Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested.

Methods: We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines.

Results: The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6.

Conclusion: Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We suggest that the detection of these two phenotypes might help the selection of patients enrolled in virally-mediated gene therapy trials.

Figure 1

Interferon related genes expression profile. Supervised cluster expression analysis of 76 selected interferon related genes, represented by 112 probesets, in 8 xenografted primary pancreatic adenocarcinomas (X-PDAC), 3 pancreatic adenocarcinoma bulk tissues (PDAC), 3 chronic pancreatitis (CP) and 3 normal pancreas (Normal). The analysis distinguished a cluster comprising the 11 adenocarcinoma samples (cluster 2) from the normal and pancreatitis samples that clustered together (cluster 1). Among the cancer samples there were two phenotypes, 2a and 2b, the former being closer to the cluster of normal and pancreatitis. The list of probesets corresponding to up regulated genes in group 2b is listed in red while those corresponding to down regulated genes are in green.

Figure 2

Genes differentially expressed between clusters 2a and 2b xenografts. Left panel, cluster analysis of 1,203 differentially expressed genes between the clusters 2a and 2b of Figure 1 (red indicates up-regulation while green down-regulation). Right panel, canonical pathway analysis of the 1,203 genes using the Ingenuity Pathway Analysis software. The 3 most significantly modulated pathways are indicated; the stacked bars represent the proportion of differentially expressed genes over the total number of genes involved in the specific pathway (number on top of the bars).

Figure 3

MxA protein expression in xenografted primary pancreatic adenocarcinomas. A) MxA expression level in microarray data analysis expressed as log2 ratio; orange and blue colors represent higher and lower expression transcript, respectively. B) Western Blot analysis of MxA in 11 xenografted primary pancreatic adenocarcinomas (X-PDAC). C) Example of MxA immuno positive (X-PDAC 4) and MxA immuno negative (X-PDAC 6) samples. D) Correlation of MxA immunohistochemistry, Western Blot and microarray data.

Figure 4

MxA protein expression in primary pancreatic adenocarcinoma tissues. Immunohistochemical (A) and Western blot (B) analysis of MxA in four primary pancreatic adenocarcinomas (PDAC).

Figure 5

Endogenous MxA expression in PDAC cell lines and resistance to viral infection. A) MxA expression in PDAC cell lines by Western Blot analysis. B) Citotopathic effect of Adenovirus wt on MxA+ (orange) versus MxA- (blu) PDAC cell lines. The vertical arrow indicates increased viral concentration, from 10⁶, 10⁷, 10⁸ DNA particles of Ad5. C) Number of viral particles measured by real time PCR after Adeno5 wt infection in MxA+ and MxA- PDAC cell lines (Ad5 DNA replication efficiency). Normalised to the Ad5 DNA amount present in Panc2 at 4^th dilution considered as 1 Correlation of MxA expression with Adeno5 infection efficiency. MxA positive (HPAFI, CFPAC, PSN1, top) and MxA negative (GER, PT45, Panc1, bottom) cells were infected with 1.36 pfu/cell, 13.6 pfu/cell and 136 pfu/cell of Ad5-CMV-GFP vector. D) FACS analysis profile of different PDAC cell lines after 2 days of Adeno5-CMV-GFP infection (13.6 pfu/cell). E) Luminescence analysis for the permissivity of MxA+ and MxA- to the adeno associated infection, data are shown as relative luciferase units (RLU).

Figure 6

Silencing and infection with Adeno5 of a MxA positive cell line: PaCa44. A) Activation of ISRE promoter (gray bars) and IFNbeta promoter (black bars) in MxA+ cell lines. The Y axes express the production of the reporter gene normalized by the same cell line carrying a plasmide with non-targeting control (NC). Please add n of experiments and error bars The data were normalized using a pMet luc plasmid control. B) ISG15 expression by Western Blot after 24 hours of silencing for NFkB, IRF7, IRF3, VISA, untreated, non-targeting control (NC), respectively. C) Decreased level of ISRE regulated reporter gene expression in PaCa44 cell line after silencing with IRF3, IRF7, NFkB or non-targeting control (NC). The data were normalized using a pMet luc plasmid control. D) FACS analysis profile of GFP expression in PaCa44 cell line infected with Adeno5 CMV-GFP virus after silencing IRF3 and IRF7. Cells were infected by using 136 pfu/cell: solid black line, 68 pfu/cell: dashed black line, 27.2 pfu/cell: dotted black line of Adeno5-CMV-GFP vector and 136 pfu/cell Adeno5-CMV-Null vector: solid grey. Numbers represent the MFI.