Bortezomib (Velcade) Induces p27Kip1 Expression through S-Phase Kinase Protein 2 Degradation in Colorectal Cancer

Abstract

S-phase kinase protein 2 (SKP2), an F-box protein, targets cell cycle regulators including cycle-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancers. We investigated the role of SKP2 and its ubiquitin-proteasome pathway in colorectal carcinoma using a panel of cell lines, clinical samples, and the NUDE mouse model. Using immunohistochemical analysis on a large tissue microarray of 448 samples, an inverse association of SKP2 expression with p27Kip1 protein levels was seen. A colorectal cancer (CRC) subset with high level of SKP2 and low level of p27Kip1 showed a decreased overall survival (P = 0.0057). Treatment of CRC cell lines with bortezomib or expression of small interfering RNA of SKP2 causes down-regulation of SKP2 and accumulation of p27Kip1. Furthermore, treatment of CRC cells with bortezomib causes apoptosis by involving the mitochondrial pathway and activation of caspases. In addition, treatment of CRC cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Finally, treatment of CRC cell line xenografts with bortezomib resulted in growth inhibition of tumors in NUDE mice via down-regulation of SKP2 and accumulation of p27Kip1. Altogether, our results suggest that SKP2 and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention for treatment of CRC. [Cancer Res 2008;68(9):3379-88]

Bortezomib Stabilizes Mitotic Cyclins and Prevents Cell Cycle Progression via Inhibition of UBE2C in Colorectal Carcinoma

“…For cell cycle analysis, cells were washed once with PBS and resus-pended in 500 L of hypotonic staining buffer and analyzed by flow cytometry as described previously. 19 For detection of apoptosis, cells were harvested and percentage apoptosis was measured by flow cytometry after staining with fluorescein-conjugated annexin V and propidium iodide (PI) (Molecular Probes, Eugene, OR).…”

“…The body weight of each mouse was monitored weekly, and tumor volume was measured as described previously. 19 After 4 weeks of treatment, mice were sacrificed and individual tumors were snap-frozen in liquid nitrogen for storage. Cell lysates were prepared from each tumor, and Western blot assays were performed to determine the status of various proteins.…”

“…19 Because our in vitro data showed that bortezomib treatment down-regulated UBE2C and subsequently induced the expression of cyclin A and cyclin B1 in the Colo-320 cell line, we tested whether proteasome inhibition in vivo altered the expression of these proteins. We therefore analyzed the UBE2C and cyclin levels in primary tumors derived from vehicle-treated mice and mice treated with bortezomib using Western blot analysis and RT-PCR.…”

“…19 -21 Bortezomib is a potential therapeutic agent in a subset of such tumors that show dysregulation in SCF-SKP2 ubiquitin proteasome pathway. 19,22 However, to date no experimental evidence exists as to whether bortezomib can be used in CRC that expresses alteration in UBE2C. Therefore, in this study, we first investigated the role of UBE2C dysregulation in Middle Eastern patients with CRC.…”

Leptin-R and its association with PI3K/AKT signaling pathway in papillary thyroid carcinoma

“…Cells (10 4 ) were incubated in triplicate in a 96-well plate in the presence or absence of indicated test doses of leptin in a final volume of 0.20 ml for 48 h. The ability of leptin to induce cell growth was determined by MTT cell proliferation assays, as previously described (Uddin et al 2008a). Replicates of six wells for each dosage including vehicle control were analyzed for each experiment.…”