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

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide (1). The major risk factors for HCC include chronic hepatitis from hepatitis B or C virus infection and exposure to carcinogens such as aflatoxin B1 (2). Despite the clinical significance of HCC, we only know the elemental basics of the molecular, cellular, and environmental mechanisms that drive disease pathogenesis, and only limited therapeutic options are available, of which many have negligible clinical benefits (3). Therefore, elucidation of the predominant molecular events underlying hepatocarcinogenesis may help identify new therapeutic targets.

Cks proteins were originally identified as subunits that interact closely with cyclin–cyclin-dependent kinase (CDK) complexes (4, 5). Recently, mammalian Cks1 (CDC28 protein kinase regulatory subunit 1B), located on chromosome 1q21.2, was identified as an essential cofactor of Skp2 in the Skp2-containing Skp1-Cullin1-F-box ubiquitin ligase complex (SCF^Skp2)–mediated ubiquitination of the tumor suppressor protein p27 that leads to the proteasomal degradation of p27 (6–8). The CDK inhibitor p27 is a hallmark of many cancers, and its reduced levels are associated with high aggressiveness and poor prognosis in various malignant tumors (9). Similarly, upregulation of Skp2 and/or Cks1 is significantly associated with poor prognosis in some cancers and, often, inversely related to p27 protein levels (10–15). Among these malignancies, HCC showed a frequent and large gain of 1q, and 1q21-22 was identified as the minimum overlapping amplified region containing candidate oncogenes for hepatocarcinogenesis (16). Moreover, some microarray studies that characterized the global gene expression in human HCC tissues and HCC-derived cell lines showed that Cks1 was upregulated in HCC (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 remodeling, and tumor progression (19). IL-8 secreted from cancer cells plays either autocrine or paracrine roles in the tumor microenvironment of various malignancies, including prostate, colon, and pancreatic cancers (20). Especially in HCC, IL-8 derived from cancer cells was significantly implicated in invasion and metastasis, rather than in tumor angiogenesis (21), and the preoperative serum IL-8 level was suggested as a biomarker of tumor invasiveness and progression (22). However, the relationship between Cks1 and IL-8 has not yet been reported.

In this study, we assessed Cks1 expression in HCC tissues and investigated the role of Cks1 in hepatocarcinogenesis, using genetic manipulation of Cks1 in HCC cell lines. Furthermore, we identified the mechanism of Cks1 oncogenesis in HCC development through analysis of gene expression profile and the relevant signaling pathways.

Fifteen pairs of HCC tissues and the corresponding nonneoplastic liver tissues were obtained from patients who underwent surgical resection at the Chonbuk National University Hospital, Jeonju, Korea (Supplementary Table S1), and the Hospital Research Ethics Committee approved the study. Written informed consent was obtained from all patients. Surgically removed tissues were sampled for histologic diagnosis and the remaining tissues were immediately cut into small pieces, snap-frozen in liquid nitrogen, and stored until use. All protocols conformed to the ethical guidelines of the Institutional Review Board.

The human HCC cell lines Huh7, SNU387, and SNU475, in addition to human foreskin fibroblasts, were purchased from the Korea Cell Line Bank (Seoul, Korea). The human HCC cell lines HepG2, Hep3B, and SK-Hep1 as well as the human fibrosarcoma cell line HT1080 were purchased from the American Type Culture Collection. The cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS and antibiotics (Invitrogen) in a humidified atmosphere of 5% CO₂ at 37°C. Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza and maintained in EGM2 (Lonza). The 293FT was purchased from Invitrogen and maintained according to the manufacturer's instructions. The cell lines have been characterized at the bank by DNA fingerprinting analysis using short tandem repeat (STR) markers. All cell lines were placed under cryostage after they were purchased from the bank and used within 6 months of thawing fresh vials.

Total RNA was isolated from cells or tissues using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Equal amounts of total RNA (10 μg/lane for tissues and 20 μg/lane for cells) were resolved on 1% denaturing formaldehyde–agarose gels and transferred to a Hybond-N membrane (GE Healthcare). The IL-8 probe was prepared from Huh7 total RNA by reverse transcription PCR using the following primers: (sense) 5′-ATGACTTCCAAGCTGGCCG-3′ and (antisense) 5′-TTATGAATTCTCAGCCCTC-3′. Probes for Cks1 and p27 were prepared with the primers used for constructing pLenti-C and pLenti-p27, respectively. Probes were labeled using the Prime-It II Random Primer Labeling Kit (Agilent Technologies) and hybridized in ExpressHyb hybridization solution (Clontech) at 60°C for 4 hours. Filters were washed with 2× SSC–0.05% SDS at room temperature for 30 minutes and with 0.1× SSC–0.1% SDS at 50°C for 30 minutes (1× SSC: 0.15 mol/L NaCl, 0.015 mol/L sodium citrate). mRNA expression was quantified by autoradiography. 18S rRNA served as a normalization control; the primers for probe preparation were as follows: (sense) 5′-GTAACCCGTTGAACCCCATT-3′ and (antisense) 5′-CCATCCAATCGGTAGTAGCG-3′.

Total RNA (10 μg), prepared using an RNeasy Mini kit and RNase-free DNase I (Qiagen), was hybridized to the CodeLink human whole genome bioarray (55K; GE Healthcare). We analyzed significant changes in gene expression by Hierarchical Clustering using Significance Analysis of Microarrays (SAM) software.

Reverse transcription was conducted with total RNA (2 μg), according to the manufacturer's instructions (SuperScript III Reverse transcriptase; Invitrogen), and the reaction mixture was used for the following PCR (ExTaq polymerase; TakaRa).

To obtain secreted IL-8 protein, 80% confluent cultures of cells were washed with PBS 3 times and incubated in DMEM for 72 hours in 100-mm culture dishes. Conditioned media were harvested and concentrated using Vivaspin 6 (5 kDa molecular weight cut-off; Sartorius-Stedim Biotech). For cellular proteins, cells were washed with PBS 3 times and lysed with lysis buffer (M-PER; Thermo Scientific) according to the manufacturer's instructions. Protein concentration was measured with a Bio-Rad Protein Assay. Approximately 20 to 30 μg of protein was separated using SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). Western blot analysis was carried out using ECL reagents (GE Healthcare). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. In addition, IL-8 was quantified by ELISA. A total of 3 × 10⁵ cells were plated in each well of a 6-well plate in DMEM containing 10% FBS. After 8-hour incubation at 37°C, cells were washed with PBS 3 times and subsequently incubated in DMEM containing 0.1% FBS for 18 hours. After pretreatment with BAY 11-7082 (a specific inhibitor of IκBα phosphorylation) or dimethyl sufloxide (vehicle) for 30 minutes, cells were further incubated in 10% FBS for 5 hours, and the amount of IL-8 secreted into the media was analyzed with an OptEIA human IL-8 ELISA kit (BD Biosciences) according to the manufacturer's instructions.

Cultured cells were incubated in DMEM containing 0.1% FBS in 6-well plates (2 × 10⁴ cells per well, in triplicate) for 18 hours at 37°C in 5% CO₂, and subsequently cultured for the indicated times in DMEM containing 10% FBS. The number of viable cells was counted using trypan blue exclusion assay.

Cultured cells were incubated for 18 hours in DMEM containing 0.1% FBS. On the day of the assay, we coated the top of Transwell polycarbonate membrane inserts (6.5-mm diameter and 8-μm pores; Corning) with Matrigel (40 μg/well; BD Biosciences). After adding DMEM containing 10% FBS into the lower wells, single-cell suspensions in DMEM containing 0.1% FBS were placed on the membrane filters (1 × 10⁵ cells/100 μL/well) and incubated for 23 hours at 37°C in 5% CO₂. Filters were washed, and the cells on the upper surface were manually removed with cotton swabs. Cells that had invaded and adhered to the lower surface were fixed with methanol for 15 minutes and stained with 0.1% (w/v) crystal violet for 15 minutes. The filters were extracted with 30% acetic acid. The cells that had invaded were indirectly quantified by determining the absorbance at 595 nm.

The cell suspension (2 × 10⁴ cells in 2 mL of DMEM supplemented with 10% FBS and 0.3% agar) was plated onto 60-mm dishes containing 5 mL of DMEM with 10% FBS and 0.5% agar. The dishes were incubated at 37°C in 5% CO₂ for 21 or 28 days. Colonies were observed under a phase-contrast microscope.

A total of 1 × 10⁶ Huh7 cells were plated in a 6-well plate on the day of transfection and were incubated for 8 hours. Cells were subsequently cotransfected with 1 μg of a luciferase reporter vector [pNF-κB-Luc (Stratagene), pAP1-Luc (Stratagene), or pIL8-Luc] and 1 μg of RSV-LacZ (a kind gift from Jae-B. Kim, Seoul National University, Seoul, Korea), using Lipofectamine 2000 (Invitrogen). Transfected cells were incubated in fresh medium. After 24 hours, both luciferase and β-galactosidase assays were conducted according to the manufacturer's instructions (Promega). The β-galactosidase assay was conducted to normalize transfection efficiencies. pIL8-Luc was constructed by inserting the minimal IL-8 promoter (∼350 bp) into the pGL3-Basic vector (Promega). The IL-8 promoter region was obtained by PCR using Huh7 genomic DNA prepared by a Wizard SV Genomic DNA Purification System (Promega) and the following primers: (sense) 5′-GATGCTAGCGATAATTCACCAAATTGTGGAG-3′ and (antisense) 5′-GATCTCGAGGTTTACACACAGTGAGATGGT-3′.

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 = 5/group) were s.c. injected with 5 × 10⁶ cells in the proximal midline of the dorsa. We measured tumor sizes every 2 to 3 days and estimated tumor volumes as width² × length × 0.52.

Data are presented as the mean ± SD or SEM. Statistical significance was calculated using the Student t test. Values of P < 0.05 were considered statistically significant.

Cks1 overexpression occurs in various cancers, including oral squamous cell, lung, and gastric carcinomas. To investigate the potential role of Cks1 in hepatocarcinogenesis, we determined the level of Cks1 in HCC tissues and cell lines by Northern blot analysis. Cks1 expression was substantially higher in HCC tissues than in the corresponding nontumor liver tissues in 13 of the 15 pairs examined (Fig. 1A; 5 representative cases; Supplementary Fig. S1 for all cases). Furthermore, Cks1 was overexpressed in human HCC cell lines, including Huh7, HepG2, Hep3B, SK-Hep1, SNU387, and SNU475, relative to the levels in human normal liver tissue and nontumorigenic HUVECs and fibroblasts (Fig. 1B). These results indicate that Cks1 overexpression may be associated with hepatocarcinogenesis.

To investigate the role of Cks1 in the regulation of cellular proliferation of HCC cells, we conducted ectopic overexpression and knockdown of Cks1 in Huh7 cells using lentiviral expression systems. Northern blot analysis confirmed the stable overexpression or knockdown of Cks1 in Huh7 cells. As shown in Fig. 2A (top), the ectopically overexpressed Cks1 mRNA transcript (V: the 6× histidine-tagged form in the C-terminus) was clearly discernible from the endogenous form. In vitro proliferation assays revealed that Cks1 overexpression significantly enhanced cellular proliferation (Fig. 2A, bottom). In Cks1 knockdown, both short hairpin RNAs (shRNA; shC-#1 and shC-#2) efficiently suppressed Cks1 mRNA expression compared with control cells (Fig. 2B, top), and the resulting Cks1-knockdown Huh7 cells showed significantly lower proliferation ability than control cells (Fig. 2B, bottom). Cks1 was also positively correlated with cellular proliferation of SK-Hep1 cells (Supplementary Fig. S2). These results imply that Cks1 plays an important role in HCC cell proliferation.

Because Cks1 is known as a cofactor of Skp2 in the SCF^Skp2-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 in the regulation of p27 degradation (Fig. 3A). Although CDK1, 2, and 4 were unaffected by Cks1 knockdown, cyclin E was slightly increased, probably because of the decrease in its SCF^Skp2-mediated ubiquitination leading to proteasomal degradation (23). Cks1 knockdown also increased active caspase 3, suggesting that this knockdown may induce cellular apoptosis. To investigate the mechanism of p27 increase by Cks1 knockdown, we treated cells with 10 μmol/L MG132 (a specific proteasome inhibitor) for 7 hours, which clearly abolished the Cks1 knockdown–induced p27 increase in Huh7 cells (Fig. 3B). This indicates that the Cks1 knockdown–induced p27 increase was likely due to decreased proteasomal degradation.

Because p27 is an established tumor suppressor, we next assessed the effects of Cks1 knockdown on Huh7 cell tumorigenicity. In a soft agar assay, Cks1-knockdown clones (shC7, shC9, and shC11) showed substantially impaired colony-forming abilities as compared with control cells (Fig. 4A), indicating 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 (Supplementary Fig. S3). Cks1 knockdown significantly 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 tumors in vivo by more than 50% as compared with tumors from mock cells (Fig. 4C; Supplementary Fig. S4 for results of immunohistochemical analysis). Thus, Cks1 overexpression may lead to hepatocarcinogenesis by directly enhancing the cellular tumorigenic potential.

To obtain genome-wide insights on Cks1 function(s) in hepatocarcinogenesis, we conducted a microarray study with mock (shM2) and Cks1-knockdown (shC9) Huh7 clones. Cks1 knockdown affected gene expression by more than 2 times in 1,432 of the total 52,486 gene probes, with 108 increased and 1,324 decreased (Supplementary Tables S2 and S3). Among the affected genes, IL-8 was one of the most downregulated genes (9.8-fold), and this was further confirmed by both Northern and Western blot analyses (Fig. 5A). To investigate whether IL-8 downregulation resulted from the loss of Cks1 function as an 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 (Supplementary Fig. S6). These results indicate that Cks1 may regulate IL-8 expression in Huh7 cells through an unknown function other than an Skp2 cofactor in p27 ubiquitination.

To investigate the contribution of IL-8 to the Cks1 oncogenesis, we carried out in vitro tumorigenicity assays with IL-8–knockdown Huh7 cells. Stable IL-8–knockdown Huh7 cells were established and confirmed by Western and Northern 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 the expression in the parental Huh7 cells. This indicates that the Cks1 knockdown–derived IL-8 reduction was not a nonspecific effect from the shRNA expression process itself in the vector system. IL-8 knockdown significantly inhibited both in vitro proliferation and invasion abilities of Huh7 cells, compared with control cells (Fig. 6B). Further evaluation showed that IL-8 also plays a critical role in the anchorage-independent Huh7 cell growth (Fig. 6C). These results suggest that IL-8 significantly contributes to hepatocarcinogenesis by mediating the oncogenic activities of Cks1.

The promoter of the IL-8 gene contains several transcription factor–binding sequences and, among these factors, NF-κB and AP1 play central roles in regulating IL-8 gene expression (24). To investigate the regulatory mechanism of IL-8 expression by Cks1, we determined the effect of Cks1 knockdown on luciferase expression under the control of NF-κB enhancer, AP1 enhancer, or minimal IL-8 promoter including both enhancers and found that Cks1 knockdown significantly decreased IL-8 promoter activity in Huh7 cells, which seemed to be derived mainly by the decreased activity of NF-κB, not AP1 (Fig. 7A; Supplementary Fig. S7). To confirm the involvement of the NF-κB pathway in the regulation of IL-8 expression, we measured IL-8 secretion in the presence or absence of BAY 11-7082 (an inhibitor of IκBα phosphorylation). BAY 11-7082 substantially decreased IL-8 expression in a dose-dependent manner, as determined by ELISA (Fig. 7B). Moreover, Western blot analysis showed that Cks1 knockdown significantly decreased the kinase subunits, both IKKα and IKKβ, and the regulatory subunit, IKKγ, of the IKK complex, whereas p27 overexpression decreased only IKKγ (Fig. 7C). This implies that the Cks1 knockdown–induced IKKγ decrease resulted from the Skp2 dysfunction-induced p27 increase. Furthermore, we examined NF-κB p65 phosphorylation status important for its transcriptional activity, but did not find any differences between control and Cks1-knockdown cells (Supplementary Fig. S8). Next, we compared IκBα phosphorylation, the rate-limiting step in NF-κB activation, between control and Cks1-knockdown cells, which showed that Cks1 knockdown significantly reduced IκBα phosphorylation compared with control cells (Fig. 7D). Therefore, it can be hypothesized that the proteasomal degradation of IκBα via IKK complex-mediated phosphorylation was suppressed by Cks1 knockdown through the downregulation of IKK complex, eventually leading to NF-κB inhibition and, thereby, IL-8 downregulation. These results revealed a novel function of Cks1 in the regulation of the NF-κB pathway.

The heterogeneous phenotypic and genetic traits of individuals with HCC and the wide range of risk factors associated with HCC have led to HCC being classified as a complex disease (25). In this article, we present a novel tumor-promoting function of Cks1 in hepatocarcinogenesis.

Cks1 reportedly correlates with proliferation of lymphoid, oral squamous cancer, and prostate cancer cells (26–28). Furthermore, Cks1 overexpression is noted in numerous cancers, including multiple myeloma, lung, gastric, and oral carcinomas (13, 27, 29, 30). Recently, Cks1, along with Skp2 and/or p27, was hypothesized to be a potential prognostic marker for some cancers (12, 27, 31, 32). In the present study, Cks1 expression was higher in human HCC tissues than in adjacent nontumor liver tissues. In addition, Cks1 was correlated with the in vitro HCC cell proliferation, and knocking down of Cks1 in Huh7 cells elicited an increase in the p27 protein. MG132 treatment of HCC cells revealed that Cks1 participates in the proteasomal degradation of p27. Consistent with the fact that p27 is a well-known tumor suppressor, Cks1 knockdown significantly inhibited the tumorigenic potential of Huh7 cells both in vitro and in vivo. The in vitro inhibitory effect of Cks1 knockdown on the tumorigenicity of cancer cells was also reported with prostate cancer (28). Recently, the Skp2–Cks1 complex was reported to be involved in the degradation of several tumor suppressor proteins other than p27 (DUSP1, RASSF1A, p130, etc.) in HCC cells (14, 33). Therefore, it may not be excluded that suppressor proteins other than p27 may also participate in the anti-HCC activities of Cks1 knockdown. Our results indicate that Cks1 overexpression may contribute to hepatocarcinogenesis by enhancing the cellular tumorigenic potential, which may be partly imparted by participating in ubiquitination-mediated proteasomal degradation of p27 as a cofactor of Skp2.

In addition to the CDK- or Skp2-dependent function, Cks1 may also function as a transcription activator for maintaining efficient transcriptional activation in Saccharomyces cerevisiae (34–36). This finding prompted us to obtain genome-wide insights on Cks1 function(s) in HCC cells. This analysis revealed that Cks1 knockdown in Huh7 cells significantly changed the expression of genes known to participate in various cellular processes, including gene transcription, cell death, apoptosis, signal transduction, stress response, immune response, cell cycle, and so on. Notably, genes related to proliferation, angiogenesis, inflammation, invasion, and metastasis were significantly downregulated by Cks1 knockdown, implying that Cks1 may play significant and diverse roles in hepatocarcinogenesis. Among these genes, IL-8 was one of the most downregulated genes in Huh7 cells, according to a microarray study and Northern and Western blot analyses.

Autocrine or paracrine roles of IL-8 in tumor growth, invasion, angiogenesis, and/or metastasis have been reported in various tumor types, such as skin, stomach, prostate, hepatic, and pancreatic cancers (37–40). In Ras-transformed cells, Ras-dependent IL-8 secretion has been shown to contribute to tumor growth by initiating tumor-associated inflammation and neovascularization as a paracrine, and not an autocrine, factor (41). In HCC patients, serum IL-8 levels significantly correlated with tumor size and tumor stage, and its preoperative levels were hypothesized to be a useful biologica marker of tumor invasiveness and progression (22). Akiba and colleagues reported IL-8 expression in HCC tissues whose cancer cells were the main source of IL-8, and hypothesized that IL-8 may have an important function in cancer invasion and metastasis, rather than in tumor angiogenesis (21). In the present study, Cks1 knockdown–induced IL-8 downregulation in Huh7 cells was not apparently associated with either Skp2 dysfunction or p27 accumulation, thereby suggesting a novel function of Cks1 in hepatocarcinogenesis. Moreover, IL-8 knockdown significantly suppressed the tumorigenicity of Huh7 cells, indicating that IL-8 may be an important autocrine factor for HCC cells and a key mediator of the Skp2- and p27-independent oncogenic function of Cks1 in hepatocarcinogenesis.

IL-8 is transcriptionally regulated mainly by NF-κB and AP1 (24). In the present study, Cks1 regulated IL-8 expression in Huh7 cells through NF-κB (not AP1)-mediated transcriptional control, although AP1 is reportedly required for constitutive IL-8 gene expression in hepatoma cells (42). IκB proteolysis, the rate-limiting step in NF-κB activation, is regulated by the IKK complex, which is composed of 2 catalytic subunits, IKKα and IKKβ, and 1 regulatory subunit, IKKγ (43). Li and colleagues reported that IKKβ-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype similar to that of mice deficient in both NF-κB p65 and NF-κB1 (p50/p105; ref. 44). Constitutive activation of NF-κB is seen in several cancers, including prostate cancer, fibrolamellar HCC, and glioblastoma (45–47). Arsura and colleagues reported that constitutive activation of NF-κB in Ras-transformed rat liver epithelial cells was dependent on IKKα and IKKβ activation (48). In addition, Factor and colleagues described an important role of the IKK complex in the constitutive NF-κB activation, which led to HCC development in double transforming growth factor-α and c-myc transgenic mice (49). In the current study, Cks1 controlled IKKα and IKKβ expression independently of p27, in contrast to the p27-dependent control of IKKγ expression, in HCC cells. The p27-induced IKKγ downregulation failed to elicit IL-8 decrease, indicating that IKKα and IKKβ, rather than IKKγ, may play an important role in the NF-κB–mediated transcriptional regulation of IL-8 in HCC cells. However, additional studies are necessary to further investigate the mechanism of how Cks1 regulates the expression of each IKK subunit and the implication of p27-mediated IKKγ regulation in Cks1 function.

In summary, we showed a significant role of Cks1 in hepatocarcinogenesis and identified IL-8 as a key autocrine mediator of the oncogenic function of Cks1. Cks1 regulated IL-8 expression in an Skp2-independent mode, probably through the specific control of IKKα and IKKβ in the NF-κB pathway. Collectively, our study uncovered a novel function of Cks1 in the NF-κB pathway regulation, which may be different from the Skp2 cofactor function in p27 ubiquitination. Thus, Cks1 may be a promising therapeutic target in HCC management.

No potential conflicts of interest were disclosed.

The authors thank J.-H. Lee and H.-K. Joo for skillful technical assistance in animal experiments.

This study was financially supported in part by the Green Cross Corporation (Grant TD03-001-00).

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 with 18 U.S.C. Section 1734 solely to indicate this fact.