Advances on the roles of tenascin-C in cancer.

The roles of the extracellular matrix molecule tenascin-C (TNC) in health and disease have been extensively reviewed since its discovery over 40 years ago. Here, we will describe recent insights into the roles of TNC in tumorigenesis, angiogenesis, immunity and metastasis. In addition to high levels of expression in tumors, and during chronic inflammation, and bacterial and viral infection, TNC is also expressed in lymphoid organs. This supports potential roles for TNC in immunity control. Advances using murine models with engineered TNC levels were instrumental in the discovery of important functions of TNC as a danger-associated molecular pattern (DAMP) molecule in tissue repair and revealed multiple TNC actions in tumor progression. TNC acts through distinct mechanisms on many different cell types with immune cells coming into focus as important targets of TNC in cancer. We will describe how this knowledge could be exploited for cancer disease management, in particular for immune (checkpoint) therapies.

Structural characterization of H-1 parvovirus: comparison of infectious virions to empty capsids.

The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel ß-barrel with large loop regions between the strands. The ß-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors.

An in-frame deletion in the NS protein-coding sequence of parvovirus H-1PV efficiently stimulates export and infectivity of progeny virions.

An in-frame, 114-nucleotide-long deletion that affects the NS-coding sequence was created in the infectious molecular clone of the standard parvovirus H-1PV, thereby generating Del H-1PV. The plasmid was transfected and further propagated in permissive human cell lines in order to analyze the effects of the deletion on virus fitness. Our results show key benefits of this deletion, as Del H-1PV proved to exhibit (i) higher infectivity (lower particle-to-infectivity ratio) in vitro and (ii) enhanced tumor growth suppression in vivo compared to wild-type H-1PV. This increased infectivity correlated with an accelerated egress of Del H-1PV progeny virions in producer cells and with an overall stimulation of the viral life cycle in subsequently infected cells. Indeed, virus adsorption and internalization were significantly improved with Del H-1PV, which may account for the earlier appearance of viral DNA replicative forms that was observed with Del H-1PV than wild-type H-1PV. We hypothesize that the internal deletion within the NS2 and/or NS1 protein expressed by Del H-1PV results in the stimulation of some step(s) of the viral life cycle, in particular, a maturation step(s), leading to more efficient nuclear export of infectious viral particles and increased fitness of the virus produced.

Production, purification, crystallization and structure determination of H-1 Parvovirus.

Crystals of H-1 Parvovirus (H-1PV), an antitumor gene-delivery vector, were obtained for DNA-containing capsids and diffracted X-rays to 2.7 Šresolution using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=255.4, b=350.4, c=271.6 Å, ß=90.34°. The unit cell contained two capsids, with one capsid per crystallographic asymmetric unit. The H-1PV structure has been determined by molecular replacement and is currently being refined.

Modulating tenascin-C functions by targeting the MAtrix REgulating MOtif, "MAREMO".

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif "MAREMO" or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis.

Impact of Tenascin-C on Radiotherapy in a Novel Syngeneic Oral Squamous Cell Carcinoma Model With Spontaneous Dissemination to the Lymph Nodes.

Radiotherapy, the most frequent treatment of oral squamous cell carcinomas (OSCC) besides surgery is employed to kill tumor cells but, radiotherapy may also promote tumor relapse where the immune-suppressive tumor microenvironment (TME) could be instrumental. We established a novel syngeneic grafting model from a carcinogen-induced tongue tumor, OSCC13, to address the impact of radiotherapy on OSCC. This model revealed similarities with human OSCC, recapitulating carcinogen-induced mutations found in smoking associated human tongue tumors, abundant tumor infiltrating leukocytes (TIL) and, spontaneous tumor cell dissemination to the local lymph nodes. Cultured OSCC13 cells and OSCC13-derived tongue tumors were sensitive to irradiation. At the chosen dose of 2 Gy mimicking treatment of human OSCC patients not all tumor cells were killed allowing to investigate effects on the TME. By investigating expression of the extracellular matrix molecule tenascin-C (TNC), an indicator of an immune suppressive TME, we observed high local TNC expression and TIL infiltration in the irradiated tumors. In a TNC knockout host the TME appeared less immune suppressive with a tendency towards more tumor regression than in WT conditions. Altogether, our novel syngeneic tongue OSCC grafting model, sharing important features with the human OSCC disease could be relevant for future anti-cancer targeting of OSCC by radiotherapy and other therapeutic approaches.

HIV transmission from infected CD4+ T cells to allogenic T and dendritic cells is inhibited by broadly neutralizing antibodies.

OBJECTIVE: In the semen, both free virus and infected cells are able to establish HIV infection during sexual intercourse. An efficient vaccine should therefore inhibit both infectious states. The aim of this study was to analyze the capacity of broadly neutralizing antibodies (bNAbs) to inhibit HIV transmission by the infected cells. DESIGN/METHODS: We developed an in-vitro model aiming to mimic mucosal HIV transmission via infected cells. PHA-activated CD4+ T cells stained with PKH26 from donor A were infected and co-cultured with CD4+ T cells and dendritic cells from donor B in the presence of bNAbs. RESULTS: We showed that dendritic cells were the preferential HIV target cells at early time points in this co-culture model. In the context of this co-culture model where infection and transmission occurred simultaneously, bNAbs efficiently inhibited HIV replication as well as HIV transmission from infected cells to allogenic dendritic cells and CD4+ T cells. CONCLUSION: Overall, our results indicate that dendritic cells, in addition to CD4+ T cells, are key cells that are efficiently infected by HIV and bNAbs are potent inhibitors of infection of both target cells. Future HIV prophylactic vaccine design should develop immune strategies able to prevent the infection of dendritic cells, in addition to the inhibition of CD4+ T-cell infection.

Mutations in the Non-Structural Protein-Coding Sequence of Protoparvovirus H-1PV Enhance the Fitness of the Virus and Show Key Benefits Regarding the Transduction Efficiency of Derived Vectors.

Single nucleotide changes were introduced into the non-structural (NS) coding sequence of the H-1 parvovirus (PV) infectious molecular clone and the corresponding virus stocks produced, thereby generating H1-PM-I, H1-PM-II, H1-PM-III, and H1-DM. The effects of the mutations on viral fitness were analyzed. Because of the overlapping sequences of NS1 and NS2, the mutations affected either NS2 (H1-PM-II, -III) or both NS1 and NS2 proteins (H1-PM-I, H1-DM). Our results show key benefits of PM-I, PM-II, and DM mutations with regard to the fitness of the virus stocks produced. Indeed, these mutants displayed a higher production of infectious virus in different cell cultures and better spreading capacity than the wild-type virus. This correlated with a decreased particle-to-infectivity (P/I) ratio and stimulation of an early step(s) of the viral cycle prior to viral DNA replication, namely, cell binding and internalization. These mutations also enhance the transduction efficiency of H-1PV-based vectors. In contrast, the PM-III mutation, which affects NS2 at a position downstream of the sequence deleted in Del H-1PV, impaired virus replication and spreading. We hypothesize that the NS2 protein-modified in H1-PM-I, H1-PM-II, and H1-DM-may result in the stimulation of some maturation step(s) of the capsid and facilitate virus entry into subsequently infected cells.

Effect of the parvovirus H-1 non-structural protein NS1 on the tumorigenicity of human gastric cancer cells.

OBJECTIVE: To investigate the in vivo oncosuppressive effect of the non-structural protein NS1 of parvovirus H-1 on human gastric cancer cell lines. METHODS: Recombinant plasmid pcDNA3.1-NS1 containing the complete NS1 gene of parvovirus H-1 was constructed and characterized by restriction enzyme digestion and sequence analysis. The human gastric cancer cell lines MKN28, SGC7901 and MKN45 were stably transfected with empty or recombinant plasmids. NS1 gene transcription and protein expression in the latter transfectants were verified by reverse transcriptase polymerase chain reaction and Western blot, respectively. The oncosuppressive effect of the parvoviral protein NS1 on the gastric cancer cell lines was tested by comparing the tumorigenicity of empty and recombinant vector-transfected cells in nude mice. RESULTS: Well differentiated gastric cancer cells (MKN28) transfected with either empty plasmid or pcDNA3.1-NS1 were tumorigenic in nude mice. Moderately (SGC7901) and poorly (MKN45) differentiated gastric cancer cells transfected with empty plasmid were also tumorigenic, but no tumor resulted from the injection when they were transfected with pcDNA3.1-NS1. This NS1-associated suppression of SGC7901 and MKN45 tumors correlated with the decreased percentage of CD44 positive cells. CONCLUSIONS: NS1 expression in poorly differentiated gastric cancer cells prevents them from forming tumors, perhaps by impairing the stem-like phenotype. The parvoviral NS1 protein warrants further investigation for its therapeutic potential against cancer.

A supraphysiological nuclear export signal is required for parvovirus nuclear export.

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis.

Vectors based on autonomous parvoviruses: novel tools to treat cancer?

Autonomous parvoviruses are small nuclear-replicating DNA viruses. The rodent parvoviruses usually are non- or weakly pathogenic in adult animals, bind to surface receptors which are expressed on most cells, and do not appear to integrate into host chromosomes during either lytic or persistent infections. Interestingly, malignant transformation of the target cells was often found to correlate with an increase in their capacity for amplifying and/or expressing the incoming parvoviral DNA, and is associated with oncolysis, i.e., the selective killing of the infected tumor cells. Moreover, the closely related parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. This finding makes these parvoviruses promising candidate vectors for therapies that require transient expression of a transduced gene. In particular, parvoviruses may be suitable to target and kill tumor cells and simultaneously deliver appropriate transgenes, e.g., genes coding for immuno-stimulatory factors. Pilot experiments performed in animals to assess whether parvovirus-based vectors carrying the interleukin 2 (IL-2) cytokine gene have reinforced anti-cancer capacity showed that these recombinant viruses suppressed tumor formation more efficiently than viruses devoid of a transgene. Strong anti-cancer effects of recombinant parvoviruses expressing interferon gamma-inducible protein 10 (IP-10) and monocyte chemotactic protein 3 (MCP-3) were also observed against established hemangiosarcomas and melanomas in immuno-competent mice, respectively. Altogether, these data illustrate the enormous potential of recombinant autonomous parvoviruses as anti-tumor agents and give hope of using them against human cancer.

The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells.

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.

Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells.

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.