Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo

Background: Low levels of p27(Kip1) expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27(Kip1) for degradation, was overexpressed and was inversely related to P27(Kip1) levels in malignant melanoma with poor prognosis. Objective: We investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27(KiP1) down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo. Methods: We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo. Results: Skp2 protein was decreased and the p27(Kip1) protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo. Conclusion: Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27(Kip1) degradation in malignant melanoma. (c) 2006 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.