Expression Of S-phase Kinase-associated Protein Research Articles

Skp2 Deteriorates the Uterine Receptivity by Interacting with HOXA10 and Promoting its Degradation.

Receptive endometrium plays a core role in successful embryo implantation, and about one-third of repeated embryo implantation failures are attributed to endometrial receptive defects. S-phase kinase-associated protein 2 (SKP2), a member of the F-box protein family, plays an important role in many cellular processes, including cell proliferation and apoptosis. However, its role in endometrial receptivity is still unclear. Here, we identified SKP2 was obviously upregulated in the patients with infertility. Functional study showed that SKP2 overexpression inhibited endometrial epithelial cell (EEC) proliferation, whereas SKP2 knockdown promoted the proliferation of EECs. In addition, the overexpression of SKP2 also repressed adhesion rate of embryonic cells to EECs. In vivo studies further suggested that the upregulation of SKP2 obviously suppressed endometrium receptivity formation and embryo implantation potential. Mechanistical study clarified that SKP2 directly interacted with HOXA10 and decreased protein stability through promoting the ubiquitin-mediated proteasome degradation of HOXA10. In conclusion, the current study documented that the high expression of SKP2 deteriorates endometrial receptivity formation by decreasing the HOXA10 expression and suggested that SKP2 may be defined as a marker of endometrial receptivity, and as a target for the diagnosis and treatment of infertility.

Tanshinone IIA Suppresses Hypoxia-induced Apoptosis in Medial Vestibular Nucleus Cells Via a Skp2/BKCa Axis.

The aim of the present study was to investigate the protective effects of Tanshinone IIA (Tan IIA) on hypoxia-induced injury in the medial vestibular nucleus (MVN) cells. An in vitro hypoxia model was established using MVN cells exposed to hypoxia. The hypoxia-induced cell damage was confirmed by assessing cell viability, apoptosis and expression of apoptosis-associated proteins. Oxidative stress and related indicators were also measured following hypoxia modeling and Tan IIA treatment, and the genes potentially involved in the response were predicted using multiple GEO datasets. The results of the present study showed that Tan IIA significantly increased cell viability, decreased cell apoptosis and decreased the ratio of Bax/Bcl-2 in hypoxia treated cells. In addition, hypoxia treatment increased oxidative stress in MVN cells, and treatment with Tan IIA reduced the oxidative stress. The expression of SPhase Kinase Associated Protein 2 (SKP2) was upregulated in hypoxia treated cells, and Tan IIA treatment reduced the expression of SKP2. Mechanistically, SKP2 interacted with large-conductance Ca2+-activated K+ channels (BKCa), regulating its expression, and BKCa knockdown alleviated the protective effects of Tan IIA on hypoxia induced cell apoptosis. The results of the present study suggested that Tan IIA had a protective effect on hypoxia-induced cell damage through its anti-apoptotic and anti-oxidative activity via an SKP2/BKCa axis. These findings suggest that Tan IIA may be a potential therapeutic for the treatment of hypoxia-induced vertigo.

High S phase kinase-associated protein 2 expression is a potential prognostic biomarker for glioma.

S phase kinase-associated protein 2 (SKP2), a substrate recognizing protein, serves an important role in promoting cell cycle progression through ubiquitination and degradation of cell cycle inhibitors. In the present study, the clinical significance of SKP2 in gliomas was studied; 395 glioma specimens and 20 non-neoplastic tissues were collected and immunohistochemical analysis was performed. χ2 test was used to assess the associations between SKP2 expression and clinical parameters. Overall survival (OS) curves were plotted according to the Kaplan-Meier method. In the tested glioma samples, SKP2 expression was mainly observed in glioblastomas (GBMs). Survival analysis demonstrated that the overall survival time of the high SKP2 expression group was lower compared with the low SKP2 expression group (median OS, 10.04 months vs. 16.50 months; P=0.003). Moreover, SKP2 was independently associated with an unfavorable prognosis in GBMs. In addition, the expression of SKP2 was associated with the expression of phosphorylated retinoblastoma protein and the epidermal growth factor receptor. A combination of SKP2 expression along with isocitrate dehydrogenase 1 (IDH1) mutations and telomerase reverse transcriptase (TERT) promoter mutations was used to classify glioma patients for survival analysis. Patients with low SKP2 expression, IDH1 mutation and wild-type TERT promoter demonstrated the longest survival time. The findings of the present study, indicate that SKP2 is a potential prognostic biomarker in patients with GBMs.

Silencing of S-phase kinase-associated protein 2 enhances radiosensitivity of esophageal cancer cells through inhibition of PI3K/AKT signaling pathway

We investigated the effect of S-phase kinase-associated protein 2 (SKP2) on radiosensitivity of esophageal cancer (EC) cells. Expression of SKP2, PI3K, AKT, Bcl-2 and Bax were assayed in EC. EC cells were transfected with SKP2-siRNA/IGF-1 to detect expression of SKP2, PI3K, AKT, Bcl-2 and Bax. At last, the radiosensitivity of cells in different doses of X (0, 2, 4, 6, 8 Gy) irradiation and cell apoptosis were also detected. EC cells displayed a higher positive expression rate of SKP2, elevated mRNA and protein expression of SKP2, PI3K, AKT, Bcl-2 and Bax, as well as higher extent of PI3K and AKT phosphorylation. SKP2 silencing downregulated mRNA and protein expression of PI3K, AKT and Bcl-2 but increased p27 protein expression, and inhibited the cell survival rate while promoting cell apoptosis. Taken together, silencing SKP2 can inhibit the PI3K/AKT signaling pathway, thereby increasing the radiosensitivity of EC cells.

S phase kinase-associated protein 1 can inhibit the proliferation and invasion of intrahepatic cholangiocarcinoma

Objective To investigate the expression of S-phase kinase-associated protein 1 (SKP1) in intrahepatic cholangiocarcinoma and to investigate its effects on proliferation, migration and invasion of intrahepatic cholangiocarcinoma. Methods Western blotting assay was used to determine the expression difference of SKP1 in intrahepatic cholangiocarcinoma and normal tissues. The SKP1 interference vector and empty vector were constructed and stably transfected into intrahepatic cholangiocarcinoma cells by lentivirus packaging to form an experimental group and a control group. The expression of SKP1 was detected by immunoblotting assay. The proliferation of experimental group and control group after transfection was detected by cell counting kit-8 (CCK-8) assay. The migration ability was detected by scratch test. The invasion ability of Transwell was detected by Transwellassay. Results The expression of SKP1 in intrahepatic cholangiocarcinoma was (1.701±0.215) times, and the difference was statistically significant (t=8.512, P<0.05). Lentiviral transfection of RBE cells decreased the expression of SKP1, and the Western blotting assay decreased the experimental group by (3.632±0.335) times, and the difference was statistically significant (t=11.376, P<0.01). The CCK-8 experiment showed that the relative survival rates of the experimental group at 24, 48, and 72 hours were (53.136±0.314)%, (42.661±0.248)% and (35.037±0.203)%, respectively, and the differences were statistically significant (t=8.594, 8.899, 9.210, P<0.01). The scratch test showed that the number of migrated cells in the experimental group at 24 and 48 hours (41.198±3.092) and (64.773±6.437) were significantly lower than those in the control group (113.487±3.514) and (168.767±8.151), and the difference was statistically significant (t=5.430, 6.224, P<0.05). The Transwell experiment showed that the number of perforated cells in the experimental group was (34.185±3.124), which was significantly lower than that in the control group (121.234±6.814), and the difference was statistically significant (t=9.918, P<0.05). Conclusion The expression of SKP1 in intrahepatic cholangiocarcinoma is higher than that in normal tissues. Decreasing SKP1 expression can inhibit the proliferation, migration and invasion of intrahepatic cholangiocarcinoma. Key words: S phase kinase associated protein-1; Intrahepatic cholangiocarcinoma; Ubiquitination; Proliferation; Migrate; Invasion

Overexpression of Mir-21-5p Induces Apoptosis and Cell Cycle Arrest By Down-Regulating SKP2 and Overcomes Bortezomib Resistance in Multiple Myeloma

High expression of SKP2 (S-Phase Kinase Associated Protein 2) is associated with tumor cell growth and drug resistance in many solid tumors and hematological malignancies. However, the role of SKP2 in drug resistance/ relapse of multiple myeloma (MM) patients has not been fully investigated. To this end, we first analyzed two publicly accessible datasets (GSE6477, GSE9782) which demonstrated that relapsed MM patients showed higher expression of SKP2 compared to newly diagnosed patients (p=0.02). Furthermore, high expression of SKP2 was correlated with shorter overall survival compared to newly diagnosed MM patients with low SKP2 expression (p=0.0002). To investigate whether miRNAs could play a role in the dysregulation of SKP2 in MM, we exploited several miRNA-target prediction algorithms (Targetscan, miRDB, microT-CDS) to generate a selective miRNA library for subsequent screening. Over all, the screening results displayed 59 miRNA candidates predicated to target SKP2, 9 miRNAs were commonly identified by all three algorithms. Among these, miR-21-5p showed the highest potential binding score for SKP2 and was found to be in an inverse correlation with SKP2 in MM patient dataset (GSE16558). To address whether miR-21-5p/SKP2 axis may play a role in drug resistance in MM, we used two drug resistant MM cell lines (RPMI-8226R5 and MM.1R) to analyze the basal expression of SKP2 and miR-21-5p compared with that in their parental lines (RPMI-8226 and MM.1S, respectively). It turned out that the resistant MM cells displayed low expression of miR-21-5p and high expression of SKP2 whereas the parental lines showed the opposite pattern. Moreover, overexpression of miR-21-5p in both MM drug resistant cell lines significantly reduced both mRNA and protein levels of SKP2 further supporting that SKP2 could be a target of miR-21-5p. SKP2 mediates the ubiquitination of cyclin kinase inhibitor p27Kip1, a central negative regulator of cell cycle. In line with this, our immunoblot analysis confirmed that high SKP2 expressing drug resistant MM cells showed a low level of p27Kip1protein and low SKP2 expressing MM parental lines showed a high level of p27kip1 protein. To investigate the functional effects of miR-21-5p overexpression, we first performed FACS to determine if this overexpression would modulate cell cycle regulation of drug resistant MM cells. miR-21-5p overexpression increased in the proportion of cells at G1/G0 phase (RPMI-8226R5: 41.6 % to 59.1%, MM.1R: 19.5 % to 39.4%), decreased in the proportion of cells in S phase (RPMI-8226R5: 30.9 % to 25.5 %, MM.1R: 44.7 % to 32.2%) and G2/M phase (RPMI-8226R5: 15.5 % to 11.5 %, MM.1R: 27.6 % to22.4% ) compared with scrambled control. At the same time, immunoblot results showed that overexpression of miR-21-5p accumulated p27Kip1 and p21 (Cip1/Waf1), and suppressed CDK6 and Cyclin E cell cycle markers. Next, we examined the effect of miR-21-5p overexpression on cell proliferation and apoptosis in MM resistant cells using both MTT cytotoxicity assay and vital exclusion staining (trypan blue). In both assays, miR-21-5p mimics-transfected MM resistant cells showed significant reduction of cell viability and increase in apoptosis in a time-dependent manner compared with scrambled-transfected cells. Overexpression of miR-21-5p upregulated apoptotic markers: cleaved PARP-1, cleaved caspase-3, PUMA and BAX and down regulated anti-apoptotic markers: BCL-2 and survivin. Subsequently, efficacy of bortezomib (BTZ) was examined on miR-21-5p overexpressed MM resistant cells which were interestingly re-sensitized to BTZ in a dose- and time-dependent manner. In addition, our FACS experiment also confirmed that combination of BTZ and miR-21-5p mimics increased the percentage of annexin-V positive cells compared to scrambled and miR-21-5p mimics in the MM resistant cells. In conclusion, miR-21-5p is suppressed in MM resistant cells, which contributes to the drug resistance in MM through upregulation of SKP2. Overexpression of miR-21-5p induces apoptosis and cell cycle arrest in MM drug resistant cells by downregulating SKP2 gene expression and overcomes BTZ resistance in these cells. Our study provides a proof-of-concept that miR-21-5p could be utilized as a SKP2-targeting molecule which in combination with BTZ would be a promising therapeutic approach to overcome MM drug resistance. Disclosures No relevant conflicts of interest to declare.

SKP2, positively regulated by circ_ODC1/miR-422a axis, promotes the proliferation of retinoblastoma.

Retinoblastoma (RB) is the most common intraocular malignancy in infants and children. S-phase kinase-associated protein 2 (SKP2) has been unmasked as an oncogene in a great many of carcinomas. The biologic function and the detailed molecular mechanism of SKP2 in RB need to be better understood. In this study, real-time quantitative polymerase chain reaction and Western blot showed the ectopic expression of SKP2 in RB tissues and cell lines. Loss of function assays showed the attenuated cell proliferation in RB as a result of SKP2 knockdown. In addition, bioinformatics analysis predicted the interaction between SKP2 and miR-422a. Luciferase reporter assay and Pearson's correlation analysis validated the negative correlation between miR-422a and SKP2. MiR-422a overexpression led to a decline of SKP2 expression and cell growth in RB. The binding capacity between miR-422a and circ_ODC1 was also predicted by bioinformatics analysis. Pearson's correlation analysis and luciferase reporter assay confirmed that circ_ODC1 is negatively correlated with miR-422a. Silencing circ_ODC1 resulted in a rise in miR-422a expression and RB cell growth. Moreover, reduced cell growth was restored by SKP2 overexpression. In a word, SKP2, induced by circ_ODC1 and miR-422a, promotes RB proliferation. Our new findings in this research might expedite the discovery of novel prognostic markers and therapeutic targets of RB.

Abstract LB-115: Preclinical assessment of retinoid X-receptor agonist, UAB110, for potential treatment of cutaneous T-cell lymphoma

Abstract Cutaneous T-cell lymphomas (CTCLs) are a group of non-Hodgkin diseases characterized by localization of neoplastic T-cells in the skin. The most common forms of CTCLs are mycosis fungoides (MF) and Sézary syndrome (SS). Bexarotene (Targretin®) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist (rexinoid) for the treatment of CTCLs. Despite its low toxicity compared to retinoids and other chemotherapeutic agents, it induces hypothyroidism and markedly elevates serum triglycerides (TGs). Thus, improved rexinoids for the treatment of this indolent disease is necessary. A potential candidate is the novel rexinoid, UAB110, which does not elevate TGs in animal studies and effectively prevents mammary cancer development. Compared to bexarotene, it has higher binding affinity for RXRα receptor and transcriptional activation activity. The purpose of this study is to assess preclinical efficacy, mechanism of action of UAB110, and compare its action with bexarotene and the rexinoid 9-cisUAB30 (UAB30) using human-derived CTCL cell lines, MyLa and HuT78, which are representative of MF and SS, respectively. Cell proliferation analysis and cell-cycle related markers were determined by hemocytometer and Western blot analysis, respectively. UAB110 is as effective as UAB30 and bexarotene in suppressing cell proliferation. Furthermore, it significantly (p &amp;lt; 0.05 relative to DMSO control) suppresses the protein expression of SKP2 (S-phase kinase-associated protein 2, a F-box protein that targets p27kip1 for degradation by 26S proteasome) and PSMA7 (proteasome subunit alpha type 7, a component of the 20S proteolytic complex in 26S proteasome). The decrease in SKP2 resulted in a concomitant up-regulation of p27kip1 protein, the major cell cycle inhibitor. The downregulating of PSMA7 impaired the 20S proteasome activity in both cell lines. These findings indicate that the novel rexinoid is effective in inhibiting cell proliferation of CTCL cells, in part, by suppressing SKP2 expression and the 20S proteasome activity resulting in increasing p27kip1 protein. In sum, UAB110 and also UAB30 with minimal effect in elevating serum TGs are both possible alternatives to bexarotene for the treatment of CTCLs. Citation Format: Chu-Fang Chou, Jason Esla, Venkatram Atigadda, Donald Muccio, Clinton Grubbs, Elmets Craig, Pi-Ling Chang. Preclinical assessment of retinoid X-receptor agonist, UAB110, for potential treatment of cutaneous T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-115.

Mechanism of Low Expression of miR-30a-5p on Epithelial-Mesenchymal Transition and Metastasis in Ovarian Cancer.

Metastasis of ovarian cancer is regulated by microRNAs. This study focused on the effects of miR-30a-5p on ovarian cancer migration and invasion. Our results showed that the miR-30a-5p and mucin type O-glycan biosynthesis are closely related to ovarian cancer, and that miR-30a-5p was downregulated in ovarian cancer cells. miR-30a-5p overexpression reduced cell viability and inhibited migration and invasion in HO-8910 and HO-8910PM cells. S phase kinase-associated protein 2 (SKP2), B cell lymphoma 9 (BCL9), and NOTHC1 are direct target genes of miR-30a-5p. MTDH, SKP2, BCL9, and NOTCH1 genes were overexpressed in ovarian cancer cells, and they are direct target genes of miR-30a-5p. miR-30a-5p overexpression inhibited epithelial-mesenchymal transition (EMT) process, while upregulation of SKP2, BCL9, and NOTCH1 gene expression levels reduced the inhibition of EMT process by miR-30a-5p. miR-30a-5p was lowly expressed in ovarian cancer, and such a phenomenon is related to ovarian cancer metastasis. miR-30a-5p might inhibit the migration and invasion of ovarian cancer cells by downregulating the expression of SKP2, BCL9, and NOTCH1 genes.

Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.

A novel thyroid hormone receptor isoform, TRβ2-46, promotes SKP2 expression and retinoblastoma cell proliferation

Retinoblastoma is a childhood retinal tumor that develops from cone photoreceptor precursors in response to inactivating RB1 mutations and loss of functional RB protein. The cone precursor's response to RB loss involves cell type-specific signaling circuitry that helps to drive tumorigenesis. One component of the cone precursor circuitry, the thyroid hormone receptor β2 (TRβ2), enables the aberrant proliferation of diverse RB-deficient cells in part by opposing the down-regulation of S-phase kinase-associated protein 2 (SKP2) by the more widely expressed and tumor-suppressive TRβ1. However, it is unclear how TRβ2 opposes TRβ1 to enable SKP2 expression and cell proliferation. Here, we show that in human retinoblastoma cells TRβ2 mRNA encodes two TRβ2 protein isoforms: a predominantly cytoplasmic 54-kDa protein (TRβ2-54) corresponding to the well-characterized full-length murine Trβ2 and an N-terminally truncated and exclusively cytoplasmic 46-kDa protein (TRβ2-46) that starts at Met-79. Whereas TRβ2 knockdown decreased SKP2 expression and impaired retinoblastoma cell cycle progression, re-expression of TRβ2-46 but not TRβ2-54 stabilized SKP2 and restored proliferation to an extent similar to that of ectopic SKP2 restoration. We conclude that TRβ2-46 is an oncogenic thyroid hormone receptor isoform that promotes SKP2 expression and SKP2-dependent retinoblastoma cell proliferation.

Sequestosome 1 protects esophageal squamous carcinoma cells from apoptosis via stabilizing SKP2 under serum starvation condition

Esophageal squamous cell carcinoma (ESCC) is one of the malignancies in digestive system, with a low 5-year survival rate. We previously revealed that Sequestosome 1 (SQSTM1/p62) protein levels were upregulated in ESCC tissues. However, it is unclear about the function of p62 and the underlying mechanism. Here, we used immunofluorescence and immunohistochemistry to investigate the expression of p62 in ESCC. Western blotting, quantitative RT-PCR, colony formation assay, flow cytometry, immunoprecipitation and xenograft tumor assay were used to analyze the role of p62 in vitro and vivo. Here, we showed that p62 serves as a regulator of cell apoptosis under serum starvation condition in ESCC cells. Through activating the protein kinase C iota (PKCiota)-S-phase kinase-associated protein 2 (SKP2) signaling pathway, p62 enhances cell apoptosis resistance and colony formation in vitro and tumor growth in mouse models. Through interaction with the domains PB1, p62 upregulated the expression of PKCiota and then depressed the ubiquitin-mediated proteasomal degradation of SKP2. p62-silencing combined with a PKCiota inhibitor ATM significantly enhanced cell apoptosis and inhibited cell survival. Immunohistochemical analysis revealed a positive association between the expression of p62 and SKP2 in primary ESCC tissues. And importantly, p62 presented a markedly cytoplasmic translocation in cancerous cells, including in 16 (30.76%) tumors at stage T1, as compared with its nuclear location in normal esophageal epithelial cells. In summary, p62 plays an anti-apoptotic role in ESCC cells via stabilizing SKP2 under serum starvation condition. These data suggest that p62 might be an early biomarker and a candidate therapeutic target of ESCC.

MicroRNA-508-5p suppresses metastasis in human gastric cancer by targeting S-phase kinase‑associated protein 2.

S-phase kinase-associated protein 2 (SKP2), a potent oncogene was revealed to be upregulated in gastric cancer (GC) tissue samples, in which SKP2 was inversely correlated with microRNA (miR)‑508‑5p transcripts. In present study, the functional effect of miR‑508‑5p on SKP2 and its metastatic potential were investigated in SGC‑7901 GC cells. Significant downregulation of the miR‑508‑5p transcript was associated with the progression of GC. Furthermore, the overexpression of miR‑508‑5p was demonstrated to inhibit the proliferation, migration and invasion of SGC‑7901 cells, as well as induced cell apoptosis and cell cycle arrest at the G0/G1 phase invitro. The overexpression of miR‑508‑5p was able to downregulate the expression of the SKP2 oncogene, through a mechanism by which miR‑508‑5p directly targeted the SKP2 gene. Thus, regulating transcriptional and post‑transcriptional SKP2 expression, as demonstrated using luciferase reporter assays, reverse transcription‑quantitative polymerase chain reaction analysis and immunoblotting assays. The results of the present study identified that miR‑508‑5p functionally affects the SKP2 gene and reduces metastatic potential in GC, suggesting a novel role of miR‑508‑5p in the regulation of SKP2 and cell cycle.

Stathmin 1 promotes the proliferation and malignant transformation of pancreatic intraductal papillary mucinous neoplasms.

Pancreatic intraductal papillary mucinous neoplasms (IPMNs) are a type of pancreatic tumor, which have been identified following improvements in diagnostic imaging. However, the malignant transformation of IPMN has been difficult to diagnose clinically. To date, the mechanisms driving the progression of IPMN to cancer remain to be fully elucidated. The present study focused on Stathmin 1 (STMN1), a protein that is associated with the development of various types of cancer. The expression of STMN1 was examined immunohistochemically in tissues from cases of IPMN. The correlation between the STMN1 staining and clinical pathological factors was evaluated, and the expression of STMN1, p27 and S-phase kinase-associated protein 2 (SKP2) were compared. High expression levels of STMN1 were significantly correlated with regions of malignancy, and was associated with high expression of SKP2, low expression of nuclear p27 and a high Ki-67 index. High expression levels of STMN1 and SKP2 were significantly correlated with the transformation of IPMN to carcinoma. In addition, within the regions of carcinoma, the expression of STMN1 was weak in regions of adenoma and high in the cancerous regions. It was concluded that the high expression of STMN1 contributed to tumor proliferation and malignant transformation in the patients with IPMN. These results suggested that characterization of the expression of STMN1 may be a promising approach for predicting malignant transformation of pancreatic intraductal papillary mucinous adenoma.

PI3K/Akt Activated by GPR30 and Src Regulates 17β-Estradiol-Induced Cultured Immature Boar Sertoli Cells Proliferation.

Sertoli cell (SC) is a key element in the process of spermatogenesis. Accumulating research show that estrogen plays an important role in regulating boar SC proliferation. However, it is unclear whether phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) is involved in this process. In the present study, the role of PI3K/Akt on the 17β-estradiol-induced piglet SC proliferation was explored. In addition, we also explained the roles of G-protein-coupled estrogen receptor (GPR30) and Sarcoma protein (Src) in this process. Our study demonstrated that, 17β-estradiol induced activation of PI3K in a time-dependent manner. Both G-15 (an antagonist of GPR30, 0.1 μmol/L) and PP2 (an inhibitor of Src, 2.0 μmol/L) inhibited 17β-estradiol-induced activation of PI3K, reduced SC proliferation, and decreased messenger RNA (mRNA) and protein expression of S-phase kinase-associated protein 2 (Skp2). We also found that 17β-estradiol induced activation of Akt in a time-dependent manner. Both LY294002 (an inhibitor of PI3K) and 10-DEBC (an inhibitor of Akt) significantly reduced 17β-estradiol-induced SC proliferation and reduced mRNA and protein expression of Skp2. In addition, LY294002 inhibited 17β-estradiol-induced activation of Akt. The results indicated that 17β-estradiol regulates SC proliferation by activating PI3K/Akt. Both GPR30 and Src are involved in 17β-estradiol-induced phosphorylation of PI3K/Akt. Activation of PI3K/Akt enhances the expression of Skp2, which promotes SC proliferation.

SiRNA KNOCKDOWN SKP2 GENE IN BREAST CANCER

The S-phase kinase-associated protein-2 (SKP2) plays a strategic role in ubiquitin-mediated proteolysis, which effects in the development of cells from inactivity to proliferative state. SKP2 is overexpressed in a variety of tumor. In this study, we used small interfering RNAs (siRNAs) to inhibit the SKP2 expression in Breast cancer cells preface investigate the role of SKP2 in breast tumorigenesis. Two Breast cancer cell lines (MCF-7 and T47D) were transfected with siRNAs targeted against SKP2. Our results showed one SKP2-siRNA specifically and efficiently reduced the levels of the SKP2 protein by 90% 48 h after transfection in MCF-7 cell line. In the transfected cells, p27 protein was accumulated inversely related to Skp2 siRNA transfected Breast cells. Skp2 siRNA inhibited the cell growth of breast cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo. Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27 degradation in breast cancer.

Proliferation and Survival of Embryonic Sympathetic Neuroblasts by MYCN and Activated ALK Signaling.

MYCN overexpression combined with activated anaplastic lymphoma kinase (ALK) is sufficient to induce neuroblastoma (NB) in mouse sympathoadrenal cells. To address cellular and molecular effects elicited by MYCN/ALK cooperation, we used cultures of chick sympathetic neuroblasts. We demonstrate that MYCN increases proliferation but not survival, whereas long-term expression of ALKF1174L elicits cell-cycle exit, differentiation, and survival of postmitotic neurons. Combined MYCN/ALKF1174L expression allows long-term proliferation and survival of neuroblasts with differentiated characteristics. In the presence of ALKF1174L signaling, MYCN induces the expression of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets p27 for degradation and is also upregulated in high-risk NB. SKP2 inhibition supports a function for SKP2 in the maintained neuroblast proliferation downstream of MYCN/ALK, which may represent an early step toward tumorigenesis.

Inhibition of SKP2 Sensitizes Bromocriptine-Induced Apoptosis in Human Prolactinoma Cells.

PurposeProlactinoma (prolactin-secreting pituitary adenoma) is one of the most common estrogen-related functional pituitary tumors. As an agonist of the dopamine D2 receptor, bromocriptine is used widely to inhibit prolactinoma progression. On the other hand, it is not always effective in clinical application. Although a dopamine D2 receptor deficiency contributes to the impaired efficiency of bromocriptine therapy to some extent, it is unknown whether there some other underlying mechanisms leading to bromocriptine resistance in prolactinoma treatment. That is the main point addressed in this project.Materials and MethodsHuman prolactinoma samples were used to analyze the S-phase kinase associated protein 2 (SKP2) expression level. Nutlin-3/adriamycin/cisplatin-treated GH3 and MMQ cells were used to analyze apoptosis in SKP2 overexpression or knockdown cells. SKP2 expression and the interaction partners of SKP2 were also detected after a bromocriptine treatment in 293T. Apoptosis was analyzed in C25 and bromocriptine-treated GH3 cells.ResultsCompared to normal pituitary samples, most prolactinoma samples exhibit higher levels of SKP2 expression, which could inhibit apoptosis in a p53-dependent manner. In addition, the bromocriptine treatment prolonged the half-life of SKP2 and resulted in SKP2 overexpression to a greater extent, which in turn compromised its pro-apoptotic effect. As a result, the bromocriptine treatment combined with C25 (a SKP2 inhibitor) led to the maximal apoptosis of human prolactinoma cells.ConclusionThese findings indicated that SKP2 inhibition sensitized the prolactinoma cells to bromocriptine and helped promote apoptosis. Moreover, a combined treatment of bromocriptine and C25 may contribute to the maximal apoptosis of human prolactinoma cells.

Increased expression of SKP2 is an independent predictor of locoregional recurrence in cervical cancer via promoting DNA-damage response after irradiation.

Although radiation therapy was known to be effective to cervical cancer, loco-regional recurrences are frequently found in patients. We aimed to identify a molecular marker predicting the response of cervical cancer to radiotherapy. We included the patients (n = 149) with cervical cancer who had undergone radiotherapy from 2004 to 2006. Tumor samples were collected to examine the association between the expression of S-phase kinase-associated protein 2 (SKP2) and prognosis in cervical cancer. We found higher expression of SKP2 associated with recurrence (HRs: 2.52, p < 0.001), death (HRs: 2.01, p < 0.001) and higher locoregional recurrence rate (HRs: 3.76, p < 0.001). Cervical cancer cell lines with higher expression of SKP2 showed higher colony formation, cell survival rate and fewer DNA damages after irradiation. SKP2-C25, an inhibitor for SKP2 activity, dose-dependently decreased cell viability after irradiation and knockdown of SKP2 impaired DNA-damage response and sensitized the cervical cancer cells to irradiation. Our data showed the SKP2 represents a promising tool to identify patients with cervical cancer who have a higher risk of locoregional recurrence after radiotherapy. Targeting SKP2 may serve as a potential radiosensitizer for developing effective therapeutic strategies against cervical cancer.

The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1 inhibition in myeloma cells

The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells We investigated the effect of stable suppression of hypoxia inducible factor (HIF)-1α in myeloma cells on sensitivity to lenalidomide (LEN) in vivo. We found that the in vivo anti-tumoral effect of LEN is enhanced by HIF-1α suppression in myeloma cells. It has been reported that HIF-1α is over-expressed by myeloma cells 1-4 and that HIF-1α suppression significantly blocks myeloma-induced angiogenesis, and reduces both tumoral burden and bone destruction in vivo in multiple myeloma (MM) mouse models. 3 The potential effects of HIF-1α modulation on drug sensitivity in MM cells are not known and are currently under investigation. The immunomodulatory drugs (IMiDs®), including LEN, are a class of drugs derived from thalidomide 4 able to exert anti-myeloma effects by the selective Cereblon-dependent destruction of IKZF proteins, 5,6 either through a direct action on MM cell proliferation and survival, 7 or through indirect immunomodulatory and anti-angiogenic effects. 7 Previous data indicated that HIF-1α inhibition did not increase the anti-proliferative in vitro effect of bortezomib on MM cells; 2,3 this drug induces a strong downregula-tion of HIF-1α in MM cells. 2 We recently reported that HIF-1α knockdown in the human myeloma cell line (HMCL) JJN3 potentiated the in vitro effect of LEN treatment (48-72 h) on cell proliferation through a significant upregulation of p27 without changing cell viability. 3 It has been consistently reported that LEN only slightly down-regulated HIF-1α expression in MM cells. 2 Such evidence has provided the rationale to investigate the effect of stable suppression of HIF-1α in myeloma cells on LEN sensitivity in vivo. Therefore, in the present study, we first inhibited HIF-1α in three HMCLs (JJN3, OPM2 and H929) using an anti-HIF1α lentiviral shRNA pool, as previously described. 3 H929 showed a very high mortality rate after anti-HIF1α lentiviral infection (data not shown) and were not used for the subsequent in vivo experiments. We assessed the effect of LEN in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous in vivo mouse model. 3 Different groups of animals (5 animals in each group) of two sets of independent experiments were injected with JJN3 pLKO.1 (empty vector) or JJN3 anti-HIF1α. When tumors became palpable (approx. 7-10 days after injection) mice were treated with LEN 5 mg/kg (Selleckchem, Houston, TX, USA) or vehicle (DMSO) using the intraperitoneal route. The same mouse model, grouping strategy and treatment conditions were used for OPM2 pLKO.1 or OPM2 anti-HIF1α. After three weeks, we evaluated tumor volume and weight, as previously described, 3 and immunohisto-chemistry was used to evaluate the microvascular density (MVD) and checked by CD34 immunostaining (Santa Cruz, Dallas, TX, USA). 3 In addition, in the first set of mice experiments, the expression of p27 (Abcam, Cambridge, UK) was evaluated by immunohistochem-istry. The expression of S-phase kinase-associated protein 2 (SKP2), a p27 inhibitor, expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasma-cytoma lysates by western blot using the following anti-bodies: SKP2, Casp-3 (Santa Cruz, Dallas, TX, USA), HK2, pERK 1/2, (Cell Signaling, Danvers, MA, USA). b-actin was used as internal control (Millipore,