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Jin Y, Cao J, Cheng H, Hu X. LncRNA POU6F2-AS2 contributes to malignant phenotypes and paclitaxel resistance by promoting SKP2 expression in stomach adenocarcinoma. J Chemother 2023; 35:638-652. [PMID: 36797828 DOI: 10.1080/1120009x.2023.2177807] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Revised: 12/12/2022] [Accepted: 02/01/2023] [Indexed: 02/18/2023]
Abstract
This study aimed to investigate the role and mechanism of POU6F2-AS2 in the development of gastric cancer. POU6F2-AS2 expression was considerably higher in clinical stomach adenocarcinoma (STAD) tissues and gastric cancer cell lines (MKN-28 and MGC-803) than in neighbouring normal tissues and gastric mucosa epithelial cells (GES-1). POU6F2-AS2 overexpression resulted in a low overall survival probability, progression-free survival probability and post progression survival probability, as well as increased cell viability, migration and invasion of gastric cancer cells, thereby inhibiting apoptosis. Based on RNA pull-down, cycloheximide and MG132 incubation experiments, POU6F2-AS2 promoted SKP2 by stabilizing NONO expression. In addition, in vivo silencing of POU6F2-AS2 in gastric cancer cells can inhibit tumour progression and produce a synergistic antitumour effect when combined with paclitaxel. POU6F2-AS2 is overexpressed in STAD, which is attributed to a bad prognosis. In vitro and in vivo experiments have confirmed that the POU6F2-AS2/NONO/SKP2 axis promotes STAD progression, and that the silencing of POU6F2-AS2 plays a synergistic antitumour effect when combined with paclitaxel. Therefore, POU6F2-AS2 may be potentially developed as a target to inhibit STAD and reduce chemoresistance.
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Affiliation(s)
- Yanzhao Jin
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Jiaqing Cao
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Hua Cheng
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Xiaoyun Hu
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
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Liang H, Zhang F, Hong Y, Wu Y, Xie H, Zhang C, Wang Z, Lu Z, Yang H. Synergistic Silencing of Skp2 by siRNA Self-Assembled Nanoparticles as a Therapeutic Strategy for Advanced Prostate Cancer. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2022; 18:e2106046. [PMID: 35182014 DOI: 10.1002/smll.202106046] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 01/21/2022] [Indexed: 06/14/2023]
Abstract
Advanced prostate cancer, harboring multiple mutations of tumor suppressor genes, is refractory to conventional therapies. Knockout of the Skp2 gene blocks pRb/p53 doubly deficient prostate cancer in mice, which inspired the authors to develop an approach for delivering siRNA that would efficiently silence Skp2 (siSkp2) in vivo. Here, a facile strategy is reported to directly assemble siSkp2 with the natural compound quercetin (Que) into supramolecular nanoparticles (NPs). This carrier-free siSkp2 delivery system could effectively protect siSkp2 from degradation in serum and enhance its cellular internalization. Furthermore, the siSkp2/Que NPs exhibit synergistic effects in Skp2 silencing, because they can degrade the mRNA and protein of Skp2 simultaneously. Indeed, siSkp2/Que NPs remarkably diminish the Skp2 abundance and further inhibit the proliferation and migration of TMU cells (RB1/TP53/KRAS triple mutations) in vitro. The in vivo results further show that i.v. administration of siSkp2/Que NPs efficiently accumulates in tumor sites and strongly inhibits the growth of TMU tumors in nude mice. Importantly, the siSkp2/Que NPs do not induce any abnormality in the treated mice, which suggests satisfactory biocompatibility. Collectively, this study describes a tractable siRNA self-assembled strategy for Skp2 silencing, which might be a promising nanodrug to cure multitherapy-resistant advanced prostate cancer.
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Affiliation(s)
- Hong Liang
- College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, P. R. China
- Fujian Key Laboratory of Functional Marine Sensing Materials, Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, 350108, P. R. China
| | - Fangming Zhang
- College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, P. R. China
| | - Yannv Hong
- College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, P. R. China
| | - Yue Wu
- College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, P. R. China
| | - Huanzhang Xie
- Fujian Key Laboratory of Functional Marine Sensing Materials, Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, 350108, P. R. China
| | - Chen Zhang
- Fujian Key Laboratory of Functional Marine Sensing Materials, Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, 350108, P. R. China
| | - Zonghua Wang
- Fujian Key Laboratory of Functional Marine Sensing Materials, Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, 350108, P. R. China
| | - Zhonglei Lu
- College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, P. R. China
| | - Huanghao Yang
- MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350108, P. R. China
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Effects of Polygonatum sibiricum Polysaccharides (PSP) on Human Esophageal Squamous Cell Carcinoma (ESCC) via NF-κB Signaling Pathway. INT J POLYM SCI 2019. [DOI: 10.1155/2019/3852879] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Objective. To explore the effects of different concentrations of Polygonatum sibiricum polysaccharides (PSP) on human esophageal squamous cell carcinoma (ESCC) cell line Eca109 and explore the new approach for the treatment of ESCC. Methods. Eca109 cells were divided into 5 groups, including one control group and 4 experimental groups where the concentrations of PSP used were 50, 100, 200, and 400 μg/mL. The proliferation rate of Eca109 cells in each group was measured with the CCK8 assay, and the apoptosis rate in each group was analyzed by flow cytometry; the in vitro scratch assay was used to determine the migration ability of Eca109 cells after PSP treatment; the expression levels of IL-1, IL-6, IL-10, TNF-α, and TGF-β were measured by RT-PCR, and the expression levels of TLR4 and proteins that are related to NF-κB signaling pathways were determined by Western blot. Results. PSP significantly inhibited the proliferation of Eca109 cells (p<0.05) on a time- and dose-dependent manner; the apoptosis rates of Eca109 cells in experimental groups were significantly increased after 48 h of culture (p<0.05); PSP significantly reduced the migration and invasion ability of Eca109 cells (p<0.05); RT-PCR results showed that the expression of IL-10 in Eca109 cells increased significantly after treatment with PSP (p<0.05), while the expression of IL-1, IL-6, TNF-α, and TGF-β decreased significantly (p<0.05). Compared with the control group, the expression level of TLR4, NF-κB/p50, and NF-κB/p65 protein in each experimental group was significantly lower than that in the control group (p<0.05). Conclusions. PSP significantly inhibited the proliferation, invasion, and migration of Eca109 cells and promoted cell apoptosis. These observed effects were probably due to the PSP’s inhibition on the NF-κB signaling pathway in Eca109 cells via the regulation of the TLR4 expression.
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Prognostic significance of CDC25C in lung adenocarcinoma: An analysis of TCGA data. Cancer Genet 2019; 233-234:67-74. [PMID: 31109596 DOI: 10.1016/j.cancergen.2019.04.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2019] [Revised: 03/16/2019] [Accepted: 04/03/2019] [Indexed: 01/01/2023]
Abstract
OBJECTIVE Cell division cycle 25C (CDC25C) is involved in the regulation of the G2/M phase transition and is associated with various cancers, including non-small cell lung cancer. We evaluated its prognostic value in lung adenocarcinoma (LUAD) based on data from The Cancer Genome Atlas (TCGA). METHODS Kruskal-Wallis test, Wilcoxon signed-rank test, and logistic regression were used to evaluate relationships between clinical-pathologic features and CDC25C expression. Cox regression analyses and the Kaplan-Meier method were used to evaluate factors contributing to prognosis. Gene set enrichment analysis (GSEA) was performed. RESULTS High CDC25C expression in LUAD was associated with a high tumor extent (odds ratio (OR) = 2.23 (1.52-3.29), P < 0.001), regional lymph node invasion (OR = 2.18 (1.48-3.22), P < 0.001), OR = advanced stage (OR = 2.47 (1.72-3.59), P < 0.001), and poor status (OR = 1.87 (1.19-2.96), P = 0.007). A univariate analysis showed that high CDC25C expression is associated with a short overall survival (OS) (HR: 1.873; 95% CI: 1.385-2.535; P < 0.001) and poor progression-free survival (HR: 1.503; 95% CI: 1.173-1.926; P = 0.0012). In a multivariate analysis, high CDC25C expression was associated with poor OS (HR = 2.193; CI: 1.394-3.452, P = 0.001). GSEA showed the enrichment of cell cycle, apoptosis, p53-dependent G1 DNA damage response, S-phase, mitotic M-M G1 phases, and FA-mediated cell death in the CDC25C high-expression phenotype. CONCLUSIONS CDC25C predicts poor prognosis in LUAD and may function in cell cycle regulation and FAS-mediated apoptosis.
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Yu B, Gao W, Zhou H, Miao X, Chang Y, Wang L, Xu M, Ni G. Propofol induces apoptosis of breast cancer cells by downregulation of miR-24 signal pathway. Cancer Biomark 2018; 21:513-519. [PMID: 29103019 DOI: 10.3233/cbm-170234] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND AND OBJECTIVE Propofol, an intravenous anesthetic agent, has been found to inhibit growth of breast cancer cells. However, the mechanisms underlying the antitumor are not known. A recent report has found that propofol could significantly downregulate miR-24 expression in the human malignant cancers. In breast cancer cells, overexpression of miR-24 promotes cell proliferation and inhibits cell apoptosis by downregulation of p27. The miR-24 has been reported to be overexpressed in breast cancer and breast cancer cell lines. In the present study, we hypothesized that propofol induces apoptosis of breast cancer cells by miR-24/p27 signal pathway. METHODS Breast cancer MDA-MB-435 cells were exposed to propofol (10 μM) for 6 hr and cell death was assessed using TUNEL staining, Flow cytometry and cleaved caspase-3 expression. microRNA-24 (miR-24) expression was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). miR-24 was overexpressed using a miR-24 mimic. P27 was knocked down using a small interfering RNA. p27 and cleaved caspase-3 expression was assessed by Western blot. RESULTS MDA-MB-435 exposed to propofol showed a significant increase in apoptotic cells, followed by the downregulation of miR-24, upregulation of p27 expression and cleaved caspase-3 expression. Targeting p27 inhibits propofol-induced cell apoptosis; miR-24 overexpression decreased propofol-induced cell apoptosis, cleaved caspase-3 and p27 expression. CONCLUSIONS Propofol inducescell death in MDA-MB-435 cells via inactivation of miR-24/p27 signal pathway.
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Affiliation(s)
- Benxia Yu
- Department of Imaging, Yantai Yuhuangding Hospital, Yantai, Shandong, China.,Department of Imaging, Yantai Yuhuangding Hospital, Yantai, Shandong, China
| | - Wei Gao
- Department of Imaging, Yantai Yuhuangding Hospital, Yantai, Shandong, China.,Department of Imaging, Yantai Yuhuangding Hospital, Yantai, Shandong, China
| | - Hui Zhou
- Department of Anesthesiology, Yantai Yuhuangding Hospital, Yantai, Shandong, China
| | - Xia Miao
- Department of Clinical Laboratory, The Affiliated Hospital of Weifang Medical College, Weifang, Shandong, China
| | - Yuan Chang
- Department of Anesthesiology, Yantai Yuhuangding Hospital, Yantai, Shandong, China
| | - Liping Wang
- Department of Imaging, Yantai Yuhuangding Hospital, Yantai, Shandong, China
| | - Miao Xu
- Department of Clinical Laboratory, The Affiliated Hospital of Weifang Medical College, Weifang, Shandong, China
| | - Guangzhen Ni
- Department of Clinical Laboratory, The Affiliated Hospital of Weifang Medical College, Weifang, Shandong, China
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Liu Z, Wu Y, Tao Z, Ma L. E3 ubiquitin ligase Hakai regulates cell growth and invasion, and increases the chemosensitivity to cisplatin in non‑small‑cell lung cancer cells. Int J Mol Med 2018; 42:1145-1151. [PMID: 29786107 DOI: 10.3892/ijmm.2018.3683] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Accepted: 04/26/2018] [Indexed: 11/06/2022] Open
Abstract
Hakai was originally identified as an E3 ubiquitin ligase of the E‑cadherin complex implicated in cell adhesion and invasion. Recently, emerging evidence has strongly suggested that Hakai serves a pivotal role in the tumorigenesis of certain tumors. However, the role of Hakai in non‑small‑cell lung cancer (NSCLC) and its underlying molecular mechanism have not been clarified. In the present study, it was observed that Hakai was highly expressed in NSCLC cell lines compared with human normal bronchial epithelial cells, and transfection with Hakai small interfering RNA significantly inhibited the growth of A549 and NCI‑H460 NSCLC cells. In addition, the inhibition of Hakai suppressed NSCLC cell migration and invasion through upregulation of E‑cadherin and downregulation of N‑cadherin. Notably, it was also revealed that knockdown of Hakai led to a decrease in the expression of phosphorylated AKT (Ser473), and a significant enhancement of chemosensitivity to cisplatin was observed following Hakai suppression. In conclusion, the present study demonstrated for the first time that knockdown of Hakai inhibited the proliferation, migration and invasion of NSCLC cells, and sensitized NSCLC cells to cisplatin. Thus, Hakai may serve as a potential therapeutic target for the treatment of NSCLC.
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Affiliation(s)
- Zi Liu
- Department of Chemical Biology and Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan, Anhui 243002, P.R. China
| | - Yuqing Wu
- Department of Chemical Biology and Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan, Anhui 243002, P.R. China
| | - Zijian Tao
- Department of Pathology, Ma'anshan Municipal People's Hospital, Ma'anshan, Anhui 243000, P.R. China
| | - Liang Ma
- Department of Chemical Biology and Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan, Anhui 243002, P.R. China
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Upregulation of kinesin family member 4A enhanced cell proliferation via activation of Akt signaling and predicted a poor prognosis in hepatocellular carcinoma. Cell Death Dis 2018; 9:141. [PMID: 29396392 PMCID: PMC5833581 DOI: 10.1038/s41419-017-0114-4] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2017] [Revised: 09/21/2017] [Accepted: 10/30/2017] [Indexed: 02/07/2023]
Abstract
Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related death worldwide, and the molecular pathogenesis and development of HCC are largely unknown. In the present study, we found that KIF4A expression was upregulated in HCC (678 samples, P = 2.03E-8) based on a meta-analysis of Oncomine database. We further confirmed that both KIF4A mRNA and protein expressions were overexpressed in human HCC tumour tissues as well as cancer cell lines. Higher KIF4A expression was correlated with poorer overall survival (P < 0.0001) and disease-free survival (P < 0.0337) in HCC patients. We constructed in vitro KIF4A overexpression and depletion HCC cell models. KIF4A overexpression significantly enhanced cellular proliferation and clonogenic abilities, whereas KIF4A depletion caused a dramatic increase of cells with abnormal chromosome segregation and subsequently resulted in augmentation of apoptosis in HCC cells. In addition, we demonstrated that KIF4A depletion was related to inhibition of Akt kinase activity and induction of intrinsic apoptosis signaling pathway. Taken together, KIF4A may act as a prognostic biomarker and potential therapeutic target in human HCC.
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Matrine derivative YF-18 inhibits lung cancer cell proliferation and migration through down-regulating Skp2. Oncotarget 2017; 8:11729-11738. [PMID: 28036296 PMCID: PMC5355299 DOI: 10.18632/oncotarget.14329] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2016] [Accepted: 12/16/2016] [Indexed: 12/02/2022] Open
Abstract
Lung cancer is the leading cause of cancer related death which needs novel drugs to improve patient outcomes. In this study, we examined the ability of YF-18, a novel matrine derivative to inhibit the growth and migration of lung cancer cells. By cell cycle analysis, wound healing and transwell assays, we found that YF-18 induced G2/M cell cycle arrest and inhibited migration of lung cancer cells in a dose-dependent manner. Further results indicated that YF-18 inhibited cell proliferation and migration through down-regulating Skp2 and up-regulating its substrates, p27 and E-cadherin. Moreover, YF-18 inhibited A549-luciferase cell xenograft tumor growth in a dose-dependent manner. The findings indicate that YF-18 bears therapeutic potentials for lung cancer.
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RalGPS2 Is Essential for Survival and Cell Cycle Progression of Lung Cancer Cells Independently of Its Established Substrates Ral GTPases. PLoS One 2016; 11:e0154840. [PMID: 27149377 PMCID: PMC4858283 DOI: 10.1371/journal.pone.0154840] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2016] [Accepted: 04/20/2016] [Indexed: 11/19/2022] Open
Abstract
The human genome contains six genes coding for proteins validated in vitro as specific activators of the small GTPases “Ras-related protein Ral-A” and “Ras-related protein Ral-B”, generically named Ral-guanine nucleotide exchange factors (RalGEF). Ral proteins are important contributors to Ras oncogenic signaling, and RAS oncogenes are important in human Non-Small Cell Lung Carcinoma (NSCLC). Therefore in this work, RalGEF contribution to oncogenic and non-oncogenic features of human NSCLC cell lines, as anchorage-dependent and independent growth, cell survival, and proliferation, was investigated. Among all human RalGEF, silencing of RGL1 and RALGPS1 had no detectable effect. However, silencing of either RGL2, RGL3, RALGDS or, to a larger extent, RALGPS2 inhibited cell population growth in anchorage dependent and independent conditions (up to 90 and 80%, respectively). RALGPS2 silencing also caused an increase in the number of apoptotic cells, up to 45% of the cell population in transformed bronchial BZR cells. In H1299 and A549, two NSCLC cell lines, RALGPS2 silencing caused an arrest of cells in the G0/G1-phase of cell cycle. Furthermore, it was associated with the modulation of important cell cycle regulators: the E3 Ubiquitin Protein Ligase S-phase kinase-associated protein 2 (Skp2) was strongly down-regulated (both at mRNA and protein levels), and its targets, the cell cycle inhibitors p27 and p21, were up-regulated. These molecular effects were not mimicked by silencing RALA, RALB, or both. However, RALB silencing caused a modest inhibition of cell cycle progression, which in H1299 cells was associated with Cyclin D1 regulation. In conclusion, RALGPS2 is implicated in the control of cell cycle progression and survival in the in vitro growth of NSCLC cell lines. This function is largely independent of Ral GTPases and associated with modulation of Skp2, p27 and p21 cell cycle regulators.
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Matsu-ura T, Sasaki H, Okada M, Mikoshiba K, Ashraf M. Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells. Stem Cell Res Ther 2016; 7:30. [PMID: 26880084 PMCID: PMC4754927 DOI: 10.1186/s13287-016-0286-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2015] [Revised: 11/26/2015] [Accepted: 01/20/2016] [Indexed: 12/25/2022] Open
Abstract
BACKGROUND Pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, have a great potential for regenerative medicine. Induced pluripotent stem cells, in particular, are suitable for replacement of tissue by autologous transplantation. However, tumorigenicity is a major risk in clinical application of both embryonic stem cells and induced pluripotent stem cells. This study explores the possibility of manipulating the cell cycle for inhibition of tumorigenicity. METHODS We genetically modified mouse induced pluripotent stem cells (miPSCs) to overexpress p27 tumor suppressor and examined their proliferation rate, gene expression, cardiac differentiation, tumorigenicity, and therapeutic potential in a mouse model of coronary artery ligation. RESULTS Overexpression of p27 inhibited cell division of miPSCs, and that inhibition was dependent on the expression level of p27. p27 overexpressing miPSCs had pluripotency characteristics but lost stemness earlier than normal miPSCs during embryoid body and teratoma formation. These cellular characteristics led to none or smaller teratoma when the cells were injected into nude mice. Transplantation of both miPSCs and p27 overexpressing miPSCs into the infarcted mouse heart reduced the infarction size and improved left ventricular function. CONCLUSIONS The overexpression of p27 attenuated tumorigenicity by reducing proliferation and earlier loss of stemness of miPSCs. The overexpression of p27 did not affect pluripotency and differentiation characteristics of miPSC. Therefore, regulation of the proliferation rate of miPSCs offers great therapeutic potential for repair of the injured myocardium.
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Affiliation(s)
- Toru Matsu-ura
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, 45267-0529, USA.
| | - Hiroshi Sasaki
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, 45267-0529, USA.
| | - Motoi Okada
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, 45267-0529, USA.
| | - Katsuhiko Mikoshiba
- Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama, 351-0198, Japan.
| | - Muhammad Ashraf
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, 45267-0529, USA. .,Department of Pharmacology, University of Illinois at Chicago College of Medicine, 835 South Wolcott Ave, Chicago, IL, 60612, USA.
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Yu WN, Nogueira V, Sobhakumari A, Patra KC, Bhaskar PT, Hay N. Systemic Akt1 Deletion after Tumor Onset in p53(-/-) Mice Increases Lifespan and Regresses Thymic Lymphoma Emulating p53 Restoration. Cell Rep 2015; 12:610-21. [PMID: 26190111 DOI: 10.1016/j.celrep.2015.06.057] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Revised: 05/18/2015] [Accepted: 06/17/2015] [Indexed: 01/14/2023] Open
Abstract
Akt is frequently activated in human cancers. However, it is unknown whether systemic inhibition of a single Akt isoform could regress cancer progression in cancers that are not driven by Akt activation. We systemically deleted Akt1 after tumor onset in p53(-/-) mice, which develop tumors independently of Akt activation. Systemic Akt1 deletion regresses thymic lymphoma in p53(-/-) mice emulating p53 restoration. Furthermore, pharmacological inhibition of Akt selectively kills thymic lymphoma cells and not primary thymocytes. Mechanistically, Akt1 inhibition in p53(-/-) thymic lymphoma inhibits Skp2 expression and induces FasL, which is the primary cause of cell death. Skp2 exerts resistance to cell death by antagonizing the induction of FasL and reducing FAS expression, which is linked to cyclin D1 expression. The results established a paradigm whereby systemic Akt1 inhibition is sufficient to regress tumors that are not driven by Akt activation and a mechanism of cell survival by Skp2.
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Affiliation(s)
- Wan-Ni Yu
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA
| | - Veronique Nogueira
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA
| | - Arya Sobhakumari
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA
| | - Krushna C Patra
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA
| | - Prashanth T Bhaskar
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA
| | - Nissim Hay
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA; Research & Development Section, Jesse Brown VA Medical Center, Chicago, IL 60612, USA.
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Yan J, Yao Z, Hu K, Zhong Y, Li M, Xiong Z, Deng M. Hepatitis B Virus Core Promoter A1762T/G1764A (TA)/T1753A/T1768A Mutations Contribute to Hepatocarcinogenesis by Deregulating Skp2 and P53. Dig Dis Sci 2015; 60:1315-24. [PMID: 25567052 DOI: 10.1007/s10620-014-3492-9] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2014] [Accepted: 12/11/2014] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND AIM Hepatitis B virus core promoter (CP) mutations can increase risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been associated with hepatocarcinogenesis. The cyclin kinase inhibitor P53 is an important regulator of cell cycle progression. We determined whether HBx mutants that result from mutations in the CP deregulate P53. METHODS A HBx combination (combo) mutant with changes in the CP region that corresponded to A1762T/G1764A (TA), T1753A, and T1768A was constructed and expressed in L-02 and Hep3B cells. The effects of CP mutations on expression and degradation of P53, and the effects on cell cycle progression and proliferation were analyzed. RESULTS The combo mutant decreased levels of P53 and increased cyclin D1 expression, accelerated P53 degradation in L-02 cells, accelerated cell cycle progression, and increased expression of S-phase kinase-associated protein 2 (Skp2) in L-02 and Hep3B cells. Silencing of Skp2 abrogated the effects of CP mutations on P53 expression. The kinetics of P53 expression correlated with changes in cell cycle distribution. CONCLUSIONS The HBx mutant with a combination of CP mutations can up-regulate Skp2, which then down-regulates P53 via ubiquitin-mediated proteasomal degradation, increasing the risk of hepatocellular carcinoma.
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Affiliation(s)
- Jian Yan
- Hepatobiliary Surgery Department, The Third Affiliated Hospital of Sun Yat-Sen University, No. 600, TianHe Road, TianHe District, Guangzhou City, 510630, China
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Kittur H, Weaver W, Di Carlo D. Well-plate mechanical confinement platform for studies of mechanical mutagenesis. Biomed Microdevices 2014; 16:439-47. [PMID: 24619125 DOI: 10.1007/s10544-014-9846-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Limited space for cell division, perhaps similar to the compressed microenvironment of a growing tumor, has been shown to induce phenotypic and karyotypic changes to a cell during mitosis. To expand understanding of this missegregation of chromosomes in aberrant multi-daughter or asymmetric cell divisions, we present a simple technique for subjecting mammalian cells to adjustable levels of confinement which allows subsequent interrogation of intracellular molecular components using high resolution confocal imaging. PDMS micropatterned confinement structures of subcellular height with neighboring taller media reservoir channels were secured on top of confluent cells with a custom compression well-plate system. The system improved ease of use over previous devices since confined cells could be initially grown on glass coverslips in a 12-well plate, and subsequently be imaged by high resolution confocal imaging, or during compression by live cell imaging. Live cell imaging showed a significant increase in abnormal divisions of confined cells across three different cell lines (HeLa, A375, and A549). Immunofluoresecence stains revealed a significant increase in cell diameter and chromosome area of confined cells, but no significant increase in centrosome-centromere distance upon division when compared to unconfined cells. The developed system could open up studies more broadly on confinement effects on mitotic processes, and increase the throughput of such studies.
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Affiliation(s)
- H Kittur
- University of California - Los Angeles, Los Angeles, CA, USA
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14
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Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma. Exp Mol Med 2014; 46:e97. [PMID: 24854087 PMCID: PMC4044675 DOI: 10.1038/emm.2014.23] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2013] [Revised: 11/26/2013] [Accepted: 12/06/2013] [Indexed: 01/19/2023] Open
Abstract
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.
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15
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Snoek BC, Wilt LHAMD, Jansen G, Peters GJ. Role of E3 ubiquitin ligases in lung cancer. World J Clin Oncol 2013; 4:58-69. [PMID: 23936758 PMCID: PMC3708064 DOI: 10.5306/wjco.v4.i3.58] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/03/2012] [Revised: 01/10/2013] [Accepted: 06/06/2013] [Indexed: 02/06/2023] Open
Abstract
E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome. Therefore, E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation, proliferation and apoptosis. E3 ubiquitin ligases are often found overexpressed in human cancers, including lung cancer, and their deregulation has been shown to contribute to cancer development. However, the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting. In this review, we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer. Furthermore, we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets. By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis, we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.
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16
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Histone acetyltransferase hMOF promotes S phase entry and tumorigenesis in lung cancer. Cell Signal 2013; 25:1689-98. [DOI: 10.1016/j.cellsig.2013.04.006] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2013] [Revised: 04/18/2013] [Accepted: 04/18/2013] [Indexed: 12/26/2022]
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17
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Sistrunk C, Kim SH, Wang X, Lee SH, Kim Y, Macias E, Rodriguez-Puebla ML. Skp2 deficiency inhibits chemical skin tumorigenesis independent of p27(Kip1) accumulation. THE AMERICAN JOURNAL OF PATHOLOGY 2013; 182:1854-64. [PMID: 23474082 DOI: 10.1016/j.ajpath.2013.01.016] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/26/2012] [Revised: 12/18/2012] [Accepted: 01/14/2013] [Indexed: 01/11/2023]
Abstract
S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.
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Affiliation(s)
- Christopher Sistrunk
- Department of Molecular Biomedical Sciences and the Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA
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18
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Wei Z, Jiang X, Liu F, Qiao H, Zhou B, Zhai B, Zhang L, Zhang X, Han L, Jiang H, Krissansen GW, Sun X. Downregulation of Skp2 inhibits the growth and metastasis of gastric cancer cells in vitro and in vivo. Tumour Biol 2013; 34:181-192. [PMID: 23229098 DOI: 10.1007/s13277-012-0527-8] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2012] [Accepted: 09/17/2012] [Indexed: 01/14/2023] Open
Abstract
S-phase kinase-associated protein-2 (Skp2) is overexpressed in human cancers and associated with poor prognosis. Skp2 acts as an oncogenic protein by enhancing cancer cell growth and tumor metastasis. The present study has demonstrated that small hairpin RNA (shRNA)-mediated downregulation of Skp2 markedly inhibits the viability, proliferation, colony formation, migration, invasion, and apoptosis of human gastric cancer MGC803 cells, which express a high level of Skp2. In contrast, Skp2 shRNA had only a slight effect on the above properties of BGC823 cells, which express a low level of Skp2. In accord, knockdown of Skp2 suppressed the ability of MGC803 cells to form tumors and to metastasize to the lungs of mice, and the growth of established tumors, by inhibiting cell proliferation and enhancing cell apoptosis. In contrast, overexpression of Skp2 promoted tumorigenesis of BGC823 cells in mice. Skp2 depletion induced cell cycle arrest in the G(1)/S phase by upregulating p27, p21, and p57 and downregulating cyclin E and cyclin-dependent kinase 2. Skp2 depletion also increased caspase-3 activity, impeded the ability of cells to form filopoidia and locomote, upregulated RECK (reversion-inducing cysteine-rich protein with kazal motifs), and downregulated matrix metalloproteinase (MMP)-2 and MMP-9 activity and expression. The results suggest that downregulating Skp2 warrants investigation as a promising strategy to treat gastric cancers that express high levels of Skp2.
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Affiliation(s)
- Zheng Wei
- The Hepatosplenic Surgery Center, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China
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19
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Jiang D, Wang X, Liu X, Li F. Gene delivery of cyclin-dependent kinase inhibitors p21Waf1 and p27Kip1 suppresses proliferation of MCF-7 breast cancer cells in vitro. Breast Cancer 2013; 21:614-23. [PMID: 23338153 DOI: 10.1007/s12282-012-0438-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2012] [Accepted: 12/21/2012] [Indexed: 11/24/2022]
Abstract
BACKGROUND Because tumorigenesis depends on a variety of oncogenes, symphyseal study of combined genes may lead to more significant knowledge about tumorigenesis and progression. Combined deficiency of p21 and p27 proteins in mice is linked to more aggressive spontaneous tumorigenesis. We investigated the effect of the transfected p21 (Waf1) -p27 (Kip1) gene on centrosome duplication, cell proliferation, and apoptosis of MCF-7, a breast cancer cell line. METHODS The pIRES-p21 (Waf1) , pIRES-p27 (Kip1) , and pIRES-p21 (Waf1) -p27 (Kip1) genes were transfected into MCF-7 cells by lipofection. The effect on proliferation was evaluated by MTT assay and clone-formation assay. Cell cycle and apoptosis were analyzed by flow cytometry. Apoptosis was tested by flow cytometry and TUNEL assay. Centrosome duplication was detected by use of indirect immunofluorescence microscopy. RESULTS The results showed that the pIRES-p21 (Waf1) , pIRES-p27 (Kip1) , and pIRES-p21 (Waf1) -p27 (Kip1) significantly inhibited proliferation of MCF-7 cells, followed by accumulation of MCF-7 cells in cycle G1, induced apoptosis, and a decrease in the proportion of MCF-7 cells which contained abnormal centrosomes. Compared with p21 (Waf1) or p27 (Kip1) alone, combination of p21 (Waf1) and p27 (Kip1) had a much more significant effect (P < 0.05). CONCLUSION Altogether, these results indicate that the p21 (Waf1) -p27 (Kip1) gene combination has a more obvious antitumor effect than p21 (Waf1) or p27 (Kip1) alone. This study provides preclinical evidence that combination of p21 (Waf1) and p27 (Kip1) could be a novel and promising therapeutic approach to treatment of breast cancer with suppressed p21 (Waf1) and p27 (Kip1) expression.
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Affiliation(s)
- Dandan Jiang
- Breast Surgery, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, 266003, Shandong, China
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20
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Yang S, Chen Y, Ahmadie R, Ho EA. Advancements in the field of intravaginal siRNA delivery. J Control Release 2013; 167:29-39. [PMID: 23298612 DOI: 10.1016/j.jconrel.2012.12.023] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2012] [Revised: 12/14/2012] [Accepted: 12/17/2012] [Indexed: 12/17/2022]
Abstract
The vaginal tract is a suitable site for the administration of both local and systemic acting drugs. There are numerous vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. Despite the potential in drug delivery, the vagina is a complex and dynamic organ that requires greater understanding. The recent discovery that injections of double stranded RNA (dsRNA) in Caenorhabditis elegans (C. elegans) results in potent gene specific silencing, was a major scientific revolution. This phenomenon known as RNA interference (RNAi), is believed to protect host genome against invasion by mobile genetic elements such as transposons and viruses. Gene silencing or RNAi has opened new potential opportunities to study the function of a gene in an organism. Furthermore, its therapeutic potential is being investigated in the field of sexually transmitted infections such as human immunodeficiency virus (HIV) and other diseases such as age-related macular degeneration (AMD), diabetes, hypercholesterolemia, respiratory disease, and cancer. This review will focus on the therapeutic potential of siRNA for the treatment and/or prevention of infectious diseases such as HIV, HPV, and HSV within the vaginal tract. Specifically, formulation design parameters to improve siRNA stability and therapeutic efficacy in the vaginal tract will be discussed along with challenges, advancements, and future directions of the field.
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Affiliation(s)
- Sidi Yang
- Faculty of Pharmacy, University of Manitoba, 750 McDermot Ave, Winnipeg, Manitoba, Canada
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21
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Chen L, Tweddle DA. p53, SKP2, and DKK3 as MYCN Target Genes and Their Potential Therapeutic Significance. Front Oncol 2012; 2:173. [PMID: 23226679 PMCID: PMC3508619 DOI: 10.3389/fonc.2012.00173] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2012] [Accepted: 11/01/2012] [Indexed: 12/15/2022] Open
Abstract
Neuroblastoma is the most common extra-cranial solid tumor of childhood. Despite significant advances, it currently still remains one of the most difficult childhood cancers to cure, with less than 40% of patients with high-risk disease being long-term survivors. MYCN is a proto-oncogene implicated to be directly involved in neuroblastoma development. Amplification of MYCN is associated with rapid tumor progression and poor prognosis. Novel therapeutic strategies which can improve the survival rates whilst reducing the toxicity in these patients are therefore required. Here we discuss genes regulated by MYCN in neuroblastoma, with particular reference to p53, SKP2, and DKK3 and strategies that may be employed to target them.
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Affiliation(s)
- Lindi Chen
- Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University Newcastle, UK
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22
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Chan JY. A clinical overview of centrosome amplification in human cancers. Int J Biol Sci 2011; 7:1122-44. [PMID: 22043171 PMCID: PMC3204404 DOI: 10.7150/ijbs.7.1122] [Citation(s) in RCA: 288] [Impact Index Per Article: 20.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2011] [Accepted: 10/06/2011] [Indexed: 01/11/2023] Open
Abstract
The turn of the 21st century had witnessed a surge of interest in the centrosome and its causal relation to human cancer development - a postulate that has existed for almost a century. Centrosome amplification (CA) is frequently detected in a growing list of human cancers, both solid and haematological, and is a candidate "hallmark" of cancer cells. Several lines of evidence support the progressive involvement of CA in the transition from early to advanced stages of carcinogenesis, being also found in pre-neoplastic lesions and even in histopathologically-normal tissue. CA constitutes the major mechanism leading to chromosomal instability and aneuploidy, via the formation of multipolar spindles and chromosomal missegregation. Clinically, CA may translate to a greater risk for initiation of malignant transformation, tumour progression, chemoresistance and ultimately, poor patient prognosis. As mechanisms underlying CA are progressively being unravelled, the centrosome has emerged as a novel candidate target for cancer treatment. This Review summarizes mainly the clinical studies performed to date focusing on the mechanisms underlying CA in human neoplasia, and highlights the potential utility of centrosomes in the diagnosis, prognosis and treatment of human cancers.
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Kruck S, Merseburger AS, Hennenlotter J, Scharpf M, Eyrich C, Amend B, Sievert KD, Stenzl A, Bedke J. High cytoplasmic expression of p27(Kip1) is associated with a worse cancer-specific survival in clear cell renal cell carcinoma. BJU Int 2011; 109:1565-70. [PMID: 21981759 DOI: 10.1111/j.1464-410x.2011.10649.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
UNLABELLED What's known on the subject? and What does the study add? The loss of p27(Kip1) correlates with poor prognosis in various human cancers, and was postulated as a biomarker in RCC. Up to now p27(Kip1) analysis in RCC was focused on its nuclear localization. We confirmed higher p27(Kip1) expression in the nucleus and cytoplasm of RCC and correlated high cytoplasmic p27(Kip1) with an unfavourable clinic and a reduced survival. OBJECTIVES To analyse the cytoplasmic and nuclear differences of p27(Kip1) expression and localization in benign and clear cell renal cell carcinoma (ccRCC) with regard to overall survival (OS) and cancer-specific survival (CSS). p27(Kip1) is considered to contribute to the progression of ccRCC and is targeted by next generation dual-therapies. PATIENTS AND METHODS In 140 patients, ccRCC and corresponding benign kidney tissue were analyzed for nuclear and cytoplasmic staining of p27(Kip1) by immunohistochemistry using a tissue microarray technique. Staining intensity and percentage of positive stained cells are given as expression scores. p27(Kip1) expression was categorized as high if ccRCC tissue stained stronger than the respective level of the corresponding benign tissue and categorized as low if ccRCC tissue stained less than or equal to the corresponding benign tissue. Differences in OS and CSS were analyzed by log-rank analysis and expression levels were correlated with tumour and patient characteristics using Fisher's exact test. RESULTS Cytoplasmatic (mean [sd]: 13.8% [1.2%] vs 10.7% [1.7%]; P= 0.04) and nuclear (mean [sd]: 75.6% [2.7%] vs 13.6% [2.1%]; P < 0.001) staining of p27(Kip1) were significantly stronger in ccRCC tissues compared to benign tissue. High cytoplasmic p27(Kip1) expression was significantly associated with a higher T and N stage, Fuhrman grade and the presence of metastatic disease (P < 0.001). The median follow-up time was 38.2 months. There was no difference in OS between the low and high expression groups, although CSS was significantly lower in patients with high cytoplasmic p27(Kip1) (P < 0.001) and CSS heavily tended to be lower in the nuclear low expression group (P= 0.069). CONCLUSIONS High cytoplasmic p27(Kip1) levels in renal cancer tissues are associated with advanced disease and reduced cancer specific survival, whereas low nuclear expression levels appear to be beneficial. The present study corroborates the consideration of cytoplasmic p27(Kip1) for future diagnostic and targeted therapeutic approaches in RCC establishing a potential protective shift of p27(Kip1) from the cytoplasm to the nucleus.
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Affiliation(s)
- Stephan Kruck
- Department of Urology Pathology, Eberhard-Karls-University Tuebingen, Tuebingen, Germany
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24
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Jia L, Sun Y. SCF E3 ubiquitin ligases as anticancer targets. Curr Cancer Drug Targets 2011; 11:347-56. [PMID: 21247385 DOI: 10.2174/156800911794519734] [Citation(s) in RCA: 118] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2010] [Accepted: 12/27/2010] [Indexed: 11/22/2022]
Abstract
The SCF multisubunit complex (Skp1, Cullins, F-box proteins) E3 ubiquitin ligase, also known as CRL (Cullin-RING ubiquitin Ligase) is the largest E3 ubiquitin ligase family that promotes the ubiquitination of various regulatory proteins for targeted degradation, thus regulating many biological processes, including cell cycle progression, signal transduction, and DNA replication. The efforts to discover small molecule inhibitors of a SCF-type ligase or its components were expedited by the FDA approval of Bortezomib (also known as Velcade or PS-341), the first (and only) class of general proteasome inhibitor, for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma. Although Bortezomib has demonstrated a certain degree of cancer cell selectivity with measurable therapeutic index, the drug is, in general, cytotoxic due to its inhibition of overall protein degradation. An alternative and ideal approach is to target a specific E3 ligase, known to be activated in human cancer, for a high level of specificity and selectivity with less associated toxicity, since such inhibitors would selectively stabilize a specific set of cellular proteins regulated by this E3. Here, we review recent advances in validation of SCF E3 ubiquitin ligase complex as an attractive anti-cancer target and discuss how MLN4924, a small molecule inhibitor of NEDD8-activating enzyme, can be developed as a novel class of anticancer agents by inhibiting SCF E3 ligase complex via removal of cullin neddylation. Finally, we discuss under future perspective how basic research on SCF biology will direct the drug discovery efforts surrounding this target.
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Affiliation(s)
- L Jia
- Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, 4424B Medical Science-I, 1301 Catherine Street, Ann Arbor, MI 48109, USA
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25
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Fotovati A, Abu-Ali S, Nakayama K, Nakayama KI. Impaired ovarian development and reduced fertility in female mice deficient in Skp2. J Anat 2011; 218:668-77. [PMID: 21450015 DOI: 10.1111/j.1469-7580.2011.01370.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
p27 is a major negative regulator of somatic cellular proliferation, and its down-regulation has been shown to be associated with cancer development. Targeted disruption ofp27 results in complete loss of fertility in female mice, suggesting that it plays a significant role in the development of female gametes and the surrounding environment. We have now investigated the effect of loss of Skp2, an F-box protein that mediates ubiquitin-dependent degradation of p27, on female gamete production. The female Skp2-deficient mice showed accumulation of p27 in the ovary and severely compromised gamete development from the embryonic stage to follicular growth in the adult ovary, eventually leading to a decreased functional gamete reserve. Additional deletion of p27 resulted in relatively normal ovarian folliculogenesis, suggesting that accumulating p27 is primarily responsible for the compromised ovarian development. Embryonic ovaries of Skp2(-/-) mice manifested massive apoptosis as evidenced by cleavage of pro-caspase 3 and poly(ADP-ribose) polymerase-1. This in turn resulted in a significant decrease in the remaining pool of functional gametes in Skp2(-/-) mice shortly after sexual maturity and premature ovarian failure. The increased apoptosis seemed to be attributable to the polyploidy of granulosa cells. These results suggest that proper progression of the cell cycle, regulated by the p27-Skp2 axis, is pivotal for the maintenance of fertility, and that defects in this system may underlie the pathogenesis of abnormal gamete production and premature ovarian failure during the reproductive life of women.
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Affiliation(s)
- Abbas Fotovati
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka, Japan.
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26
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Hershko DD. Cyclin-dependent kinase inhibitor p27 as a prognostic biomarker and potential cancer therapeutic target. Future Oncol 2010; 6:1837-47. [PMID: 21142858 DOI: 10.2217/fon.10.144] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The prognosis and clinical management of patients with cancer is commonly determined by traditional clinical and pathological factors. Nevertheless, patients may present with significantly different clinical outcomes despite similar clinicopathological features. This has prompted intense research to find biological markers that may closely reflect tumor biology and thereby clinical outcome. This article presents the current knowledge on the prognostic significance of p27 expression in cancer and its potential role as a target for future therapy.
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Affiliation(s)
- Dan D Hershko
- Department of Surgery & Breast Health Institute, Rambam Health Care Campus & the Technion – Israel Institute of Technology, Haifa 31096, Israel
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27
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Jiang F, Todd NW, Li R, Zhang H, Fang H, Stass SA. A Panel of Sputum-Based Genomic Marker for Early Detection of Lung Cancer. Cancer Prev Res (Phila) 2010; 3:1571-8. [DOI: 10.1158/1940-6207.capr-10-0128] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase. J Virol 2010; 84:437-44. [PMID: 19828607 DOI: 10.1128/jvi.01162-09] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The human papillomavirus type 18 (HPV-18) E2 gene is inactivated in cervical carcinoma after integration of the viral DNA into the host cellular genome. Since E2 represses the transcription of the two viral oncogenes E6 and E7, integration which allows their strong expression is considered a major step in transformation by HPV. We show here that E2 is specifically degraded at the end of the G(1) phase in a Brd4-independent manner, implying that its regulatory functions are cell cycle dependent. Degradation of E2 occurs via the Skp1/Cullin1/F-box Skp2 (SCF(Skp2)) ubiquitin ligase, since silencing of Skp2 induces stabilization of E2. In addition, the amino-terminal domain of E2 can interact with Skp2 as shown by coimmunoprecipitation experiments. We previously showed that E2 inhibits the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, leading to accumulation of several of its substrates. We demonstrate here that Skp2, which is a known APC/C substrate in G(1), is also stabilized by E2. Therefore, by negative feedback, SCF(Skp2) activation could lead to E2 degradation and E6/E7 expression specifically in the late G(1) phase. Moreover, since the SCF(Skp2) can trigger S-phase entry and Skp2 itself is a known oncogene, we believe that E2-mediated accumulation of Skp2, together with E2 degradation leading to putative release of E6 and E7 inhibition, could induce premature S-phase entry in HPV-infected cells, pointing to a direct role of E2 in the early steps of HPV-mediated transformation.
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29
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Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L, Wang H, Liu Z, Su Y, Stass SA, Katz RL. Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res 2009; 7:330-8. [PMID: 19276181 PMCID: PMC4255559 DOI: 10.1158/1541-7786.mcr-08-0393] [Citation(s) in RCA: 622] [Impact Index Per Article: 38.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features of CSCs, including capacities for proliferation, self-renewal, and differentiation, resistance to chemotherapy, and expressing CSC surface marker CD133. In vivo experiments show that the ALDH1-positive cells could generate tumors that recapitulate the heterogeneity of the parental cancer cells. Immunohistochemical analysis of 303 clinical specimens from three independent cohorts of lung cancer patients and controls show that expression of ALDH1 is positively correlated with the stage and grade of lung tumors and related to a poor prognosis for the patients with early-stage lung cancer. ALDH1 is therefore a lung tumor stem cell-associated marker. These findings offer an important new tool for the study of lung CSCs and provide a potential prognostic factor and therapeutic target for treatment of the patients with lung cancer.
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Affiliation(s)
- Feng Jiang
- Department of Pathology, Greenbaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201-1192, USA.
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He S, Zhang D, Cheng F, Gong F, Guo Y. Applications of RNA interference in cancer therapeutics as a powerful tool for suppressing gene expression. Mol Biol Rep 2009; 36:2153-63. [PMID: 19117119 DOI: 10.1007/s11033-008-9429-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2008] [Accepted: 12/08/2008] [Indexed: 01/07/2023]
Abstract
Cancer poses a tremendous therapeutic challenge worldwide, highlighting the critical need for developing novel therapeutics. A promising cancer treatment modality is gene therapy, which is a form of molecular medicine designed to introduce into target cells genetic material with therapeutic intent. The history of RNA interference (RNAi) has only a dozen years, however, further studies have revealed that it is a potent method of gene silencing that has developed rapidly over the past few years as a result of its extensive importance in the study of genetics, molecular biology and physiology. RNAi is a natural process by which small interfering RNA (siRNA) duplex directs sequence specific post-transcriptional silencing of homologous genes by binding to its complementary mRNA and triggering its elimination. RNAi has been extensively used as a novel and effective gene silencing tool for the fundamental research of cancer therapeutics, and has displayed great potential in clinical treatment.
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Affiliation(s)
- Song He
- Molecular Medicine & Tumor Research Center, Chongqing Medical University, Chongqing, China.
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Zhao J, Yang CL, Zhang H, Ding WZ, Liu ZP, Liu JY. Expression of SKP2 protein in lung carcinoma. Chin J Cancer Res 2008. [DOI: 10.1007/s11670-008-0216-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
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Fang L, Hu Q, Hua Z, Li S, Dong W. Growth inhibition of a tongue squamous cell carcinoma cell line (Tca8113) in vitro and in vivo via siRNA-mediated down-regulation of skp2. Int J Oral Maxillofac Surg 2008; 37:847-52. [DOI: 10.1016/j.ijom.2008.05.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2007] [Revised: 01/02/2008] [Accepted: 05/29/2008] [Indexed: 10/21/2022]
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Kitagawa M, Lee SH, McCormick F. Skp2 suppresses p53-dependent apoptosis by inhibiting p300. Mol Cell 2008; 29:217-31. [PMID: 18243116 DOI: 10.1016/j.molcel.2007.11.036] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2007] [Revised: 07/25/2007] [Accepted: 11/16/2007] [Indexed: 10/22/2022]
Abstract
The F box protein Skp2 is oncogenic, and its frequent amplification and overexpression correlate with the grade of malignancy of certain tumors. Conversely, depletion of Skp2 decreases cell growth and increases apoptosis. Here, we show that Skp2 counteracts the transactivation function of p53 and suppresses apoptosis mediated by DNA damage or p53 stabilization. We demonstrate that Skp2 forms a complex with p300 through the CH1 and the CH3 domains of p300 to which p53 is thought to bind and antagonizes the interaction between p300 and p53 in cells and in vitro. As Skp2 antagonizes the interaction between p300 and p53, Skp2 suppresses p300-mediated acetylation of p53 and the transactivation ability of p53. Conversely, ectopic expression of p300 rescues the transactivation function of p53 in cells overexpressing Skp2. Taken together, our results indicate that Skp2 controls p300-p53 signaling pathways in cancer cells, making Skp2 a potential molecular target for cancer therapy.
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Affiliation(s)
- Mayumi Kitagawa
- Cancer Research Institute and Comprehensive Cancer Center, University of California-San Francisco, San Francisco, CA 94115, USA
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Targeting the p27 E3 ligase SCF(Skp2) results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy. Blood 2008; 111:4690-9. [PMID: 18305219 DOI: 10.1182/blood-2007-09-112904] [Citation(s) in RCA: 221] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Decreased p27(Kip1) levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCF(Skp2) E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27(Kip1), agents inhibiting SCF(Skp2) may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCF(Skp2) ligase function in vitro, and induced specific accumulation of p21 and other SCF(Skp2) substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCF(Skp2) ligase, and induced G(1)/S cell-cycle arrest as well as SCF(Skp2)- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCF(Skp2) inhibitors as a novel class of antitumor agents.
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Feng L, Barnhart JR, Seeger RC, Wu L, Keshelava N, Huang SH, Jong A. Cdc6 knockdown inhibits human neuroblastoma cell proliferation. Mol Cell Biochem 2008; 311:189-97. [DOI: 10.1007/s11010-008-9709-5] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2007] [Accepted: 01/10/2008] [Indexed: 01/11/2023]
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Ji P, Sun D, Wang H, Bauzon F, Zhu L. Disrupting Skp2-cyclin A interaction with a blocking peptide induces selective cancer cell killing. Mol Cancer Ther 2007; 6:684-91. [PMID: 17308064 DOI: 10.1158/1535-7163.mct-06-0538] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Skp2 fulfills the definition of an oncoprotein with its frequent overexpression in cancer cells and oncogenic activity in various laboratory assays and therefore is a potential cancer therapy target. The best-known function of Skp2 is that of an F-box protein of the SCF(Skp2)-Roc1 E3 ubiquitin ligase targeting the cyclin-dependent kinase inhibitor p27(Kip1). Knockdown of Skp2 generally leads to accumulation of p27 but its effects on cancer cells are less certain. Another function of Skp2 is its stable interaction with cyclin A, which directly protects cyclin A from inhibition by p27 in in vitro kinase assays. Here, we report that an 18-residue blocking peptide of Skp2-cyclin A interaction can indirectly inhibit cyclin A/Cdk2 kinase activity dependent on the presence of p27 in in vitro kinase assays. Transmembrane delivery of this blocking peptide can induce cell death in a panel of four cancer cell lines in which Skp2 knockdown only have mild inhibitory effects. This Skp2-cyclin A interaction blocking peptide can synergize with a previously identified E2F1-derived LDL peptide, which blocks its access to cyclin A, in killing cancer cells. IC(50) of the Skp2-cyclin A blocking peptide correlated with abundance of Skp2, its intended target, in cancer cells. These results suggest that Skp2-cyclin A interaction plays an important role in cancer cell survival and is an attractive target for cancer drug discovery.
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Affiliation(s)
- Peng Ji
- Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room U-521, Bronx, NY 10461, USA
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Fan T, Li R, Todd NW, Qiu Q, Fang HB, Wang H, Shen J, Zhao RY, Caraway NP, Katz RL, Stass SA, Jiang F. Up-regulation of 14-3-3zeta in lung cancer and its implication as prognostic and therapeutic target. Cancer Res 2007; 67:7901-6. [PMID: 17699796 DOI: 10.1158/0008-5472.can-07-0090] [Citation(s) in RCA: 97] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
A functional genomic approach integrating microarray and proteomic analyses done in our laboratory has identified 14-3-3zeta as a putative oncogene whose activation was common and driven by its genomic amplification in lung adenocarcinomas. 14-3-3zeta is believed to function in cell signaling, cycle control, and apoptotic death. Following our initial finding, here, we analyzed its expression in lung tumor tissues obtained from 205 patients with various histologic and stage non-small cell lung cancers (NSCLC) using immunohistochemistry and then explored the effects of specific suppression of the gene in vitro and in a xenograft model using small interfering RNA. The increased 14-3-3zeta expression was positively correlated with a more advanced pathologic stage and grade of NSCLCs (P = 0.001 and P = 0.006, respectively) and was associated with overall and cancer-specific survival rates of the patients (P = 0.022 and P = 0.018, respectively). Down-regulation of 14-3-3zeta in lung cancer cells led to a dose-dependent increased sensitivity to cisplatin-induced cell death, which was associated with the inhibition of cell proliferation and increased G2-M arrest and apoptosis. The result was further confirmed in the animal model, which showed that the A549 lung cancer cells with reduced 14-3-3zeta grew significantly slower than the wild-type A549 cells after cisplatin treatment (P = 0.008). Our results suggest that 14-3-3zeta is a potential target for developing a prognostic biomarker and therapeutics that can enhance the antitumor activity of cisplatin for NSCLC.
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Affiliation(s)
- Tao Fan
- Department of Pathology, University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201-1192, USA
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Nickeleit I, Zender S, Kossatz U, Malek NP. p27kip1: a target for tumor therapies? Cell Div 2007; 2:13. [PMID: 17488529 PMCID: PMC1872022 DOI: 10.1186/1747-1028-2-13] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2007] [Accepted: 05/09/2007] [Indexed: 11/27/2022] Open
Abstract
The cyclin kinase inhibitor p27kip1 acts as a potent tumor supressor protein in a variety of human cancers. Its expression levels correlate closely with the overall prognosis of the affected patient and often predict the outcome to different treatment modalities. In contrast to other tumor suppressor proteins p27 expression levels in tumor cells are frequently regulated by ubiquitin dependent proteolysis. Re-expression of p27 in cancer cells therefore does not require gene therapy but can be achieved by interfering with the protein turnover machinery. In this review we will summarize experimental results which highlight the potential use of p27 as a target for oncological therapies.
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Affiliation(s)
- Irina Nickeleit
- Institute for Molecular Biology, Hannover Medical School, Hannover, Germany
| | - Steffen Zender
- Institute for Molecular Biology, Hannover Medical School, Hannover, Germany
| | - Uta Kossatz
- Institute for Molecular Biology, Hannover Medical School, Hannover, Germany
| | - Nisar P Malek
- Institute for Molecular Biology, Hannover Medical School, Hannover, Germany
- Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
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Kloth JN, Oosting J, van Wezel T, Szuhai K, Knijnenburg J, Gorter A, Kenter GG, Fleuren GJ, Jordanova ES. Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer. BMC Genomics 2007; 8:53. [PMID: 17311676 PMCID: PMC1805756 DOI: 10.1186/1471-2164-8-53] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2006] [Accepted: 02/20/2007] [Indexed: 11/10/2022] Open
Abstract
Background Cervical carcinoma develops as a result of multiple genetic alterations. Different studies investigated genomic alterations in cervical cancer mainly by means of metaphase comparative genomic hybridization (mCGH) and microsatellite marker analysis for the detection of loss of heterozygosity (LOH). Currently, high throughput methods such as array comparative genomic hybridization (array CGH), single nucleotide polymorphism array (SNP array) and gene expression arrays are available to study genome-wide alterations. Integration of these 3 platforms allows detection of genomic alterations at high resolution and investigation of an association between copy number changes and expression. Results Genome-wide copy number and genotype analysis of 10 cervical cancer cell lines by array CGH and SNP array showed highly complex large-scale alterations. A comparison between array CGH and SNP array revealed that the overall concordance in detection of the same areas with copy number alterations (CNA) was above 90%. The use of SNP arrays demonstrated that about 75% of LOH events would not have been found by methods which screen for copy number changes, such as array CGH, since these were LOH events without CNA. Regions frequently targeted by CNA, as determined by array CGH, such as amplification of 5p and 20q, and loss of 8p were confirmed by fluorescent in situ hybridization (FISH). Genome-wide, we did not find a correlation between copy-number and gene expression. At chromosome arm 5p however, 22% of the genes were significantly upregulated in cell lines with amplifications as compared to cell lines without amplifications, as measured by gene expression arrays. For 3 genes, SKP2, ANKH and TRIO, expression differences were confirmed by quantitative real-time PCR (qRT-PCR). Conclusion This study showed that copy number data retrieved from either array CGH or SNP array are comparable and that the integration of genome-wide LOH, copy number and gene expression is useful for the identification of gene specific targets that could be relevant for the development and progression in cervical cancer.
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Affiliation(s)
- Judith N Kloth
- Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
| | - Jan Oosting
- Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
| | - Tom van Wezel
- Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
| | - Karoly Szuhai
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
| | - Jeroen Knijnenburg
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
| | - Arko Gorter
- Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
| | - Gemma G Kenter
- Department of Gynecology, Leiden University Medical Center, Leiden, The Netherlands
| | - Gert Jan Fleuren
- Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
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Abstract
RNA interference (RNAi) is a naturally occurring cellular defense mechanism against viral infections and transposon invasion. Short double-stranded RNA molecules, so-called small-interfering (si)RNAs, bind their complementary mRNA leading to the mRNA's degradation. During the past few years, RNAi has become a valuable tool for transient as well as stable repression of gene expression rendering the time-consuming production of knockout animals superfluous. In this chapter the usability of the RNAi technology in cancer research will be described, focusing on the application of large-scale screens for identification of new components in cancer-relevant signal pathways (e.g., p53, RAS). The screens are especially helpful in the detection of potential anticancer drug targets or siRNAs with therapeutic potential.
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Affiliation(s)
- Uta Fuchs
- Dr. von Haunersches Kinderspital, Ludwig Maximilians Universität München, München, Germany
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Wei Q, Li M, Fu X, Tang R, Na Y, Jiang M, Li Y. Global analysis of differentially expressed genes in androgen-independent prostate cancer. Prostate Cancer Prostatic Dis 2007; 10:167-74. [PMID: 17199135 DOI: 10.1038/sj.pcan.4500933] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Progression to androgen independent (AI) is the main cause of death in prostate cancer, and the mechanism is still unclear. By reviewing the expression profiles of 26 prostate cancer samples in a holistic view, we found a group of genes differentially expressed in AI compared with androgen-dependent groups (P-value<0.01, t-test). Focusing on apoptosis, proliferation, hormone and angiogenesis, we found a group of genes such as thioredoxin domain containing 5 , tumor necrosis factor receptor superfamily, member 10a , ribosomal protein S19 and Janus kinase 2 upregulated in AI prostate cancer, could play important roles in the transition from AD to AI and could be biomarkers of prognosis.
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Affiliation(s)
- Q Wei
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, PR China
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Pateras IS, Apostolopoulou K, Koutsami M, Evangelou K, Tsantoulis P, Liloglou T, Nikolaidis G, Sigala F, Kittas C, Field JK, Kotsinas A, Gorgoulis VG. Downregulation of the KIP family members p27(KIP1) and p57(KIP2) by SKP2 and the role of methylation in p57(KIP2) inactivation in nonsmall cell lung cancer. Int J Cancer 2006; 119:2546-56. [PMID: 16988944 DOI: 10.1002/ijc.22214] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Knowing the status of molecules involved in cell cycle control in cancer is vital for therapeutic approaches aiming at their restoration. The p27(KIP1) and p57(KIP2) cyclin-dependent kinase inhibitors are nodal factors controlling normal cell cycle. Their expression in normal lung raises the question whether they have a mutual exclusive or redundant role in nonsmall cell lung cancer (NSCLC). A comparative comprehensive analysis was performed in a series of 70 NSCLCs. The majority of cases showed significantly reduced expression of both members compared to normal counterparts. Low KIP protein levels correlated with increased proliferation, which seems to be histological subtype preponderant. At mechanistic level, degradation by SKP2 was demonstrated, in vivo and in vitro, by siRNA-methodology, to be the most important downregulating mechanism of both KIPs in NSCLC. Decreased p57(KIP) (2)-transcription complements the above procedure in lowering p57(KIP2)-protein levels. Methylation was the main cause of decreased p57(KIP) (2)-mRNA levels. Allelic loss and imprinting from LIT1 mRNA contribute also to decreased p57(KIP2) transcription. In vitro recapitulation of the in vivo findings, in A549 lung cells (INK4A-B((-/-))), suggested that inhibition of the SKP2-degradation mechanism restores p27(KIP1) and p57(KIP2) expression. Double siRNA treatments demonstrated that each KIP is independently capable of restraining cell growth. An additional demethylation step is required for complete reconstitution of p57(KIP2) expression in NSCLC.
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Affiliation(s)
- Ioannis S Pateras
- Molecular Carcinogenesis Group, Laboratory of Histology-Embryology, Medical School, University of Athens, Athens, Greece
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He G, Kuang J, Huang Z, Koomen J, Kobayashi R, Khokhar AR, Siddik ZH. Upregulation of p27 and its inhibition of CDK2/cyclin E activity following DNA damage by a novel platinum agent are dependent on the expression of p21. Br J Cancer 2006; 95:1514-24. [PMID: 17088910 PMCID: PMC2360737 DOI: 10.1038/sj.bjc.6603448] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Revised: 09/20/2006] [Accepted: 09/27/2006] [Indexed: 12/12/2022] Open
Abstract
The cisplatin analogue 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP) is a DNA-damaging agent that will be entering clinical trials for its potent cytotoxic effects against cisplatin-resistant tumour cells. This cytotoxicity may reside in its ability to selectively activate G1-phase checkpoint response by inhibiting CDKs via the p53/p21 pathway. We have now evaluated the role of another CDK inhibitor p27 as a contributor to DAP-mediated inhibition of G1-phase CDK2 activity. Our studies in ovarian A2780 tumour cells demonstrate that p27 levels induced by DAP are comparable to or greater than those seen for p21. The induction of p27 is not through a transcriptional mechanism, but rather is due to a four-fold increase in protein stabilisation through a mechanism dependent on p21. Moreover, DAP-induced p21 promoted the selective increase of p27 in the CDK2 complex, but not in CDK4 complex, and this selective increase contributed to inhibition of the CDK2 kinase activity. The inhibited complex contained either p27 or p21, but not both, with the relative levels of cyclin E associated with p27 and p21 indicating that about 25% of the inhibition of CDK2 activity was due to p27 and 75% due to p21. This study provides the first evidence that p27 upregulation is directly attributable to activation of the p53/p21 pathway by a DNA-damaging agent, and promulgates p53/p21/p27 axis as a significant component of checkpoint response.
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Affiliation(s)
- G He
- Department of Experimental Therapeutics, Unit 353, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
| | - J Kuang
- Department of Experimental Therapeutics, Unit 353, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
| | - Z Huang
- Department of Experimental Therapeutics, Unit 353, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
| | - J Koomen
- Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - R Kobayashi
- Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - A R Khokhar
- Department of Experimental Therapeutics, Unit 353, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
| | - Z H Siddik
- Department of Experimental Therapeutics, Unit 353, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
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Abstract
E3 ubiquitin ligases are a large family of proteins that are engaged in the regulation of the turnover and activity of many target proteins. Together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of biologically significant protein substrates for targeted degradation through the 26S proteasome, as well as for nonproteolytic regulation of their functions or subcellular localizations. E3 ubiquitin ligases, therefore, play an essential role in the regulation of many biologic processes. Increasing amounts of evidence strongly suggest that the abnormal regulation of some E3 ligases is involved in cancer development. Furthermore, some E3 ubiquitin ligases are frequently overexpressed in human cancers, which correlates well with increased chemoresistance and poor clinic prognosis. In this review, E3 ubiquitin ligases (such as murine double minute 2, inhibitor of apoptosis protein, and Skp1-Cullin-F-box protein) will be evaluated as potential cancer drug targets and prognostic biomarkers. Extensive study in this field would lead to a better understanding of the molecular mechanism by which E3 ligases regulate cellular processes and of how their deregulations contribute to carcinogenesis. This would eventually lead to the development of a novel class of anticancer drugs targeting specific E3 ubiquitin ligases, as well as the development of sensitive biomarkers for cancer treatment, diagnosis, and prognosis.
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Affiliation(s)
- Yi Sun
- Division of Cancer Biology, Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109-0936, USA.
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Abstract
Regulated protein turnover via the ubiquitin-proteasome system (UPS) underlies a wide variety of signalling pathways, from cell-cycle control and transcription to development. Recent evidence that pharmacological inhibition of the proteasome can be efficacious in the treatment of human cancers has set the stage for attempts to selectively inhibit the activities of disease-specific components of the UPS. Here, we review recent advances linking UPS components with specific human diseases, most prominently cancer and neurodegenerative disorders, and emphasize potential sites of therapeutic intervention along the regulated protein-degradation pathway.
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Affiliation(s)
- Grzegorz Nalepa
- Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA
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Li R, Wang H, Bekele BN, Yin Z, Caraway NP, Katz RL, Stass SA, Jiang F. Identification of putative oncogenes in lung adenocarcinoma by a comprehensive functional genomic approach. Oncogene 2006; 25:2628-35. [PMID: 16369491 DOI: 10.1038/sj.onc.1209289] [Citation(s) in RCA: 97] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Amplification and overexpression of putative oncogenes confer growth advantages for tumor development. We used a functional genomic approach that integrated simultaneous genomic and transcript microarray, proteomics, and tissue microarray analyses to directly identify putative oncogenes in lung adenocarcinoma. We first identified 183 genes with increases in both genomic copy number and transcript in six lung adenocarcinoma cell lines. Next, we used two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify 42 proteins that were overexpressed in the cancer cells relative to normal cells. Comparing the 183 genes with the 42 proteins, we identified four genes - PRDX1, EEF1A2, CALR, and KCIP-1 - in which elevated protein expression correlated with both increased DNA copy number and increased transcript levels (all r > 0.84, two-sided P < 0.05). These findings were validated by Southern, Northern, and Western blotting. Specific inhibition of EEF1A2 and KCIP-1 expression with siRNA in the four cell lines tested suppressed proliferation and induced apoptosis. Parallel fluorescence in situ hybridization and immunohistochemical analyses of EEF1A2 and KCIP-1 in tissue microarrays from patients with lung adenocarcinoma showed that gene amplification was associated with high protein expression for both genes and that protein overexpression was related to tumor grade, disease stage, Ki-67 expression, and a shorter survival of patients. The amplification of EEF1A2 and KCIP-1 and the presence of overexpressed protein in tumor samples strongly suggest that these genes could be oncogenes and hence potential targets for diagnosis and therapy in lung adenocarcinoma.
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Affiliation(s)
- R Li
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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49
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Sugihara E, Kanai M, Saito S, Nitta T, Toyoshima H, Nakayama K, Nakayama KI, Fukasawa K, Schwab M, Saya H, Miwa M. Suppression of centrosome amplification after DNA damage depends on p27 accumulation. Cancer Res 2006; 66:4020-9. [PMID: 16618721 DOI: 10.1158/0008-5472.can-05-3250] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21Cip1 and p27Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after gamma-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27-/- and p27+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.
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Affiliation(s)
- Eiji Sugihara
- Nagahama Institute of Bio-Science and Technology, Shiga, Japan
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50
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Katagiri Y, Hozumi Y, Kondo S. Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo. J Dermatol Sci 2006; 42:215-24. [PMID: 16504485 DOI: 10.1016/j.jdermsci.2005.12.016] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2005] [Revised: 12/16/2005] [Accepted: 12/20/2005] [Indexed: 11/21/2022]
Abstract
BACKGROUND Low levels of p27Kip1 expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27Kip1 for degradation, was overexpressed and was inversely related to p27Kip1 levels in malignant melanoma with poor prognosis. OBJECTIVE We investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27Kip1 down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo. METHODS We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo. RESULTS Skp2 protein was decreased and the p27Kip1 protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo. CONCLUSION Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27Kip1 degradation in malignant melanoma.
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Affiliation(s)
- Yoshiyuki Katagiri
- Department of Dermatology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, 990-9585 Yamagata, Japan.
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