br We observed that the infection of pancreatic cancer cells
We observed that the infection of pancreatic cancer cells with AdwtE miR-99b and AdwtE miR-485 was associated with higher expression of viral genes encoding structural proteins (namely, hexon, penton, and fiber) and the E1A gene, both at the RNA and protein levels, without changes in the content of total viral genomes. This suggests that the miR-99b and miR-485 effects were probably related to the transcrip-tional activation of viral genes. This induction was superior in the late genes, probably because the increase in E1A expression facilitates the transactivation of late genes through its binding to the MLP.26
Through bioinformatics analysis, we identified potential miRNA target genes for both miR-99b and miR-485 belonging to the tran-scription factor family of proteins. In the current study, ELF4 was validated as a target gene of miR-99b and KLF8 as that of both miR-99b and miR-485. MDM2 has already been described as a vali-dated target for miR-485. Infection of pancreatic cancer cells with AdwtE miR-99b and AdwtE miR-485 resulted in decreased expres-sion of these three proteins, in line with their regulation by miR-
99b and/or miR-485. Interestingly, MDM2 is an adenoviral limiting factor.7,20,27 Importantly, we show that genetic downregulation of
ELF4 or KLF8 resulted in enhanced release of adenoviral particles, pointing to a role for them in adenoviral activity modulation. KLF8 is a CACCC (GT-box)-binding dual-transcription factor, and its Caerulein have been identified at or next to the promoters of different adenoviral genes, including two sites at the left ITR (L-ITR) and two in the MLP. In this study, we could validate that KLF8 reduces transcription from the adenoviral L-ITR/E1A promoter and MLP. Promoter reporter assays showed higher tran-scriptional activity in cells with downregulated KLF8 than in cells ex-pressing KLF8 (Figure S8). These results support the view that, through the recruitment of the C-terminal-binding protein (CtBP), KLF8 represses the expression of these genes.28 Indeed, CtBP2 has been previously identified as an adenoviral limiting factor.29,30
Furthermore, KLF8 recruits p300 and PCAF co-activators to pro-moters.31 To promote productive virus infection, E1A removes
CtBP2 from repressed promoters, sequesters p300 from transcrip-tional active regions, and redirects it together with Rb to selected host genes, thereby repressing their expression.29,32 In those cases, high levels of KLF8 might compete with E1A and lead to reduced adenoviral yields. ELF4 is an ETS transcription factor that becomes activated during the antiviral response and in cells with oncogenic Ras.33 ELF4 promotes MDM2 expression, which could explain, at least in part, why MDM2 is downregulated in AdwtE miR-99b-in-fected cells even though it is not a target for this miRNA.21 The ef-fects of ELF4 on the adenoviral activity might be explained by its regulation of MDM2. In fact, different hypotheses have been pro-posed to explain MDM2 antiviral activity. MDM2 might send early adenoviral proteins (E1A and E1B-55K) or cellular proteins
required for a productive viral cycle (PP2A) to proteasomal degra-dation,27,34 but it can also induce the expression of CtBP235 or act as a transcriptional repressor by interacting with the 34K subunit of TFIIE.36 Thus, we propose that the downregulation of KLF8, ELF4, and MDM2 through miR-99b or miR-485 expression pro-motes the expression of the adenoviral late genes and E1A, which in turn facilitates the adenoviral life cycle.
The expression of miR-99b and miR-485 may also provide pancreatic cancer with tumor suppressor activities. Interestingly, we observed that miR-99b and miR-485 were poorly expressed in PDAC samples and that their reduced expression correlated with poorer survival rates of patients. Thus, the enhanced antitumor activity of ICOVIR15 miR viruses might be the result of miR-99b and miR-485 contributing to improve the adenoviral fitness and their tumor-suppressive effects.
Our work was restricted to pancreatic cancer; however, other authors have identified a contribution of miR-99b and miR-485 in the pro-gression of different neoplasias. Low miR-99b expression has been associated with advanced stages of glioma and with the presence of lymphatic metastasis in cervical cancer.37,38 A metastatic inhibitor
role has been proposed for miR-485 in lung adenocarcinoma and breast cancer.39,40 Also, low miR-485 expression has been found to correlate with poor prognosis in gastric cancer.41 Moreover, high expression levels of either KLF8 or ELF4 have been associated with pro-tumorigenic processes, suggesting that their downregulation may also be beneficial for controlling tumor growth.42–44 Altogether, these findings indicate that the expression of miR-99b and miR-485 from the adenoviral genome produces a significant antitumor effect as a consequence of the enhanced adenoviral activity, with a potential contribution of their tumor suppressor activity. The similarities in miR-99b and/or miR-485 alterations between cancer cells of different origins allow us to speculate that the improved antitumoral response of ICOVIR15-miR99b and ICOVIR15-miR-485 observed in PDAC