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Constitutive NF-κB Activation is Amplified by VSV in Aggressive PC3 Prostate Cancer Cells that Resist Viral Oncolysis
Alaa A Abdelmageed1, Jack Smerczynski2, Lute Douglas2
1University of Rochester School of Medicine and Dentistry.
Abstract:
Cancer cells often have defects in antiviral pathways, making them susceptible to oncolytic viruses like vesicular stomatitis virus (VSV). However, some cancer cells resist viral infection through the constitutive expression of interferon-stimulated genes. This study examined whether NF-κB activation and NF-κB-dependent antiviral signaling contributes to resistance to VSV infection in the PC3 cell line, derived from an aggressive metastatic prostate cancer (PrCa) tumor. We found that NF-κB localized to the nucleus in VSV-infected PC3 cells, but not in the VSV-susceptible LNCaP PrCa cell line. Analysis of the upstream NF-κB inhibitor IκB-α revealed higher levels of both total and phosphorylated IκB-α in PC3 cells compared to LNCaP cells, indicating constitutive activation of the NF-κB pathway via an IκB-α-dependent mechanism. Notably, VSV infection did not alter IκB-α phosphorylation in PC3 cells, suggesting that VSV may amplify NF-κB signaling through an IκB-α-independent pathway. Furthermore, PC3 cells displayed elevated levels of the NF-κB p65 protein subunit compared to LNCaP cells, with its phosphorylated form significantly increased upon VSV infection. These results from phosphorylation assays confirm that multiple steps in the NF-κB pathway are differentially activated in PC3 and LNCaP cells. Additionally, the expression of several NF-κB-dependent cytokine and proinflammatory genes, including IL12 and IL6, were upregulated following VSV infection in PC3 cells, as compared to LNCaP cells. Blocking the NF-κB pathway using a pharmaceutical inhibitor resulted in increased PC3 cell death with VSV infection. Collectively, these findings suggest that enhanced NF-κB signaling may underlie the resistance of PC3 cells to VSV oncolysis, potentially offering new insights into therapeutic strategies targeting NF-κB in resistant prostate cancers.
Insights
Nuclear factor-kappa B (NF-κB) pathway activation confers resistance to vesicular stomatitis virus (VSV) oncolytic therapy in aggressive prostate cancer cells. Blocking this pathway enhances cancer cell death, suggesting new therapeutic targets for resistant prostate cancers.
Area of Science:
- Molecular Biology
- Oncology
- Virology
Background:
- Cancer cells often exhibit defects in antiviral pathways, rendering them susceptible to oncolytic viruses.
- However, constitutive expression of interferon-stimulated genes can confer resistance to viral infections in some cancer cells.
- The role of nuclear factor-kappa B (NF-κB) signaling in mediating resistance to oncolytic viruses in prostate cancer remains incompletely understood.
Purpose of the Study:
- To investigate the involvement of NF-κB activation and NF-κB-dependent antiviral signaling in resistance to vesicular stomatitis virus (VSV) infection.
- To compare NF-κB pathway activation in resistant PC3 prostate cancer cells versus susceptible LNCaP prostate cancer cells.
- To determine if targeting the NF-κB pathway can enhance the efficacy of VSV oncolysis in resistant prostate cancer models.
Main Methods:
- Western blot analysis to assess levels of NF-κB subunits (p65) and inhibitors (IκB-α) and their phosphorylation states.
- Immunofluorescence microscopy to determine NF-κB nuclear localization in response to VSV infection.
- Quantitative real-time PCR to measure the expression of NF-κB-dependent genes (e.g., IL12, IL6).
- In vitro cell death assays following VSV infection in the presence or absence of an NF-κB pathway inhibitor.
Main Results:
- PC3 cells exhibited constitutive NF-κB pathway activation, evidenced by nuclear localization of NF-κB and elevated IκB-α levels, compared to LNCaP cells.
- VSV infection led to increased phosphorylation of the NF-κB p65 subunit in PC3 cells, suggesting amplification of signaling.
- VSV infection upregulated NF-κB-dependent pro-inflammatory genes (IL12, IL6) in PC3 cells.
- Pharmacological inhibition of the NF-κB pathway significantly increased PC3 cell death during VSV infection.
Conclusions:
- Enhanced NF-κB signaling contributes to the resistance of aggressive prostate cancer cells (PC3) to VSV oncolysis.
- The NF-κB pathway is differentially activated in prostate cancer cells with varying susceptibility to VSV.
- Targeting the NF-κB pathway represents a potential therapeutic strategy to overcome resistance to oncolytic virotherapy in prostate cancer.
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