It has been reported that melanoma metastases display a heterogeneous phenotype in vivo that could be segregated according to the coordinate expression of an inflammatory signature including cytokines, chemokines and angiogenic factors [16
]. The expression of these genes followed a modular behavior and was coordinated among them resulting in two cutaneous melanoma metastases phenotypes. Modular "operon-like" gene expression has been recognized to be a relatively common feature in several immune pathologies [20
] and may offer a bottom up view of complex diseases and their interaction with the host. The original observation described for metastatic melanoma could not separate the identified modular patterns between those related to the host's response to cancer cells and those primarily due to potential taxonomic differences between two molecular subsets of cutaneous melanoma [33
The present study confirms this phenomenon, and in addition suggests that 1) the two phenotypes ("inflammatory" vs "quiescent") are not limited to cutaneous melanoma but are also present in pancreatic adenocarcinoma, suggesting that it could be possibly a widespread phenomenon among cancers; 2) the activation of ISGs is due to two independent taxonomies of cancer cells and not to the host's reaction to the cancer as it is was observed in xenografts growing in immune deficient animals and in in vitro cultured cell lines; 3) the two phenotypes reflect a true "anti-viral" state capable of inhibiting replication of at least two families of viruses (adeno viruses and adeno associated viruses); 4) the two cancer taxonomies described here may bear relevant biological characteristics that might affect treatment of cancer with viral vectors or with immunotherapy.
It remains to be elucidated why these two phenotypes exist. One possibility is that the cancer cells bearing the "anti-viral" state are chronically infected with a latent virus that could induce endogenous activation of innate cellular immune responses. Alternatively, it might represent an endogenous activation of anti-viral pathways associated with the mutagenic process. This phenomenon has been clearly described for Epstein-Barr virus or papilloma virus related cancers and could apply to other viruses as well [34
]. However, two observations mitigate against this interpretation. First, no genes encoding for any known type I IFNs were observed to be up-regulated in association with the "anti-viral state" or the down-stream activation of ISGs; although type one IFN expression is not an absolute requirement for ISG activation during cytomegalovirus infection [36
], this IFN-independent activation of ISGs remains to be demonstrated in other viral models in which IFN production at mRNA and protein levels are believed to be crucial [30
]. Second, in a preliminary analysis, we compared a number of cancer cell lines bearing either phenotype by hybridizing their mRNA to a commercially available pathogen chip containing probes for all known viruses (Agilent Technology) and we could not identify any viral sequence in the cell lines (Worschech A et al., unpublished observation).
Thus, the "anti-viral state" is a characteristic molecular phenotype of a subset of pancreatic cancers that may be the result of a specific mutational profile of cancer cells which is difficult to be understood at this time [38
]. Epigenetic level control, such as methylation, may represent an additional mechanism since a strict correlation exists between demethylation and enhancements in STAT-1 phosphorylation followed by an increase in ISG expression [39
]. From the gene ontology analysis it was interesting to observe the participation of hypoxia pathways in cancer cells with the "anti-viral" state as this can clearly affect tumor biology and responsiveness to chemotherapy [40
] and likely immunotherapy of immune responsive cancers such as renal cell carcinoma [41
] and melanoma [42
We could also speculate that the constitutive activation of antigen presentation pathways might be significant in modulating T cells responses and be responsible for their heterogeneity in various cancers; this may explain the immunogenicity of some melanomas compared with other melanomas [43
] and may become a tool to stratify cancer patients to be treated with T cell-directed vaccines. Whether cancer cells with an active "anti-viral" state bear an enhancement in the presentation of endogenous proteins needs to be evaluated in future studies.
The existence of cancer cells with "anti-viral" capacity has potential relevance to viral gene therapy approaches. Adenoviruses and Adeno-Associated viruses are used to deliver genes to tumor cells with the goal of modifying the phenotype, as for example, by introducing suicide genes [44
]. Particularly in the case of incurable solid tumors such as pancreatic adenocarcinoma, trials have been initiated with third generation adenoviral vectors [46
]. The present study suggests that gene delivery by adenoviral vectors might be hampered in some patients; this information can be important in the selection of patients undergoing virally-related gene therapy and could provide important insights into the interpretation of clinical results.
Brunicardi's group [48
] demonstrated that gene therapy using Adenovirus subtype 5 mediates rat insulin promoter directed thymidine kinase (A-5-RIP-TK)/ganciclovir (GCV) gene therapy resulting in significantly enhanced cytotoxicity to both Panc1 and MiaPaCa2 pancreatic cancer cells in vitro
]. An in vivo
study from the same group showed that systemically administered A-5-RIP-TK/GCV is an effective treatment for pancreatic cancer [50
]. These studies are based on a rat PDAC model in which the pancreatic tumors were derived from Panc1 and MiaPaca2 cell lines. In this model they found a very tight correlation among A-5-RIP-TK/GCV cytotoxicity to malignant cells, adenoviral dose and length of GCV treatment [48
]. Interestingly, all the experiments were performed on cell lines that were negative for the MxA expression. These findings are in full accordance with our theory of a possible effect of interferon associated gene up regulation and its relationship to gene therapy outcome.
If these findings are confirmed in humans, positivity for MxA at diagnosis might become important exclusion criteria and might consequently increase the efficacy of viral-mediated gene therapy for those who test MxA negative.
The observation that both Adenovirus and Adeno Associated viruses were similarly affected by the anti-viral state suggests that this phenomenon is at least partially independent of viral idiosyncrasies related to specific receptors or other restricted properties of each individual virus but rather is a general phenomenon that can apply to several oncolytic delivery systems. Of course, work needs to be done to assess the relevance of this phenotype in other viral systems.
The existence of either phenotype in xenografted primary cancers and in vitro models provides evidence that the antiviral state phenotype is stable. Since most of those genes are expressed only during viral infection in non cancer patients, this observation makes some of the product of those inducible genes, for example ones that codify for membrane proteins, new markers and new possible therapeutic target.