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Cell-Cycle Regulator Cks1 Promotes Hepatocellular Carcinoma by Supporting NF-Kb–Dependent Expression of Interleukin-8

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Cell-Cycle Regulator Cks1 Promotes Hepatocellular Carcinoma by Supporting NF-Kb–Dependent Expression of Interleukin-8 Published OnlineFirst September 14, 2011; DOI: 10.1158/0008-5472.CAN-10-4356 Cancer Tumor and Stem Cell Biology Research Cell-Cycle Regulator Cks1 Promotes Hepatocellular Carcinoma by Supporting NF-kB–Dependent Expression of Interleukin-8 Eun-Kyoung Lee1,2, Dae-Ghon Kim3, Jang-Seong Kim1, and Yeup Yoon1 Abstract The cell-cycle regulator Cks1 has recently been implicated in Skp2-mediated ubiquitination of the tumor suppressor protein p27. In this article, we report that Cks1 exerts a Skp2-independent regulation of NF-kB that promotes interleukin-8 (IL-8) expression, which is critical to hepatocellular carcinoma (HCC) growth. Cks1 was upregulated frequently in human HCC tissues and cell lines. Cks1 knockdown in HCC cells elevated p27 levels and decreased tumorigenicity in a manner that was also associated with a strong downregulation of IL-8 expression. IL-8 downregulation was not phenocopied by either RNAi-mediated knockdown of Skp2 or ectopic over- expression of p27. However, attenuation of IL-8 expression itself was sufficient to blunt HCC growth. Mechanistic investigations revealed that IL-8 was controlled at a transcriptional level by Cks1 targeting of the NF-kB regulator IkBa, which led to NF-kB activation and IL-8 expression, through a p27-independent regulation of IkB kinase complex components. Collectively, our findings support the hypothesis that Cks1 supports hepatocarcinogenesis by NF-kB–mediated regulation of IL-8 expression, broadening the function of Cks1 in cancer beyond its role as a Skp2 cofactor in p27 ubiquitination. Cancer Res; 71(21); 1–9. Ó2011 AACR. Introduction was identified as an essential cofactor of Skp2 in the Skp2- containing Skp1-Cullin1-F-box ubiquitin ligase complex Hepatocellular carcinoma (HCC) is the third most common (SCFSkp2)–mediated ubiquitination of the tumor suppressor cause of cancer-related deaths worldwide (1). The major risk protein p27 that leads to the proteasomal degradation of p27 factors for HCC include chronic hepatitis from hepatitis B or C (6–8). The CDK inhibitor p27 is a hallmark of many cancers, virus infection and exposure to carcinogens such as aflatoxin and its reduced levels are associated with high aggressiveness B1 (2). Despite the clinical significance of HCC, we only know and poor prognosis in various malignant tumors (9). Similarly, the elemental basics of the molecular, cellular, and environ- upregulation of Skp2 and/or Cks1 is significantly associated mental mechanisms that drive disease pathogenesis, and only with poor prognosis in some cancers and, often, inversely limited therapeutic options are available, of which many have related to p27 protein levels (10–15). Among these malignan- negligible clinical benefits (3). Therefore, elucidation of the cies, HCC showed a frequent and large gain of 1q, and 1q21-22 predominant molecular events underlying hepatocarcinogen- was identified as the minimum overlapping amplified region esis may help identify new therapeutic targets. containing candidate oncogenes for hepatocarcinogenesis Cks proteins were originally identified as subunits that (16). Moreover, some microarray studies that characterized interact closely with cyclin–cyclin-dependent kinase (CDK) the global gene expression in human HCC tissues and HCC- complexes (4, 5). Recently, mammalian Cks1 (CDC28 protein derived cell lines showed that Cks1 was upregulated in HCC kinase regulatory subunit 1B), located on chromosome 1q21.2, (17, 18). Together, these findings indicate a significant role of Cks1 in hepatocarcinogenesis. Interleukin-8 (IL-8), a proinflammatory CXC chemokine, plays important roles in angiogenesis, mitosis, tissue remodel- Authors' Affiliations: 1New Biologics Team, Mogam Biotechnology Research Institute, Yongin, Kyonggi-do; 2Laboratory of Biophysics, School ing, and tumor progression (19). IL-8 secreted from cancer cells of Biological Sciences, Seoul National University, Seoul; and 3Department plays either autocrine or paracrine roles in the tumor micro- of Internal Medicine, Chonbuk National University Medical School and Hospital, Jeonju, Republic of Korea environment of various malignancies, including prostate, colon, and pancreatic cancers (20). Especially in HCC, IL-8 Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). derived from cancer cells was signi cantly implicated in inva- sionandmetastasis,ratherthanintumorangiogenesis(21),and Corresponding Author: Yeup Yoon, Mogam Biotechnology Research Institute, 341 Bojeong-dong, Giheung-gu, Yongin, Gyeonggi-do 446- the preoperative serum IL-8 level was suggested as a biomarker 799, Republic of Korea. Phone: 82-31-260-9833; Fax: 82-31-260-9808; of tumor invasiveness and progression (22). However, the E-mail: [email protected] relationship between Cks1 and IL-8 has not yet been reported. doi: 10.1158/0008-5472.CAN-10-4356 In this study, we assessed Cks1 expression in HCC tissues Ó2011 American Association for Cancer Research. and investigated the role of Cks1 in hepatocarcinogenesis, www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst September 14, 2011; DOI: 10.1158/0008-5472.CAN-10-4356 Lee et al. using genetic manipulation of Cks1 in HCC cell lines. Further- sion was quantified by autoradiography. 18S rRNA served as a more, we identified the mechanism of Cks1 oncogenesis in normalization control; the primers for probe preparation were HCC development through analysis of gene expression profile as follows: (sense) 50-GTAACCCGTTGAACCCCATT-30 and and the relevant signaling pathways. (antisense) 50-CCATCCAATCGGTAGTAGCG-30. Materials and Methods Microarray study Total RNA (10 mg), prepared using an RNeasy Mini kit and Tissue samples RNase-free DNase I (Qiagen), was hybridized to the CodeLink Fifteen pairs of HCC tissues and the corresponding non- human whole genome bioarray (55K; GE Healthcare). We neoplastic liver tissues were obtained from patients who analyzed significant changes in gene expression by Hierarchi- underwent surgical resection at the Chonbuk National Uni- cal Clustering using Significance Analysis of Microarrays versity Hospital, Jeonju, Korea (Supplementary Table S1), and (SAM) software. the Hospital Research Ethics Committee approved the study. Written informed consent was obtained from all patients. Reverse transcription PCR Surgically removed tissues were sampled for histologic diag- Reverse transcription was conducted with total RNA nosis and the remaining tissues were immediately cut into (2 mg), according to the manufacturer's instructions (Super- small pieces, snap-frozen in liquid nitrogen, and stored until Script III Reverse transcriptase; Invitrogen), and the reaction use. All protocols conformed to the ethical guidelines of the mixture was used for the following PCR (ExTaq polymerase; Institutional Review Board. TakaRa). Cell lines and culture conditions Western blot analysis and ELISA The human HCC cell lines Huh7, SNU387, and SNU475, in To obtain secreted IL-8 protein, 80% confluent cultures addition to human foreskin fibroblasts, were purchased of cells were washed with PBS 3 times and incubated in from the Korea Cell Line Bank (Seoul, Korea). The human DMEM for 72 hours in 100-mm culture dishes. Conditioned HCC cell lines HepG2, Hep3B, and SK-Hep1 as well as the media were harvested and concentrated using Vivaspin 6 (5 human fibrosarcoma cell line HT1080 were purchased from kDa molecular weight cut-off; Sartorius-Stedim Biotech). the American Type Culture Collection. The cell lines were For cellular proteins, cells were washed with PBS 3 times maintained in Dulbecco's Modified Eagle's Medium and lysed with lysis buffer (M-PER; Thermo Scientific) (DMEM) supplemented with FBS and antibiotics (Invitro- according to the manufacturer's instructions. Protein con- gen) in a humidified atmosphere of 5% CO2 at 37 C. Human centration was measured with a Bio-Rad Protein Assay. umbilical vein endothelial cells (HUVEC) were obtained Approximately 20 to 30 mg of protein was separated using from Lonza and maintained in EGM2 (Lonza). The 293FT SDS-PAGE and transferred onto nitrocellulose membranes was purchased from Invitrogen and maintained according (Bio-Rad Laboratories). Western blot analysis was carried to the manufacturer's instructions. The cell lines have been out using ECL reagents (GE Healthcare). Glyceraldehyde-3- characterized at the bank by DNA fingerprinting analysis phosphate dehydrogenase (GAPDH) served as a loading using short tandem repeat (STR) markers. All cell lines control. In addition, IL-8 was quantified by ELISA. A total were placed under cryostage after they were purchased of 3 Â 105 cells were plated in each well of a 6-well plate in from the bank and used within 6 months of thawing fresh DMEM containing 10% FBS. After8-hourincubationat37C, vials. cells were washed with PBS 3 times and subsequently incubated in DMEM containing0.1%FBSfor18hours.After Northern blot pretreatment with BAY 11-7082 (a specific inhibitor of IkBa Total RNA was isolated from cells or tissues using TRIzol phosphorylation) or dimethyl sufloxide (vehicle) for 30 min- Reagent (Invitrogen) according to the manufacturer's instruc- utes, cells were further incubated in 10% FBS for 5 hours, and tions. Equal amounts of total RNA (10 mg/lane for tissues and the amount of IL-8 secreted into the media was analyzed 20 mg/lane for cells) were resolved on 1% denaturing formal- with an OptEIA human IL-8 ELISA kit (BD Biosciences) dehyde–agarose
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