Antonio Marchini

Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species

ABSTRACT The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.

Transcriptional and translational regulation of the Leri-Weill and Turner syndrome homeobox gene SHOX

Regulation of gene expression is particularly important for gene dosage-dependent diseases and the phenomenon of clinical heterogeneity frequently associated with these phenotypes. We here report on the combined transcriptional and translational regulatory mechanisms controlling the expression of the Léri-Weill and Turner syndrome gene SHOX. We define an alternative promotor within exon 2 of the SHOX gene by transient transfections of mono- and bicistronic reporter constructs and demonstrate substantial differences in the translation efficiency of the mRNAs transcribed from these alternative promotors by in vitro translation assays and direct mRNA transfections into different cell lines. Although transcripts generated from the intragenic promotor (P2) are translated with high efficiencies, mRNA originating from the upstream promotor (P1) exhibit significant translation inhibitory effects due to seven AUG codons upstream of the main open reading frame (uAUGs). Site-directed mutagenesis of these uAUGs confers full translation efficiency to reporter mRNAs in different cell lines and after injection of Xenopus embryos. In conclusion, our data support a model where functional SHOX protein levels are regulated by a combination of transcriptional and translational control mechanisms.

Molecular Pathways: Rodent Parvoviruses—Mechanisms of Oncolysis and Prospects for Clinical Cancer Treatment

Rodent parvoviruses (PV) are recognized for their intrinsic oncotropism and oncolytic activity, which contribute to their natural oncosuppressive effects. Although PV uptake occurs in most host cells, some of the subsequent steps leading to expression and amplification of the viral genome and production of progeny particles are upregulated in malignantly transformed cells. By usurping cellular processes such as DNA replication, DNA damage response, and gene expression, and/or by interfering with cellular signaling cascades involved in cytoskeleton dynamics, vesicular integrity, cell survival, and death, PVs can induce cytostasis and cytotoxicity. Although productive PV infections normally culminate in cytolysis, virus spread to neighboring cells and secondary rounds of infection, even abortive infection or the sole expression of the PV nonstructural protein NS1, is sufficient to cause significant tumor cell death, either directly or indirectly (through activation of host immune responses). This review highlights the molecular pathways involved in tumor cell targeting by PVs and in PV-induced cell death. It concludes with a discussion of the relevance of these pathways to the application of PVs in cancer therapy, linking basic knowledge of PV–host cell interactions to preclinical assessment of PV oncosuppression. Clin Cancer Res; 18(13); 3516–23. ©2012 AACR.

SHOX at a glance: from gene to protein.

Abstract The Short Stature Homeobox-containing Gene SHOX was identified as the genetic cause of the short stature phenotype in patients with Turner Syndrome and in certain patients with idiopathic short stature. Shortly after, SHOX mutations were also associated with the growth failure and skeletal deformities seen in patients with Léri – Weill dyschondrosteosis and Langer mesomelic dysplasia. Today it is estimated that SHOX mutations occur with an incidence of roughly 1:1000 in newborns, making mutations of this gene one of the most common genetic defects leading to growth failure in humans. This review summarises the involvement of SHOX in several short stature syndromes and describes recent advances in our understanding of SHOX functions and regulation. We also discuss the current evidence in the literature that points to a role of this protein in growth and bone development. These studies have improved our knowledge of the SHOX gene and protein functions, and have given insight into the etiopathogenesis of short stature. However, the exact role of SHOX in bone development still remains elusive and poses the next major challenge for researchers in this field.

Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas

The rat parvovirus H‐1PV has oncolytic and tumour‐suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co‐treating cancer cells with H‐1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H‐1PV/VPA co‐treatment strongly inhibits tumour growth promoting complete tumour remission in all co‐treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1‐mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H‐1PV/VPA co‐treatment against cervical and pancreatic ductal carcinomas.

Overcoming Barriers in Oncolytic Virotherapy with HDAC Inhibitors and Immune Checkpoint Blockade

Oncolytic viruses (OVs) target and destroy cancer cells while sparing their normal counterparts. These viruses have been evaluated in numerous studies at both pre-clinical and clinical levels and the recent Food and Drug Administration (FDA) approval of an oncolytic herpesvirus-based treatment raises optimism that OVs will become a therapeutic option for cancer patients. However, to improve clinical outcome, there is a need to increase OV efficacy. In addition to killing cancer cells directly through lysis, OVs can stimulate the induction of anti-tumour immune responses. The host immune system thus represents a “double-edged sword” for oncolytic virotherapy: on the one hand, a robust anti-viral response will limit OV replication and spread; on the other hand, the immune-mediated component of OV therapy may be its most important anti-cancer mechanism. Although the relative contribution of direct viral oncolysis and indirect, immune-mediated oncosuppression to overall OV efficacy is unclear, it is likely that an initial period of vigorous OV multiplication and lytic activity will most optimally set the stage for subsequent adaptive anti-tumour immunity. In this review, we consider the use of histone deacetylase (HDAC) inhibitors as a means of boosting virus replication and lessening the negative impact of innate immunity on the direct oncolytic effect. We also discuss an alternative approach, aimed at potentiating OV-elicited anti-tumour immunity through the blockade of immune checkpoints. We conclude by proposing a two-phase combinatorial strategy in which initial OV replication and spread is maximised through transient HDAC inhibition, with anti-tumour immune responses subsequently enhanced by immune checkpoint blockade.

Oncolytic parvoviruses: from basic virology to clinical applications.

Accumulated evidence gathered over recent decades demonstrated that some members of the Parvoviridae family, in particular the rodent protoparvoviruses H-1PV, the minute virus of mice and LuIII have natural anticancer activity while being nonpathogenic to humans. These studies have laid the foundations for the launch of a first phase I/IIa clinical trial, in which the rat H-1 parvovirus is presently undergoing evaluation for its safety and first signs of efficacy in patients with glioblastoma multiforme. After a brief overview of the biology of parvoviruses, this review focuses on the studies which unraveled the antineoplastic properties of these agents and supported their clinical use as anticancer therapeutics. Furthermore, the development of novel parvovirus-based anticancer strategies with enhanced specificity and efficacy is discussed, in particular the development of second and third generation vectors and the combination of parvoviruses with other anticancer agents. Lastly, we address the key challenges that remain towards a more rational and efficient use of oncolytic parvoviruses in clinical settings, and discuss how a better understanding of the virus life-cycle and of the cellular factors involved in virus infection, replication and cytotoxicity may promote the further development of parvovirus-based anticancer therapies, open new prospects for treatment and hopefully improve clinical outcome.

Impairment of SHOX nuclear localization as a cause for Leri-Weill syndrome

We report the characterization of the nuclear localization signal (NLS) of the short stature homeobox gene SHOX. Mutations within the SHOX gene cause Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LD) as well as idiopathic short stature (ISS). Furthermore, haploinsufficiency of SHOX has also been implicated in Turner syndrome. SHOX has been shown to be a cell-type-specific transcriptional activator that localizes to the nucleus. The SHOX protein contains a central homeodomain that together with its transactivation domain regulates the transcription of its target sequences within the nucleus. The sequences for its nuclear localization have not been identified yet. Experimental characterization of SHOX-NLS by deletion mapping identified a non-classic type basic signal, AKCRK, in the recognition helix of the homeodomain. Fusion of this stretch of five amino acids to a cytoplasmic reporter protein resulted in its nuclear translocation. Functional analysis of a missense mutation R173C (C517T) affecting the identified SHOX-NLS in two families with LWS and LD showed that the mutated SHOX protein is unable to enter the nucleus. Conversely, we can demonstrate that insertion of the identified signal adjacent to the mutant site can restore its nuclear translocation. These results establish impairment of nuclear localization as a mechanistic basis for SHOX-related diseases.

Retargeting of Rat Parvovirus H-1PV to Cancer Cells through Genetic Engineering of the Viral Capsid

ABSTRACT The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds αvβ3 and αvβ5 integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing αvβ5 integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

A novel point mutation A170P in the SHOX gene defines impaired nuclear translocation as a molecular cause for Léri–Weill dyschondrosteosis and Langer dysplasia

1-3 and encodes a paired related homeodomain transcription factor. Nominal levels of the SHOX protein have been implicated in bone development and longitudinal body growth, as heterozygous and homozygous loss of SHOX functions result in Leri-Weill dyschondrosteosis (LWD) and Langer dysplasia (LD), respectively. 45 Apart from mesomelic short stature and a characteristic deformity of the forearm leading to a limited mobility of the wrist (Madelung deformity), some individuals with LWD and LD present with a subset of clinical stigmata frequently observed in females with Turner syndrome, including high arched palate, curvature of radius/ulna/tibia, and short fourth metacarpals. 6-8 These clinical features are variable, leading to a significant phenotypic heterogeneity particularly among persons with LWD. 9 In addition, SHOX mutations are a major cause of isolated short stature conditions, with an estimated incidence of 2-3% in children presenting with idiopathic growth retardation. 10 11 This is higher than the incidence of achondroplasia and growth hormone deficiency together. 12 13