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 overexpression 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. 2011 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 2011 American Association for Cancer Research. and investigated the role of Cks1 in hepatocarcinogenesis,

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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 3 105 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 gels and transferred to a Hybond-N mem- according to the manufacturer's instructions. brane (GE Healthcare). The IL-8 probe was prepared from Huh7 total RNA by reverse transcription PCR using the fol- Cell proliferation assay lowing primers: (sense) 50-ATGACTTCCAAGCTGGCCG-30 and Cultured cells were incubated in DMEM containing 0.1% (antisense) 50-TTATGAATTCTCAGCCCTC-30. Probes for Cks1 FBS in 6-well plates (2 104 cells per well, in triplicate) for and p27 were prepared with the primers used for constructing 18 hours at 37 Cin5%CO2, and subsequently cultured for pLenti-C and pLenti-p27, respectively. Probes were labeled the indicated times in DMEM containing 10% FBS. The using the Prime-It II Random Primer Labeling Kit (Agilent number of viable cells was counted using trypan blue Technologies) and hybridized in ExpressHyb hybridization exclusion assay. solution (Clontech) at 60C for 4 hours. Filters were washed with 2 SSC–0.05% SDS at room temperature for 30 minutes In vitro invasion assay and with 0.1 SSC–0.1% SDS at 50C for 30 minutes (1 SSC: Cultured cells were incubated for 18 hours in DMEM con- 0.15 mol/L NaCl, 0.015 mol/L sodium citrate). mRNA expres- taining 0.1% FBS. On the day of the assay, we coated the top of

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Transwell polycarbonate membrane inserts (6.5-mm diameter Results and 8-mm pores; Corning) with Matrigel (40 mg/well; BD Biosciences). After adding DMEM containing 10% FBS into Cks1 is overexpressed in human HCC the lower wells, single-cell suspensions in DMEM containing Cks1 overexpression occurs in various cancers, including 0.1% FBS were placed on the membrane filters (1 105 cells/ oral squamous cell, lung, and gastric carcinomas. To investi- 100 mL/well) and incubated for 23 hours at 37 Cin5%CO2. gate the potential role of Cks1 in hepatocarcinogenesis, we Filters were washed, and the cells on the upper surface were determined the level of Cks1 in HCC tissues and cell lines by manually removed with cotton swabs. Cells that had invaded Northern blot analysis. Cks1 expression was substantially and adhered to the lower surface were fixed with methanol for higher in HCC tissues than in the corresponding nontumor 15 minutes and stained with 0.1% (w/v) crystal violet for 15 liver tissues in 13 of the 15 pairs examined (Fig. 1A; 5 repre- minutes. The filters were extracted with 30% acetic acid. The sentative cases; Supplementary Fig. S1 for all cases). Further- cells that had invaded were indirectly quantified by determin- more, Cks1 was overexpressed in human HCC cell lines, ing the absorbance at 595 nm. including Huh7, HepG2, Hep3B, SK-Hep1, SNU387, and SNU475, relative to the levels in human normal liver tissue In vitro colony-forming assay and nontumorigenic HUVECs and fibroblasts (Fig. 1B). These The cell suspension (2 104 cells in 2 mL of DMEM results indicate that Cks1 overexpression may be associated supplemented with 10% FBS and 0.3% agar) was plated onto with hepatocarcinogenesis. 60-mm dishes containing 5 mL of DMEM with 10% FBS and in vitro 0.5% agar. The dishes were incubated at 37 Cin5%CO2 for 21 Cks1 correlates with proliferation rates or 28 days. Colonies were observed under a phase-contrast of HCC cells microscope. To investigate the role of Cks1 in the regulation of cellular proliferation of HCC cells, we conducted ectopic overexpres- Promoter activity assay sion and knockdown of Cks1 in Huh7 cells using lentiviral fi A total of 1 106 Huh7 cells were plated in a 6-well plate expression systems. Northern blot analysis con rmed the on the day of transfection and were incubated for 8 hours. stable overexpression or knockdown of Cks1 in Huh7 cells. Cells were subsequently cotransfected with 1 mgofalucif- As shown in Fig. 2A (top), the ectopically overexpressed Cks1 erase reporter vector [pNF-kB-Luc (Stratagene), pAP1-Luc mRNA transcript (V: the 6 histidine-tagged form in the C- (Stratagene), or pIL8-Luc] and 1 mgofRSV-LacZ(akindgift terminus) was clearly discernible from the endogenous form. In vitro from Jae-B. Kim, Seoul National University, Seoul, Korea), proliferation assays revealed that Cks1 overexpression fi using Lipofectamine 2000 (Invitrogen). Transfected cells signi cantly enhanced cellular proliferation (Fig. 2A, bottom). were incubated in fresh medium. After 24 hours, both In Cks1 knockdown, both short hairpin RNAs (shRNA; shC-#1 fi luciferase and b-galactosidase assays were conducted and shC-#2) ef ciently suppressed Cks1 mRNA expression according to the manufacturer's instructions (Promega). compared with control cells (Fig. 2B, top), and the resulting fi The b-galactosidase assay was conducted to normalize Cks1-knockdown Huh7 cells showed signi cantly lower pro- transfection efficiencies. pIL8-Luc was constructed by liferation ability than control cells (Fig. 2B, bottom). Cks1 was inserting the minimal IL-8 promoter (350 bp) into the also positively correlated with cellular proliferation of SK-Hep1 pGL3-Basic vector (Promega). The IL-8 promoter region was cells (Supplementary Fig. S2). These results imply that Cks1 obtained by PCR using Huh7 genomic DNA prepared by a plays an important role in HCC cell proliferation. Wizard SV Genomic DNA Purification System (Promega) and the following primers: (sense) 50-GATGCTAGCGA- TAATTCACCAAATTGTGGAG-30 and (antisense) 50-GAT- CTCGAGGTTTACACACAGTGAGATGGT-30.

Implantation of tumors Male 5-week-old athymic BALB/c nu/nu nude mice (Charles River Laboratories Japan Inc.) were used for xenograft studies. All mice were fed a commercial diet, given water ad libitum, and subjected to a 12-hour light/12-hour dark cycle. Mice (n ¼ 5/group) were s.c. injected with 5 106 cellsintheproximalmidlineofthedorsa.Wemeasured tumor sizes every 2 to 3 days and estimated tumor volumes 2 as width length 0.52. Figure 1. Cks1 is overexpressed in human HCC. A, Cks1 expression in human HCC tissues. Primary HCC tissues (T) and the matched Statistical analysis surrounding nontumor liver tissues (N) of 15 patients were analyzed for Cks1 expression by Northern blot analysis. Five representative cases are Data are presented as the mean SD or SEM. Statistical – ﬁ t shown (#1 5). B, Cks1 expression in human HCC cell lines, compared signi cance was calculated using the Student test. Values of with human normal liver tissue and nontumorigenic cells. 18S rRNA P < 0.05 were considered statistically signiﬁcant. served as a loading control.

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Figure 2. Cks1 correlates with in vitro proliferation rates of Huh7 ABHuh7 Mock Cks1 Huh7 shMock shC-#1 shC-#2 cells. Construction of (A) Cks1- Cks1 V Cks1 overexpressing and (B) Cks1- C knockdown Huh7 cells and in vitro 18S 18S proliferation assays (V, lentivirally overexpressed form; C, endogenous form). Cks1 level was 7e+5 1e+6 + determined by Northern blot Start Start 8 days 6e+5 8 days analysis. shC-#1 and shC-#2 8e+5 indicate shRNA I and II, respectively. 5e+5 For proliferation assay, cells were 6e+5 4e+5 incubated for 8 days in DMEM containing10% FBS afterstarvation 3e+5 4e+5 ** in DMEM containing 0.1% FBS for 18 hours at 37 Cin5%CO2. The Cell number Cell number 2e+5 * number of viable cells was counted 2e+5 1e+5 using trypan blue exclusion assay. 0 The mean SD (n ¼ 3) is shown. 0 þ Start Mock Cks1 Start shMock shC-#1 shC-#2 , P < 0.05; , P < 0.0005; , P < 0.002 (vs. Mock and shMock, respectively).

Cks1 knockdown increases p27 expression and mock (shM2) and Cks1-knockdown (shC9) Huh7 clones. Cks1 decreases HCC cell tumorigenicity knockdown affected gene expression by more than 2 times in Because Cks1 is known as a cofactor of Skp2 in the SCFSkp2- 1,432 of the total 52,486 gene probes, with 108 increased and mediated ubiquitination of p27, we examined the effect of Cks1 knockdown on p27 level in Huh7 cells by Western blot analysis. Cks1 knockdown increased p27 levels, indicating a role of Cks1 A Huh7 shM shC7 shC9 shC11 in the regulation of p27 degradation (Fig. 3A). Although CDK1, 2, and 4 were unaffected by Cks1 knockdown, cyclin E was Cks1 slightly increased, probably because of the decrease in its Skp2 SCF -mediated ubiquitination leading to proteasomal deg- p27 radation (23). Cks1 knockdown also increased active caspase 3, suggesting that this knockdown may induce cellular apoptosis. Cyclin E To investigate the mechanism of p27 increase by Cks1 knockdown, we treated cells with 10 mmol/L MG132 (a speciﬁc proteasome inhibitor) for 7 hours, which clearly abolished the CDK1 Cks1 knockdown–induced p27 increase in Huh7 cells (Fig. 3B). This indicates that the Cks1 knockdown–induced p27 increase CDK2 was likely due to decreased proteasomal degradation. Because p27 is an established tumor suppressor, we next CDK4 assessed the effects of Cks1 knockdown on Huh7 cell tumorigenicity. In a soft agar assay, Cks1-knockdown clones (shC7, Active caspase 3 shC9, and shC11) showed substantially impaired colony-forming abilities as compared with control cells (Fig. 4A), indicating GAPDH that Cks1 contributes to the anchorage-independent growth of Huh7 cells. Furthermore, SK-Hep1 cells exhibited a positive correlation between Cks1 and in vitro colony-forming activity B (Supplementary Fig. S3). Cks1 knockdown signiﬁcantly MG132 (10 μmol/L, 7 h) decreased the in vitro invasion ability of Huh7 cells stimulated –+ by 10% FBS (Fig. 4B). In mouse xenograft tumor experiments, Cks1 knockdown substantially inhibited the growth of solid M2 M4 C7 C9 M2 M4 C7 C9 tumors in vivo by more than 50% as compared with tumors from mock cells (Fig. 4C; Supplementary Fig. S4 for results of p27 immunohistochemical analysis). Thus, Cks1 overexpression may lead to hepatocarcinogenesis by directly enhancing the GAPDH cellular tumorigenic potential.

Cks1 regulates IL-8 expression in an Skp2-independent Figure 3. Cks1 knockdown increases p27 in Huh7 cells. Western blot – þ mode analysis of cell-cycle related proteins (A) and p27 in the presence ( )or absence () of MG132 (B). GAPDH served as a loading control. shM To obtain genome-wide insights on Cks1 function(s) in indicates mock cells; shC7, shC9, and shC11 indicate Cks1-knockdown hepatocarcinogenesis, we conducted a microarray study with clones; and MG132, a speciﬁc proteasome inhibitor.

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Western blot analyses (Fig. 5A). To investigate whether IL-8 A downregulation resulted from the loss of Cks1 function as an Huh7 shMock Skp2 cofactor, we prepared Skp2-knockdown and p27-overexpressing Huh7 cells, as confirmed by Western blot analysis (Fig. 5B and C, left). However, neither Skp2-knockdown nor p27-overexpressing Huh7 cells showed IL-8 downregulation (Fig. 5B and C, right). This may be partly attributable to the fact that, unlike Cks1 knockdown, either p27 overexpression or Skp2 knockdown was not accompanied by Cks1 decrease, as assessed by Western blot analysis (Supplementary Fig. S5). In contrast to IL-8, osteopontin (OPN), another protein substantially downregulated by Cks1 knockdown in Huh7 cells, showed p27-dependent expression in Huh7 cells, showing a different regulation mechanism from that of IL-8 (Supplemen- shC7 shC9 shC11 tary Fig. S6). These results indicate that Cks1 may regulate IL-8 expression in Huh7 cells through an unknown function other B than an Skp2 cofactor in p27 ubiquitination. 1.0 IL-8 contributes to the oncogenic activities of Cks1 0.8 To investigate the contribution of IL-8 to the Cks1 oncogenesis, we carried out in vitro tumorigenicity assays with IL-8– 0.6 knockdown Huh7 cells. Stable IL-8–knockdown Huh7 cells were established and confirmed by Western and Northern 0.4 blot analyses (Fig. 6A). Neither the empty vector (pLL3.7B) nor the nonspecific shRNA-expressing vector (pLL3.7B) affected * IL-8 expression (shM and shM, respectively), compared with Relative invasion 0.2 ** the expression in the parental Huh7 cells. This indicates that – 0.0 the Cks1 knockdown derived IL-8 reduction was not a non- fi NS Huh7shMock shC7 shC9 shC11 speci c effect from the shRNA expression process itself in the vector system. IL-8 knockdown significantly inhibited both in C vitro proliferation and invasion abilities of Huh7 cells, com- 2,500 pared with control cells (Fig. 6B). Further evaluation showed shM2 that IL-8 also plays a critical role in the anchorage-independent

) shM4 3 Huh7 cell growth (Fig. 6C). These results suggest that IL-8 2,000 shC7 signiﬁcantly contributes to hepatocarcinogenesis by mediat- shC9 1,500 shC11 ing the oncogenic activities of Cks1.

1,000 Cks1 controls IL-8 expression through the unique k ** regulation of the NF- B pathway 500 The promoter of the IL-8 gene contains several transcription

Tumor volume (mm volume Tumor * factor–binding sequences and, among these factors, NF-kB and ** 0 AP1 play central roles in regulating IL-8 gene expression (24). 0102030 To investigate the regulatory mechanism of IL-8 expression by Days after implantation Cks1, we determined the effect of Cks1 knockdown on luciferase expression under the control of NF-kB enhancer, AP1 enhancer, or minimal IL-8 promoter including both enhancers Figure 4. Cks1 knockdown decreases the tumorigenicity of Huh7 cells. A, and found that Cks1 knockdown significantly decreased IL-8 soft agar assay. In vitro colony-forming activities were compared after 21 promoter activity in Huh7 cells, which seemed to be derived n ¼ in vitro days of incubation ( 3). B, invasion assay. Invasion was mainly by the decreased activity of NF-kB, not AP1 (Fig. 7A; stimulated by 10% FBS for 23 hours after 18-hour starvation in DMEM Supplementary Fig. S7). To confirm the involvement of the NF- containing 0.1% FBS at 37 Cin5%CO2. The mean SD (n ¼ 3) is shown. , P < 0.0005 (vs. shMock). C, in vivo tumor growth. Cells were implanted kB pathway in the regulation of IL-8 expression, we measured s.c. in the proximal midline of the dorsa of nude mice. The mean SEM IL-8 secretion in the presence or absence of BAY 11-7082 (an (n ¼ 5) is shown. , P < 0.0005; , P < 0.02 [vs. shM2 (mock clone 2)]. inhibitor of IkBa phosphorylation). BAY 11-7082 substantially decreased IL-8 expression in a dose-dependent manner, as determined by ELISA (Fig. 7B). Moreover, Western blot anal- 1,324 decreased (Supplementary Tables S2 and S3). Among the ysis showed that Cks1 knockdown significantly decreased affected genes, IL-8 was one of the most downregulated genes the kinase subunits, both IKKa and IKKb, and the regu- (9.8-fold), and this was further confirmed by both Northern and latory subunit, IKKg, of the IKK complex, whereas p27

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Figure 5. Cks1 regulates IL-8 A Huh7 shM1 shM2 shC7 shC9 shC11 Huh7 shM1 shM2 shC7 shC9 shC11 expression in an Skp2-independent Cks1 Cks1 mode in Huh7 cells. A, effect of Cks1 knockdown on IL-8 IL-8 GAPDH expression. Northern (left) and Western (right) blot analysis. B and 18S IL-8(s) C, effect of p27 overexpression (B) or Skp2 knockdown (C) on IL-8 Huh7 Mock 27-1 27-2 27-4 27-5 27-6 expression. Each genetic B Huh7 Mock p27 p27 modiﬁcation was conﬁrmed by p27 Western blot analysis (left). IL-8(s) GAPDH indicates IL-8 protein secreted into GAPDH the serum-free culture media (right). IL-8(s) The 27-1, 27-2, 27-4, 27-5, and 27- 6 indicate p27-overexpressing Huh7 shM S2 S3 S4 S5 S6 S7 S8 C Huh7 shM shSKP2 clones. S2 to S8 indicate Skp2- Skp2 Skp2 knockdown clones. 18S rRNA and GAPDH served as loading controls GAPDH GAPDH for Northern and Western blotting, IL-8(s) respectively.

overexpression decreased only IKKg (Fig. 7C). This implies that Discussion the Cks1 knockdown–induced IKKg decrease resulted from the Skp2 dysfunction-induced p27 increase. Furthermore, we The heterogeneous phenotypic and genetic traits of indivi- examined NF-kB p65 phosphorylation status important for duals with HCC and the wide range of risk factors associated its transcriptional activity, but did not ﬁnd any differences with HCC have led to HCC being classiﬁed as a complex disease between control and Cks1-knockdown cells (Supplementary (25). In this article, we present a novel tumor-promoting Fig. S8). Next, we compared IkBa phosphorylation, the rate- function of Cks1 in hepatocarcinogenesis. limiting step in NF-kB activation, between control and Cks1- Cks1 reportedly correlates with proliferation of lymphoid, knockdown cells, which showed that Cks1 knockdown signif- oral squamous cancer, and prostate cancer cells (26–28). icantly reduced IkBa phosphorylation compared with control Furthermore, Cks1 overexpression is noted in numerous can- cells (Fig. 7D). Therefore, it can be hypothesized that the cers, including multiple myeloma, lung, gastric, and oral car- proteasomal degradation of IkBa via IKK complex-mediated cinomas (13, 27, 29, 30). Recently, Cks1, along with Skp2 and/or phosphorylation was suppressed by Cks1 knockdown through p27, was hypothesized to be a potential prognostic marker for the downregulation of IKK complex, eventually leading to NF- some cancers (12, 27, 31, 32). In the present study, Cks1 kB inhibition and, thereby, IL-8 downregulation. These results expression was higher in human HCC tissues than in adjacent revealed a novel function of Cks1 in the regulation of the NF-kB nontumor liver tissues. In addition, Cks1 was correlated with pathway. the in vitro HCC cell proliferation, and knocking down of Cks1

1.4e+6 A B Start Huh7 shM shM* shIL-8 shI1 shI2 1.2e+6 8 days IL-8(s) 1.0e+6 Figure 6. IL-8 contributes to the 8.0e+5 tumorigenicity of Huh7 cells. A, Huh7 shM shM* shIL-8 shI1 shI2 shI3 shI4 construction of IL-8–knockdown 6.0e+5 * Huh7 cells [Western (top) and IL-8 4.0e+5 Northern blotting (bottom)]. B, in Cell number 18S 2.0e+5 vitro proliferation (top) and invasion 0.0 (bottom) assays. The mean SD n ¼ StartHuh7 shM* shIL-8 ( 3) is shown. NS, not stimulated. C, soft agar assay. In vitro colony- C 0.8 forming activities were compared after 28 days of incubation (n ¼ 3). Huh7 shM shM* shIL-8 0.6 shM indicates nonspeciﬁc shRNA- expressing mock cells. shI1–4 0.4 indicate IL-8–knockdown clones. ** ** þ , P < 0.002; , P < 0.000002; 0.2 + , P < 0.005 (vs. shM ).

Relative invasion 0.0 Huh7NSshM* shIL-8 shI-1 shI-2

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AC1.2 IL-8 NF-κB 1.0 AP1 Huh7 shM1 shM2 shC7 shC9 shC11 Figure 7. Cks1 controls IL-8 Cks1 expression via NF-kB pathway in 0.8 Huh7 cells. A, promoter activity p27 assay. shM2 indicates mock clone 0.6 ** IL-8(s) 2 and shC9 indicates Cks1- 0.4 knockdown clone 9. Each data * IKKα point was normalized by the 0.2 corresponding control data from β

Relative luciferase activity Relative luciferase IKK 0.0 shM2. B, effect of BAY 11-7082 on shM2 shC9 IKKγ IL-8 expression. Cells were treated ﬁ B with BAY 11-7082 (a speci c 5e+4 IκBα inhibitor of IkBa phosphorylation) for 5 hours at concentrations of 0 4e+4 NF-κB (p50) (B0), 25 (B25), and 50 mmol/L (B50), NF-κB (p65) and secreted IL-8 was measured by 3e+4 ELISA. The mean SD (n ¼ 3) is GAPDH shown. , P < 0.002; , P < 0.01; 2e+4 * þ, P < 0.000005 (vs. shM2 and B0, IIL-8 (pg/mL) respectively). C, effects of Cks1 1e+4 + knockdown (top) or p27 Huh7 Mock 27-1 27-2 27-4 overexpression (bottom) on the 0 p27 components of the NF-kB pathway Huh7 B0 B25 B50 (Western blotting). D, Western blot IKKα analysis of IkBa phosphorylation D IKKβ when stimulated with 10% FBS for Huh7 shM1 shM2 shC7 shC9 shC11 15 minutes after 18-hour starvation p-IκBα IKKγ in DMEM containing 0.1% FBS. IκBα IκBα

GAPDH GAPDH

in Huh7 cells elicited an increase in the p27 protein. MG132 response, cell cycle, and so on. Notably, genes related to treatment of HCC cells revealed that Cks1 participates in the proliferation, angiogenesis, inflammation, invasion, and proteasomal degradation of p27. Consistent with the fact that metastasis were significantly downregulated by Cks1 knock- p27 is a well-known tumor suppressor, Cks1 knockdown down, implying that Cks1 may play significant and diverse roles significantly inhibited the tumorigenic potential of Huh7 cells in hepatocarcinogenesis. Among these genes, IL-8 was one of both in vitro and in vivo. The in vitro inhibitory effect of Cks1 the most downregulated genes in Huh7 cells, according to a knockdown on the tumorigenicity of cancer cells was also microarray study and Northern and Western blot analyses. reported with prostate cancer (28). Recently, the Skp2–Cks1 Autocrine or paracrine roles of IL-8 in tumor growth, invasion, complex was reported to be involved in the degradation of angiogenesis, and/or metastasis have been reported in various several tumor suppressor proteins other than p27 (DUSP1, tumor types, such as skin, stomach, prostate, hepatic, and RASSF1A, p130, etc.) in HCC cells (14, 33). Therefore, it may not pancreatic cancers (37–40). In Ras-transformed cells, Ras- be excluded that suppressor proteins other than p27 may also dependent IL-8 secretion has been shown to contribute to participate in the anti-HCC activities of Cks1 knockdown. Our tumor growth by initiating tumor-associated inflammation and results indicate that Cks1 overexpression may contribute to neovascularization as a paracrine, and not an autocrine, factor hepatocarcinogenesis by enhancing the cellular tumorigenic (41). In HCC patients, serum IL-8 levels significantly correlated potential, which may be partly imparted by participating in with tumor size and tumor stage, and its preoperative levels ubiquitination-mediated proteasomal degradation of p27 as a were hypothesized to be a useful biologica marker of tumor cofactor of Skp2. invasiveness and progression (22). Akiba and colleagues In addition to the CDK- or Skp2-dependent function, Cks1 reported IL-8 expression in HCC tissues whose cancer cells may also function as a transcription activator for maintaining were the main source of IL-8, and hypothesized that IL-8 may efficient transcriptional activation in Saccharomyces cerevisiae have an important function in cancer invasion and metastasis, (34–36). This finding prompted us to obtain genome-wide rather than in tumor angiogenesis (21). In the present study, insights on Cks1 function(s) in HCC cells. This analysis Cks1 knockdown–induced IL-8 downregulation in Huh7 cells revealed that Cks1 knockdown in Huh7 cells significantly was not apparently associated with either Skp2 dysfunction or changed the expression of genes known to participate in p27 accumulation, thereby suggesting a novel function of Cks1 various cellular processes, including gene transcription, cell in hepatocarcinogenesis. Moreover, IL-8 knockdown significant- death, apoptosis, signal transduction, stress response, immune ly suppressed the tumorigenicity of Huh7 cells, indicating that

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Lee et al.

IL-8 may be an important autocrine factor for HCC cells and a gate the mechanism of how Cks1 regulates the expression of key mediator of the Skp2- and p27-independent oncogenic each IKK subunit and the implication of p27-mediated IKKg function of Cks1 in hepatocarcinogenesis. regulation in Cks1 function. IL-8 is transcriptionally regulated mainly by NF-kB and AP1 In summary, we showed a significant role of Cks1 in hepa- (24). In the present study, Cks1 regulated IL-8 expression in tocarcinogenesis and identified IL-8 as a key autocrine medi- Huh7 cells through NF-kB (not AP1)-mediated transcriptional ator of the oncogenic function of Cks1. Cks1 regulated IL-8 control, although AP1 is reportedly required for constitutive expression in an Skp2-independent mode, probably through IL-8 gene expression in hepatoma cells (42). IkB proteolysis, the the specific control of IKKa and IKK b in the NF-kB pathway. rate-limiting step in NF-kB activation, is regulated by the IKK Collectively, our study uncovered a novel function of Cks1 in complex, which is composed of 2 catalytic subunits, IKKa and the NF-kB pathway regulation, which may be different from the IKKb, and 1 regulatory subunit, IKKg (43). Li and colleagues Skp2 cofactor function in p27 ubiquitination. Thus, Cks1 may reported that IKKb-deficient mice die at mid-gestation from be a promising therapeutic target in HCC management. uncontrolled liver apoptosis, a phenotype similar to that of mice deficient in both NF-kB p65 and NF-kB1 (p50/p105; Disclosure of Potential Conflict of Interest ref. 44). Constitutive activation of NF-kB is seen in several fi cancers, including prostate cancer, brolamellar HCC, and No potential conflicts of interest were disclosed. glioblastoma (45–47). Arsura and colleagues reported that constitutive activation of NF-kB in Ras-transformed rat liver Acknowledgments epithelial cells was dependent on IKKa and IKKb activation (48). In addition, Factor and colleagues described an important The authors thank J.-H. Lee and H.-K. Joo for skillful technical assistance in animal experiments. role of the IKK complex in the constitutive NF-kB activation, which led to HCC development in double transforming growth factor-a and c-myc transgenic mice (49). In the current study, Grant Support Cks1 controlled IKKa and IKKb expression independently of This study was financially supported in part by the Green Cross Corporation p27, in contrast to the p27-dependent control of IKKg expres- (Grant TD03-001-00). sion, in HCC cells. The p27-induced IKKg downregulation The costs of publication of this article were defrayed in part by the payment of advertisement failed to elicit IL-8 decrease, indicating that IKKa and IKKb, page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rather than IKKg, may play an important role in the NF-kB– mediated transcriptional regulation of IL-8 in HCC cells. Received December 2, 2010; revised July 21, 2011; accepted August 17, 2011; However, additional studies are necessary to further investi- published OnlineFirst September 14, 2011.

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

Eun-Kyoung Lee, Dae-Ghon Kim, Jang-Seong Kim, et al.

Cancer Res Published OnlineFirst September 14, 2011.

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