Cks1 Expression Research Articles

Aberrant expression of Cks1 and Cks2 contributes to prostate tumorigenesis by promoting proliferation and inhibiting programmed cell death

The mammalian Cks family consists of 2 well-conserved small proteins, Cks1 and Cks2. Cks1 has been shown to promote cell-cycle progression by triggering degradation of p27(kip1). The function of Cks2 in somatic mammalian cells is not well understood although it is required for the first metaphase/anaphase transition during the meiosis. Emerging evidence shows that elevated expression of Cks1 and Cks2 is often found in a variety of tumors, and is correlated with poor survival rate of the patients. Here we demonstrated that expression of Cks1 and Cks2 were elevated in prostate tumors of human and animal models, as well as prostatic cancer cell lines. Forced expression of Cks1 and Cks2 in benign prostate tumor epithelial cells promoted cell population growth. Knockdown of Cks1 expression in malignant prostate tumor cells inhibited proliferation, anchorage-independent growth, and migration activities, whereas knockdown of Cks2 expression induced programmed cell death and inhibited the tumorigenicity. Collectively, the data suggest that elevated expression of Cks1 contributes to the tumorigenicity of prostate tumor cells by promoting cell growth and elevated expression of Cks2 protects the cells from apoptosis. Thus, the finding suggests a novel therapeutic strategy for prostatic cancer based on inhibiting Cks1 and Cks2 activity.

Distinct roles for p107 and p130 in Rb-independent cellular senescence

Telomere attrition, DNA damage and constitutive mitogenic signaling can all trigger cellular senescence in normal cells and serve as a defense against tumor progression. Cancer cells may circumvent this cellular defense by acquiring genetic mutations in checkpoint proteins responsible for regulating permanent cell cycle arrest. A small family of tumor suppressor genes encoding the retinoblastoma susceptibility protein family (Rb, p107, p130) exerts a partially redundant control of entry into S phase of DNA replication and cellular proliferation. Here we report that activation of the p53-dependent DNA damage response has been found to accelerate senescence in human prostate cancer cells lacking a functional Rb protein. This novel form of irradiation-induced premature cellular senescence reinforces the notion that other Rb family members may compensate for loss of Rb protein in the DNA damage response pathway. Consistent with this hypothesis, depletion of p107 potently inhibits the irradiation-induced senescence observed in DU145 cells. In contrast, p130 depletion triggers a robust and unexpected form of premature senescence in unirradiated cells. The dominant effect of depleting both p107 and p130, in the absence of Rb, was a complete blockade of irradiation-induced cellular senescence. Onset of the p107-dependent senescence was temporally associated with p53-mediated stabilization of the cyclin-dependent kinase inhibitor p27 and decreases in c-myc and cks1 expression. These results indicate that p107 is required for initiation of accelerated cellular senescence in the absence of Rb and introduces the concept that p130 may be required to prevent the onset of terminal growth arrest in unstimulated prostate cancer cells lacking a functional Rb allele.

Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCF(Skp2) ubiquitin ligase. In these studies, we explored its roles in estrogen receptor-positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G(1) by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCF(Skp2) target. Heregulin beta1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCF(Skp2) target, p27(Kip1). Remarkably, however, Cks1-depleted cells not only exhibit slowed G(1) progression, but also accumulate in G(2)-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G(2)-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin beta1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer.

Increased SKP2 and CKS1 Gene Expression Contributes to the Progression of Human Urothelial Carcinoma

SKP2 and CKS1 promote aggressive tumor behavior via the regulation of p27 degradation. Our previous DNA microarray analysis of human urothelial carcinoma and normal urothelial epithelium showed that in urothelial carcinoma the 2 most highly up-regulated genes among SKP2-p27 interaction related genes are SKP2 (4.7-fold) and CKS1 (2.2-fold). We hypothesized that SKP2 and CKS1 gene expression is associated with urothelial carcinoma invasiveness and prognosis. A total of 84 urothelial carcinoma specimens from patients with bladder (71) and upper urinary tract (13) cancer were examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study. Real-time reverse transcriptase-polymerase chain reaction showed that the average mRNA expression level of SKP2 and CKS1 significantly correlated with tumor stage, that is superficial vs invasive urothelial carcinoma (SKP2 and CKS1, p<0.001 and 0.006) and grade (p<0.001 and 0.009, respectively). Of the superficial urothelial carcinomas examined the SKP2 and CKS1 expression level was significantly higher in pT1 than in pTa samples (p=0.005 and 0.017, respectively). Immunohistochemical expression patterns of SKP2 and CKS1 also significantly correlated with tumor stage (p<0.001 and 0.048) and grade (p=0.003 and 0.025, respectively). In contrast, p27 expression inversely correlated with tumor stage and grade (p<0.001 and 0.011, respectively). Logistic regression analysis revealed that while SKP2 mRNA expression was a significant dependent predictor of p27 expression (p=0.021), there was no correlation between CKS1 mRNA expression and p27 (p=0.748). Kaplan-Meier curves and log rank tests revealed that the high mRNA expression levels of SKP2 and CKS1 had a significant adverse effect on prognosis (p=0.043 and 0.003, respectively). Our results suggest that SKP2 has a major role in the regulation of p27 degradation and CKS1 has a supporting role for SKP2 function in human urothelial carcinoma.

Myc targets Cks1 to provoke the suppression of p27Kip1, proliferation and lymphomagenesis

Reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1) connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Emu-Myc transgenic mice, p27(Kip1) expression is markedly reduced. We show that the transcription of the Cks1 component of the SCF(Skp2) complex that is necessary for p27(Kip1) ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Emu-Myc B cells, and high levels of Cks1 are also a hallmark of Emu-Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Emu-Myc B cells elevates p27(Kip1) levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27(Kip1) expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.

Gene expression of cyclin-dependent kinase subunit Cks2 is repressed by the tumor suppressor p53 but not by the related proteins p63 or p73

Cks2 proteins are essential components of cyclin/cyclin-dependent kinase complexes and contribute to cell cycle control. We identify Cks2 as a transcriptional target downregulated by the tumor suppressor p53. Cks2 expression was found to be repressed by p53 both at the mRNA and the protein levels. p53 downregulates transcription from the Cks2 promoter in a dose-dependent manner and in all cell types tested. This repression appears to be independent of p53 binding to the Cks2 promoter. In contrast to p53, neither p63 nor p73 proteins can repress Cks2 transcription. Thus p53, rather than its homologues p63 and p73, may contribute to control of the first metaphase/anaphase transition of mammalian meiosis by downregulation of Cks2 expression.

Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling

Large-scale gene expression profiling is an effective strategy for understanding the progression of bladder cancer (BC). The aim of this study was to identify genes that are expressed differently in the course of BC progression and to establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n = 10) or invasive (n = 11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected to microarray analysis. The validity of the microarray results was verified by real-time RT-PCR. Of the 136 up-regulated genes we detected, 21 were present in all 14 BCs examined (100%), 44 in 13 (92.9%), and the other 71 in 12 BCs (85.7%). Of 69 down-regulated genes, 25 were found in all 14 BCs (100%), 22 in 13 (92.9%), and the other 22 in 12 BCs (85.7%). Functional annotation revealed that of the up-regulated genes, 36% were involved in metabolism and 14% in transcription and processing; 25% of the down-regulated genes were linked to cell adhesion/surface and 21% to cytoskeleton/cell membrane. Real-time RT-PCR confirmed the microarray results obtained for the 6 most highly up- and the 2 most highly down-regulated genes. Among the 6 most highly up-regulated genes, CKS2 was the only gene with a significantly greater level of up-regulation in invasive than in superficial BC (p = 0.04). To confirm this result, we subjected all 21 BC samples to real-time PCR assay for CKS2. We found a considerable difference between superficial and invasive BC (p = 0.001). Interestingly, there was a considerable difference between the normal bladder and invasive BC (p = 0.001) and less difference between the normal bladder and superficial BC (p = 0.005). We identified several genes as promising candidates for diagnostic biomarkers of human BC and the CKS2 gene not only as a potential biomarker for diagnosing, but also for staging human BC. This is the first report demonstrating that CKS2 expression is strongly correlated with the progression of human BC.

Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

Levels of cyclins and cyclin-dependent kinase (Cdk) inhibitors are tightly controlled during normal cell proliferation and are frequently dysregulated in cancerous cells. In melanoma, cyclin D1 is highly expressed and downregulation of the Cdk inhibitor, p27(Kip1), is associated with a poor prognosis. Mutant B-RAF is frequently expressed in melanoma and overrides growth factor and matrix adhesion control of cyclin D1 and p27(Kip1) levels in human melanocytes. Here, we demonstrate that p27(Kip1) expression is regulated by multiple mechanisms in melanoma cells. B-RAF regulates p27(Kip1) mRNA abundance independently of cyclin D1. Additionally, B-RAF and cyclin D1 control the levels of S-phase kinase-associated protein 2 (Skp2) that directs ubiquitin-mediated proteolysis of p27(Kip1). The cofactor for Skp2, Cdc kinase subunit 1 (Cks1) controls levels of Skp2 in melanoma cells and acts jointly with Skp2 to regulate p27(Kip1) levels. Importantly, expression of Cks1 is regulated by B-RAF and cyclin D1 at the mRNA level. Reduced Cks1 or Skp2 expression and enhanced p27(Kip1) levels inhibit melanoma cell growth. In summary, p27(Kip1) expression in melanoma is regulated by B-RAF at the mRNA level, and via B-RAF and cyclin D1 control of Cks1/Skp2-mediated proteolysis.

The expression of the ubiquitin ligase subunit Cks1 in human breast cancer

IntroductionLoss of the cell-cycle inhibitory protein p27Kip1 is associated with a poor prognosis in breast cancer. The decrease in the levels of this protein is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits S-phase kinase protein 2 (Skp2) and cyclin-dependent kinase subunit 1 (Cks1). Skp2 was recently found to be overexpressed in breast cancers, but the role of Cks1 in these cancers is unknown. The present study was undertaken to examine the role of Cks1 expression in breast cancer and its relation to p27Kip1 and Skp2 expression and to tumor aggressiveness.MethodsThe expressions of Cks1, Skp2, and p27Kip1 were examined immunohistochemically on formalin-fixed, paraffin-wax-embedded tissue sections from 50 patients with breast cancer and by immunoblot analysis on breast cancer cell lines. The relation between Cks1 levels and patients' clinical and histological parameters were examined by Cox regression and the Kaplan–Meier method.ResultsThe expression of Cks1 was strongly associated with Skp2 expression (r = 0.477; P = 0.001) and inversely with p27Kip1 (r = -0.726; P < 0.0001). Overexpression of Cks1 was associated with loss of tumor differentiation, young age, lack of expression of estrogen receptors and of progesterone receptors, and decreased disease-free (P = 0.0007) and overall (P = 0.041) survival. In addition, Cks1 and Skp2 expression were increased by estradiol in estrogen-dependent cell lines but were down-regulated by tamoxifen.ConclusionThese results suggest that Cks1 is involved in p27Kip1 down-regulation and may have an important role in the development of aggressive tumor behavior in breast cancer.

The prognostic impact of the ubiquitin ligase subunits Skp2 and Cks1 in colorectal carcinoma

Loss of the cell-cycle inhibitory protein p27Kip1 is associated with poor prognosis in colorectal carcinoma. The decrease in p27Kip1 levels is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits S-phase kinase protein (Skp) 2 and cyclin-dependent kinase subunit (Cks) 1. Recently, Skp2 and Cks1 expression were found to be increased in some colorectal carcinomas, but their potential role as prognostic markers for survival is unknown. The present study was undertaken to assess the prognostic value of both Skp2 and Cks1 in colorectal carcinoma. The expression of Skp2, Cks1, and p27Kip1 was examined by immunohistochemistry using highly specific antibodies on formalin-fixed, paraffin-embedded tissue sections from 80 patients with colorectal carcinoma. Overexpression of Skp2 and Cks1 strongly correlated with loss of p27Kip1 and loss of tumor differentiation. A significant decrease in overall survival was observed in patients expressing high Skp2 or Cks1 levels, and in particular, patients with Stage II and III disease. Each protein provided significant additional prognostic information to that given by disease stage, tumor grade, or p27Kip1 expression. Results suggest that overexpression of Skp2 or Cks1 is strongly associated with poor prognosis and may thus be used as prognostic markers for overall survival in colorectal carcinoma.

Role of Cks1 Overexpression in Oral Squamous Cell Carcinomas: Cooperation with Skp2 in Promoting p27 Degradation

Down-regulation of p27 is frequently observed in various cancers due to an enhancement of its degradation. Skp2 is required for the ubiquitination and consequent degradation of p27 protein. Another protein called Cks1 is also required for p27 ubiquitination in the SCF(Skp2) ubiquitinating machinery. In the present study, we examined Cks1 expression and its correlation with p27 in oral squamous cell carcinoma (OSCC) derived from tongue and gingiva. By immunohistochemical analysis, high expression of Cks1 was present in 62% of OSCCs in comparison with 0% of normal mucosae. In addition, 65% of samples with low p27 expression displayed high Cks1 levels. Finally, Cks1 expression was well correlated with Skp2 expression and poor prognosis. To study the role of Cks1 overexpression in p27 down-regulation, we transfected Cks1 with or without Skp2 into OSCC cells. Cks1 transfection could not induce a p27 down-regulation by itself, but both Cks1 and Skp2 transfection strongly induced. Moreover, we inhibited Cks1 expression by small interference RNA (siRNA) in OSCC. Cks1 siRNA transfection induced p27 accumulation and inhibited the growth of OSCC cells. These findings suggest that Cks1 overexpression may play an important role for OSCC development through Skp2-mediated p27 degradation, and that Cks1 siRNA can be a novel modality of gene therapy.

The ubiquitin ligase subunits Skp2 and Cks1 are novel independent prognostic markers for survival in colorectal cancer

Introduction: Loss of the cell-cycle inhibitory protein p27 is associated with aggressive tumor behavior and poor prognosis in colorectal cancer. The decrease in p27 levels is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits Skp2 and Cks1. We recently found that overexpression of Skp2 and Cks1 in colorectal cancer correlated with low p27 levels and poor tumor differentiation. The potential role of Skp2 and Cks1 as independent prognostic markers, however, is unknown. Methods: Tissue samples from 100 patients operated for colorectal cancer at 1997 were subjected to Western blot analysis and immunohistochemistry using highly specific monoclonal antibodies against p27, Skp2 and Cks1. Results were plotted against patients’ characteristics, disease stage and overall survival using Cox analysis and the Kaplan-Meier method. Results: Skp2 and Cks1 expression strongly correlated with overall survival (HR 7.672; p < 0.001 and HR 5.026; p < 0.001, respectively). Thus, high levels of these proteins, alone or in combination, accurately predicted poor prognosis whereas low levels predicted good overall survival rates. The strength of these proteins as independent prognostic markers was also valid after controlling for age, sex, grade and stage. Moreover, after having stratified for p27 levels, both Skp2 and Cks1 expression significantly enhanced the predictive value for survival (HR 4.530; p < 0.001 and HR 4.236; p < 0.001, respectively). The strongest additive effect was observed in patients with stage II disease. Conclusions: Skp2 and Cks1 expression strongly correlate with overall survival and may thus be used as novel prognostic markers in colorectal cancer.