and by a rearrangement of the actin cytoskeleton. The treatment of cells with the PI3-K inhibitor, LY294002, counteracted the pro-migratory activity of IGF-I. Experiments were then performed to clarify whether the integrin, alphavbeta3, could be involved in the action of IGF-I. We demonstrated that: a) the IGF-I-induced migration of cells is completely antagonized by an antibody specifically blocking the function of alphavbeta3; b) IGF-I increases alphavbeta3 immunofluorescence at the level of cell membranes, and this effect is counteracted by LY294002; and c) IGF-I increases alphavbeta3 protein levels. Our results demonstrate that IGF-I promotes the motility of androgen-independent prostate cancer cells by modulating alphavbeta3 integrin activation/expression; these effects are mediated by the PI3-K/Akt signaling pathway. This study: a) supports a crucial role for IGF-I in the progression of the pathology towards the highly metastatic phase; and b) provides an additional rationale basis for the development of therapeutic strategies directed at the IGF-I/IGF-IR system in the treatment of androgen-independent prostate cancer.