Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines

Abstract

Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3-and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCEDespite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts.

An engineered avian-origin influenza A virus for pancreatic ductal adenocarcinoma virotherapy

“…To test the feasibility of this approach, we engineered a new H7N3 NS1-77 virus in which the genes expressing the surface antigens haemagglutinin (HA) and neuraminidase (NA) were replaced with those of the well characterized human strain H1N1 A/ Puerto Rico/8/1934 (PR8). In vitro experiments performed using this 6 : 2 recombinant virus, termed PR8 H1/N1-2962 NS1-77, confirmed that the exchange of surface antigens did not alter the virus' ability to trigger apoptosis in BxPC-3 cells at levels similar to the parental strains and thus, in line with previous data (Kasloff et al, 2014), at higher levels compared to the H1N1 PR8 virus (Fig. 6a).…”

“…Avian-origin influenza A viruses (IAVs) were previously shown to infect and induce potent apoptosis of various PDA cell lines, including BxPC-3 cells (Kasloff et al, 2014) in which different viruses displayed reduced oncolytic effect compared to other malignant pancreatic cells (Lee et al, 2014;Murphy et al, 2012). These observations suggested that avian influenza isolates might be a useful treatment option for specific subclasses of PDA.…”

“…Conversely to many human IAVs, which are able to cope with Mx gene product, thanks to mutation within specific recognition sites on the NP protein, in general, avian IAVs are more sensitive to MxA activity and in order to be stably introduced in mammals often they have to acquire adaptive mutations to escape this restriction factor (Dittmann et al, 2008;Manz et al, 2013;Zimmermann et al, 2011). Because the H7N3 NP protein displays a typical avian signature (Riegger et al, 2015), it is possible that the expression of IFNstimulated genes (ISGs), such as MxA, together with IFN, might account for the low growth rate and ensuing oncolytic activity (Kasloff et al, 2014) displayed by the wild-type H7N3 virus in HPDE6 cells.…”

“…These observations suggested that avian influenza isolates might be a useful treatment option for specific subclasses of PDA. As such, the low pathogenicity (LP) avian IAV H7N3 A/turkey/Italy/2962/03, whose rapidity and potency of cell death induction in different PDA cell lines repeatedly outperformed other IAV isolates (Kasloff et al, 2014), including the human H1N1 PR8 virus currently tested as oncolytic candidate for other tumour types (Sturlan et al, 2010;van Rikxoort et al, 2012;Wolschek et al, 2011), was selected for further development.…”

“…Due to their genetic heterogeneity, PDA cells are highly variable in their permissiveness to various OVs. The avian influenza A virus (IAV) H7N3 A/turkey/Italy/2962/03 is a potent inducer of apoptosis in PDA cells previously shown to be resistant to other OVs (Kasloff et al, 2014), suggesting that it might be effective against specific subclasses of pancreatic cancer. To improve the selectivity of the avian influenza isolate for PDA cells, here confirmed deficient for IFN response, we engineered a truncation in the NS1 gene that is the major virusencoded IFN antagonist.…”