Gessica Filocamo

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2–CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild‐type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1‐deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR‐BI). E2–SR‐BI interaction is very selective since neither mouse SR‐BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR‐BI was competed out in an isolate‐specific manner both on the hepatoma cell line and on the human SR‐BI‐transfected cell line by an anti‐HVR1 monoclonal antibody.

Dissecting the Biological Functions of Drosophila Histone Deacetylases by RNA Interference and Transcriptional Profiling

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, we have used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G2 arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3 (Pile, L. A., Spellman, P. T., Katzenberger, R. J., and Wassarman, D. A. (2003) J. Biol. Chem. 278, 37840–37848). We conclude the following. 1) DHDAC1 and -3 have distinct functions in the control of gene expression. 2) Under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells. 3) The anti-proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1. 4) The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

Loss of Histone Deacetylase 4 Causes Segregation Defects during Mitosis of p53-Deficient Human Tumor Cells

We investigated the role of histone deacetylase 4 (HDAC4) using RNA interference (RNAi) and knockout cells to specifically address its role in cell cycle progression in tumor and normal cells. Ablation of HDAC4 led to growth inhibition in human tumor cells but not to detectable effects in normal human dermal fibroblasts (NHDF) or myelopoietic progenitors. HDAC4-/+ or HDAC4-/- murine embryonic fibroblasts showed no detectable growth defects. On the other hand, HDAC4 RNAi in HeLa cells produced mitotic arrest followed by caspase-dependent apoptosis. Mitotically arrested cells showed chromosome segregation defects. Even though the growth of both p53-wild-type and p53-null tumor cells were affected by HDAC4 ablation, segregation defects were observed only in p53-null cells. HDAC4 associates with the PP2A-B56 regulatory subunit, which is known to be involved in chromosome segregation, and RNAi of either the structural subunit A or the regulatory subunit B56 of PP2A also caused chromosome segregation defects. We conclude that HDAC4 is required for cell cycle progression of tumor cells by multiple mechanisms, one of which seems to be specific to p53-deficient cells through chromosome segregation defects. On the contrary, HDAC4 is not required for the progression of NHDF. We therefore suggest that systemic selective interference with the expression or function of HDAC4 is expected to have a significant therapeutic window, in particular, for p53-deficient tumors.

In Vivo Selection of Protease Cleavage Sites by Using Chimeric Sindbis Virus Libraries

ABSTRACT Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

Smoothened antagonists: a promising new class of antitumor agents

Background: Hedgehog signaling is essential for the development of most metazoans. In recent years, evidence has accumulated showing that many human tumors aberrantly re-activate this developmental signaling pathway and that interfering with it may provide a new strategy for the development of novel anti-cancer therapeutics. Smoothened is a G-protein coupled receptor-like protein that is essentially involved in hedgehog signal transduction and small molecule antagonists of Smoothened have started to show antitumor activity in preclinical models and in clinical trials. Objective: We critically review the role of hedgehog signaling in normal development and in human malignancies, the available drug discovery tools and the classes of small molecule inhibitors that are in development. We further aim to address the potential impact that pathway antagonists may have on the treatment options of cancer patients. Methods: Literature, patents and clinical trial results from the past 5 years were analyzed. Conclusions: 1) A large body of evidence suggests a frequent reactivation of hedgehog signaling in human cancer. 2) Smoothened is an attractive, highly druggable target with extensive preclinical and initial clinical validation in basal cell carcinoma. Several promising novel classes of Smoothened antagonists have been discovered and are being developed as anticancer agents. 3) Our knowledge of the biology of hedgehog signaling in cancer is still very incomplete and significant efforts will be required to understand how to use the emerging novel agents in the clinic.

MK-4101 - a potent inhibitor of the hedgehog pathway - is highly active against medulloblastoma and basal cell carcinoma

Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/− mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for the treatment of medulloblastoma and BCC. Results clearly demonstrated a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/− mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidated the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in tumor cells, showing the maximum inhibitory effect on Gli1. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF, and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity. Mol Cancer Ther; 15(6); 1177–89. ©2016 AACR.

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of haematopoietic, osteogenic and neural progenitors. Very high expression of this factor is present in cerebellum and particularly in the granule layer of neonatal cerebellum, that contains candidate cells-of-origin of medulloblastoma. Here we have explored the possible involvement of ZNF521 in the development of this tumour. As an experimental system we used the human medulloblastoma cell line, DAOY, and primary cells from medulloblastomas occurring in Ptc1-/+ mice. To investigate the effect of ZNF521 on the growth and tumourigenic potential of these cells, its expression was modulated using lentiviral vectors carrying the ZNF521 cDNA, or containing shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY cells, that normally produce relatively low amounts of this protein, was associated with a significant increase in their proliferation rate. This was mirrored by an increase in the ability to grow as spheroids and clonogenicity in single-cell cultures and in semisolid media, and accompanied by an enhanced migratory capacity in wound-healing assays. Finally, ZNF521-expressing DAOY cells demonstrated a greatly enhanced tumourigenic potential in nude mice. All these activities required the presence of an N-terminal domain of ZNF521 that recruits the nucleosome remodeling and histone deacetylase (NuRD) complex. Consistently with the effects of ZNF521 overexpression in DAOY, silencing of Zfp521 in Ptc1-/+ medulloblastoma cells resulted in a drastic decrease in their proliferation and tumourigenic potential, lending further support to the notion that zinc finger protein 521 may contribute to the generation and/or maintenance of the cancer-initiating cell compartment in this cancer. Preliminary experiments detected a selective up-regulation of HES5 mRNA in DAOY overexpressing ZNF521, raising the possibility that some of the effects illustrated here may at least in part be mediated by the co-operation of ZNF521 with the Notch pathway. Citation Format: Raffaella Spina, Gessica Filocamo, Enrico Iaccino, Stefania Scicchitano, Michela Lupia, Emanuela Chiarella, Tiziana Mega, Daniela Pelaggi, Maria Mesuraca, Eli E. Bar, Heather M. Bond, Charles G. Eberhart, Christian Steinkuhler, Gianni Morrone. Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5045. doi:10.1158/1538-7445.AM2013-5045 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.