Approximately 20% of human cancers worldwide have been linked to infection (28
). MCV as a new cause for human cancer is particularly difficult to establish because (i) MCC is relatively rare (5
), (ii) MCV infection is near-ubiquitous among adults (10
), and (iii) not all MCC tumors are infected with the virus (4
). Nonetheless, direct detection of viral proteins is strictly associated with and specific for MCC tumors (39
). While MCV infection is widespread, MCV-positive MCC patients have markedly higher titers of antibody to late viral antigens than MCV-negative MCC patients, consistent with persistent antigen stimulation (29
). Finally, clonal MCV integration and the presence of specific TA mutations for tumor-derived MCV provide unambiguous evidence that MCV was present prior to tumor cell genesis and that the virus is not an incidental or passenger infection (14
Analysis of MCC tumors revealed a strong selective pressure within tumors to silence independent DNA replication from the integrated viral genomes in MCC cancer cells. MCV LT encodes carboxyl-terminal origin-binding and helicase domains that are required for viral DNA replication (25
), but most MCC-derived MCV LT DNA sequences harbor stop codon mutations truncating these domains (40
). These tumor-specific mutations do not affect amino-terminal Rb1 interaction and DnaJ domains, although they may eliminate a putative p53-binding domain. The MCV sT protein, encoded by an alternative reading frame 5′ to the hypermutable LT region, remains unaffected by tumor-specific mutations.
The observational evidence for MCV causality in MCC is directly supported by the experimental studies presented. By means of shRNA knockdown, we demonstrate that MCV-positive MCC cell lines are “addicted” to expression of the viral TAs. We achieved efficient TA knockdown in five different MCV-positive MCC cell lines, using three different exon 1 target sequences and two independent selection methods in order to rule out off-target effects or artifacts due to the method used. In each case, MCV-positive cells initiated growth arrest and/or underwent cell death with the TA exon 1-specific vectors but not with scrambled shRNA vectors. MCV-negative cell lines, however, were unaffected by MCV TA-targeting shRNA, further indicating that this is not likely to be an off-target effect. These results show that MCV TA is required for MCC cell survival among those tumors infected with the virus. Since exon 1 is common to all TA isoforms, all early MCV transcripts were inhibited in our study. Dissecting the contributions of each TA isoform (e.g., LT, sT, and 57kT) to the transformed MCC phenotype will be important for future investigations.
The dependency of the transformed phenotype on the expression of TAs has been shown to be time dependent. In transgenic mice with inducible expression of SV40 TAs in the submandibular gland, hyperplasia was reversed upon the silencing of TA expression after 4 months but persisted when expression was shut down after 7 months (12
). Similarly, adenovirus-transformed hamster cells have been reported to lose the previously integrated transforming viral DNA while retaining the oncogenic phenotype (30
). It is conceivable that such a phenomenon may also apply to some of the MCV-negative cases of MCC; however, for MCV-positive cell lines, we show clear dependence on MCV TA expression.
Since cell death induced by TA knockdown is not associated with caspase activation, PARP cleavage, major shifts in phosphatidylserine location, or alterations in p53 or Bcl-2 family protein expression, it lacks important features of classical apoptosis. Alternatively, cell death may occur through autophagy, a process of cellular self-degradation involving the lysosomal machinery that has recently attracted increasing attention in cancer research (6
). Necrosis, a process distinct from either apoptosis or autophagy, may also cause cell death (for a review, see reference 24
). The observed cell cycle arrest upon T-antigen knockdown can be more directly explained by alterations of the Rb-E2F pathway: Rb family proteins are the master regulators of S-phase entry, and they are the prominent cellular targets for polyomavirus T antigens (13
Despite the fact that MCV was discovered only recently, both observational data and the experimental studies described here demonstrate that it is the likely infectious trigger for most human MCC. While polyomaviruses have been studied extensively in animal cancer models, MCV is the first polyomavirus for which strong evidence supports a causal role in human cancer (32
). Identifying specific cellular pathways targeted by putative MCV TA oncoproteins can lead directly to new, more effective, and less toxic therapies for this human cancer.