Overexpression Of Skp2 Research Articles

Characterization of Gene Amplification–Driven SKP2 Overexpression in Myxofibrosarcoma: Potential Implications in Tumor Progression and Therapeutics

Myxofibrosarcoma remains obscure in molecular determinants of clinical aggressiveness, for which we elucidated implications of SKP2 amplification. Array comparative genomic hybridization was applied on samples and cell lines (NMFH-1 to OH931) to search causal genes of tumor progression. SKP2 gene dosage was determined in 82 independent tumors for clinical correlates. Stable SKP2 knockdown was achieved in myxofibrosarcoma cells to assess its oncogenic attributes and candidate mediators in prometastatic function. Pharmacologic assays were evaluated in vitro and in vivo for the therapeutic relevance of bortezomib. DNA gains frequently involved 5p in which three amplicons were differentially overrepresented in samples behaving unfavorably, encompassing mRNA-upregulated TRIO, SKP2, and AMACR genes. Detected in NMFH-1 cells and 38% of tumors, SKP2 amplification was associated with SKP2 immunoexpression and adverse prognosticators and independently predictive of worse outcomes. Nevertheless, SKP2-expressing OH931 cells and 14% of such tumors lacked gene amplification. Knockdown of SKP2 suppressed proliferation, anchorage-independent growth, migration, and invasion of sarcoma cells and downregulated motility-promoting genes, including ITGB2, ACTN1, IGF1, and ENAH. In vitro, bortezomib downregulated SKP2 expression at the mRNA level with p27(kip1) accumulation, induced caspase activation, and decreased cell viability in myxofibrosarcoma cells but not in fibroblasts. In vivo, bortezomib inhibited growth of NMFH-1 xenografts, the cells of which displayed decreased SKP2 expression but increased p27(kip1) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). As a predominant mechanism driving protein overexpression, SKP2 amplification confers tumor aggressiveness in myxofibrosarcoma. The sensitivity of myxofibrosarcoma cells to bortezomib with SKP2-repressing effect indicates the potentiality of ubiquitin-proteasome pathway as a therapeutic target.

Stat3 induces oncogenic Skp2 expression in human cervical carcinoma cells

Dysregulated Skp2 function promotes cell proliferation, which is consistent with observations of Skp2 over-expression in many types of human cancers, including cervical carcinoma (CC). However, the molecular mechanisms underlying elevated Skp2 expression have not been fully explored. Interleukin-6 (IL-6) induced Stat3 activation is viewed as crucial for multiple tumor growth and metastasis. Here, we demonstrate that Skp2 is a direct transcriptional target of Stat3 in the human cervical carcinoma cells. Our data show that IL-6 administration or transfection of a constitutively activated Stat3 in HeLa cells activates Skp2 mRNA transcription. Using luciferase reporter and ChIP assays, we show that Stat3 binds to the promoter region of Skp2 and promotes its activity through recruiting P300. As a result of the increase of Skp2 expression, endogenous p27 protein levels are markedly decreased. Thus, our results suggest a previously unknown Stat3–Skp2 molecular network controlling cervical carcinoma development.

The key role of ubiquitination and sumoylation in signaling and cancer: a research topic

EDITORIAL article Front. Oncol., 05 December 2012 | https://doi.org/10.3389/fonc.2012.00187

Overexpression of SKP2 promotes the radiotherapy resistance of esophageal squamous cell carcinoma

Overexpression of SKP2 promotes the radiotherapy resistance of esophageal squamous cell carcinoma

Regulation of Hematopoietic Cell Engraftment and Myeloid Differentiation by the E3 Ubiquitin Ligase F-Box Protein SKP2,

Abstract 3397The precise molecular mechanisms that coordinate, at cellular level, the cell fate decisions of self-renewal and differentiation during normal adult hematopoiesis are not yet well defined. SKP2 protein is necessary for ubiquitination and degradation of several cell cycle inhibitors, such as p27Kip1, p21Cip1 and p57Kip2. SKP2 overexpression causes quiescent cells to enter the cell cycle and its downregulation is critical for cell cycle arrest. SCFSKP2 is undoubtedly the major ubiquitin ligase regulating the abundance of cell cycle regulatory proteins at the G1-S transition. Thus, we sought to explore its role in regulating cell cycle entry of hematopoietic cells.By using a mouse model of SKP2 deletion, we show that SKP2 inactivation retards entry of HSC and progenitors into cell cycle, resulting in enhanced HSC quiescence, increased pool size and increased HSC maintenance. These effects were accompanied by increased cellular levels of p27Kip1, p21Cip1 and p57Kip2 in HSC and progenitors. Competitive repopulation assays and serial bone marrow (BM) transplantations showed that loss of SKP2 improved hematopoietic engraftment at long-term. Conversely, the slower cell cycle entry induced by SKP2 deletion greatly impaired hematopoietic engraftment at short-term. We also analyzed the effect of SKP2 deletion on myeloid differentiation. Although the loss of SKP2 did not affect significantly the representation of Gr1+Mac1+ cells in the BM of SKP2 null mice at steady-state, SKP2 null BM lineage negative cells showed an accelerated myeloid differentiation in vitro. Similar results were obtained by using a shRNA anti-SKP2. This effect required p27Kip1 expression, as deletion of SKP2 did not increased differentiation in a p27Kip1 null background, and was, surprisingly, cell cycle-independent.Taken together, these results demonstrate a previously unrecognized role for SKP2 in regulating rates of hematopoietic engraftment and myeloid differentiation in the BM. The identification of SKP2 as a physiologic regulator of cell cycle progression of HSC and progenitors has significant implications in the biology of hematopoietic stem cells. We believe that these results may contribute to a better understanding of the dynamics of hematopoietic recovery during BM transplantation, as well as of the mechanisms involved in the HSC exhaustion driving BM failure syndromes and may provide new insights for therapeutic applications. Disclosures:No relevant conflicts of interest to declare.

Overexpression of SKP2 Promotes the Radiation Resistance of Esophageal Squamous Cell Carcinoma

SKP2 is the substrate recognition subunit of the SCF(SKP2) ubiquitin ligase complex. It is implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase (CDK) inhibitor p27(KIP1) and positively regulates the G(1)/S transition. Overexpression of SKP2 has been found in many kinds of tumors. In the present study, we found that SKP2 expression levels increased in esophageal squamous cell carcinoma tissues. Elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). Moreover, a significantly negative correlation was found between SKP2 expression and the survival of patients who received radiotherapy (P < 0.05). At the molecular level, induced expression of SKP2 promoted the radioresistance of EC9706 cells. Knockdown of SKP2 expression sensitized cancer cells to radiation, and a wobble mutant of SKP2 that was resistant to SKP2 siRNA was able to rescue this effect. Increased or decreased expression levels of SKP2 had effects on Rad51 expression after irradiation. These results demonstrate for the first time that overexpression of SKP2 was correlated with the increased radioresistance of esophageal squamous cell carcinoma. Elevated expression of SKP2 promoted the radioresistance of cancer cells, and this effect was mediated at least in part by the Rad51 pathway.

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

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-κB 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-κB regulator IκBα, which led to NF-κB activation and IL-8 expression, through a p27-independent regulation of IκB kinase complex components. Collectively, our findings support the hypothesis that Cks1 supports hepatocarcinogenesis by NF-κB-mediated regulation of IL-8 expression, broadening the function of Cks1 in cancer beyond its role as a Skp2 cofactor in p27 ubiquitination.

Transcriptional Regulation of S Phase Kinase-associated Protein 2 by NR4A Orphan Nuclear Receptor NOR1 in Vascular Smooth Muscle Cells

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27(KIP1) through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation.

Androgen suppresses proliferation of castration‐resistant LNCaP 104‐R2 prostate cancer cells through androgen receptor, Skp2, and c‐Myc

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoration of testosterone levels could suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets were implanted. Of 33 tumors, 24 adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen. We then studied the molecular mechanism underlying the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G(1) cell cycle arrest through reduction of S-phase kinase-associated protein 2 (Skp2) and c-Myc, and induction of p27(Kip1). 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with a slower growth rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27(Kip1). Nuclear AR and prostate-specific antigen expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype; knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells through AR, Skp2, and c-Myc.

Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation

Sirtuin deacetylases and FOXO (Forkhead box, class O) transcription factors have important roles in many biological pathways, including cancer development. SIRT1 and SIRT2 deacetylate FOXO factors to regulate FOXO function. Because acetylation and ubiquitination both modify the ɛ-amino group of lysine residues, we investigated whether FOXO3 deacetylation by SIRT1 or SIRT2 facilitates FOXO3 ubiquitination and subsequent proteasomal degradation. We found that SIRT1 and SIRT2 promote FOXO3 poly-ubiquitination and degradation. Proteasome-inhibitor treatment prevented sirtuin-induced FOXO3 degradation, indicating that this process is proteasome dependent. In addition, we demonstrated that E3 ubiquitin ligase subunit Skp2 binds preferentially to deacetylated FOXO3. Overexpression of Skp2 caused poly-ubiquitination of FOXO3 and degradation, whereas knockdown of Skp2 increased the amount of FOXO3 protein. We also present evidence that SCF-Skp2 ubiquitinates FOXO3 directly in vitro. Furthermore, mutating four known acetylated lysine residues (K242, K259, K290 and K569) of FOXO3 into arginines to mimic deacetylated FOXO3 resulted in enhanced Skp2 binding but with inhibition of FOXO3 ubiquitination; this suggests that some or all of these four lysine residues are likely the sites for ubiquitination. In the livers of mice deficient in SIRT1, we detected increased expression of FOXO3, indicating SIRT1 regulates FOXO3 protein levels in vivo. Furthermore, we found that the elevation of SIRT1 and Skp2 expression in malignant PC3 and DU145 prostate cells is responsible for the downregulation of FOXO3 protein levels in these cells. Taken together, our data support the notion that deacetylation of FOXO3 by SIRT1 or SIRT2 facilitates Skp2-mediated FOXO3 poly-ubiquitination and proteasomal degradation.

Skp2 Overexpression Is Associated with Loss of BRCA2 Protein in Human Prostate Cancer

BRCA2 (breast cancer 2, early onset) is a tumor suppressor gene that confers increased susceptibility for prostate cancer (PCa). Previous in vitro experiments demonstrated that Skp2, an E3 ubiquitin ligase aberrantly overexpressed in PCa, is involved in the proteolytic degradation of BRCA2 in PCa cells, suggesting that the BRCA2-Skp2 interaction may play a role in prostate tumorigenesis. Herein, we investigated BRCA2 and Skp2 expression during PCa development using a prostate TMA. Although luminal and basal benign prostate epithelium exhibited moderate to strong nuclear BRCA2 immunostaining, the intensity and number of positive nuclei decreased significantly in high-grade prostatic intraepithelial neoplasia and PCa. Decreased frequency and intensity of nuclear BRCA2 labeling were inversely correlated with Skp2 expression in high-grade prostatic intraepithelial neoplasia and PCa. To functionally assess the effects of BRCA2 and Skp2 expression on prostate malignant transformation, we overexpressed Skp2 in normal immortalized prostate cells. Skp2 overexpression reduced BRCA2 protein and promoted cell growth and migration. A similar phenotype was observed after reduction of BRCA2 protein levels using specific BRCA2 small-interfering RNA. Forced BRCA2 expression in Skp2-overexpressing stable transfectants inhibited the migratory and growth properties by >60%. These results show that loss of BRCA2 expression during prostate tumor development is strongly correlated with both migratory behavior and cancer growth and include Skp2 as a BRCA2 proteolytic partner in vivo.

Correlation of Skp2 overexpression to prognosis of patients with nasopharyngeal carcinoma from South China

S-phase kinase-associated protein 2 (Skp2), which plays a role in cell cycle regulation, is commonly overexpressed in a variety of human cancers and associated with poor prognosis. However, its role in nasopharyngeal carcinoma (NPC) is not well understood. In this study, we examined the clinical significance of Skp2, with a particular emphasis on overall survival (OS) and disease-free survival (DFS), in NPC cases in South China, where NPC is an epidemic. Additionally, we explored the function of Skp2 in maintaining a cancer stem cell-like phenotype in NPC cell lines. Skp2 expression was assessed for 127 NPC patients using tissue microarrays and immunohistochemistry and analyzed together with clinicopathologic features, OS, and DFS. Skp2 expression was detectable, or positive, in 75.6% of patients. Although there was no correlation between Skp2 and any clinicopathologic factor, Skp2 expression significantly portended inferior OS (P = 0.013) and DFS (P = 0.012). In the multivariate model, Skp2 expression remained significantly predictive of poor OS [P = 0.009, risk ratio (RR) = 4.06] and DFS (P = 0.008, RR = 3.56), and this was also true for clinical stage (P = 0.012 and RR=3.201 for OS; P = 0.002 and RR=1.94 for DFS) and sex (P = 0.016 and RR=0.31 for OS; P = 0.006 and RR = 0.27 for DFS). After Skp2 knockdown, a colony formation assay was used to evaluate the self-renewal property of stem-like cells in the NPC cell lines CNE-1 and CNE-2. The colony formation efficiency in CNE-1 and CNE-2 cells was decreased. In Skp2-transfected CNE-1 and CNE-2 cells, side population (SP) proportion was increased as detected by flow Cytometry. Skp2 is an independent prognostic marker for OS and DFS in NPC. Skp2 may play a role in maintaining the cancer stem cell-like phenotype of NPC cell lines.

CDC28 protein kinase regulatory subunit 1B (CKS1B) expression and genetic status analysis in oral squamous cell carcinoma.

CKS1B is a member of the highly conserved cyclin kinase subunit 1 (CKS1) protein family which interacts with cyclin-dependent kinases and plays a critical role in cell cycle progression. In oral squamous cell carcinoma (OSCC), as in other malignancies, CKS1B overexpression has been correlated with reduced survival. To our knowledge, no studies evaluating the genetic status of CKS1B gene in OSCC have been reported. Herein, genetic and protein status of CKS1B were analyzed by immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) techniques in a series of primary OSCC (n=51) and lymph node OSCC metastases samples (n=14). The observed results were compared with those obtained in either inflammatory (oral lichen planus [OLP]) (n=13) and premalignant oral mucosal lesions (oral leukoplakia) (n=16). A significant CKS1B overexpression was observed in OSCC and lymph node metastases samples than in OLP and oral leukoplakia (mean 70% vs 35%, p<0.001). CKS1B overexpression correlated with p27 loss of expression (p=0.0013) and SKP2 overexpression (p<0.00). FISH study disclosed statistical differences in both gene amplifications and gains between samples corresponding to OSCC and metastases from those of OLP and leukoplakia (p<0.001). Amplifications were present in 53% of OSCC samples and 33% of lymph node metastases vs 14% of oral leukoplakia and 0% of OLP biopsy specimens (p=0.002). Polysomies of chromosome 1 were seen in 46% of OSCC, 33% of ganglionar metastases, 14% of oral leukoplakia and 10% of OLP (p=0.036). Correlation of CKS1B over-expression and gains (both polysomies and amplifications) determined by FISH was statistically significant (p<0.001). Our results indicate that a high CKS1B expression is a common finding in primary OSCC which correlates with p27 low expression and SKP2 overexpression. This phenomenon may be due either to numerical (chromosome 1 polysomy) or structural (amplifications) CKS1B genetic abnormalities. This phenotypical and cytogenetic profile is not observed in premalignant or inflammatory oral mucosal lesions.

Clinical relevance of SKP2 alterations in metastatic melanoma

In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2.

A Novel Target of Mizoribine Inhibiting Mesangial Cell Proliferation: S Phase Kinase-Associated Protein 2

Background/Aims: Mizoribine (MZR) can inhibit mesangial cell (MC) proliferation, but the mechanism remains unknown. In this study, we investigated the inhibitory effect of MZR on MC proliferation via a cell cycle regulatory protein-dependent mechanism. Methods: We investigated the effect of MZR on MC proliferation and expression of cell cycle regulatory proteins such as cyclin D1, cyclin-dependent kinase 2 and p27^kip1 in primary cultured rat MCs. We further focused on analyzing the effects of MZR on S phase kinase-associated protein 2 (Skp2), which played a crucial role in p27^kip1 degradation. Results: MZR effectively inhibited MC proliferation in primary cultured rat MCs, reduced the expression of cyclin D1 and cyclin-dependent kinase 2, while it dramatically increased the protein level of p27^kip1, maintained the nucleus location of p27^kip1 and induced G1/S arrest. In contrast to the protein level, MZR produced no changes in p27^kip1 mRNA abundance. MZR impaired p27^kip1 degradation through downregulating the expression of Skp2 and this effect was not dependent on its inhibition on DNA synthesis. Skp2 overexpression abolished MZR-induced p27^kip1 accumulation. Conclusion: These results suggested that MZR-induced p27^kip1 accumulation was at least partly mediated by Skp2, and that Skp2 might be a novel target of MZR inhibiting MC proliferation.

Cumulative alterations of p27Kip1‐related cell‐cycle regulators in the development of endometriosis‐associated ovarian clear cell adenocarcinoma

To identify the key cell-cycle dysregulations in the development of endometriosis-associated ovarian clear cell adenocarcinoma (CCA). Expression of p27(Kip1)-interacting cell-cycle regulators, such as p27(Kip1) itself, Skp2, cyclin-dependent kinase subunit 1 (Cks1), cyclin A and cyclin E, and Ki67 labelling index (LI), were analysed by immunohistochemistry in 23 CCAs with 36 endometriotic or atypical endometriotic lesions adjacent to CCA from a cohort of 23 patients, and in 31 cases of solitary endometriosis. The cell-cycle regulators examined were overexpressed (Skp2, Cks1, cyclin A and cyclin E; P < 0.01, each) or down-regulated (p27(Kip1), P = 0.044) significantly more frequently in the CCAs than in the adjacent endometriosis. The frequency of Skp2 overexpression was significantly higher in atypical endometriosis than in endometriosis, and the frequency of Skp2 and cyclin A overexpression was significantly higher in CCA than in atypical endometriosis (P < 0.01, each). Mean Ki67 LI increased from endometriosis (8.4%) through atypical endometriosis (21.4%) to CCA (46.9%), with statistical significance between each component (P < 0.01, each). The frequency of cell-cycle regulator expression and mean Ki67 LIs were not significantly different between solitary endometriosis and endometriosis adjacent to CCA. Alteration of the p27(Kip1)-interacting cell-cycle regulators appeared strongly involved in the progression of endometriosis-associated ovarian clear cell carcinogenesis through increasing cell proliferative activity.

Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2

Skp2 is the substrate binding subunit of the SCFSkp2 ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APCCdh1, which targets Skp2 for degradation in early G1 and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCFSkp2 ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.

Regulation of Skp2 Expression and Activity and Its Role in Cancer Progression

The regulation of cell cycle entry is critical for cell proliferation and tumorigenesis. One of the key players regulating cell cycle progression is the F-box protein Skp2. Skp2 forms a SCF complex with Skp1, Cul-1, and Rbx1 to constitute E3 ligase through its F-box domain. Skp2 protein levels are regulated during the cell cycle, and recent studies reveal that Skp2 stability, subcellular localization, and activity are regulated by its phosphorylation. Overexpression of Skp2 is associated with a variety of human cancers, indicating that Skp2 may contribute to the development of human cancers. The notion is supported by various genetic mouse models that demonstrate an oncogenic activity of Skp2 and its requirement in cancer progression, suggesting that Skp2 may be a novel and attractive therapeutic target for cancers.

CKS1B Mediates SKP2/p27Kip1-Independent Myeloma Cell Survival and Disease Progression through Activation of MEK/ERK and JAK/STAT3 Signaling Pathways.

Abstract 126We previously reported that CKS1B may influence myeloma (MM) cell growth and survival through SKP2/p27Kip1-dependent and -independent mechanisms. However, there is still no direct evidence to prove that CKS1B has a role in MM cell proliferation and disease progression. The present study was performed to establish its functional role and define CKS1B-mediated SKP2/p27Kip1-independent down-stream signaling pathways. CKS1B was over-expressed in OCI-MY5 and XG1 MM cell lines by lentivirus. Western blots confirmed CKS1B over-expression. Cells were cultured in medium containing 1% fetal bovine serum for 7 days. CKS1B-transfection resulted in increased cell proliferation compared to empty-vector (EV)-transfected controls. We also examined the role of CKS1B in myeloma resistance to the general used chemotherapeutic drugs, such as bortezomib (5nM), doxorubicin (100nM) and etoposide (100nM). Untreated cells and empty-vector (EV)-transfected cells with or without drug treatments served as controls. Significant less inhibition of cell growth and cell death was observed after drug treatment in CKS1B-transfected cells compared with controls (P <.05). To screen down-stream signaling pathways associated with cell growth and survival in OCI-MY5, MS28PE and XG-1 cells were transfected with specific CKS1B-shRNA, which resulted in decreased phosphorylation of MEK1/2, ERK1/2, STAT3, MCL1 and BCL2 compared to wild-type and control cells, transfected with scrambled CKS1B-shRNA. To confirm these results, we examined the alteration of STAT3, MEK/ERK and BCL2 signaling pathways in OCI-MY5 and XG1 cells after forced over-expression of CKS1B. Increased levels of p-MEK1/2, p-ERK1/2, p-STAT3, MCL1 and p-BCL2 were observed compared to the EV-transfected controls, confirming that CKS1B activates STAT3, MEK/ERK and BCL2 signaling pathways. In Contrast, SKP2 over-expression or p27Kip1 inhibition resulted in inhibition of STAT3 and MEK/ERK pathways with no remarkable changes inBCL2. Further investigation showed that BCL2 is a downstream target of MEK/ERK signaling. Stimulation of STAT3, MEK/ERK and BCL2 signaling pathways only partially abrogated MM cell death and growth inhibition induced by CKS1B knockdown. Targeting either the STAT3, MEK/ERK or BCL2 signaling pathway with specific inhibitors induced significant MM cell death and growth inhibition in CKS1B-over-expressing MM cells; their combination had a synergistic effect on cell death and growth inhibition. Our findings provide a rationale for targeting STAT3 and MEK/ERK/BCL2 signaling in the therapy of aggressive CKS1B-overexpressing MM, which shows increased proliferation and drug-resistance Disclosures:No relevant conflicts of interest to declare.

CKS1B Overexpression Implicates Clinical Aggressiveness of Hepatocellular Carcinomas but Not p27Kip1 Protein Turnover: an Independent Prognosticator with Potential p27Kip1-Independent Oncogenic Attributes?

Through data mining the Stanford Microarray Database, the CKS1B transcript was found to be frequently upregulated in hepatocellular carcinomas (HCCs) with low alpha-fetal protein (AFP) expression. Together with SKP2, CKS1B is known to implicate p27(Kip1) protein turnover promoting cell-cycle progression. CKS1B, p27(Kip1), and SKP2 were immunostained in 75 HCCs and correlated with clinicopathological features, local recurrence-free survival (LRFS), and overall survival (OS). Silencing of CKS1B and SKP2 with interference short-hairpin RNA (shRNA) was performed in SK-Hep1 and Hep-3B cell lines. Immunohistochemically, increased CKS1B and SKP2, and attenuated p27(Kip1) were all associated with tumor multiplicity (P < 0.05) and increasing American Joint Committee on Cancer (AJCC) stage (P < 0.05). Overexpression of CKS1B significantly correlated with advanced Okuda stages (P = 0.048) and SKP2 overexpression (P = 0.047). Neither CKS1B nor SKP2 was inversely related to p27(Kip1), which was reinforced by no alteration in p27(Kip1) abundance in HCC-derived cells with CKS1B or SKP2 silencing. Both CKS1B overexpression (P = 0.0011 and P = 0.0017) and p27(Kip1) attenuation (P = 0.0079 and P = 0.0085) were predictive of OS and LRFS, respectively, while SKP2 overexpression was associated with worse OS alone (P = 0.0043). Combined assessment of CKS1B and p27(Kip1) was able to robustly distinguish three prognostically different groups (P < 0.0001). In multivariate comparison, CKS1B overexpression represented the strongest independent adverse prognosticator [OS, P = 0.0235, hazard ratio (HR): 4.193; LRFS, P = 0.0204, HR: 4.262], followed by p27(Kip1) attenuation (OS, P = 0.0320, HR: 2.553; LRFS, P = 0.0262, HR: 2.533). CKS1B protein overexpression in HCCs is implicated in clinical aggressiveness but not in p27(Kip1) turnover, implying presence of p27(Kip1)-independent oncogenic attributes. The combined assessment of CKS1B and p27(Kip1) immunoexpressions effectively risk-stratifies HCCs with different prognoses, which may aid in the management of this deadly malignancy.