Índices de aprovechamiento de los alumnos

164

Tumor dormancy in EOC is thought to allow the persistence of microscopic tumor lesions after front-line treatment with chemotherapeutic drugs. In the context of metastatic ovarian cancer, the ability to target and kill dormant cancer cells is essential to eradicate disease recurrence, and decrease the high proportion of patients which experience relapse. Multi-cellular spheroids are not only a delivery mechanism to promote metastasis, but a reservoir for supporting tumor cell dormancy in EOC [1, 2]. In our assessment of oncolytic virus efficacy in dormant EOC spheroids, we observed significant reduction in the replication efficiency and induction of lysis from large poxviruses, including MYXV and vvDD. However smaller viruses, like MRBV were able to efficiently replicate, albeit to a slightly reduced capacity in EOC spheroids made from cell lines [3]. Given that the oncolysis by MYXV and vvDD was induced immediately upon spheroid reattachment rather than requiring multiple days to complete replication as was observed in adherent cell infections, it is likely that viral replication is progressing within spheroids but is restricted at a late stage such that completion of oncolysis can only be observed upon reattachment. Similarly, expression of GFP by MYXV and vvDD in both adherent and spheroid EOC cells implies at least early gene expression of the viruses in both culture conditions as viral GFP expression is under the control of a synthetic early/late promoter. Given that the first wave of poxvirus transcription is driven by RNA polymerases and transcription factors packaged with the virus, it is logical that virus replication in spheroids may be restricted in later stages that are more heavily dependent on host cell factors [4-8]. This would imply restriction possibly during intermediate or late stages of the viral life cycle perhaps during, morphogenesis, packaging, or egress in both MYXV and vvDD.

165

During morphogenesis, assembling virions require abundant lipid and protein within cytoplasmic viral factories to enclose the virus core components [9]. We have previously documented the role of autophagy in the degradation of many cellular components during quiescence in spheroids [10]. It is possible that the digestion of cellular organelles and proteins that are non-essential to cell survival through autophagy is sequestering vital components required for membrane wrapping of maturing virions. Similarly, intracellular acquisition of poxvirus envelopes requires trafficking through golgi-derived membranes. However, golgi-derived membranes are commonly redirected for the formation of autophagosomes during autophagy and this may curtail viral envelope incorporation in spheroids [11]. Electron microscopy based studies of the intracellular components of spheroids is one future experiment which could be performed to determine the stage of poxvirus replication within spheroids.

Independent of the potential affect that autophagy may have on late stage viral replication in spheroids, it is possible that poxviruses do not represent an effective oncolytic therapy for the treatment of dormant cancer cells regardless of their morphology (adherent or spheroid). It is likely that poxvirus replication is highly dependent on cell cycle progression [12, 13]. For instance, effective replication of MYXV requires the activation of mammalian p21-activated kinase 1 (PAK1) which is phosphorylated during mitosis and is important for cell cycle regulation [14]. Decreased phosphorylation of PAK1 does not impact virus entry or early virus gene expression but impedes MYXV before late gene expression and viral DNA replication, thus restricting the production of virus progeny [15]. Furthermore, a number of poxviruses have been shown to encode genes which act as viral mimetics of cellular anaphase-promoting protein complexes [13].

166

This regulates the progression of the host cell through mitosis and promotes poxvirus growth, thus emphasizing the significance of cellular replication for poxvirus replication. Dependence of MYXV on AKT signaling and vvDD on EGFR/ERK activation and abundant deoxynucleotide pools are unfavorable requirements for virus replication in dormant cells, particularly for viruses with sizeable genomes [16, 17]. Unfortunately, not only are these unfavorable conditions found in EOC spheroids but both AKT and ERK are among the most common signaling pathways to be suppressed in many dormant and resilient forms of pancreatic, colorectal, head and neck, and breast cancer metastases [18- 21]. Likewise, the expression of both host cell TK (on which vvDD is dependent) and ribonucleotide reductase, which is integral for the synthesis of dNTP, are nearly

undetectable in resting G0 cells, and this represents as an important rate limiting step in the

synthesis of the large DNA genomes of progeny virions [22, 23]. dNTP pools required for DNA synthesis are 20x lower in resting cells than those that are actively replicating [22]. Thus, although there may be potential clinical therapeutic benefits of these larger poxviruses for highly proliferative cancer cells, they may be subject to the same limitations as chemotherapeutic drugs in indolent cancers.

Interestingly, MRBV oncolysis of spheroids was relatively resistant to tumor dormancy. Thus far, no host cell factors have been identified for MRBV replication and its replication may be relatively independent of the host cell cycle. Genome-wide siRNA screens to identify potential host factors necessary for VSV replication did not link any genes associated with cell cycle [24]. Furthermore, efficient replication of MRBV in dormant EOC ascites-derived spheroids suggests complete independence of MRBV from host cell replication. The comparatively small genome size of MRBV implies a reduced

167

dependence on abundant ribonucleic acid pools for mRNA synthesis and genome replication for each progeny virion, potentially allowing for more virus progeny to be produced per round of infection, as we observed. Thus, it is likely MRBV represents a more viable therapeutic option for the treatment of slow growing, indolent cancers which are associated with greater chemotherapeutic resistance.