Novel biomarkers of resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus

Abstract

Vesicular stomatitis virus (VSV) based recombinant viruses (such as VSV-ΔM51) are effective oncolytic viruses (OVs) against a majority of pancreatic ductal adenocarcinoma (PDAC) cell lines. However, some PDAC cell lines are highly resistant to VSV-ΔM51. We recently showed that treatment of VSV-resistant PDAC cells with ruxolitinib (JAK1/2 inhibitor) or TPCA-1 (IKK-β inhibitor) breaks their resistance to VSV-ΔM51. Here we compared the global effect of ruxolitinib or TPCA-1 treatment on cellular gene expression in PDAC cell lines highly resistant to VSV-ΔM51. Our study identified a distinct subset of 22 interferon-stimulated genes (ISGs) downregulated by both ruxolitinib and TPCA-1. Further RNA and protein analyses demonstrated that 4 of these genes (MX1, EPSTI1, XAF1, and GBP1) are constitutively co-expressed in VSV-resistant, but not in VSV-permissive PDACs, thus serving as potential biomarkers to predict OV therapy success. Moreover, shRNA-mediated knockdown of one of such ISG, MX1, showed a positive effect on VSV-ΔM51 replication in resistant PDAC cells, suggesting that at least some of the identified ISGs contribute to resistance of PDACs to VSV-ΔM51. As certain oncogene and tumor suppressor gene variants are often associated with increased tropism of OVs to cancer cells, we also analyzed genomic DNA in a set of PDAC cell lines for frequently occurring cancer associated mutations. While no clear correlation was found between such mutations and resistance of PDACs to VSV-ΔM51, the analysis generated valuable genotypic data for future studies.

Keywords: biomarker of resistance; interferon-stimulated gene; oncolytic virus; pancreatic cancer; vesicular stomatitis virus.

Figure 1. Phenotypes of VSV-permissive and VSV-resistant PDAC cell lines

VSV-ΔM51 replication A. and VSV-ΔM51-mediated oncolysis B. in 4 different human PDAC cell lines. Cells were infected with VSV-ΔM51 at 3 different MOIs (0.001, 1, or 10) or mock-treated, and (A) virus replication driven GFP fluorescence was monitored through 90 h p.i. and (B) cell viability was analyzed by MTT assay at 90 h p.i. and is plotted as percentage of the uninfected control. The assays were done in triplicate and data represent the mean±SEM. Statistical analysis was performed using GraphPad Prism Software, using one-way ANOVA with Bonferroni post-test for comparison to the control. (*) P<0.01; (***) P<0.0001 (*) indicate statistical significance between infected and uninfected cells within the same cell line.

Figure 2. RT-PCR analysis of gene expression of putative biomarkers of resistance in PDAC cell lines

Resistant cells (HPAF-II and Hs766T) and permissive cells (MIA PaCa-2 and Capan-1) were mock treated or treated with ruxolitinib (2.5 μM) for 24 h prior to mock treatment or infection with VSV-ΔM51 at MOI 10 (based on virus titer on BHK-21 cells). Virus was aspirated after 1 hour absorption and replaced with growth media containing 5% FBS. Total RNA was extracted 12 h p.i. and reverse transcribed. Gene specific primers (Supplementary Table S1) were used to amplify cDNA. PCR products were run on a 2% agarose gel. All reactions were run in duplicate/triplicate, and one representative sample is shown for each condition.

Figure 3. Western blot analysis of putative biomarkers of resistance in PDAC cell lines

Protein expression following ruxolitinib treatment and VSV-ΔM51 infection. Cells were mock treated or treated with ruxolitinib (2.5 μM) for 24 h prior to mock treatment or infection with VSV-ΔM51 at an MOI of 10 (based on virus titer on BHK-21 cells). At 16 h p.i., cell lysates were prepared and analyzed by Western blot for the indicated protein. Protein sizes (kDa) are indicated on the right. Actin protein levels and Coomassie Blue staining of total protein demonstrate equal loading of protein.

Figure 4. Western blot analysis of putative biomarkers of resistance in 10 PDAC cell lines

A. Protein expression following mock-treatment or VSV-ΔM51 infection. Cells were mock treated or infected with VSV-ΔM51 at an MOI of 5 (based on virus titer on Suit-2 cells, which have an average permissiveness to VSV-ΔM51). At 8 h p.i., cell lysates were prepared and analyzed by Western blot for the indicated protein. “NL” – not loaded, the well #18 was skipped. B. Cells were seeded and protein was isolated 24 h later. Protein sizes (kDa) are indicated on the right. Actin protein levels and Coomassie Blue staining of total protein demonstrate equal loading of protein.

Figure 5. Effect of MX1 knockdown on VSV-ΔM51 replication and oncolysis

A. HPAF-II based cell line clones were generated using a lentivector system with the vector genomes carrying MX1-shRNA1 or MX1-shRNA3 sequences. Multiple cell clones for each shRNA construct were puromycin selected and cell lysates were prepared for Western blot analysis of MX1 expression. B. Protein expression following ruxolitinib treatment and/or VSV-ΔM51 infection. Cells were mock treated or treated with VSV-ΔM51 at an MOI of 10 (based on virus titer on BHK-21 cells) and/or ruxolitinib (2.5 μM). At 48 h p.i., cell lysates were prepared and analyzed by Western blot for the indicated protein. Protein sizes (kDa) are indicated on the right. C. HPAF-II and HPAF-II based clones MX1-1b, MX1-3c and SCRA (scramble shRNA) were mock treated or treated with ruxolitinib (2.5 μM) for 24 h prior to infection with VSV-ΔM51 at an MOI of 0.001, 0.05, or 1 (based on virus titer on HPAF-II cells; 1 MOI_HPAF equates 1500 MOI_BHK-21). MTT cell viability analysis was conducted 96 h p.i. The MTT assay was done in triplicate and data represent the mean±SEM. Statistical analysis was performed using GraphPad Prism Software, using multiple t-tests for comparison to uninfected control. (*) indicates statistical significance (p<0.05) between infected and uninfected cells within the same cell line.