Davide Genini

Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway.

Adenine deoxynucleosides, such as 2-chlorodeoxyadenosine (2CdA) and fludarabine, induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. We previously demonstrated that that the 5′-triphosphate metabolite of 2CdA (2CdATP), similar to dATP, can cooperate with cytochrome c and apoptosis protein-activating factor-1 (APAF-1) to trigger a caspase pathway in a HeLa cell-free system. We used a fluorometry-based assay of caspase activation to extend the analysis to several other clinically relevant adenine deoxynucleotides in B-chronic lymphocytic leukemia extracts. The nucleotide-induced caspase activation displayed typical Michaelis-Menten kinetics. As estimated by theV max/K m ratios, the relative efficiencies of different nucleotides were Ara-ATP > 9-fluoro-9-β-d-arabinofuranosyladenine 5′-triphosphate > dATP > 2CdATP > 9-β-d-arabinofuranosylguanine 5′-triphosphate > dADP > ATP. In contrast to dADP, both ADP and its nonhydrolyzable α,β-methylphosphonate analog were strong inhibitors of APAF-1-dependent caspase activation. The hierarchy of nucleotide activation was confirmed in a fully reconstituted system using recombinant APAF-1 and recombinant procaspase-9. These results suggest that the potency of adenine deoxynucleotides as co-factors for APAF-1-dependent caspase activation is due both to stimulation by the 5′-triphosphates and lack of inhibition by the 5′-diphosphates. The capacity of adenine deoxynucleoside metabolites to activate the apoptosome pathway may be an additional biochemical mechanism that plays a role in the chemotherapy of indolent lymphoproliferative diseases.

Artificial antigen-presenting cells as a tool to exploit the immune `synapse'

Recent progress in molecular medicine has provided important tools to identify antigen-specific T cells. In most cases, the approach is based on oligomeric combinations of recombinant major histocompatibility complex–peptide complexes fixed to various rigid supports available for binding by the T-cell receptor. These tools have greatly increased our insight into mechanisms of immune responses mediated by CD8+ T cells. Examples of the diverse fields of application for this technology include immunization, viral infections and oral tolerance induction.

HIV induces lymphocyte apoptosis by a p53-initiated, mitochondrial-mediated mechanism

HIV‐1 induces apoptosis and leads to CD4+ T‐lymphocyte depletion in humans. It is still unclear whether HIV‐1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV‐1–induced apoptosis, we infected human CD4+ T cells with HIV‐1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro‐caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis‐inducing factor (AIF) from mitochondria was observed in HIV‐infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. HIV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up‐regulation of surface FasL expression, accompanied by a down‐regulation of Fas‐associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.

Polyclonal Proliferation and Apoptosis of CCR5+ T Lymphocytes during Primary Human Immunodeficiency Virus Type 1 Infection: Regulation by Interleukin (IL)-2, IL-15, and Bcl-2

We measured apoptosis of subsets of T lymphocytes by single-cell analysis of caspase activation, to confirm high turnover of chemokine receptor CCR5(+) T cells in subjects with acute, primary human immunodeficiency virus type 1 (HIV-1) infection (PHI). High levels of spontaneous apoptosis, consisting mainly of CD8(+) T lymphocytes, were closely associated with increases in the activation markers Ki-67, CD38, and the HIV coreceptor CCR5 and with decreases in Bcl-2 and the interleukin (IL)-7 receptor at the single-cell level. Increased expression of Ki-67 and CCR5 ex vivo, as well as increased apoptosis, was seen in all T cell receptor beta-chain variable region (TCRBV) subfamilies studied. The addition of IL-2 or IL-15, but not IL-7, significantly inhibited caspase activation, increased Bcl-2 expression, and rapidly initiated proliferation in vitro of CD8(+) T cells expressing CCR5 and multiple TCRBV subfamilies. Furthermore, IL-15 receptor alpha-chain messenger RNA levels were increased in peripheral blood mononuclear cells during PHI. These results suggest that CCR5(+)Ki-67(+)Bcl-2(dim) activated T cells generated during PHI traffic via blood to tissue sites, where the cells may survive and/or further proliferate under the local influence of IL-2 or IL-15. Understanding cytokine effects on CCR5(+) T cells will be important in understanding chronic HIV-1 replication and pathogenesis.

Block of Nuclear Receptor Ubiquitination A MECHANISM OF LIGAND-DEPENDENT CONTROL OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR δ ACTIVITY

Peroxisome proliferator-activated receptor δ (PPARδ) is a ligand-activated transcription factor involved in many physiological and pathological processes. PPARδ is a promising therapeutic target for metabolic, chronic inflammatory, and neurodegenerative disorders. However, limited information is available about the mechanisms that control the activity of this nuclear receptor. Here, we examined the role of the ubiquitinproteasome system in PPARδ turnover. The receptor was ubiquitinated and subject to rapid degradation by the 26 S proteasome. Unlike most nuclear receptors that are degraded upon ligand binding, PPARδ ligands inhibited the ubiquitination of the receptor, thereby preventing its degradation. Ligand binding was required for inhibition of the ubiquitination since disruption of the ligand binding domain abolished the effect. Site-directed mutagenesis showed that the DNA binding domain was also required, indicating that ligands preferentially stabilized the DNA-bound receptor. In contrast, the activation function-2 domain and co-repressor binding site were not involved in ligand-induced stabilization. Block of ubiquitination by ligands may be an essential step to avoid rapid degradation of a receptor, like PPARδ, with a very short half-life and sustain its transcriptional activity once it is engaged in transcriptional activation complexes.

Transcriptional and Non-Transcriptional Functions of PPARβ/δ in Non-Small Cell Lung Cancer

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a nuclear receptor involved in regulation of lipid and glucose metabolism, wound healing and inflammation. PPARβ/δ has been associated also with cancer. Here we investigated the expression of PPARβ/δ and components of the prostaglandin biosynthetic pathway in non-small cell lung cancer (NSCLC). We found increased expression of PPARβ/δ, Cox-2, cPLA2, PGES and VEGF in human NSCLC compared to normal lung. In NSCLC cell lines PPARβ/δ activation increased proliferation and survival, while PPARβ/δ knock-down reduced viability and increased apoptosis. PPARβ/δ agonists induced Cox-2 and VEGF transcription, suggesting the existence of feed-forward loops promoting cell survival, inflammation and angiogenesis. These effects were seen only in high PPARβ/δ expressing cells, while low expressing cells were less or not affected. The effects were also abolished by PPARβ/δ knock-down or incubation with a PPARβ/δ antagonist. Induction of VEGF was due to both binding of PPARβ/δ to the VEGF promoter and PI3K activation through a non-genomic mechanism. We found that PPARβ/δ interacted with the PI3K regulatory subunit p85α leading to PI3K activation and Akt phosphorylation. Collectively, these data indicate that PPARβ/δ might be a central element in lung carcinogenesis controlling multiple pathways and representing a potential target for NSCLC treatment.

Abstract 953: Novel inhibitors of signal transducer and activator of transcription 3 (STAT3) show potent activity in cell cultures and tumor xenografts

The transcription factor (TF) STAT3 is an attractive target for development of anticancer drugs. STAT3 is over-expressed and activated in many human malignancies and has an important role in multiple oncogenic signaling pathways affecting proliferation, survival and metabolic adaptation of cancer cells. It has been difficult, however, to develop effective inhibitors of STAT3. In this study, we performed an in-depth analysis of the mechanism of action of two compounds, OPB-31121 and OPB-51602, which are currently undergoing clinical testing. We combined computational docking (CD), molecular dynamic simulation (MDS) and in vitro binding assays to study the compounds interaction with STAT3. CD predicted that OPB-31121 and OPB-51602 could bind to a common pocket in the STAT3 SH2 domain, which was not shared with other STAT3 inhibitors. MDS and in silico mutational analysis allowed refinement of the binding site predictions and an estimate of the relative binding affinities. Isotermal titration calorimetry (ITC) studies confirmed that OPB-31121 and OPB-51602 bound with high affinity (Kd, 5-10 nM) to recombinant STAT3 SH2 domain. Binding of the two compounds was disrupted by mutations of aminoacid residues in the predicted binding pocket and was mutually exclusive in competition assays. In contrast, OPB-31121 and OPB-51602 did not compete for binding with other STAT3 inhibitors, confirming that they occupied distinct pockets in the SH2 domain. In cell culture assays, OPB-31121 and OPB-51602 interfered with both Tyr705 and Ser727 phosphorylation, which are required for full transcriptional activity of STAT3. Proliferation of cancer cells was strongly affected in vitro by OPB-51602 and OPB-31121 in anchorage-dependent growth and soft-agar assays. In these assays OPB-51602 and OPB-31121 were active at low nanomolar concentrations (IC50, 5-10 nM). Interestingly, the compounds were more effective in metabolic stress conditions (e.g., nutrient and glucose depletion), suggesting that STAT3 inhibition interfered with relevant metabolic functions in cancer cells. In vivo treatment with OPB-51602 (PO, 20-40 mg/kg, 3-5 days) reduced Tyr705 and Ser727 phosphorylation in tumor xenografts. Growth of DU145 prostate tumor xenografts was almost completely arrested by daily treatment with OPB-51602 (PO, 20-40 mg/kg, 2 weeks). Interestingly, tumor growth did not resume after discontinuation of the 2-weeks treatment, indicating a persistent impairment of tumor-initiating capability. Altogether, this study demonstrates that STAT3 is the relevant intracellular target of OPB-51602 and OPB-31121. The two compounds bound with high affinity to a distinct pocket in the SH2 domain of STAT3 and interfered with STAT3 functions both in cells and tumor xenografts. These features resulted in distinctive biological activity and pharmacological properties of these novel compounds. Citation Format: Davide Genini, Lara Brambilla, Erik Laurini, Gianluca Civenni, Sandra Pinton, Manuela Sarti, Ramon Garcia-Escudero, Laurent Perez, Giuseppina M. Carbone, Sabrina Pricl, Carlo V. Catapano. Novel inhibitors of signal transducer and activator of transcription 3 (STAT3) show potent activity in cell cultures and tumor xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 953. doi:10.1158/1538-7445.AM2014-953

Abstract C180: Novel small molecule inhibitors of signal transducer and activator of transcription (STAT3) for cancer treatment.

Transcription factors (TFs) represent attractive targets for cancer therapy. However, very few direct TF inhibitors are currently in the clinic. STAT3 is overexpressed and activated in many human cancers promoting proliferation, survival and metabolic adaptation of cancer cells. In this study we investigated the mechanism of action of two new anticancer compounds, OPB-31121 and OPB-51602, currently evaluated in phase I clinical trials. Computational docking and molecular dynamic simulation (MDS) showed that the two compounds bound to the SH2 domain of STAT3 with an estimated binding affinity 2-3 orders of magnitude lower than other known STAT3 inhibitors (STAT3i). Furthermore, the compounds shared a common binding pocket that did not overlap with that of the other STAT3i. Binding assays using isothermal titration calorimetry (ITC) confirmed that OPB-31121 and OPB-51602 bound to recombinant STAT3-SH2 with Kd in the nM range. Binding of the two compounds was disrupted by mutations of amino acids in the predicted binding pocket. Interestingly, competition assays demonstrated that OPB-31121 and OPB-51602 did not prevent binding of other STAT3i, in agreement with the MDS predictions. OPB-51602 and OPB-31121 blocked both Tyr705 and Ser727 phosphorylation, which are required for full STAT3 activation, and inhibited the activity of STAT3 dependent luciferase reporter in human cancer cells. Both compounds strongly reduced anchorage-dependent growth and colony formation in soft-agar. Intriguingly, both compounds were more effective in conditions of metabolic stress (e.g., nutrient and glucose depletion), suggesting that they affected STAT3 functions relevant for metabolic adaptation of cancer cells. In vivo OPB-51602 (20-40 mg/kg PO daily for 2 weeks) arrested growth of DU145 prostate tumor xenografts and concomitantly reduced Tyr705 and Ser727 phosphorylation in tumor tissues. Interestingly, growth inhibition persisted after discontinuation of the treatment, suggesting a stable impairment of tumor-initiating capability and tumor regrowth. Altogether, this study identifies STAT3 as a relevant target of these new compounds. We show that both drugs interact directly with STAT3 and interfere with STAT3 functions in cells and tumor xenografts. Notably, the two compounds bind with high affinity to a unique pocket in the STAT3-SH2 domain that is not shared by other known STAT3i. These features might be relevant for their ability to block specific STAT3 functions and protein interactions resulting in distinctive biological activity and pharmacological properties of these novel compounds.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):C180.nnCitation Format: Lara Brambilla, Davide Genini, Erik Laurini, Gianluca Civenni, Sandra Pinton, Manuela Sarti, Laurent Perez, Sabrina Pricl, Giuseppina M. Carbone, Carlo V. Catapano. Novel small molecule inhibitors of signal transducer and activator of transcription (STAT3) for cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C180.

Abstract 1451: MicroRNAs regulated by ESE3/EHF control important mediators of epithelial cell differentiation and stemness in prostate tumors

Deregulated expression of ETS transcription factors has emerged as an important event in prostate cancer pathogenesis. We found that loss of the ETS factor ESE3/EHF induced a broad dedifferentiation program associated with epithelial-to-mesenchymal transition (EMT) and induction of metastatic and cancer stem-like cell properties. To understand the mechanism by which ESE3/EHF controls differentiation of prostate epithelial cells, we examined microRNA (miRNA) expression in a cohort of primary prostate tumors and prostate epithelial normal and cancer cells using miRNA gene arrays. A distinct set of miRNAs was specifically deregulated in cancer cells and tumors with low ESE3/EHF expression. Bioinformatic analysis indicated that the deregulated miRNAs controlled many genes involved in EMT and cell stemness. Interestingly, we found that miR-424 was at the top list of the miRNAs up-regulated in ESE3 low expressing tumors and cell lines. This finding was confirmed by qRT-PCR both in cells and human tumors. Functional assays showed that ESE3/EHF controlled directly miR-424 by binding to the pre-miRNA promoter and repressing its transcription. Inhibition of miR-424 using an antimiR reduced anchorage-independent growth and cell migration in cancer cells with low ESE3 expression and high miR-424 level. On the contrary, stable expression of pre-miR-424 in cells with low endogenous miR-424 level increased anchorage-independent growth and cell migration. Furthermore, modulation of miR-424 expression affected in vitro prostatosphere formation, a phenotype associated with cancer stem-like cell properties. Consistently, inhibition of miR-424 in DU145 prostate cancer cells reduced growth of tumor xenografts in immunodeficient mice. Integrating bioinformatic analyses of the predicted targets and gene profiling of cells with miR-424 overexpression we found that miRNA-424 controlled several factors involved in protein degradation. Collectively, these results show for the first time that ESE3/EHF controls a distinct network of miRNAs with both oncogenic and tumor suppressor functions. Loss of ESE3/EHF resulted specifically in increased expression of miR-424, which has oncogenic properties in prostate epithelial cells. This represents a novel mechanism by which deregulation of ESE3/EHF impact on prostate tumorigenesis. Thus, targeting miR-424 could be a novel therapeutic strategy for prostate cancer. Citation Format: Cecilia Dallavalle, Domenico Albino, Gianluca Civenni, Paola Ostano, Davide Genini, Ramon Garcia-Escudero, Laura Curti, Sandra Pinton, Manuela Sarti, Giovanna Chiorino, Carlo V. Catapano, Giuseppina M. R. Carbone. MicroRNAs regulated by ESE3/EHF control important mediators of epithelial cell differentiation and stemness in prostate tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1451. doi:10.1158/1538-7445.AM2014-1451

Abstract 4965: The epithelial-specific ETS transcription factor ESE1 links inflammation with prostate cancer transformation and progression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnAltered expression of ETS transcription factors by chromosomal translocation is a frequent event in prostate cancers. However, deregulated expression of other oncogenic ETS factors can be important for the pathogenesis of this disease. By analyzing the expression of multiple ETS genes in prostate cancer and normal prostate by genomic profiling and quantitative real-time PCR we found that the epithelial-specific ETS factor ESE1 was highly expressed (>4 fold) in 44% of prostate tumors compared to normal prostate. ESE1 has been previously shown to be induced by pro-inflammatory stimuli and to regulate the expression of genes involved in inflammation. Treatment of immortalized prostate epithelial cells (LHS) and prostate cancer cells 22RV1 with the pro-inflammatory cytokine IL-1beta induced ESE1 at RNA and protein levels. Concomitantly we observed that cells acquired ability to grow in poly-hema coated plates and an EMT phenotype. To define the role of ESE1 in prostate cancer progression we established 22RV1 prostate cancer cells stably expressing ESE1 (22RV1-ESE1). ESE1 expressing cells were capable to grow in poly-hema coated plates and when injected in nude mice formed larger subcutaneous tumors compared to control cells. Global gene expression profiles indicate robust changes with many genes differentially expressed in 22RV1-ESE1 cells compared to control cells, including key genes involved in cell invasion and metastasis such as COX2 and MMP-10. Consistently, 22RV1-ESE1 cells had greater ability to migrate compared to parental cells in Boyden Chamber and wound-healing assays. Furthermore, when implanted in mice via tail vein injection, 22RV1-ESE1 cells displayed greater ability to form lung metastasis compared to control cells. These results suggest that ESE1 contribute to the acquisition of transforming properties such as the ability to invade and metastasize. Induction of ESE1 during chronic inflammation could be an essential step for prostate cancer initiation and may co-operate with other ETS alterations for prostate cancer progression.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4965.