br Original Article br Bioselection Reveals
15 Original Article
Bioselection Reveals miR-99b and miR-485 as Enhancers of Adenoviral Oncolysis in Pancreatic Cancer
Maria Rovira-Rigau,1,2 Giulia Raimondi,1,2 Miguel Ángel Marín,1 Meritxell Gironella,1,3 Ramon Alemany,4 and Cristina Fillat1,2,5
1Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain; 2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 08036 Barcelona, Spain; 3Gastrointestinal & Pancreatic Oncology Group, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), 08036 Barcelona, Spain; 4Institut Català d’Oncologia-IDIBELL, 08907 L’Hospitalet de Llobregat, Spain; 5Facultat de Medicina i Ciències de la Salut. Universitat de Barcelona (UB), 08036 Barcelona, Spain
Oncolytic viruses are designed for cancer treatment. Cell-virus interactions are key determinants for successful viral replica-tion. Therefore, the extensive reprogramming of gene expres-sion that occurs in tumor VX 765 might create a hurdle for viral propagation. We used a replication-based approach of a microRNA (miRNA) adenoviral library encoding up to 243 hu-man miRNAs as a bioselection strategy to identify miRNAs that facilitate adenoviral oncolytic activity in pancreatic ductal adenocarcinoma. We identify two miRNAs, miR-99b and miR-485, that function as enhancers of adenoviral oncolysis by improving the intra- and extracellular yield of mature virions. An increased adenoviral activity is the consequence of enhanced E1A and late viral protein expression, which is prob-ably mediated by the downregulation of the transcriptional repressors ELF4, MDM2, and KLF8, which we identify as miR-99b or miR-485 target genes. Arming the oncolytic adeno-virus ICOVIR15 with miR-99b or miR-485 enhances its fitness and its antitumoral activity. Our results demonstrate the po-tential of this strategy to improve oncolytic adenovirus po-tency, and they highlight miR-99b and miR-485 as sensitizers of adenoviral replication. r> INTRODUCTION
Viruses have evolved a number of strategies to hijack cellular re-sources, forcing them to synthesize new virions for a successful viral propagation.1,2 These strategies have been acquired as a conse-quence of an adaptive process in specific cellular contexts. While the use of viruses in cancer treatments has long been studied,3 tu-mor cells present a number of genetic alterations that elicit massive reprogramming of cellular gene expression, creating a novel context in which viruses have not adapted to replicate.4 The inherent complexity of tumors makes the oncolytic viral activity highly dependent on the specific characteristics of tumor cells. In fact, the differential expression of certain genes in cancer cells can nega-tively impact viral replication.5–7 Nevertheless, molecular mecha-nisms explaining viral replication heterogeneity in cancer cells remain to be fully appraised.8
MicroRNAs (miRNAs) are post-transcriptional negative regulators of gene expression that modulate a variety of cellular physiological pro-cesses. miRNAs also play important roles in host-virus interactions.9 Viral infections can modulate the cellular miRNome with changes induced either by the virus, to promote a proviral environment, or by the antiviral cellular response, to limit the viral life cycle.10–12 Although the mechanisms of miRNA-mediated regulation of viral infection have not been completely clarified, it is clear that host miRNAs can regulate viral infections.
In the context of tumorigenesis, many miRNAs with essential roles in cancer-associated pathways are dysregulated.13 Participation of miRNAs has been demonstrated in central cancer processes, such as cell proliferation and death, cell differentiation, and maintenance of stem cell potency.14 Aberrant expression of miRNAs has been well documented for pancreatic ductal adenocarcinoma (PDAC), and specific miRNA signatures have been associated with diagnosis, staging, progression, prognosis, and treatment response.15,16 Hence, when viruses are used as oncolytic agents, they face an already per-turbed miRNome in cancer cells. These miRNA profile changes may not necessarily favor viral replication and propagation within the tumor; indeed, the downregulation of certain miRNAs could induce the overexpression of viral-limiting factors.
Adenoviruses (Ads) are non-enveloped icosahedral viruses with dou-ble-stranded DNA genomes. The viral genome is sub-divided into regions termed early and late according to when transcription activa-tion initiates. Early genes remodel the intracellular environment to prepare the cell for viral replication and activate the expression of other viral genes. Late genes primarily constitute viral structural pro-teins. Adenoviruses, and particularly adenovirus serotype 5 (Ad5), are