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Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MOLECULAR PATHWAYS: RODENT PARVOVIRUSES: MECHANISMS OF ONCOLYSIS AND PROSPECTS FOR CLINICAL CANCER TREATMENT Jürg P.F. Nüesch, Jeannine Lacroix, Antonio Marchini, and Jean Rommelaere Authors’ Affiliation: Program „Infection and Cancer“, Division F010, German Cancer Research Center (DKFZ), Heidelberg, Germany. Corresponding author: Jean Rommelaere, Program „Infection and Cancer“, Abt. F010, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: +(49) 6221 424960 ; Fax: +(49) 6221 424962 ; E-mail: [email protected] Running Title: Parvovirus-tumor cell interplay Key Words: rodent parvovirus, oncotropism, oncolysis, cancer virotherapy Disclosure of Potential Conflicts of Interest: J. Rommelaere and J. Lacroix have commercial research grants from Oryx GmbH & Co.KG. No potential conflicts of interest were disclosed by other authors 1 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Rodent parvoviruses (PVs) are recognized for their intrinsic oncotropism and oncolytic activity contributing to their natural oncosuppressive effects. While 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. While 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). The present review aims to highlight 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. 2 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Background Replication-competent oncolytic viruses Over the last three decades, clinical research conducted with a view to optimizing multimodal therapeutic strategies has led to increasing the 5-year survival rate of cancer patients by about 20% (from 49% survival for the 1975-1977 diagnosis period, to 68% survival for the period 2001-2007) (SEER Cancer Statistics Review 1975-2008). Yet to further improve the outcome of cancer treatment, new therapeutic approaches are needed. In this perspective, replicationcompetent oncolytic viruses (OV) appear to be promising. OVs are characterized by their ability to infect, propagate in and kill tumor cells while sparing non-transformed cells, and thereby prime an antitumor immune response. Some of these agents (e.g. vesicular stomatitis virus, newcastle disease virus, reovirus, parvovirus) show inherent preference for replication in cancer cells due to their dependence on neoplasia-associated features, including deficiency of interferon response and aberrant activation of permissiveness factors. The oncotropism of other OVs (e.g. adenovirus, vaccinia virus, measles virus, herpes simplex virus) has been engineered by modifying/deleting viral genes or regulatory elements so as to make virus expression/replication/entry conditional on the malignant phenotype. Furthermore, the oncosuppressive potential of OVs can be boosted by arming them with transgenes/genetic motifs that confer an additional therapeutic effector function (e.g. immune-stimulation, prodrug activation) to recombinant viruses (1). Oncolytic parvoviruses Rodent parvoviruses (PV) are of particular interest in this regard, due to their natural oncotropism, lack of pre-existing antiviral immunity in most of the human population, and excellent safety profile (2). This suppressive activity of PVs against various rodent and human cancers has been documented in both in vitro systems and animal models (3, 4). PVs distinguish themselves by their dual oncolytic and immunostimulating activities, broad range of target tumors, ability to circumvent tumor cell resistance to conventional death inducers and suitability for both local and systemic applications. Furthermore, their cytopathic effects can be recapitulated in vitro through the expression of a single parvoviral product, the nonstructural protein NS1, which functions as an oncogenic transformation-dependent toxin (2). 3 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. PVs belong to the genus Parvovirus of the family Parvoviridae. Among the PVs studied for their anticancer properties, two species are particularly characterized the minute virus of mice (MVM) and H-1 PV whose natural hosts are mice and rats, respectively. PVs are nonenveloped icosahedral protein particles 24 nm in diameter containing a single-stranded linear DNA genome of about 5.1 kb. Their coding capacity is limited to the production of two capsid proteins (VP1 and VP2) and five nonstructural regulatory polypeptides (NS1, three NS2 splice variants and SAT). Expression of the NS1/NS2 genes is controlled by the early P4 promoter; that of the VP1/VP2 and SAT gene is programmed by the late (NS1-inducible) P38 promoter (5). The restricted number (and size) of parvoviral proteins is compensated for, to a certain extent by the multifunctionality of some of these products. This is exemplified by the NS1 protein which exerts various functions ranging from viral DNA amplification and transcription regulation to cytotoxicity. These activities are timely regulated through NS1 post-translational modification and/or subcellular distribution. In particular, the phosphorylation of distinct NS1 domains controls both enzymatic (ATPase, helicase) and non-enzymatic (DNA and protein binding) functions of the viral product, and varies during infection (6). Besides being intrinsically multifunctional, NS1 interacts with a number of cellular partner proteins which further broaden the range of NS1-mediated activities (see below). These features make PV strongly dependent on host cell factors. More importantly, PVs require S-phase factors to start replication and viral protein production. Unlike tumor viruses, PVs are unable to promote the progression of quiescent cells into S-phase, despite their ability to enter quiescent cells (5). This dependency restricts PV infections to rapidly proliferating tissues, and together with other parameters (see Fig. 1), makes tumor cells preferential targets for PV replication and cytopathic effects. Cell permissiveness and PV oncotropism As depicted in Fig. 1, PV oncotropism is due in part to viral dependence on cellular replication and transcription factors whose synthesis, activity, or both correlate with cell cycling and oncogenic transformation. Furthermore, the interplay between PVs and transformation-sensitive cell proteins raises the possibility that transformation might have an impact on late events of the PV life cycle, e.g. cytopathic effects and virus release. The presence of these factors is not sufficient, however, to ensure virus propagation. A restriction 4 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. can occur, for instance, at the level of delivery of incoming viral genomes to the nucleus, a prerequisite to their conversion to double-stranded transcription templates (5). This may account for the heterogeneity of tumor cells as regards permissiveness towards PV replication. It represents a target for optimization. An additional barrier to the PV life cycle is created by the type-I-interferon-mediated antiviral response, of which PVs were recently shown to be both triggers and targets (7, 8). PV oncotropism might depend on whether many tumor cells become deficient in the IFN response or on the capability of PVs to counteract this antiviral defense mechanism in transformed cells. In addition to regulation of their synthesis, PV proteins are subject to functional modulations resulting from posttranslational modifications and alterations of subcellular distribution (6). These modulations may also contribute to oncotropism, as some of the cellular effector proteins involved, e.g. NS1-activating PRKCH/PKCη (9), are overexpressed and/or altered in cancer cells (10). Cell-transformation-dependent modifications of the PV protein NS1 can thus be considered responsible for its specific toxicity in oncogene-transformed cells at concentrations that are innocuous to non-transformed cells (3). Analysis of PV interactions with cell proteins (Fig. 2) has revealed a striking interplay between PVs and cellular components of various metabolic pathways related to gene expression or DNA replication and repair. Some of these components are direct binding partners of PV proteins, as exemplified by the formation of complexes between NS1 and cellular transcription factors (TBP, TFIIA, SP1/3) or replication factors (RPA) (11-13). Others are known effectors and/or targets of the PV life-cycle, which are not found in complexes with PV proteins. Interestingly, a number of these physical and/or functional partners of PVs get together in specialized areas of infected cells. In particular, many constituents of the cell DNA replication machinery (RPA, POLA1/POLα, PCNA, RF-C) and DNA damage response (RPA-P32, γH2AX, NBS1-P, ATM) are recruited to subnuclear PV replication centers called APAR-bodies (14-16). This redistribution is expected to promote PV multiplication and contributes to host DNA synthesis shut-off. These various cell factors are established or potential actors in the PV life cycle, or may contribute to reshaping the intracellular environment to make it adequate for virus replication and spread. This cellconditioning effect is illustrated by the phosphorylation chain of cellular protein kinases that 5 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. is triggered by PV infection and leads to PDK-1 driven activation of PKCλ and PKCη (17). As stated above, both members of the PKC family are essential for modifying the viral NS1 protein and enabling it to exert its replicative functions. This phosphorylation cascade forms a positive feedback loop which is thought to be initiated by the PV-induced nuclear export of PKCλ, as a possible result of the interaction of the viral product NS2 with the cellular transporter XPO1/CRM1 (Fig. 1) (6). It is noteworthy that the major metabolic pathways interconnecting with PVs are subject to modulation through malignant transformation. In particular, overexpression of c-MYC and/or activated Ha-RAS renders normally resistant cells permissive towards PV propagation and NS1-induced killing (18). Both oncoproteins are known to have profound effects on the intracellular environment by dysregulating molecular pathways that are also involved in cell permissiveness towards PV infection. Factors controlling transcription (E2F, ETS, ATF/CREB, NF-YA) (19-21) or replication (CCNA1/cyclinA, DNA damage response effectors) (14-16, 22), and protein kinases (PRKCH/PKCη, PKB/AKT1) [(9); Nüesch unpublished] are among the cell proteins acting as modulators of the PV life cycle, belonging to this signaling network, and whose expression or activation is affected by malignant transformation. Cell disturbances and PV oncolysis PV infection of permissive cells leads to shut-off of host macromolecule syntheses, notably causing early inhibition of cell DNA replication (5, 23), due to the sequestration of components of the cell DNA replication machinery in viral replication centers (see above). Stalled replication forks (24) and ssDNA genomes of PV-related adeno-associated viruses (25) are known to activate the DNA damage response (DDR). Another DDR-activating mechanism triggered by PVs is NS1-induced production of reactive oxygen species (ROS) causing DNA damage (26). PV-induced DDR signaling is mediated by activation of ATM but not ATR, is characterized by phosphorylation of H2AX, NBS1, RPA32, CHK2, and P53, and results in accumulation of DNA repair proteins in viral replication centers (15, 16). Consequently, cell cycle arrest after PV infection is observed at the S/G2 transition or G2 phase, depending on the virus and cell type (26, 27). It is associated with upregulation of cellcycle control proteins such as CDK1, CCNB1/cyclinB1, CDKN1A/p21/Cip1, CDKN1B/p27/Kip1, and RLB2/p130 (26, 28, 29). Arrested cells are proposed to offer a more 6 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. favorable terrain for PV replication. Interestingly, expression of the NS1 protein is alone sufficient to induce cell cycle arrest (26, 28, 29), in keeping with the fact that NS1 is the major effector of virus cytotoxicity. Virus-induced cytotoxicity can involve mechanisms ranging from cytostasis (eventually leading to suicide of the infected/transfected cell) to direct induction of cell death programs. This is apparent from analysis of the PV-host cell interactome, revealing the interplay of PVs with cellular stress responses and with regulation of the cell cycle and death (Fig. 2). Whatever the nature of the initiating event, cell death and eventual lysis are the final outcome when a PV infects permissive malignant cells. Depending on the cell type and metabolic status, PV-infected cells can die through different mechanisms (see Fig. 1). H-1PV is reported to induce apoptotic cell death in various cancer cell lines (26, 30-32), and this is reflected in the statistically very significant interaction of PVs with components of the apoptosisregulating pathway (Fig. 2). Interestingly, NS1 expression is sufficient to trigger caspase-9and caspase-3-mediated apoptosis via oxidative stress and DNA damage (26). Alternatively, PV infection can induce a lysosomal type of death involving multiplication and permeabilization of acidic vesicles and repression of cathepsin inhibitors, resulting in the cytosolic accumulation of functional cathepsins to levels the cell cannot tolerate. It is noteworthy that by mobilizing this pathway, PVs can circumvent the mechanism of resistance to apoptosis developed by many tumor cells, and thereby kill cancer cells that dodge proapoptotic treatments (33). The mechanisms by which PVs affect cell growth and cell death pathways are still elusive but likely to be multiple, including viral usurpation of, and interference with the cellular gene expression and DNA replication machineries (Fig. 2). Furthermore, the NS1 protein appears to exert at least part of its toxic activity in the cytosol. This has been traced back to the illegitimate phosphorylation of cell substrates by a complex containing NS1 and the catalytic subunit of a cellular protein kinase (CKIIα in the case of MVM NS1) (34). PV-infected cells and cells expressing the NS1 protein alone undergo morphological changes that typically lead to their shrinkage and rounding (35). This has been ascribed to targeting of cytoskeletal microfilaments. Actin filaments are destroyed through NS1/CKIIα-mediated phosphorylation and activation of the severing protein gelsolin in conjunction with repression of the polymerization protein N-WASP (36, 37). This may have a greater impact on cancer cells, which lack the most rigid actin fibers. Filamentous tropomyosin is also a direct target of the 7 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. NS1/CKIIα complex. Differential NS1/CKIIα-driven phosphorylation of tropomyosins 2 and 5 leads to perinuclear restructuring and decay of these filaments (38). These alterations are likely to be especially deleterious to cancer cells, where tropomyosin 1 is underrepresented (39). Interestingly, artificial polypeptides combining the targeting domain of NS1 with CKIIα (or a binding site thereof) have been found to cause the death of transformed cells (38). This has led us to propose that the toxic activity of the NS1 protein depends at least partly on its ability to act as a cytosolic adaptor allowing a cellular kinase or kinases to phosphorylate illegitimate substrates. It is also worth mentioning that timely induction of cell death is necessary after PV infection in order to allow virus multiplication prior to cell collapse. There is evidence suggesting that premature death of PV-infected cells is prevented by NS1-mediated activation of the PDK1/PKB survival pathway [(17); Bär et al., in prep], providing enough time for the formation of progeny virions and their eventual release through active vesicular transport to the cell surface. This transport appears important for the maturation of progeny particles and the eventual lysis of infected tumor cells (Bär et al., in prep). Direct killing of tumor cells is not the only way oncolytic viruses can contribute to cancer suppression. That they might pass the job on to the immune system is actually expected, since a productive lytic viral infection of cancer cells should promote the display or release of both tumor-associated antigens and immunostimulating viral or cellular molecular patterns. Adoptive transfer, rechallenge, and immunodepletion experiments indicate that the host immune system takes part in PV-mediated oncosuppression (40, 41). The active role of PVs in inducing this anti-cancer immune response is further supported by the observation that PV infection of tumor cells enhances their ability to serve as an autologous vaccine (41, 42) and to stimulate both natural killer (43) and dendritic (44) cells with which they come into contact. Interestingly, the vesicular egress of progeny PVs may contribute to exposing intracellular tumor-associated antigens (37). Altogether, these data point to synergies between virus- and immune-cell-mediated oncolysis in PV-initiated tumor suppression. 8 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Clinical-Translation Advances Implications for cancer parvovirotherapy As discussed above, both activation of cellular helper functions and defects in cell defense mechanisms contribute to the permissiveness of cancer cells towards PV replication and toxicity. In contrast, the mildness of PV attack against normal tissues in adult animals results in clinically unapparent PV infections (45). This tolerance is assumed also to exist in humans and is the subject of a current clinical study. This trial involves patients with glioblastoma multiforma (GBM), the most malignant brain tumors against which standard treatments, including surgery and radio-chemotherapy, are inefficient. Preclinical evidence of strong cytotoxic and oncosuppressive effects of H-1PV against glioma cells led to the launch, in October 2011, of a first phase I/IIa study of H-1PV in patients with recurrent GBM (ParvOryx 01 – ClincialTrials.govIdentifier:NCT01301430). ParvOryx 01 is an open, non-controlled, dose escalation, single center trial (46). H-1PV is administered before resection by either intratumoral or intravenous route, and again into the walls of the tumor cavity during tumor removal. The objectives of the trial are related primarily to local/systemic safety and tolerability, and secondarily to proof of concept. Activation of (proto)oncogenes leads to changes in the intracellular environment which boost the parvoviral life cycle, as documented for c-myc (18). Overexpression of MYC drives malignant transformation in Burkitt’s lymphoma, medulloblastoma, and a large proportion of breast cancers with poor prognosis (47). In GBM stem cells, MYC is involved in regulating malignant progenitor cell differentiation, self-renewal, and tumorigenic potential (48). Parvovirus H-1PV efficiently suppresses these MYC-dependent tumors in preclinical models [(49, 50); Lacroix, in prep]. In response to PV infection, MYC expression is repressed in various cancer cell lines of different origins [(30, 51); Lacroix et al.,in prep]. These facts strongly suggest that cancers associated with MYC alterations are good targets for PVinduced oncolysis, and argue in favor of evaluating MYC as a predictive marker associated with tumor responsiveness to H-1PV treatment. In the absence of maternal immunity, PVs can infect rodent embryos, where they propagate in a variety of cell types, while maternal tissues remain unaffected (45). This has prompted us to hypothesize that malignant cells having embryonic features may show similar permissiveness. Accordingly, neuroblastoma and 9 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. pediatric medulloblastoma cell lines prove to be very susceptible to H-1PV killing in vitro, and we have observed suppression of xenotransplants in animal models [(32); Lacroix, in prep]. Besides these hypothesis-based suggestions, needs-driven consideration of cancers with desolate prognosis should also influence the choice of tumor targets for subsequent PV clinical trials. In this regard, it is noteworthy that H-1PV synergizes with gemcitabine to kill pancreatic carcinoma cells, and improves gemcitabine-based therapy of pancreatic tumors in animal models (52) Within a given cancer entity, the heterogeneity of tumors calls for individualized treatments based on prior identification of patients most likely to respond. It is therefore important to discover biomarkers that can serve as predictors of individual tumor sensitivity to PV infection. For PDAC, a first candidate is the transcription factor SMAD4, which controls P4 promoter activity and hence nonstructural protein expression and toxicity (53). Other candidates should emerge from “omic” approaches allowing the correlative analysis of PV sensitivity and interactome status in tumors from large cohorts of patients. Future developments Although PVs can cure cancers in animals and open new prospects for cancer therapy in humans, optimizing their oncosuppressive capacity is likely required in order to reach effectiveness in humans. The mere fact that some PVs have been isolated from human tumor xenotransplants indicates that their oncolytic activity is not always sufficient to arrest tumor growth and induce remission. This may be due to limitations on PV infectiousness, cytotoxicity, spread, and/or immunostimulation. A portfolio of second-generation therapeutics based on replication-competent PVs is under development to circumvent these limitations. Different complementary strategies might be used to achieve this goal. Virus variants with an enhanced ability to replicate lytically and spread in tumor cell populations are being isolated through natural selection or site-directed mutagenesis (54, 55). To minimize PV sequestration by normal tissues, PV capsids are first detargeted and then retargeted to cancer cells through the display of specific peptide ligands (56). The ability of PVs to act as adjuvants to boost the host anti-tumor response can also be increased by arming them with immunostimulatory molecular patterns (42). Furthermore, PVs can be combined with ionizing radiation (57), the cytostatic drug gemcitabine (52), the cytokine interferon γ (40) or oncolytic reovirus (58) to 10 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. achieve additive or even synergistic therapeutic effects. Indeed, the non-PV component of these combination treatments can be administered so as to minimize interference with PV replication and instead to potentiate PV oncolytic or immunomodulating activities. These various improvements of replicative PV-based treatments should pave the way for future clinical testing of anticancer efficacy, after the hoped-for confirmation of H-1PV safety in the current clinical trial. 11 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. References 1. Wollmann G, Ozduman K, van den Pol AN. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer Journal. 2012;18:69-81. 2. Rommelaere J, Geletneky K, Angelova AL, Daeffler L, Dinsart C, Kiprianova I, et al. 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The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Research. 2011;39:D561-8. 60. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols. 2009;4:44-57. 15 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure legends Figure 1. Parvovirus-induced cell disturbances. Viral genome replication and gene expression take place in the nucleus and depend strictly on S-phase-associated cellular factors. Conversion of the single-stranded viral DNA to a double-stranded transcription template is achieved by cellular replication factors (RF) under the control of cyclin A {1}. After conversion, S-phase (E2F) and transformation-sensitive (ATF/CREB, ETS, NF-Y) transcription factors (TF) activate the parvovirus early promoter P4 controlling expression of the nonstructural proteins NS1 and NS2 {2}. NS1 activates the late parvovirus promoter P38 programming capsid gene expression, and drives viral DNA amplification as an initiator protein and helicase (not illustrated in the Figure). NS1 also interferes with cell survival at many levels. Direct interactions of NS1 with components of the DNA replication (RPA 1-3) and transcription (TBP, TFIIA, SP1) machineries play a role both in viral DNA replication and transcription and in deregulating DNA/RNA metabolic processes in infected cells {3, 4}. Other virus protein-cell protein interactions such as NS1-CKII {5} or NS2-XPO1 {6} interfere, respectively, with host cell signaling and nuclear export. These events affect the host cell dramatically by causing oxidative stress, DNA damage, cell cycle arrest, cytoskeleton structure re-arrangements, mitochondrial membrane depolarization, and/or lysosome permeabilization. Cell death ensues, and ectopic NS1 expression alone is sufficient to cause it. Productive PV infections are characterized by virus-induced cytolysis followed by virus dissemination and subsequent rounds of infection. Besides providing permissiveness factors, transformed cells fail to mount an efficient anti-PV type I interferon response, which also contributes to the oncotropism of these agents. The oncoselectivity of PVs is thus attributed to both features of cancer cells: the presence of positive permissiveness factors and the inability to antagonize viral infection, as indicated with asterisks in the Figure. Abbreviations: CKII, casein kinase II alpha; ds, double-stranded; GSN: gelsolin; GTF2A/TFIIA: general transcription factor 2A; NS1, PV nonstructural protein 1; NS2, PV nonstructural protein 2; PV, parvovirus; ROS, reactive oxygen species; RPA: replicator protein A; SP1/3: Sp1/3 transcription factor; TBP; TATA binding protein; RF, replication factors; TF, transcription factors; TPM2/5: tropomyosin 2/5; VP, PV capsid proteins; XPO1 exportin 1 (CRM1 yeast homolog). 16 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 2. Parvovirus-host cell interplay. (A) The PV interactome. Cellular factors involved in PV propagation and spread and/or subject to modulation upon PV infection are represented as a network. The interconnections between the different genes are based on direct (physical) and indirect (functional) protein interactions according to STRING analysis (59). Most of these factors belong to known cellular pathways, as indicated by color codes. (B) Cellular metabolic pathways whose targeting by PVs is of highest significance (P-values). Pathway analysis enrichment was performed using the gene functional classification tool of DAVID (60). Resulting pathways are marked in different colors referring to the associated proteins identified under (A). PV impacts on the host cell are indicated on the right. 17 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Cell membrane Parvoviral particle Antiviral response Nucleus NS1 6 Nuclear trafficking NS2 Other viral components 5 CKII? XPO1 NS1 Expression TF NS1 2 NS1 TBP, GTF2A, SP1/3 3 NS VPs Virus/cell gene transcription P4 P38 PV-genome RF Target cellular proteins DNA replication/repair 1 NS1 NS1 RPA 4 Virus/cell DNA amplification PV-ds DNA DNA damage Cell DNA GSN Cell cycle arrest NS1 ROS CKII? TPM2/5 Permeabilization Depolarization NS1 Rearrangement NS1 Lysosome Cytoskeleton Mitochondrion Lysis Cell membrane © 2012 American Association for Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. PV-cell interactome A RPA2 RPA3 PRKCI H2AFX PIF1 PRKCH RPA1 SP3 POLA1 PDPK1 YWHAQ CCNB1 SMN1 POLD1 PCNA CDKN1B CCNA1 CD E2F1 XPO1 ATF1 CDK1 YBX1 MYC SMAD1 CSNK2A1 ID1 CASP9 CREB1 SP1 NFYA CASP3 SGTA JUN TBP EZR MSN HMGB1 GMEB2 CTSB GTF2A1 RDX TPM2 CSTB Involvement in: Response to DNA damage stimulus B DNA metabolic processes and transcription regulation Regulation of apoptosis Regulation of cell cycle Other pathways Pathways analysis and major perturbances for the host cell Dysregulation of transcription Regulation of transcription Regulation of apoptosis Cell death Regulation of cell cycle Cell cycle arrest DNA metabolic process Dysregulation of replication DNA replication Cellular response to stress Activation of cell stress responses Response to DNA damage stimulus 1.00E+00 1.00E-02 1.00E-04 1.00E-06 1.00E-08 (P-value) © 2012 American Association for Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. RODENT PARVOVIRUSES: MECHANISMS OF ONCOLYSIS AND PROSPECTS FOR CLINICAL CANCER TREATMENT Jürg P.F. Nüesch, Jeannine Lacroix, Antonio Marchini, et al. Clin Cancer Res Published OnlineFirst May 7, 2012. Updated version Author Manuscript E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-11-2325 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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