VCP (p97) Regulates NFKB Signaling Pathway, Which Is Important for Metastasis of Osteosarcoma Cell Line

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Abstract
In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin‐containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor‐αBα (IαBα), an inhibitor of nuclear factor‐αB (NFαB), whether VCP influences NFαB activation or not was examined by using VCP‐transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor‐α (TNFα), Dunn/VCPs showed constantly activated NFαB, although in the original Dunn cells and control vector transfectant (Dunn/Dunn‐c) NFαB activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated IαBα (p‐IKBα) in the cytoplasm of confluent Dunn/Dunn‐c cells compared to that of Dunn/VCPs. Therefore, decrease of p‐IKBα degrading activity might be responsible for the decrease in NFKB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn‐c cells after TNFα stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP‐1 inoculated male C3H mice compared to those in Dunn/Dunn‐c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti‐apoptotic potential in these cells owing to constant NFKB activation via efficient cytoplasmic p‐IKBα degrading activity may explain the increased metastatic potential of these cells.