Aspartate ß-hydroxylase targeting in castration-resistant prostate cancer modulates the NOTCH/HIF1α/GSK3ß crosstalk.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) ß hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3ß by ASPH silencing demonstrates that ASPH regulates GSK3ß activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

FAS-mediated apoptosis impairment in patients with ALPS/ALPS-like phenotype carrying variants on CASP10 gene.

Autoimmune lymphoproliferative syndrome (ALPS) is a congenital disorder that results in an apoptosis impairment of lymphocytes, leading to chronic lymphoproliferation and autoimmunity, mainly autoimmune cytopenias. FAS gene defects are often responsible for the disease, the phenotype of which can vary from asymptomatic/mild forms to severe disease. More rarely, defects are associated to  other genes involved in apoptosis pathway, such as CASP10. Few data are available on CASP10-mutated patients. To date, two CASP10 mutations have been recognized as pathogenic (I406L and L258F) and others have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l). In this study, we evaluated apoptosis function in patients with an ALPS/ALPS-like phenotype carrying CASP10 variants. Molecular findings were obtained by next generation sequencing analysis of genes involved in immune dysregulation syndromes. Functional studies were performed after inducing apoptosis by FAS-ligand/TRIAL stimulation and analysing cell death and the function of CASP10, CASP8 and PARP proteins. We identified 6 patients with an ALPS (n = 2) or ALPS-like (n = 4) phenotype, carrying I406L (n = 1),V410l (n = 2),Y446C (n = 1) heterozygous CASP10 variants or the L522l polymorphisms (n = 2) associated with another polymorphic homozygote variant on CASP8 or a compound heterozygous mutation on TNFRSF13C. Apoptosis was impaired in all patients showing that such variants may play a role in the development of clinical phenotype.

Adaptive phenotype drives resistance to androgen deprivation therapy in prostate cancer.

BACKGROUND: Prostate cancer (PCa), the second most common cancer affecting men worldwide, shows a broad spectrum of biological and clinical behaviour representing the epiphenomenon of an extreme heterogeneity. Androgen deprivation therapy is the mainstay of treatment for advanced forms but after few years the majority of patients progress to castration-resistant prostate cancer (CRPC), a lethal form that poses considerable therapeutic challenges. METHODS: Western blotting, immunocytochemistry, invasion and reporter assays, and in vivo studies were performed to characterize androgen resistant sublines phenotype in comparison to the parental cell line LNCaP. RNA microarray, mass spectrometry, integrative transcriptomic and proteomic differential analysis coupled with GeneOntology and multivariate analyses were applied to identify deregulated genes and proteins involved in CRPC evolution. RESULTS: Treating the androgen-responsive LNCaP cell line for over a year with 10 µM bicalutamide both in the presence and absence of 0.1 nM 5-α-dihydrotestosterone (DHT) we obtained two cell sublines, designated PDB and MDB respectively, presenting several analogies with CRPC. Molecular and functional analyses of PDB and MDB, compared to the parental cell line, showed that both resistant cell lines were PSA low/negative with comparable levels of nuclear androgen receptor devoid of activity due to altered phosphorylation; cell growth and survival were dependent on AKT and p38MAPK activation and PARP-1 overexpression; their malignant phenotype increased both in vitro and in vivo. Performing bioinformatic analyses we highlighted biological processes related to environmental and stress adaptation supporting cell survival and growth. We identified 15 proteins that could direct androgen-resistance acquisition. Eleven out of these 15 proteins were closely related to biological processes involved in PCa progression. CONCLUSIONS: Our models suggest that environmental factors and epigenetic modulation can activate processes of phenotypic adaptation driving drug-resistance. The identified key proteins of these adaptive phenotypes could be eligible targets for innovative therapies as well as molecules of prognostic and predictive value.

Aminobisphosphonates prevent the inhibitory effects exerted by lymph node stromal cells on anti-tumor Vδ 2 T lymphocytes in non-Hodgkin lymphomas.

In this study, we analyzed the influence of mesenchymal stromal cells derived from lymph nodes of non-Hodgkin's lymphomas, on effector functions and differentiation of Vdelta (δ)2 T lymphocytes. We show that: i) lymph-node mesenchymal stromal cells of non-Hodgkin's lymphoma inhibit NKG2D-mediated lymphoid cell killing, but not rituximab-induced antibody-dependent cell-mediated cytotoxicity, exerted by Vδ2 T lymphocytes; ii) pre-treatment of mesenchymal stromal cells with the aminobisphosphonates pamidronate or zoledronate can rescue lymphoma cell killing via NKG2D; iii) this is due to inhibition of transforming growth factor-ß and increase in interleukin-15 production by mesenchymal stromal cells; iv) aminobisphosphonate-treated mesenchymal stromal cells drive Vδ2 T-lymphocyte differentiation into effector memory T cells, expressing the Thelper1 cytokines tumor necrosis factor-α and interferon-γ. In non-Hodgkin's lymphoma lymph nodes, Vδ2 T cells were mostly naïve; upon co-culture with autologous lymph-node mesenchymal stromal cells exposed to zoledronate, the percentage of terminal differentiated effector memory Vδ2 T lymphocytes increased. In all non-Hodgkin's lymphomas, low or undetectable transcription of Thelper1 cytokines was found. In diffused large B-cell lymphomas and in a group of follicular lymphoma, transcription of transforming growth factor ß and interleukin-10 was enhanced compared to non-neoplastic lymph nodes. Thus, in non-Hodgkin lymphomas mesenchymal stromal cells interfere with Vδ2 T-lymphocyte cytolytic function and differentiation to Thelper1 and/or effector memory cells, depending on the prominent in situ cytokine milieu. Aminobisphosphonates, acting on lymph-node mesenchymal stromal cells, can push the balance towards Thelper1/effector memory and rescue the recognition and killing of lymphoma cells through NKG2D, sparing rituximab-induced antibody-dependent cell-mediated cytotoxicity.

SB202190 Predicts BRAF-Activating Mutations in Primary Colorectal Cancer Organoids via Erk1-2 Modulation.

The p38 inhibitor SB202190 is a necessary component of the medium used for normal colorectal mucosa cultures. Sato et al. suggested that the primary activity of SB202190 may be EGFR signaling stabilization, causing an increased phosphorylation of Erk1-2 sustaining organoid proliferation. However, the growth of some colorectal cancer (CRC)-derived organoid cultures is inhibited by this molecule via an unknown mechanism. We biochemically investigated SB202190 activity on a collection of 25 primary human CRC organoids, evaluating EGFR, Akt and Erk1-2 activation using Western blot. We found that Erk1-2 phosphorylation was induced by SB202190 in 20 organoid cultures and inhibited in 5 organoid cultures. A next-generation sequencing (NGS) analysis revealed that the inhibition of p-Erk1-2 signaling corresponded to the cultures with BRAF mutations (with four different hits, one being undescribed), while p-Erk1-2 induction was apparently unrelated to other mutations involving the EGFR pathway (Her2, KRAS and NRAS). We found that SB202190 mirrored the biochemical activity of the BRAF inhibitor Dabrafenib, known to induce the paradoxical activation of p-Erk1-2 signaling in BRAF wild-type cells. SB202190 was a more effective inhibitor of BRAF-mutated organoid growth in the long term than the specific BRAF inhibitors Dabrafenib and PLX8394. Overall, SB202190 can predict BRAF-activating mutations in patient-derived organoids, as well as allowing for the identification of new BRAF variants, preceding and enforcing NGS data.

Xanthohumol impairs human prostate cancer cell growth and invasion and diminishes the incidence and progression of advanced tumors in TRAMP mice.

Despite recent advances in understanding the biological basis of prostate cancer, management of the disease, especially in the phase resistant to androgen ablation, remains a significant challenge. The long latency and high incidence of prostate carcinogenesis provides the opportunity to intervene with chemoprevention to prevent or eradicate prostate malignancies. In this study, we have used human hormone-resistant prostate cancer cells, DU145 and PC3, as an in vitro model to assess the efficacy of xanthohumol (XN) against cell growth, motility and invasion. We observed that treatment of prostate cancer cells with low micromolar doses of XN inhibits proliferation and modulates focal adhesion kinase (FAK) and AKT phosphorylation leading to reduced cell migration and invasion. Oxidative stress by increased production of reactive oxygen species (ROS) was associated with these effects. Transgenic adenocarcinoma of the mouse prostate (TRAMP) transgenic mice were used as an in vivo model of prostate adenocarcinoma. Oral gavage of XN, three times per week, beginning at 4 wks of age, induced a decrease in the average weight of the urogenital (UG) tract, delayed advanced tumor progression and inhibited the growth of poorly differentiated prostate carcinoma. The ability of XN to inhibit prostate cancer in vitro and in vivo suggests that XN may be a novel agent for the management of prostate cancer.

Pathogen-induced interleukin-1beta processing and secretion is regulated by a biphasic redox response.

In this study, we show that IL-1beta processing and secretion induced by pathogen-associated molecular pattern (PAMP) molecules in human monocytes is regulated by a biphasic redox event including a prompt oxidative stress and a delayed antioxidant response. Namely, PAMPs induce an early generation of reactive oxygen species (ROS) followed by increase of intracellular thioredoxin and release of reduced cysteine: this antioxidant phase is paralleled by secretion of mature IL-1beta. ROS production and antioxidant response are both required, because either inhibitors of NADPH oxidase and of thioredoxin reductase impair IL-1beta secretion. These inhibitors also hinder cysteine release and consequently prevent reduction of the extracellular medium: addition of exogenous reducing agents restores IL-1beta secretion. Not only silencing of thioredoxin, but also of the ROS scavenger superoxide dismutase 1 results in inhibition of IL-1beta secretion. Thus, PAMP-induced ROS trigger an antioxidant response involving intracellular redox enzymes and release of cysteine, ultimately required for IL-1beta processing and secretion.

The chemopreventive retinoid 4HPR impairs prostate cancer cell migration and invasion by interfering with FAK/AKT/GSK3beta pathway and beta-catenin stability.

BACKGROUND: Prostate cancer shows an extremely slow progression, appearing in its metastatic, hormone refractory phenotype mostly in elderly men. The chemopreventive targeting of this tumor could accordingly delay its malignancy over life expectancy. The cancer chemopreventive retinoid N-(4 hydroxyphenyl)retinamide (4HPR) has already been shown to restrain prostate cancer growth in vitro and in vivo, though its mechanisms of action are only partially explained. RESULTS: We found that 4HPR impairs DU145 and PC3 prostate cancer cells migration and invasion by down-regulating FAK and AKT activation and by enhancing beta-catenin degradation, causing the downregulation of target genes like cyclin D1, survivin and VEGF. This non-migratory phenotype was similarly produced in both cell lines by stable silencing of beta-catenin. 4HPR was able to decrease AKT phosphorylation also when powerfully upregulated by IGF-1 and, consequently, to impair IGF-1-stimulated cell motility. Conversely, the expression of constitutively active AKT (myr-AKT) overcame the effects of 4HPR and beta-catenin-silencing on cell migration. In addition, we found that BMP-2, a 4HPR target with antiangiogenic activity, decreased prostate cancer cell proliferation, migration and invasion by down-regulating the pathway described involving AKT phosphorylation, beta-catenin stability and cyclin D1 expression. CONCLUSION: These data point to 4HPR as a negative regulator of AKT phosphorylation, effectively targeting the beta-catenin pathway and inducing a relatively benign phenotype in prostate cancer cells, limiting neoangiogenesis and cell invasion.

Defective FAS-Mediated Apoptosis and Immune Dysregulation in Gaucher Disease.

BACKGROUND: Gaucher disease (GD) is a rare disorder characterized by defective function of ß-glucocerebrosidase, which leads to progressive accumulation of its substrate in various organs, particularly the mononuclear phagocyte system. Hepatosplenomegaly and cytopenia represent the disease's most common features, but patients with GD also show hyperinflammation, hypergammaglobulinemia, and immune dysregulation involving B, T, and natural killer cells. As clinical phenotype can be underhand, symptoms can overlap with autoimmune lymphoproliferative syndrome (ALPS) or other ALPS-like disorders. OBJECTIVE: To evaluate the ALPS-like immunological pattern and apoptosis function in patients with GD. METHODS: We evaluated lymphocyte subsets and immunophenotypic and serological features of ALPS (double-negative T cells [DNTs], B220+DNTs, CD27+, T-reg/HLA-DR ratio, IL-10, IL-18, vitamin B12) in a population of patients with GD. Moreover, we tested FAS/TRAIL-induced apoptosis and CASP8/CASP10/PARP function in patients showing an immune-dysregulation pattern. RESULTS: A total of 41 patients (33 treated, 8 treatment-naïve) were studied. Nine (21%) and 7 (17%) of 41 patients had high DNT and B220+DNT counts, respectively. Overall, 10 of 41(24%) patients showed immunological features suggestive of ALPS that were more frequent in treatment-naïve subjects (P = .040 vs P = .031) and in those with early onset of the disease (P = .046 vs P = .011), respectively. FAS-induced apoptosis and caspase activation were further evaluated in these 10 patients and were found to be defective in 7 of them. CONCLUSIONS: We show that patients with GD may have ALPS-like features and FAS-mediated apoptosis defects that are more pronounced in treatment-naïve subjects and in patients with early onset of the disease. Therefore, diagnostic workup of patients with an ALPS-like phenotype should include screening for GD.

Evaluation of Glycosylated PTGS2 in Colorectal Cancer for NSAIDS-Based Adjuvant Therapy.

Observational/retrospective studies indicate that prostaglandin-endoperoxide synthase-2 (PTGS2) inhibitors could positively affect colorectal cancer (CRC) patients' survival after diagnosis. To obtain an acceptable cost/benefit balance, the inclusion of PTGS2 inhibitors in the adjuvant setting needs a selective criterion. We quantified the 72 kDa, CRC-associated, glycosylated form of PTGS2 in 100 frozen CRC specimens and evaluated PTGS2 localization by IHC in the same tumors, scoring tumor epithelial-derived and stroma-derived fractions. We also investigated the involvement of interleukin-1 beta (IL1ß) in PTGS2 induction, both in vitro and in CRC lysates. Finally, we used overall survival (OS) as a criterion for patient selection. Glycosylated PTGS2 can be quantified with high sensibility in tissue lysates, but the expression in both tumor and stromal cells limits its use for predictive purposes. Immunohistochemistry (IHC) analysis indicates that stromal PTGS2 expression could exert a protective role on patient OS. Stromal PTGS2 was prevalently expressed by cancer-associated fibroblasts exerting a barrier function near the gut lumen, and it apparently favored the antitumor M1 macrophage population. IL1ß was directly linked to gPTGS2 expression both in vitro and in tumors, but its activity was apparently prevalent on the stromal cell population. We suggest that stromal PTGS2 could exert a positive effect on patients OS when expressed in the luminal area of the tumor.

Anti-angiogenic properties of chemopreventive drugs: fenretinide as a prototype.

Several cancer chemopreventive agents have been demonstrated to exert antiangiogenic effects. Blocking tumor angiogenesis, a process critical for tumor mass expansion and metastasis, represents an intriguing approach not only to cancer therapy, but also to cancer chemoprevention. We found that angiogenesis is a common and key target of many chemopreventive molecules, where they most likely suppress the angiogenic switch in premalignant tumors, a concept we termed "angioprevention." In this manuscript we use as an example the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR), a molecule with confirmed clinical applications in breast cancer adjuvant therapy to prevent cancer recurrence and under evaluation in neuroblastoma and glioblastoma treatment.

Human Gut-Associated Natural Killer Cells in Health and Disease.

It is well established that natural killer (NK) cells are involved in both innate and adaptive immunity. Indeed, they can recognize molecules induced at the cell surface by stress signals and virus infections. The functions of NK cells in the gut are much more complex. Gut NK cells are not precisely organized in lymphoid aggregates but rather scattered in the epithelium or in the stroma, where they come in contact with a multitude of antigens derived from commensal or pathogenic microorganisms in addition to components of microbiota. Furthermore, NK cells in the bowel interact with several cell types, including epithelial cells, fibroblasts, macrophages, dendritic cells, and T lymphocytes, and contribute to the maintenance of immune homeostasis and development of efficient immune responses. NK cells have a key role in the response to intestinal bacterial infections, primarily through production of IFNγ, which can stimulate recruitment of additional NK cells from peripheral blood leading to amplification of the anti-bacterial immune response. Additionally, NK cells can have a role in the pathogenesis of gut autoimmune inflammatory bowel diseases (IBDs), such as Crohn's Disease and Ulcerative Colitis. These diseases are considered relevant to the generation of gastrointestinal malignancies. Indeed, the role of gut-associated NK cells in the immune response to bowel cancers is known. Thus, in the gut immune system, NK cells play a dual role, participating in both physiological and pathogenic processes. In this review, we will analyze the known functions of NK cells in the gut mucosa both in health and disease, focusing on the cross-talk among bowel microenvironment, epithelial barrier integrity, microbiota, and NK cells.

Novel cell death pathways induced by N-(4-hydroxyphenyl)retinamide: therapeutic implications.

We previously reported that N-(4-hydroxyphenyl)retinamide (4HPR) inhibits retinoblastoma tumor growth in a murine model in vivo and kills Y79 retinoblastoma cells in vitro. In this work, we assayed different cell death-related parameters, including mitochondrial damage and caspase activation, in Y79 cells exposed to 4HPR. 4HPR induced cytochrome c release from mitochondria, caspase-3 activation, and oligonucleosomal DNA fragmentation. However, pharmacologic inactivation of caspases by the pan-caspase inhibitor BOC-D-fmk, or specific caspase-3 inhibition by Z-DEVD-fmk, was not sufficient to prevent cell death, as assessed by loss of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, disruption of mitochondrial transmembrane potential (Deltapsi(m)), and ATP depletion. We found that 4HPR causes lysosomal membrane permeabilization and cytosolic relocation of cathepsin D. Pepstatin A partially rescued cell viability and reduced DNA fragmentation and cytosolic cytochrome c. The antioxidant N-acetylcysteine attenuated cathepsin D relocation into the cytosol, suggesting that lysosomal destabilization is dependent on elevation of reactive oxygen species and precedes mitochondrial dysfunction. Activation of AKT, which regulates energy level in the cell, by the retinal survival facto]r insulin-like growth factor I was impaired and insulin-like growth factor I was ineffective against ATP and Deltapsi(m) loss in the presence of 4HPR. Lysosomal destabilization, associated with mitochondrial dysfunction, was induced by 4HPR also in other cancer cell lines, including PC3 prostate adenocarcinoma and the vascular tumor Kaposi sarcoma KS-Imm cells. The novel finding of a lysosome-mediated cell death pathway activated by 4HPR could have implications at clinical level for the development of combination chemoprevention and therapy of cancer.

Prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), a complex target for colorectal cancer prevention and therapy.

A plentiful literature has linked colorectal cancer (CRC) to inflammation and prostaglandin-endoperoxide synthase (PTGS)2 expression. Accordingly, several nonsteroidal antiinflammatory drugs (NSAIDs) have been tested often successfully in CRC chemoprevention despite their different ability to specifically target PTGS2 and the low or null expression of PTGS2 in early colon adenomas. Some observational studies showed an increased survival for patients with CRC assuming NSAIDs after diagnosis, but no clinical trial has yet demonstrated the efficacy of NSAIDs against established CRC, where PTGS2 is expressed at high levels. The major limits for the application of NSAIDs, or specific PTGS2 inhibitors, as adjuvant drugs in CRC are (1) a frequent confusion about the physiological role of PTGS1 and PTGS2, reflecting in CRC pathology and therapy; (2) the presence of unavoidable side effects linked to the intrinsic function of these enzymes; (3) the need of established criteria and markers for patient selection; and (4) the evaluation of the immunomodulatory potential of PTGS2 inhibitors as possible adjuvants for immunotherapy. This review has been written to rediscover the multifaceted potential of PTGS2 targeting, hoping it could act as a starting point for a new and more aware application of NSAIDs against CRC.

Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells.

Shedding of ADAM10 substrates, like TNFα, MICA or CD30, is reported to affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. Soluble forms of these molecules and ADAM10 can be carried and spread in the microenvironment by exosomes released by tumor cells. We reported new ADAM10 inhibitors able to prevent MICA shedding in Hodgkin lymphoma (HL), leading to recognition of HL cells by cytotoxic lymphocytes. In this paper, we show that the mature bioactive form of ADAM10 is released in exosome-like vesicles (ExoV) by HL cells and lymph node mesenchymal stromal cells (MSC). We demonstrate that ADAM10 inhibitors are released in ExoV by MSC or HL cells, endocytosed by bystander cells and localized in the endolysosomal compartment in HL MSC. ExoV released by HL cells can enhance MICA shedding by MSC, while ExoV from MSC induce TNFα or CD30 shedding by HL cells. Of note, ADAM10 sheddase activity carried by ExoV is prevented with the ADAM10 inhibitors LT4 and CAM29, pretreating either the ExoV-producing or the ExoV-receiving cells. In particular, both inhibitors reduce CD30 shedding maintaining the anti-tumor effects of the ADC Brentuximab-Vedotin or the anti-CD30 Iratumumab on HL cells. Thus, spreading of ADAM10 activity due to ExoV can result in the release of cytokines, like TNFα, a lymphoma growth factor, or soluble molecules, like sMICA or sCD30, that potentially interfere with host immune surveillance or immunotherapy. ADAM10 blockers can interfere with this process, allowing the development of anti-lymphoma immune response and/or efficient ADC-based or human antibody-based immunotherapy.

Targeting the Epidermal Growth Factor Receptor Can Counteract the Inhibition of Natural Killer Cell Function Exerted by Colorectal Tumor-Associated Fibroblasts.

Mesenchymal stromal cells (MSC) present in the tumor microenvironment [usually named tumor-associated fibroblasts (TAF)] can exert immunosuppressive effects on T and natural killer (NK) lymphocytes, favoring tumor immune escape. We have analyzed this mechanism in colorectal carcinoma (CRC) and found that co-culture of NK cells with TAF can prevent the IL-2-mediated NKG2D upregulation. This leads to the impairment of NKG2D-mediated recognition of CRC cells, sparing the NK cell activation through DNAM1 or FcγRIIIA (CD16). In situ, TAF express detectable levels of epidermal growth factor receptor (EGFR); thus, the therapeutic anti-EGFR humanized antibody cetuximab can trigger the antibody-dependent cellular cytotoxicity of TAF, through the engagement of FcγRIIIA on NK cells. Importantly, in the tumor, we found a lymphoid infiltrate containing NKp46+CD3- NK cells, enriched in CD16+ cells. This population, sorted and cultured with IL-2, could be triggered via CD16 and via NKG2D. Of note, ex vivo NKp46+CD3- cells were able to kill autologous TAF; in vivo, this might represent a control mechanism to reduce TAF-mediated regulatory effect on NK cell function. Altogether, these findings suggest that MSC from the neoplastic mucosa (TAF) of CRC patients can downregulate the immune cell recognition of CRC tumor cells. This immunosuppression can be relieved by the anti-EGFR antibody used in CRC immunotherapy.

Inhibition of a vascular ocular tumor growth by IL-12 gene transfer.

Ocular tumors such as retinoblastoma and uveal melanoma have devastating effects on vision. Patients with uveal melanoma also have low 5-year survival rates, thus new therapeutic modalities are necessary. As both retinoblastoma and uveal melanoma are highly vascular, we tested application of a gene transduction approach with a potent TH1 cytokine also endowed with strong anti-angiogenic activity, Interleukin-12 (IL-12). Gene transfer into murine 99E1 uveal melanoma-like cells, while having no effects on growth in vitro, essentially blocked subcutaneous tumor growth in vivo without evident signs of toxicity. Orthotopic intraocular injection resulted in invasive tumors that destroyed ocular architecture by the control cells while the IL-12 transduced cells rarely formed tumors. Histological analysis revealed highly invasive and angiogenic tumor growth in the controls and poorly vascularized tumors in the presence of IL-12. The tumor repression effect could be reproduced by a systemic anti-angiogenic effect, where controlateral injection of IL-12 expressing cells strongly repressed growth in tumors formed by parental 99E1 cells. This was associated with significantly lowered tumor vessel densities, a trend toward lower VEGF levels in the lesion, and significantly decreased NK cells in the parental tumors exposed to systemic IL-12. Taken together, our data suggest that IL-12 gene transfer can provide anti-angiogenic effects without toxicity and may be particularly suited for therapy of vascularized ocular tumors.

Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kappaB and Akt as targets.

Xanthohumol (XN), the principal flavonoid of the hop plant (Humulus lupulus L.) and a constituent of beer, has been suggested to have potential cancer chemopreventive activities. We have observed that most cancer chemopreventive agents show antiangiogenic properties in vitro and in vivo, a concept we termed "angioprevention." Here we show for the first time that XN can inhibit growth of a vascular tumor in vivo. Histopathology and in vivo angiogenesis assays indicated that tumor angiogenesis inhibition was involved. Further, we show the mechanisms for its inhibition of angiogenesis in vivo and related endothelial cell activities in vitro. XN repressed both the NF-kappaB and Akt pathways in endothelial cells, indicating that components of these pathways are major targets in the molecular mechanism of XN. Moreover, using in vitro analyses, we show that XN interferes with several points in the angiogenic process, including inhibition of endothelial cell invasion and migration, growth, and formation of a network of tubular-like structures. Our results suggest that XN can be added to the expanding list of antiangiogenic chemopreventive drugs whose potential in cancer prevention and therapy should be evaluated.

The ErbB family and androgen receptor signaling are targets of Celecoxib in prostate cancer.

Inflammation plays a central role in prostate cancer (PCa) development through significant crosstalk between the COX-2-ErbB family receptor network and androgen receptor (AR)-EGFR signaling pathways. The purpose of this work was to determine the ability of the COX-2 inhibitor Celecoxib to modulate the EGFR-AR signaling pathway in androgen-dependent PCa cells and to provide a rationale for its beneficial use in chemopreventive strategies. Functional studies of Celecoxib activity were performed on LNCaP prostate cancer cells. Western blotting, gene expression analysis, dual-luciferase reporter assay and ELISA were applied to assess the Celecoxib mechanisms of action. We found that Celecoxib, through EGF and amphiregulin (AREG) induction, caused EGFR and ErbB2 activation and consequent degradation associated with the inhibition of androgenic signaling. By upregulating the E3 ubiquitin ligase Nrdp1, Celecoxib also efficiently downregulated ErbB3, which is strongly implicated in castration-resistant prostate cancer. Lastly, Celecoxib directly regulated AR transcription and translation independent of ErbB activation by downregulating the RNA binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K). The simultaneous suppression of ErbB kinases and androgen signaling by Celecoxib represents a novel strategy to interrupt the vicious cycle of AR/ErbB cross-talk with the primary purpose of undermining their resilient signaling in prostate cancer progression. Our data provide important premises for the chemopreventive use of Celecoxib in the clinical management of prostate cancer.