Inhibition Of Skp2 Research Articles

S-phase kinase-associated protein 2 is involved in epithelial-mesenchymal transition in methotrexate-resistant osteosarcoma cells.

Osteosarcoma (OS), a common worldwide primary aggressive bone malignancy, arises from primitive transformed cells of mesenchymal origin and usually attacks adolescents and young adults. Methotrexate (MTX) is the anti-folate drug used as a pivotal chemotherapeutic agent in the treatment of OS. However, patients with OS often develop drug resistance, leading to poor treatment outcomes. In the present study, in order to explore the underlying mechanisms responsible for MTX resistance, we established MTX-resistant OS cells using the U2OS and MG63 cell lines and examined whether MTX-resistant OS cells underwent epithelial-mesenchymal transition (EMT) by Transwell assay, wound healing assay, MTT assay, RT-PCR and western blot analysis. We found that the viability of the MTX-resistant cells remained relatively unaltered following further treatment with MTX compared to the parental cells. The resistant cells appeared to possess a mesenchymal phenotype, with an elongated and more spindle-like shape, and acquired enhanced invasive, migratory and attachment abilities. The measurement of EMT markers also supported EMT transition in the MTX-resistant OS cells. Our result further demonstrated that the overexpression of S-phase kinase-associated protein 2 (Skp2) was closely involved in the resistance of OS cells to MTX and in the acquirement of EMT properties. Thus, the pharmacological inhibition of Skp2 may prove to be a novel therapeutic strategy with which to overcome drug resistance in OS.

Inhibiting Skp2 E3 Ligase Suppresses Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and no curative therapies. SCF-Skp2 E3 ligase is a target for cancer therapy, but there have been no reports about Skp2 as a target for IPF. Here we demonstrate that Skp2 is a promising therapeutic target for IPF. We examined whether disrupting Skp2 suppressed pulmonary fibrosis in a bleomycin (BLM)-induced mouse model and found that pulmonary fibrosis was significantly suppressed in Skp2-deficient mice compared with controls. The pulmonary accumulation of fibrotic markers such as collagen type 1 and fibronectin in BLM-infused mice was decreased in Skp2-deficient mice. Moreover, the number of bronchoalveolar lavage fluid cells accompanied with pulmonary fibrosis was significantly diminished. Levels of the Skp2 target p27 were significantly decreased by BLM-administration in wild-type mice, but recovered in Skp2−/− mice. In vimentin-positive mesenchymal fibroblasts, the decrease of p27-positive cells and increase of Ki67-positive cells by BLM-administration was suppressed by Skp2-deficency. As these results suggested that inhibiting Skp2 might be effective for BLM-induced pulmonary fibrosis, we next performed a treatment experiment using the Skp2 inhibitor SZL-P1-41. As expected, BLM-induced pulmonary fibrosis was significantly inhibited by SZL-P1-41. Moreover, p27 levels were increased by the SZL-P1-41 treatment, suggesting p27 may be an important Skp2 target for BLM-induced pulmonary fibrosis. Our study suggests that Skp2 is a potential molecular target for human pulmonary fibrosis including IPF.

Chemical probes of Skp2-mediated p27 ubiquitylation and degradation.

Skp2 is a member of the F-box family of proteins that serve as substrate-specific adaptors in Skp1-CUL1-ROC1-F-box (SCF) E3 ubiquitin ligases. Skp2 (Fbxl1) directly binds to the tumor suppressor p27 in the context of the SCFSkp2 E3 ubiquitin ligase to ubiquitylate and target-phosphorylated p27 for proteasomal degradation. As p27 is a powerful suppressor of growth in a variety of cells, and as Skp2 is also overexpressed in many human cancers, Skp2 is considered an oncogene and an intriguing drug target. However, despite 20 years of investigation, a valid chemical inhibitor of Skp2-mediated degradation of p27 has not been identified. Recently, an increasing number of compounds designed to have this bioactivity have been reported. Here, we conduct a meta-analysis of the evidence regarding bioactivity, structure, and medicinal chemistry in order to evaluate and compare these Skp2 inhibitor compounds. Despite chemically diverse compounds with a wide array of Skp2-mediated p27 ubiquitylation inhibition properties reported by several independent groups, no current chemical probe formally qualifies as a validated pharmaceutical hit compound. This finding suggests that our knowledge of the structural biochemistry of the Skp2-p27 complex remains incomplete and highlights the need for novel modes of inquiry.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0–20 μM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

Genistein inhibits the S-phase kinase-associated protein 2 expression in breast cancer cells.

Breast cancer is one of the most lethal cancers affecting women worldwide and was estimated to account for ~30% of all new cancer diagnoses in women. Although available evidence has proved the tumor suppressor role of genistein in cancer, the underling mechanisms have remained to be fully elucidated. S-phase kinase-associated protein 2 (Skp2) has been revealed to critically enhance the pathogenesis of multiple human cancers. The present study determined whether genistein exerts its anti-tumor function by suppressing Skp2 in breast cancer cells. Genistein significantly inhibited the proliferation, invasion and migration of breast cancer cells. Furthermore, genistein treatment also induced marked apoptosis and a typical cell cycle arrest in G2/M phase. Mechanistically, genistein treatment was identified to cause a significant downregulation of Skp2. Two crucial tumor suppressors, p21 and p27, were upregulated in genistein-treated breast cancer cells. The present results revealed that genistein exerted its tumor suppressor effect at least partially via inhibition of Skp2 and promotion of its downstream targets p21 and p27. Therefore, inactivation of Skp2 by genistein may be a promising approach for breast cancer treatment.

Abstract 2108: Imatinib-induced downregulation of Skp2 inhibits cell growth in human squamous cell carcinoma

Abstract It is apparent that Skp2(S-phase kinase associated protein) plays a crucial role in tumorigenesis of various human cancers. Imatinib, inhibitor of BCR-ABL tyrosine kinase, KIT and PDGF receptor, has been approved for the treatment and investigation of chronic myeloid leukemia, gastrointestinal stromal tumors and additional various solid tumors. But An area of skin cancer has not been yet investigated. The aim of this study was to explore the role of the Skp2 in Imatinib antitumor effect on human skin cancer. To explore Imatinib effect on growth of A431 cells (squamous cell carcinoma / skin cancer) we conducted CCK8 assay and Flow cytometry. Western blot assay was performed for determining the functions and molecular mechanism of Imatinib in A431 cells. we found that Imatinib significantly inhibited cell growth and induced cell cycle arrest at G0/G1 phase. Furthermore, we observed that depletion of Skp2 triggered cell cycle arrest and colony formation inhibition by Imatinib. Mechanistically we identified that Imatinib markedly downregulated Skp2 protein expression and subsequently upregulated p21 expression via increased protein stability. In conclusion, Imatinib exerts its antitumor activity via inhibition of Skp2 - p21 axis and these findings suggest that targeting Skp2 by Imatinib could be promising therapeutic approach for Squamous cell carcinoma therapy. Note: This abstract was not presented at the meeting. Citation Format: Hyo Jin Jeong. Imatinib-induced downregulation of Skp2 inhibits cell growth in human squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2108. doi:10.1158/1538-7445.AM2017-2108

Skp2 deficiency restricts the progression and stem cell features of castration-resistant prostate cancer by destabilizing Twist.

Castration-resistant prostate cancer (CRPC) remains a major clinical challenge due to the lack of effective targeted therapy for its treatment. The mechanism underlying how CRPC gains resistance towards hormone depletion and other forms of chemotherapy is poorly understood. Research on understanding the factors that drive these processes is desperately needed to generate new therapies to cure the disease. Here, we discovered a fundamental role of S-phase protein kinase 2 (Skp2) in the formation and progression of CRPC. In transgenic adenocarcinoma mouse prostate model, Skp2 depletion leads to a profound repression of prostate tumor growth and distal metastasis and substantially prolonged overall survival. We revealed that Skp2 regulates CRPC through Twist-mediated oncogenic functions including epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) acquisitions. Mechanistically, Skp2 interacted with Twist and promoted the non-degradative ubiquitination of Twist. Consequently, Skp2 stabilized Twist protein expression by preventing proteasomal degradation of Twist by β-TrCP. We found that Twist overexpression augments CSC self-renewal and population and that Skp2 inhibition reverts Twist’s effects on CSC regulation. Furthermore, genetically depleting or pharmacologically inactivating Skp2 synergistically re-sensitized CRPC cells towards chemotherapies such as paclitaxel or doxorubicin. Together, the current study uncovering Skp2-mediated Twist stabilization and oncogenic functions in CRPC offers new knowledge on how CRPC progresses and acquires chemoresistance during tumor progression. It provides prove-of principle that Skp2 targeting is a promising approach to combat metastatic CRPC by targeting Twist and CSCs.

Inhibition of Skp2 suppresses the proliferation and invasion of osteosarcoma cells

Osteosarcoma (OS) is a common bone tumor that mainly affects children and young adults. S-phase kinase‑associated protein 2 (Skp2) has been characterized to play a critical oncogenic role in a variety of human malignancies. However, the biological function of Skp2 in OS remains largely obscure. In the present study, we elucidated the role of Skp2 in cell growth, cell cycle, apoptosis and migration in OS cells. We found that depletion of Skp2 inhibited cell growth in both MG-63 and SW1353 cells. Moreover, we observed that depletion of Skp2 triggered cell apoptosis in two OS cell lines. Furthermore, downregulation of Skp2 induced cell cycle arrest in the G0/G1phase in OS cells. Notably, our wound healing assay results revealed that inhibition of Skp2 suppressed cell migration in OS cells. Invariably, our western blot results demonstrated that depletion of Skp2 in OS cells inhibited activation of pAkt and increased p27 expression in OS cells, suggesting that Skp2 exerted its oncogenic function partly through the regulation of Akt and p27. Our findings revealed that targeting Skp2 could be a promising therapeutic strategy for the treatment of OS.

Inhibition of Skp2 sensitizes lung cancer cells to paclitaxel.

S-phase kinase-associated protein 2 (Skp2) is an E3 ubiquitin ligase and plays an important role in the control of cell cycle progression. Skp2 is upregulated in several cancers, including lung cancers, but the role of Skp2 in the tumorigenesis and anticancer drug resistance in human lung cancer remains to be determined. We report here that Skp2 positively regulated mitotic arrest deficient 2 (MAD2) expression and that inhibition of Skp2 sensitizes human lung cancer cells to paclitaxel. Knockdown of Skp2 by small interfering RNA (siRNA) decreased Mad2 messenger RNA (mRNA) and protein levels in A549 and NCI-H1975 cells, accompanied with upregulation of p27 but decrease of the phosphorylation of retinoblastoma (Rb). In contrast, ectopic overexpression of Skp2 increased Mad2 mRNA and protein levels and phosphorylation of Rb, while it decreased p27. Pharmacological inhibition of CDK1/2 by flavopiridol or E2F1 with HLM006474 led to downregulation of Mad2 expression and prevented the increase of Mad2 expression by Skp2. Most importantly, pharmacological inhibition of Skp2 sensitized A549 and NCI-H1299 cells to paclitaxel. Our results demonstrated that SKP2 positively regulates the gene expression of MAD2 through p27-CDKs-E2F1 signaling pathway and that inhibition of Skp2 sensitizes A549 and NCI-H1299 cells to paclitaxel, suggesting that small molecule inhibitors of Skp2 are potential agents for the treatment of lung cancer with upregulation of Skp2.

Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer.

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option.

Rottlerin exerts its anti-tumor activity through inhibition of Skp2 in breast cancer cells.

Studies have investigated the tumor suppressive role of rottlerin in carcinogenesis. However, the molecular mechanisms of rottlerin-induced anti-tumor activity are largely unclear. Skp2 (S-phase kinase associated protein 2) has been validated to play an oncogenic role in a variety of human malignancies. Therefore, inactivation of Skp2 could be helpful for the treatment of human cancers. In the current study, we explore whether rottlerin could inhibit Skp2 expression, leading to inhibition of cell growth, migration and invasion in breast cancer cells. We found that rottlerin treatment inhibited cell growth, induced apoptosis and cell cycle arrest. We also revealed that rottlerin suppressed cell migration and invasion in breast cancer cells. Mechanically, we observed that rottlerin significantly down-regulated the expression of Skp2 in breast cancer cells. Importantly, overexpression of Skp2 abrogated rottlerin-mediated tumor suppressive activity, whereas down-regulation of Skp2 enhanced rottlerin-triggered anti-tumor function. Strikingly, we identified that rottlerin exhibited its anti-tumor potential partly through inactivation of Skp2 in breast cancer. Our findings indicate that rottlerin could be a potential safe agent for the treatment of breast cancer.

Inhibiting the role of Skp2 suppresses cell proliferation and tumorigenesis of human gastric cancer cells via the upregulation of p27kip1.

Gastric cancer is a malignant disease of the digestive system with high rates of incidence and mortality. S‑phase kinase‑associated protein 2 (Skp2) is a novel oncogene, which has been identified to be important in tumor progression and metastasis. In order to clarify the role of Skp2 in human gastric cancer, the present study detected the expression of Skp2 in human gastric cancer tissues, and investigated the molecular mechanism of Skp2 in the progression of gastric carcinoma. The results of the initial bioinformatics analysis showed that Skp2 was significantly upregulated in 31specimens of primary gastric cancer from a UK patient cohort, and in 10 gastric cancer lines of a side population, compared with normal gastric tissues (P<0.01). Specimens from 47patients with gastric cancer and 19 normal gastric tissue specimens were obtained and analyzed using western blot analysis. The positive rate of expression of Skp2 was 87.2%, indicating that the expression of Skp2 was observed in 41specimens of the detected gastric cancer samples, whereas the positive rate of the expression of Skp2 was 5.6% in the normal gastric samples (P<0.01). In the human gastric cancer cell lines, the defective regulation of Skp2 or presence of an Skp2 inhibitor inhibited the proliferation of BGC‑823 and MKN‑45 cells. In addition, the Skp2 inhibitor suppressed the proliferation of gastric cancer cells in a time‑ and dose‑dependent manner. Furthermore, transfection with Skp2 short hairpin (sh)RNA or treatment with SKPinhibitorC1 for 48 and 72h led to the accumulation of p27kip1 in Hela cells. Tumorigenicity experiments involving nude mice showed that interference of the expression of Skp2 inhibited the growth of the human gastric tumor cells in the nude mice, and the tumor weights and volumes in the Skp2 shRNA group were significantly lower, compared with those in the negative control shRNA group (P<0.01) and untreated group (P<0.01). Taken together, these data suggested that Skp2 acted as an oncogene in human gastric cancer, and that Skp2‑mediated p27kip1 degradation contributed to the progression of gastric cancer. Abrogating the effects of Skp2 may effectively inhibit the growth of gastric cancer cells, which may be useful as a novel target in the clinical treatment of gastric cancer.

Skp2 is associated with paclitaxel resistance in prostate cancer cells.

Prostate cancer is the most commonly diagnosed tumor in men in the United States. Patients with hormone-refractory prostate cancer are often treated with paclitaxel, but most of them eventually develop drug resistance. S-phase kinase associated protein2 (Skp2) is a component of the SCF (Skp1-Cullin1-F-box) type of E3 ubiquitin ligase complexes. In the present study, we investigated the role of Skp2 in paclitaxel-resistant DU145-TxR or PC-3-TxR cells by Skp2 silencing or using Skp2 inhibitors. We first confirmed that Skp2 expression is up-regulated in DU145-TxR or PC-3-TxR cells compared with their parental cells DU145 or PC-3, respectively. Knockdown of Skp2 or Skp2 inhibitor treatment in DU145-TxR or PC-3-TxR cells restored paclitaxel sensitivity. E-cadherin was decreased while Vimentin was increased in PC-3-TxR or DU145-TxR cells. In addition, p27 expression was inversely correlated with Skp2 expression in DU145-TxR or PC-3-TxR cells. Moreover, p27 was found to increase in both Skp2 silencing PC-3-TxR and DU145-TxR cells. These results suggest that Skp2 is associated with prostate cancer cell resistance to paclitaxel. Skp2 may be a potential therapeutic target for drug-resistant prostate cancer.

Skp2 inhibits osteogenesis by promoting ubiquitin–proteasome degradation of Runx2

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2–Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.

A Chemical Inhibitor of the Skp2/p300 Interaction that Promotes p53‐Mediated Apoptosis

AbstractSkp2 is thought to have two critical roles in tumorigenesis. As part of the SCFSkp2 ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53‐mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity‐based high‐throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300‐mediated p53 acetylation, resulting in p53‐mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non‐proteolytic function in tumorigenesis.

A Chemical Inhibitor of the Skp2/p300 Interaction that Promotes p53-Mediated Apoptosis.

Skp2 is thought to have two critical roles in tumorigenesis. As part of the SCF(Skp2) ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53-mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity-based high-throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300-mediated p53 acetylation, resulting in p53-mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non-proteolytic function in tumorigenesis.

Abstract LB-068: Inhibitors of Skp2 E3 ligase-mediated degradation of p27kip1 as a novel therapeutic approach to malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is a highly aggressive cancer related to asbestos exposure for which there is no cure. Even with multimodal therapies median survival is only 24 months. Therefore, new therapeutic approaches are desperately needed for this cancer. MPMs are epithelioid, sarcomatoid, or composed of both patterns (biphasic); those tumors that are sarcomatous are most aggressive. Nuclear p27kip1 (p27) is a tumor suppressor critical to inhibiting cell proliferation by blocking Cdk2 activity to enforce cell cycle arrest. Cell cycle dysregulation has been shown to be involved in the pathogenesis of MPM. Accordingly, among the prognostic markers described for MPM, nuclear expression of p27 has been associated with increased progression-free survival. The levels of nuclear p27 are regulated post-translationally by the ubiquitin E3 ligase complex SCF-Skp2/Cks1, which causes its destruction. Accordingly, in many human cancers, Skp/Cks1 levels are high and associated with poor survival. Recently, we have shown that inhibitors of Skp2 E3 ligase activity block both, estrogen-induced degradation of nuclear p27 and inhibit proliferation of primary endometrial carcinoma cells in vitro as well as increase nuclear p27 and inhibit endometrial hyperplasia in vivo in estrogen-primed mice. Whereas nuclear p27 appears to be a good prognostic biomarker for MPM, the mechanistic relationship to high levels of Skp2 has not been shown. Hence, the aim of our study is to determine whether MPM is a candidate cancer for targeted Skp2 inhibitor therapy to regain cell cycle control. By gene expression profiling of 53 surgically resected MPM tumors and matched control tissue, Skp2 gene expression was elevated by 3-fold. In addition, by immunoblot analysis, we show that high levels of Skp2 protein are inversely related to p27 in four cell lines derived from MPM tumors of different tumor types. Interestingly, Skp2 expression was higher and associated with a higher proliferative rate in H2452 and HP-1 cells derived from biphasic tumors compared to those from epithelioid (H2591) or sarcomatoid (H2373) tumors and LP9 cells derived from normal pleural tissue expressed the lowest level of Skp2 and a lower proliferative rate. Similarly, by IHC of MPM tissue, Skp2 levels were inversely correlated with p27. These results suggest that Skp2 inhibitor therapy to stabilize nuclear p27 and regain growth control is a promising new approach for treatment of MPM. Citation Format: Julien Daubriac, Jonathan Melamed, Unnati Pandya, Harvey I. Pass, Leslie I. Gold. Inhibitors of Skp2 E3 ligase-mediated degradation of p27kip1 as a novel therapeutic approach to malignant pleural mesothelioma. [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-068. doi:10.1158/1538-7445.AM2015-LB-068

Abstract 3792: SKP2 supports cell proliferation and is regulated by Notch signaling in myoblasts and embryonal rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) in a pediatric tumor of myogenic origin. It includes two subtypes: embryonal and alveolar. Embryonal RMS (ERMS) cells express key myogenic factors such as MyoD and Myogenin, but proliferate indefinitely and have lost the ability to terminally differentiate into skeletal myofibers. Differently from the alveolar tumors bearing specific chromosomal translocations, ERMS has cytogenetic aberrations and molecular deregulations of pathways regulating senescence, proliferation and differentiation. It has been shown that SKP2, an F-box protein and a component of the ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box), is over-expressed in RMS primary samples and correlates with a dismal outcome. Therefore, we sought here to investigate the regulation of SKP2 and its role in ERMS. Methods: We modulated SKP2 expression through silencing, by using a siRNA validated in the literature, and forcing its expression through retroviral infection. In parallel, we investigate the effect of Notch signaling modulation on SKP2 expression. Results: SKP2 silencing resulted in cell cycle slowdown in both normal myoblasts and ERMS cells. Down-regulation of Notch1 led to SKP2 reduction while that of Notch3 supported SKP2 expression. Cosilencing SKP2 and Notch3 gave raise to myoblast-like structure formation in ERMS and facilitated myoblasts fusion. Finally, using a SKP2 inhibitor ERMS cell proliferation was completely blocked. Conclusion: Altogether, these preliminary experiments suggest that SKP2 could be regulated by Notch signaling in ERMS and that its inhibition hampers tumor cell proliferative capability. Note: This abstract was not presented at the meeting. Citation Format: Rossella Rota, Laura Adesso, Beatrice Conti, Roberta Ciarapica, Lavinia Raimondi, Maria De Salvo, Sonia Rodriguez, Nadia Carlesso, Lucio Miele, Franco Locatelli. SKP2 supports cell proliferation and is regulated by Notch signaling in myoblasts and embryonal rhabdomyosarcoma. [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 3792. doi:10.1158/1538-7445.AM2015-3792

Arsenic Trioxide Inhibits Cell Growth and Invasion via Down- Regulation of Skp2 in Pancreatic Cancer Cells.

Arsenic trioxide (ATO) has been found to exert anti-cancer activity in various human malignancies. However, the molecular mechanisms by which ATO inhibits tumorigenesis are not fully elucidated. In the current study, we explored the molecular basis of ATO-mediated tumor growth inhibition in pancreatic cancer cells. We used multiple approaches such as MTT assay, wound healing assay, Transwell invasion assay, annexin V-FITC, cell cycle analysis, RT-PCR and Western blotting to achieve our goal. We found that ATO treatment effectively caused cell growth inhibition, suppressed clonogenic potential and induced G2-M cell cycle arrest and apoptosis in pancreatic cancer cells. Moreover, we observed a significant down-regulation of Skp2 after treatment with ATO. Furthermore, we revealed that ATO regulated Skp2 downstream genes such as FOXO1 and p53. These findings demonstrate that inhibition of Skp2 could be a novel strategy for the treatment of pancreatic cancer by ATO.

Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

Colon cancer is a common type of malignancy in the digestive system. The aim of the present study was to investigate the role of S-phase kinase-associated protein2(Skp2) in colon carcinoma and to identify whether depletion of Skp2 by Skp2‑RNA interference (RNAi) attenuates the proliferation and migration of colon carcinoma. Three pairs of small interfering (si)RNA were designed based on the Skp2 gene sequence and the most effective one was used to silence the Skp2 gene in SW620 cells. Subsequent to the interference, quantitative polymerase chain reaction and western blot analysis were used for detecting the expression of Skp-2 mRNA and protein, respectively. The data demonstrated that the Skp2‑siRNA effectively inhibited proliferation (P<0.01), increased the levels of apoptosis and induced G0/G1phase arrest of the SW620 cells (P<0.01). Transfection of the Skp2 siRNA into SW620 cells effectively reduced Skp2 protein levels, while p27 protein levels increased. In the invivo experiments, a lentiviral vector of the Skp2-RNAi transfected into SW620 cells markedly inhibited Skp2 expression, as detected by immunohistochemical analysis of nude mice. Additionally, tumorigenicity experiments showed that inhibition of Skp2 significantly increased the survival rate of nude mice. Thus, the invitro and invivo results demonstrated that interference of Skp2 expression significantly inhibited the proliferation and induced the apoptosis of SW620cells. This suggests that Skp2 protein has an important role in the progression of colon cancer. Therefore, Skp2 may enable the early diagnosis of colon cancer and provide new insights into molecular targets for cancer therapy.