Dissection of theAPC-Skp2 Cascade in Breast Cancer

Purpose: Skp2 is a subunit of the SCF ubiquitin protein ligase, which plays a vital role in the control of tumorigenesis via its regulationofG1-S transition. Deregulationof Skp2 invarious types of cancers correlates with aggressive clinical behavior and poor prognosis. Recent studies suggest that cell cycle ^ dependent fluctuation of Skp2 is governed byAPC, another important E3 ligase, thereby preventing premature entry into S phase. To assess the potential role of APC in tumorigenesis through proteolysis of Skp2, we have dissected the APC-Skp2 cascade. Experimental Design:We manipulated the APC-Skp2 cascade and examined its cellular behavior using bothbreast cancer andnormal breast epithelial cells. Furthermore, applying immunohistochemistry, we analyzed the clinicopathologic significance of these molecules in patients with breast cancer. Results: Analysis of tissue arrays indicated that the percentage of samples positive for Cdh1in breast cancer was significantly lower compared with normal breast tissues (P = 0.004). Conversely, the percentage of samples scored as positive for Skp2 in cancer was significantly higher than in normal tissues (P < 0.001). Moreover, prognostic studies revealed that relatively high levels of Cdh1are associated with survivability in patients with breast cancer. In addition, depletion of Cdh1by small interfering RNA in normal breast cells resulted in increased cellular proliferation, whereas knockdownof Skp2 significantly suppressed growth in breast cancer cells. Conclusions:This study shows a correlation between Skp2 and APCin breast cancer.Thus, Cdh1may act as an important component in tumor suppression and could be considered as a novel biomarker in breast cancer. Skp2 (S phase kinase–associated protein 2) has been suggested to be a promising biological marker for several types of cancer, which could also be used as a prognostic indicator in certain types of malignancy (1, 2). Skp2 in association with SCF complex (Skp1, Cullin, and F-box) targets substrate proteins for degradation, resulting in the regulation of cell cycle progression and other cellular processes (3). The major role of Skp2 in the control of the cell cycle is to ubiquitylate p27 for proteolysis, regulating the onset of S phase (4, 5). Both Skp2 transcripts and protein oscillate in a cell cycle–dependent manner (6–10). Recent studies have revealed that APC (anaphase-promoting complex), a pivotal E3 ligase, degrades Skp2 and thus, prevents abnormal entry into S phase (8, 9). Nevertheless, the correlation between APC and Skp2 has not yet been validated in tumorigenesis. APC is a multifunctional E3 ligase, regulating several critical cellular events including mitotic progression, DNA replication, cellular differentiation, genomic integrity, and signal transduction (11–18). Activation of APC is controlled by two WD40 family proteins (e.g., Cdh1 and Cdc20). Cdh1, in association with APC, regulates APC function in G1 and postmitotic processes, whereas the interaction of Cdc20 with APC controls chromatid separation during mitosis (12, 19–22). Recognition of the substrate by the substrate-specific activator (e.g., Cdh1 and Cdc20) is facilitated by several well-characterized degrons/recognition domains including destruction box (RXXL), KEN box and, A box present in the substrate (21). Recent evidence has drawn our attention to the connection between APC function and human diseases. Pathologic and epigenetic studies have shown that dysfunction in several components of the APC pathway including APC6, Cdc16, Cdc23, and Cdh1 or Cdc20, is correlated with different types of cancer such as colon cancer, B lymphoma, gastric, and lung cancer (23–26). However, the underlying mechanism by which APC is involved in the aforementioned types of carcinogenesis remains largely unknown. Dissection of the APC pathway in human cancer will facilitate our understanding of APC in tumor progression. Overexpression of Skp2 is often correlated with malignancy. Understanding the mechanism by which Skp2 protein levels is down-regulated could provide strategies for manipulating the status of tumor cells. The notion that Cdh1 targets Skp2 for Human Cancer Biology Authors’Affiliations: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania and Department of Cancer and Thoracic Surgery, Okayama University School of Medicine, Okayama, Japan Received 6/27/07; revised11/5/07; accepted11/30/07. Grant support: NIH grants CA115943.Y.Wan is a scholar of theAmerican Cancer Society andVCancer Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Yong Wan, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Suite 2.6C, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3275; Fax: 412-623-7761; E-mail: [email protected]. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-1585 www.aacrjournals.org Clin Cancer Res 2008;14(7) April 1, 2008 1966 Research. on May 2, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from degradation suggests a potential role for Cdh1 in the suppression of tumor growth. To validate the connection between Cdh1 and Skp2 in tumor formation, we have carried out a human breast cancer tissue array and prognostic analyses with the components of the APC-Skp2 cascade. In addition, using RNA interference technology, we have further dissected the function of Cdh1 and Skp2 in suppressing or enhancing cell growth in both normal and breast cancer cells. Our study shows that Cdh1 expression is inversely correlated with the expression of Skp2 in cancer and in normal condition. Suppressing Skp2 protein levels by enhancing Cdh1 function results in the inhibition of tumor cell growth. The present results suggest that Cdh1 may function as a critical component in the suppression of breast tumor. Materials andMethods Plasmid preparation and construction of small interfering RNA–stable cell lines. pCS2-Myc-Cdh1 and pCS2-HA-Skp2 plasmids have been engineered and reported previously (11). Stable knockdown using small interfering RNA (siRNA) for Cdh1 has also been previously reported (16). Construction of the Skp2-siRNA stable cell line 1 Skp2 NH2-terminal (5¶-GAGGAGCCCGACAGTGAGA-3¶) was engineered using pSUPER system (OligoEngine). Transfection was carried out using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s protocol. Thereafter, positive clones were selected in the presence of puromycin-containing medium (Promega). Antibodies and reagent. Antibodies were Cdh1 (Calbiochem), Skp2 (Santa Cruz), p27(Santa Cruz), tubulin (Calbiochem), and horseradish peroxidase–conjugated goat anti-mouse and anti-rabbit secondary antibody (Promega). 17h-Estradiol was from Sigma. Western blot analysis was done using an enhanced chemiluminescence detection kit (Amersham). Semiquantification of data was done using an image