SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV.

Pancreatic ductal adenocarcinoma (PDAC) represents the eighth frequent solid tumor and fourth leading cause of cancer death. Because current treatments against PDAC are still unsatisfactory, new anticancer strategies are required, including oncolytic viruses. Among these, autonomous parvoviruses (PV), like MVMp (minute virus of mice) and H-1PV are being explored as candidates for cancer gene therapy. Human PDAC cell lines were identified to display various susceptibilities to an infection with H-1PV. The correlation between the integrity of the transcription factor SMAD4, mutated in 50% of all PDAC, and H-1PV permissiveness was particularly striking. Indeed, mutation or deletion of SMAD4 dramatically reduced the activity of the P4 promoter and, consequently, the accumulation of the pivotal NS1 protein. By means of DNA affinity immunoblotting, novel binding sites for SMAD4 and c-JUN transcription factors could be identified in the P4 promoter of H-1PV. The overexpression of wild-type SMAD4 in deficient cell lines (AsPC-1, Capan-1) stimulated the activity of the P4 promoter, whereas interference of endogenous SMAD4 function with a dominant-negative mutant decreased the viral promoter activity in wild-type SMAD4-expressing cells (Panc-1, MiaPaCa-2) reducing progeny virus production. In conclusion, the importance of members of the SMAD family for H-1PV early promoter P4 activity should guide us to select SMAD4-positive PDACs, which may be possible targets for an H-1PV-based cancer therapy.

The infectivity and lytic activity of minute virus of mice wild-type and derived vector particles are strikingly different.

Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.

Cancer gene therapy through autonomous parvovirus--mediated gene transfer.

Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines/chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virus-free vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines/chemokines are presented.