Overexpression Of Skp2 Research Articles

Skp2 Regulates the Expression of MMP-2 and MMP-9, and Enhances the Invasion Potential of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck regions and accounts for more than 90% of cancers in the oral cavity. S-phase kinase-associated protein-2 (Skp2) is a member of the F-box protein family and the substrate recognition subunit of the Skp1-Cullin-F box protein E3 ubiquitin ligase complex. Skp2 is oncogenic and overexpressed in human cancers. The aims of the present study were to determine the clinicopathological significance of Skp2 in OSCC and clarify its function in OSCC cell lines in vitro. Multiple methods including immunohistochemical staining, RT-PCR, western blotting, migration and invasion assays, and siRNA transfection were employed in order to investigate the clinicopathological significance and molecular function of Skp2 in OSCC. The overexpression of Skp2 was more frequent in OSCC than in the normal oral epithelium. It was also more frequently detected in cancers with higher grades according to the T classification, N classification, and pattern of invasion. The high-Skp2 expression group had a significantly poorer prognosis, at 30.1%, than that of the low-expression group, at 63.0%. The downregulation of Skp2 decreased migration and invasion potentials in HSC3 cells. Moreover, the suppression of Skp2 reduced the enzyme activities of MMP-2 and MMP-9 via Sp1. Skp2 may be a prognostic factor in OSCC patients, and may also play crucial roles in the migration and invasion potentials of OSCC cells.

E3 Ubiquitin Ligases as Molecular Targets in Human Oral Cancers.

The ubiquitin-proteasome pathway is involved in various biological processes. Several oncogenic E3 ligases target tumor suppressor proteins for ubiquitin-mediated degradation. Alternatively, some other E3 ligases play as a tumor suppressor specifically targeting oncogene products. Deregulation of these E3 ligases induces unbalance between oncogenic signal and tumor suppressor pathway and leads to cellular transformation, tumor growth and metastasis in various human malignancies including oral, and head and neck cancers. Facilitated degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) has been observed in oral, and head and neck cancers, and is correlated with their poor prognosis. SCF(Skp2), KPC complex, Pirh2 and CRL4(DDB2-Artemis) have been reported as E3 ligases targeting p27(Kip1) for degradation. In oral cancers, it is reported that overexpression of Skp2 and Pirh2 is associated with poor prognosis. Thus, chemical inhibitors against these E3 ligases are applicable for oral cancer therapy. Some potential compounds that inhibit E3 ligase activity of SCF(Skp2) have been reported. Moreover, the HECT-type E3 ligase WWP family and Smurf1 are also involved in the development and growth of human oral cancers. Therefore, small molecule inhibitors against HECT-type E3 ligases are discussed as anti-oral cancer drugs.

The unpredictable consequences of CDKN1B/p27Kip1 mutations in cancer

The unpredictable consequences of CDKN1B/p27Kip1 mutations in cancer

Abstract 729: The role of skp2 in paclitaxel-resistant prostate cancer cells

Abstract Prostate cancer is the most common cancer and the second most frequent cause of cancer-related death of men in the United States. It has been shown that the F-box protein Skp2(S-phase kinase associated protein 2) plays an important role in the development and progression of prostate cancer. The molecular mechanism responsible for paclitaxel-resistant in prostate cancer cells remain unclear. Interestingly, we found that the expression of Skp2 is higher in the paclitaxel-resistant prostate cancer cells DU145-TXR or PC-3-TXR compared with their parental cells DU145 or PC-3 respectively.This study was aimed to investigate the role of Skp2 in prostate cancer cell resistance to paclitaxel and the progress of epithelial-to-mesenchymal transition(EMT). Skp2 encoding plasmid was stably transfected into PC-3 and DU145 cells. Meanwhile, Skp2 was silenced by shRNA in DU145-TXR or PC-3-TXR cells. MTS assay was used to measure the cytotoxicity effect of Skp2 overexpression or knockdown of Skp2 in cells after treatment with paclitaxel. We found that when downregulating Skp2 in DU145-TXR and PC-3-TXR, the cells showed a more sensitive response to paclitaxel. Moreover, Overexpression of Skp2 enhances prostate cancer cells resistance to paclitaxel. EMT markers E-cadherin was decreased while vimentin was increased in PC-3-TXR or DU145-TXR cells. The further study showed that when downregulating Skp2 in PC-3 or DU145, the expression of E-cadherin was increased. However, E-cadherin has no significant change when knockdown of Skp2 in PC-3-TXR or DU145-TXR cells. CyclinD1, P21 and P27 were found to increase while CyclinB1 and CyclinE2 to decrease when downregulating Skp2 in PC-3-TXR or DU145-TXR cells. These data provide evidence that Skp2 is associated with prostate cancer cell resistance to paclitaxel. Skp2 could be a potential therapeutic target for prostate cancer. This study is supported by NSFC key project 81130046, NSFC projects 81201669, 81171993 and 81272415. Note: This abstract was not presented at the meeting. Citation Format: Yeguo Yang, Qiong Song, Yi Lu, Jian Zhang, Jiejun Fu. The role of skp2 in paclitaxel-resistant prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 729. doi:10.1158/1538-7445.AM2015-729

Abstract LB-245: Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy

Abstract The molecular events that dictate the initiation, progression and therapeutic response in multiple myeloma (MM) remain incompletely understood. The ubiquitin (Ub) ligase Skp1-Cullin-1-Skp2 (SCFSkp2) complex promotes proliferation by inducing proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1. Skp2 overexpression and reduced p27 levels are frequent events in human cancers and are associated with poor prognosis. Methods: Prospective pharmacogenomics was performed using next generation exome sequencing gene expression profiles (GEPs) compiled from the APEX/SUMMIT phase III clinical trial deposited in dataset GSE9782. 255 relapsed MM patients were randomized to bortezomib (BTZ) vs. dexamethasone (DEX) alone and progression-free (PFS) and overall survival (OS) then correlated with the pre-treatment GEPs. RPMI8226 cells were exposed for 8 months to successively escalated doses of the proteasome inhibitors BTZ, carfilzomib or ixazomib to generate chemoresistant cell lines. Side population (SP) cells were isolated from MMCLs and patients’ samples by flow cytometry sorting based upon Hoechst 33342 staining. Results: Pharmacogenomic correlation of GEPs with clinical response revealed hyperexpression of CUL1 and SKP2 in tumors from MM patients that did not respond to BTZ. CUL1 and SKP2 hyperexpression was correlated with significantly reduced PFS and OS after treatment with BTZ but not with DEX. Cullin-1 and Skp2 were elevated in cells generated with acquired PI-resistance. CUL1 or SKP2 genetic ablation significantly enhanced the sensitivity of chemoresistant cells to PIs. A high-throughput assay was then employed using GFP-tagged p27 to screen large databases and chemical libraries in order to identify novel lead compounds that inhibited the Skp2-dependent ubiquitination of p27. Treatment with DT204, a novel lead compound identified here, reduced p27 ubiquitination, its accumulation in myeloma cells and impaired Skp2 association with Cullin-1 and Cks1, a critical rate-limiting effector of Skp2 activity. The anti-myeloma effect of DT204 was diminished using a phosphorylation-defective p27 mutant. Co-treatment with DT204 enhanced the anti-myeloma effect of BTZ against MM cell lines, patient tumor cells, stem cells and the tumor-initiating clonogenic side population (SP). The in-vivo efficacy and survival benefit of DT204 as monotherapy or in combination with BTZ will be reported. Conclusion: Taken together, the results demonstrate that pharmacologics to selectively disrupt the SCFSkp2 complex-p27 axis within the UPS holds promise to overcome chemoresistance with therapeutic benefit for myeloma patients. Skp2, Cullin1 can be utilized as biomarkers to select MM patients who will benefit from early experimental therapeutics of Skp2-inhibitors. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Mohamed Abdel Malek, James J. Driscoll. Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-245. doi:10.1158/1538-7445.AM2015-LB-245

The miR-30 Family Inhibits Pulmonary Vascular Hyperpermeability in the Premetastatic Phase by Direct Targeting of Skp2.

Before metastasis, primary tumor can create a premetastatic niche in distant organ to facilitate the dissemination of tumor cells. In the premetastatic phase, the permeability of pulmonary vasculatures is increased to accelerate the extravasation of circulating tumor cells. However, it is not clear whether local miRNAs contribute to the vascular hyperpermeability of the premetastatic niche. The expression of total miRNAs was determined using microarray in series of premetastatic lungs from tumor-bearing mice. Significantly differentially expressed miRNAs were identified and validated with qRT-PCR. Vascular permeability assays, vascular mimic systems, and orthotopic tumor models were used to investigate roles of selected miRNAs and target genes in premetastatic hyperpermeability. We identified a miRNA signature in premetastatic lungs. Among these miRNAs, miR-30a, b, c, d, and e were significantly attenuated. Subsequent investigations elucidated that lung fibroblast-derived miR-30s stabilized pulmonary vessels. Overexpression of miR-30s in lungs postponed metastasis and extended overall survival of B16 tumor-bearing mice. Following studies uncovered that Skp2 was directly targeted by miR-30s. Overexpression of Skp2 could disrupt pulmonary vessels, promote lung metastasis, and decrease overall survival of B16 tumor-bearing mice. These findings illuminate a novel mechanism for the modulation of premetastatic niches by miR-30s, which suggest that miR-30s represent not only promising targets for antimetastasis therapy but also indicators for metastasis.

The Role of Skp2 and its Substrate CDKN1B (p27) in Colorectal Cancer

Colorectal cancer is one of the most frequent cancers worldwide, having the fourth mortality rate among cancers in both sexes. Numerous studies are investigating the signalling pathways and different factors involved in the development and progression of colorectal cancer. It has recently been shown that the S-phase kinase-associated protein 2 (Skp2) overexpression plays an important role in the pathogenesis of colorectal cancer. We review the role of Skp2 and its ubiquitin-proteasome pathway in colorectal cancer. The F-box protein Skp2, a component of the SCF (Skp1-Cullin 1-F-box) E3 ubiquitin-ligase complex, has been shown to regulate cellular proliferation, cancer progression and metastasis by targeting several cell cycle regulators for ubiquitination and subsequent 26S proteasome degradation. The best known protein substrate of the Skp2 is the cyclin-dependent kinase inhibitor 1B (CDKN1B), also known as p27Kip1. Overexpression of Skp2 and loss of CDKN1B (p27) was strongly associated with aggressive tumor behavior and poor clinical outcome in a variety of cancers, including colorectal cancer. An efficient interaction between Skp2 and CDKN1B (p27) requires the presence of an essential activator of the SCF-Skp2 complex, the cyclin-dependent kinase subunit 1 (Cks1) cofactor. Alterations in the Skp2, Cks1 and CDKN1B (p27) expression have major effects on colorectal carcinogenesis and may serve as an important and independent prognostic marker. Furthermore, we highlight that Skp2 may be a promising therapeutic target for colorectal cancer, and development of Skp2 inhibitors would have a great impact on colorectal cancer therapy.

Curcumin suppresses cell growth and invasion and induces apoptosis by down-regulation of Skp2 pathway in glioma cells.

Studies have demonstrated that curcumin exerts its tumor suppressor function in a variety of human cancers including glioma. However, the exact underlying molecular mechanisms remain obscure. Emerging evidence has revealed that Skp2 (S-phase kinase associated protein 2) plays an oncogenic role in tumorigenesis. Therefore, we aim to determine whether curcumin suppresses the Skp2 expression, leading to the inhibition of cell growth, invasion, induction of apoptosis, and cell cycle arrest. To this end, we conducted multiple methods such as MTT assay, Flow cytometry, Wound healing assay, invasion assay, RT-PCR, Western blotting, and transfection to explore the functions and molecular insights of curcumin in glioma cells. We found that curcumin significantly inhibited cell growth, suppressed cell migration and invasion, induced apoptosis and cell cycle arrest in glioma cells. Furthermore, we observed that overexpression of Skp2 promoted cell growth, migration, and invasion, whereas depletion of Skp2 suppressed cell growth, migration, and invasion and triggered apoptosis in glioma cells. Mechanistically, we defined that curcumin markedly down-regulated Skp2 expression and subsequently up-regulated p57 expression. Moreover, our results demonstrated that curcumin exerts its antitumor activity through inhibition of Skp2 pathway. Collectively, our findings suggest that targeting Skp2 by curcumin could be a promising therapeutic approach for glioma prevention and therapy.

Antiproliferative effects and molecular mechanisms of troglitazone in human cervical cancer in vitro.

We investigated the effects of troglitazone on human cervical cancer SiHa cells and its mechanisms of action. SiHa cells were incubated with different concentrations of troglitazone (100, 200, or 400 μg/mL) for 24, 48, and 72 hours. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay; cell cycle and apoptosis were detected by flow cytometry; and morphology of SiHa cells was observed under an inverted microscope. pcDNA3.1 and pcDNA3.1-Skp2 plasmids were constructed and then transfected into SiHa cells. Protein expression was analyzed by Western blotting. Troglitazone inhibited the proliferation of SiHa cells in a time- and concentration-dependent manner. Troglitazone caused G0/1 phase arrest but failed to reduce apoptosis in SiHa cells. Troglitazone significantly increased expression of p27 but decreased Skp2 expression. Skp2 overexpression inhibited the role of troglitazone in increasing expression of p27, and the cell cycle inhibitory effect of troglitazone. Troglitazone can inhibit SiHa cell viability by affecting cell cycle distribution but not apoptosis, and Skp2 and p27 may play a critical role.

Skp2-Dependent Ubiquitination and Activation of LKB1 Is Essential for Cancer Cell Survival under Energy Stress

LKB1 is activated by forming a heterotrimeric complex with STRAD and MO25. Recent studies suggest that LKB1 has pro-oncogenic functions, besides acting as a tumor suppressor. How the LKB1 activity is maintained and how LKB1 regulates cancer development are largely unclear. Here we show that K63-linked LKB1 polyubiquitination by Skp2-SCF ubiquitin ligase is critical for LKB1 activation by maintaining LKB1-STRAD-MO25 complex integrity. We further demonstrate that oncogenic Ras acts upstream of Skp2 to promote LKB1 polyubiquitination by activating Skp2-SCF ubiquitin ligase. Moreover, Skp2-mediated LKB1 polyubiquitination is required for energy-stress-induced cell survival. We also detected overexpression of Skp2 and LKB1 in late-stage hepatocellular carcinoma (HCC), and their overexpression predicts poor survival outcomes. Finally, we show that Skp2-mediated LKB1 polyubiquitination is important for HCC tumor growth invivo. Our study provides new insights into the upstream regulation of LKB1 activation and suggests a potential target, the Ras/Skp2/LKB1 axis, for cancer therapy.

Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1-3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4-2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1.

Saurolactam Inhibits Proliferation, Migration, and Invasion of Human Osteosarcoma Cells.

Osteosarcoma is a common type of malignant bone tumor with features of osteoid formation or osteolytic lesions of bone. New therapeutic approaches are urgently needed since it lacks response to chemotherapeutic treatments. Saurolactam, a natural compound isolated from the aerial portions of Saururus chinensis, was reported to have an anti-inflammatory activity. Here, we demonstrate that saurolactam shows anti-cancer activity against human osteosarcoma cells. Saurolactam treatment inhibited proliferation of human osteosarcoma cell lines MG-63 and HOS and decreased colony formation in soft agar in a dose-dependent manner. Intraperitoneal administration of saurolactam at 25mg/kg of body weight for 21days dramatically inhibited the growth of MG-63 xenografts in nude mice. Flow cytometric analysis indicated that saurolactam treatment (20μM) led to G1 cell cycle arrest and induced apoptosis in these two cell lines. Western analysis suggested that saurolactam treatment resulted in a reduction of Akt/PKB, phospho-Ser473-Akt, c-Myc, and S-phase kinase-associated protein 2 (Skp2) in MG-63 and HOS osteosarcoma cells. Akt overexpression significantly abolished saurolactam-induced decrease in protein and phosphorylation levels of Akt, c-Myc, and Skp2 protein levels, implying that Akt inactivation was a causal mediator of saurolactam-induced inhibition of c-Myc and Skp2. Moreover, Skp2 overexpression in MG-63 cells partly abolished the growth inhibition induced by saurolactam. Saurolactam treatment repressed migration and invasion ability, and Skp2 overexpression significantly blocked these inhibitory effects of saurolactam in MG-63 Cells. The present study indicates that saurolactam might represent a new promising agent to improve osteosarcoma treatment.

E3 ubiquitin ligase Skp2 as an attractive target in cancer therapy.

E3 ubiquitin ligase Skp2 attaches ubiquitin to its target proteins and marks them for destruction by the 26S proteasome. This mechanism participates in a number of important cellular processes such as cell proliferation, DNA replication, V(D)J recombination, gene transcription, cellular metabolism and senescence. Skp2 is oncogenic. It is overexpressed in various solid tumors and hematological malignancies. Due to the antagonistic role Skp2 plays against p27, Skp2 overexpression is frequently associated with down-regulation of p27. Importantly, Skp2 overexpression in cancer cells is prognostic of cancer progression and overall survival. Recent studies have shown that Skp2 suppression might be an excellent strategy to inhibit tumorigenesis in tumors in which tumor suppressor genes such as VHL, RB or TP53 are mutated. In this review, we also summarize early efforts in the development of Skp2 inhibitors. The implications of continued, long-term Skp2 suppression is discussed.

Prognostic role of s-phase kinase-associated protein 2 in breast cancer: A meta-analysis.

Emerging evidence has shown that the F-box protein S-phase kinase-associated protein 2 (Skp2) plays an important role in the pathogenesis of breast cancer (BC). Our study aimed to evaluate the prognostic value of Skp2 in BC patients using meta-analysis based on the published studies. Eligible studies were identified by searching the online databases such as PubMed, EMBASE, and Web of Science up to October 2015. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to clarify the correlation between Skp2 expression and indicators of BC clinical outcomes, including overall survival (OS), disease-free survival (DFS), and BC-specific survival. In total, nine studies with 1820 BC patients were included for final analysis. The meta-analysis suggested that Skp2 overexpression was associated with poor OS (HR = 2.58, 95% CI: 1.83-3.63, P = 0.000) and poor DFS (HR = 2.12, 95% CI: 1.48-3.05, P = 0.000) in BC patients. This meta-analysis indicates that enhanced Skp2 is an independent prognostic factor for poor cancer survival.

SKP2 Is Dispensable for Normal T-Cell Development but Required for T-Cell Leukemogenesis

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer. Despite the significant clinical successes in the treatment of pediatric T-ALL, leukemia relapse, refractory disease and induction failure (around 30% of patients) remain significant clinical problems, which are often life-threatening. ALL remains the second leading cause of childhood death. Thus, more effective, curative therapeutic strategies are much needed, particularly for refractory and relapse T- ALL. Recent advances in understanding the biology and the molecular alterations of acute lymphoblastic leukemias have led to identification of new molecular targets, such as the Notch signaling pathway. Constitutive activation of Notch signaling is involved in more than 50% of human T-ALL, and overexpression of activated Notch induces T-cell leukemia and lymphoma in murine tumor models. However, disruption of Notch signaling by gamma-secretase inhibitors (GSI) failed to fulfill its clinical promise and, overall, the significant advances attained in dissecting the molecular effectors in T-ALL has yet to translate into effective, curative molecular therapies for relapse patients. Furthermore, despite multiple studies on Notch signaling, little is known on the role of its downstream mediators in T-cell ALL.Previous studies in our laboratory demonstrated that Notch1 activation induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 is the main F-box protein regulating cell cycle, promoting downregulation of the CKIs (p21Cip1, p27Kip1, p57Kip2 and p130) and its overexpression accelerate cell cycle progression in hematopoietic cells. SKP2 overexpression is frequently associated with cancers, in particular lymphomas and leukemias, and correlates with poor prognosis.We found that Skp2 expression is dynamically regulated during T-cell development coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro, responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle status, whereas the loss of SKP2 impaired their ability to mount a proliferative response to IL-7 stimulation. Importantly, we observed that SKP2 expression is increased in T-ALL patient samples and that mice with Notch-induced T-cell leukemia showed 5 fold upregulation of Skp2 expression. Our hypotheses are that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of Skp2, and that selective targeting of SKP2 is a novel, effective therapeutic strategy for childhood T-ALL. To test whether SKP2 is a key downstream mediator of Notch in T-ALL, we transduced oncogenic Notch (ICN; the constitutive intracellular form) in hematopoietic cells lacking SKP2 from Skp2-/- null mice, and in controls and we determine their ability to induce leukemia in irradiated recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia and increased animal survival by 40%. Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Sulforaphane down-regulates SKP2 to stabilize p27KIP1 for inducing antiproliferation in human colon adenocarcinoma cells

Sulforaphane is a cruciferous vegetable-derived isothiocyanate with promising chemopreventive and therapeutic activities. Induction of proliferation arrest and apoptosis principally contribute to sulforaphane's anticancer activity, but the precise molecular mechanisms remain elusive. The oncoprotein SKP2 is a key component of the SKP1-CULLIN1-F-box (SCF) E3 ligase complex and is responsible for directing SCF-mediated degradation of cyclin-dependent kinase inhibitor p27(KIP1) to promote cell proliferation. We herein provide the first evidence supporting the critical involvement of the SKP2-p27(KIP1) axis in sulforaphane-induced antiproliferation in various human colon adenocarcinoma cell lines. Specifically, sulforaphane markedly suppressed the levels of bromodeoxyuridine (BrdU) incorporation and clonogenicity in all tested cell lines, illustrating the antiproliferative effect of sulforaphane. Of note, sulforaphane-induced antiproliferation was accompanied with down-regulation of SKP2, leading to the stabilization and thus up-regulation of p27(KIP1). Additionally, sulforaphane was found to down-regulate SKP2 mainly through transcriptional repression, as sulforaphane lowered SKP2 mRNA expression and the SKP2 promoter activity. Furthermore, sulforaphane treatment led to the activation of both AKT and ERK, thus ruling out the possibility that sulforaphane down-regulates SKP2 by inhibiting AKT or ERK. Notably, sulforaphane-elicited suppression of BrdU incorporation and clonogenicity were significantly rescued in the context of SKP2 overexpression or p27(KIP1) depletion, therefore highlighting the important role of SKP2 down-regulation and the ensuing stabilization of p27(KIP1) in sulforaphane-induced antiproliferation. Collectively, these data expand our molecular understanding about how sulforaphane elicits proliferation arrest, but also implicate the application of sulforaphane in therapeutic modalities targeting SKP2.

Ablation of galectin-3 induces p27(KIP1)-dependent premature senescence without oncogenic stress.

Premature senescence induced by oncogenic stimuli or tumor suppressor activation plays opposing roles in tumorigenesis. Here, we propose that galectin-3, a β-galactoside-binding lectin, regulates premature senescence without oncogenic stress. We detected premature senescence, decreased Skp2, and increased p27KIP1 expression in galectin-3 knockout MEFs and galectin-3-depleted gastric cancer cells. Interestingly, galectin-3 depletion did not affect other senescence inducers such as p14ARF, p16INK4A, and p21WAF1/CIP1, suggesting that galectin-3-regulated senescence is p27KIP1 dependent. We demonstrate that galectin-3 depletion decreases retinoblastoma protein (Rb) phosphorylation (Ser780, Ser807/811), cyclin D1 and CDK4 expression, and E2F1 transcriptional activation. Galectin-3 directly interacts with the cyclin D1/CDK4 complex and promotes hyperphosphorylation of Rb. It also blocks the inhibition of E2F1 transcription, thereby increasing the expression of Skp2 and reducing the stability of p27KIP1 to promote the proliferation of gastric cancer cells. Xenograft mice with galectin-3-depleted gastric cancer cells display tumor growth retardation that is reversed by Skp2 overexpression. Increased expression of galectin-3 is also associated with the advanced TNM (tumor, lymph node, metastasis) system, clinicopathological stage, and lymph node metastases. The probability of survival was significantly decreased in gastric cancer patients with galectin-3high p27KIP1-lowcells. Taken together, our results show that galectin-3 may accelerate gastric tumorigenesis by inhibiting premature senescence.

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma.

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Longikaurin A, a natural ent-kaurane, induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.

Skp2 regulates androgen receptor through ubiquitin‐mediated degradation independent of Akt/mTOR pathways in prostate cancer

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling.