Prognostic, Biological, and Potential Therapeutic Implications of Gene Amplification-Driven Skp2 Overexpression in Myxofibrosarcoma
CF Li, HY Kang, CK Huang, HY Huang. Chi-Mei Hospital, Tainan, Taiwan; Chang Gung Memorial Hospital, Kaohsiung, Taiwan
Background: The tumorigenesis and prognostication of myxofibrosarcoma (MFS) remains obscured, for which we previously reported Skp2 overexpression as an adverse prognosticator (Clin Cancer Res 2006). Harboring Skp2 gene on 5p13, chromosome (Chr) arm 5p is frequently gained in copy number among various sarcomas, while its specific oncogenes have never been analyzed for MFS.
Design: To search oncogenes on Chr5, we used 385K array comparative genomic hybridization (aCGH) to profile DNA copy number alterations of 12 samples and 2 MFS cell lines (NMFH1, OH931), with special attention to Skp2. Another 82 primary tumors were measured for Skp2 gene dosage by real-time PCR and immunostained for Skp2. MFS cells were transiently transfected with Skp2-specific cDNA and siRNA or treated with bortezomib (a proteasome inhibitor) to analyze the effects on various cellular processes, Skp2 abundance, and/or cytotoxicity.
Results: aCGH identified nonrandom 5p gains in NMFH1 and 5 grade II or III tumors with 2 recurrent amplicons mapped to 5p15.1-15.2 and 5p12-13.3. The latter showed DNA gain in the Skp2 locus. Present in 31 cases (38%), Skp2 amplification was strongly associated with deep location, advanced stages, higher grades, large size, tumor necrosis, and mitotic activity (all p<0.01). While gene status correlated with Skp2 abundance (p<0.001), 5 of 35 MFSs with Skp2 overexpression did not show gene amplification, corroborating lack of this aberration in Skp2-overexpressing OH931 cells. Skp2 amplification was independently predictive of adverse outcomes for local recurrence-free survival (p=0.0179, RR=2.159) along with margin status, for metastasis-free survival (p=0.0011, RR=5.064) with higher grades, and for disease-specific survival (p=0.0004, RR=10.919) with proximal location. Silencing of Skp2 expression in both MFS cell lines attenuated cell proliferation and retarded cell migration, which were reversed by ectopic Skp2 overexpression. Treatment with bortezomib at a low concentration (10nM) could remarkably suppress Skp2 expression, induce activation of caspases, and, as compared to fibroblasts, result in apparently declined cell viability in both NMFH1 and OH931 cells.
Conclusions: Skp2 amplification, the predominant but not the sole cause of protein overexpression in MFS, plays a critical role in tumor aggressiveness. The Skp2-suppressing effect of bortezomib and the relative cytotoxic sensitivity of MFS indicate the potentiality of Skp2 and ubiquitin-proteasome pathway as therapeutic targets.
Category: Bone & Soft Tissue
Monday, March 22, 2010 1:00 PM
Poster Session II # 5, Monday Afternoon