Correlation of Skp2 with carcinogenesis, invasion, metastasis, and prognosis in colorectal tumors

In colorectal tumors, S-phase kinase-associated protein 2 (Skp2) still has numerous important questions unanswered: its expression in adenomas, its correlation with key clinicopathological indices, its association with patient prognosis, its variation in lymph node metastases, and its association with many cell-cycle regulators. To answer these questions in colorectal tumors, Skp2, cyclin A, cyclin B1, cyclin E, CDK2, and Ki67 were immunohistochemically stained in 12 normal mucosa, 36 adenomas, 11 carcinomas in adenomas, 102 primary carcinomas, and 12 paired lymph node metastases; and Skp2 was examined by Western blot in 8 pairs of normal mucosa and carcinomas. Situated in nuclei, Skp2 expression significantly increased from normal mucosa through adenoma to primary carcinoma (p<0.0001), from mild through moderate to severe dysplasia in adenomas (p=0.038), from peripheral adenoma to paired central carcinoma (p=0.0033), and from primary carcinoma to lymph node metastasis (p=0.015), and these increases were confirmed by Western blot. Expression, however, relatively declined significantly in the primary carcinomas showing deep invasion (p=0.0113), lymph nodal metastases (p=0.0268), and poor prognosis for all (p=0.0104) or stage III patients (p=0.0119). High Skp2 was also significantly linked with elevated cyclin A, cyclin B1, cyclin E, CDK2 (in primary carcinomas only), and Ki67 in both adenomas and primary carcinomas. Thus, overexpression of Skp2 is associated with colorectal carcinogenesis and late metastasis to lymph nodes, whereas relative reduction of Skp2 is correlated with local invasion of primary carcinoma.     

Expression of p27Kip1, cyclin E and E2F-1 in primary and metastatic tumors of gastric carcinoma.

The cell cycle is controlled by positive and negative regulators. Gene abnormalities and aberrant expressions of various cyclins/CDKs and CDK inhibitors may play a pivotal role in stomach carcinogenesis. To clarify the role of cyclin E, CDK inhibitor p27Kip1 and their target molecule, E2F-1 in tumor metastasis, we examined immunohistochemically the expression of cyclin E, p27Kip1 and E2F-1 in 23 gastric carcinomas and metastatic tumors of the lymph node. Most of gastric carcinomas with lymph node metastasis showed reduced p27Kip1 expression. p27Kip1 was negative in 39% (9/23) of primary tumors, while it was so in 52% (12/23) of lymph node metastases. By comparison of p27Kip1 expression in primary and metastatic tumors in individual cases, metastatic tumor cells in the lymph nodes were expressed at weaker levels than in those in primary tumors in 43% (10/23) of the cases. On the other hand, over 70% (17/23) and 50% (12/23) of the cases expressed cyclin E and E2F-1 at nearly the same levels in both primary tumor and lymph node metastasis, respectively. These results suggest that tumor cells with reduced p27Kip1 expression may selectively metastasize to lymph node or distant organs.     

Cyclin E expression, a potential prognostic marker for non-small cell lung cancers.

Cyclin E is a G1 cyclin that has been shown to be one of the key regulators of the G1-S transition and could consequently be a deregulated molecule in tumors. In the present study, we have characterized cyclin E expression by immunohistochemistry in 217 resected non-small cell lung cancers (NSCLCs) and found large variations in cyclin E expression among tumors. High-level cyclin E expression (a cyclin E-labeling index > or =30%), observed in 115 (53%) of 217 NSCLCs, was more frequently found in tumors from smokers than from nonsmokers (P = 0.001), in squamous cell carcinomas than in nonsquamous cell carcinomas (P = 0.0002), and in pT2-4 tumors than in pT1 tumors (P = 0.04) by the chi2 test. Multivariate logistic regression analysis for the correlation between cyclin E expression and various characteristics showed a significant association of high-level cyclin E expression with squamous cell carcinomas (P = 0.005). Patients with tumors having high-level cyclin E expression survived a significantly shorter time than patients with tumors having low-level expression, both among the 151 patients with potentially curatively resected NSCLCs (5-year survival rates, 48 and 63%, respectively; P = 0.03) and the 103 patients with p stage I NSCLCs (5-year survival rates, 57 and 81%, respectively; P = 0.007). High-level cyclin E expression was also a significant and independent unfavorable prognostic factor in both patients with potentially curatively resected NSCLCs (P = 0.01) and in those with p stage I NSCLCs (P = 0.03) by Cox's proportional hazards model analysis. These findings indicate that cyclin E may play a pivotal role for the biological behavior of NSCLCs, and that a high level of cyclin E expression may be a new prognostic marker for NSCLCs.     

p57KIP2、cyclin D1和cyclin E在宫颈鳞癌中的表达及意义

目的探讨p57^KIP2、cyclin D1及cyclin E蛋白在宫颈癌发生、发展中的作用。方法用免疫组织化学SP法检测100例宫颈鳞癌、60例宫颈上皮内瘤变和30例正常宫颈鳞状上皮组织中p57^KIP2、cyclin D1和cyclin E蛋白的表达情况。结果cyclin D1、cyclin E蛋白在宫颈SCC与NE、CIN与NE组织中阳性表达率之间比较、cyclin E蛋白在宫颈SCC与CIN组织中阳性表达率之间比较，差异均有显著性（P〈0．01）；cyclin D1与cyclin E之间的表达呈正相关（P〈0．01）；三者表达均与组织学分级、淋巴结转移、患者年龄无关（P〉0．05）。结论p57^KIP2、cyclin D1及cyclin E蛋白共同参与了宫颈癌的发生发展。cyclin D1和cyclin E蛋白高表达可能是宫颈组织恶变的重要生物学标志，cyclin E异常表达是宫颈癌发生的早期事件。     

胃癌中E2F-1的表达及其临床意义

目的探讨胃癌中E2F-1的表达及其临床意义.方法采用免疫组化技术对49例人原发性胃癌癌组织与癌旁正常黏膜中E2F-1的表达进行检测,通过统计学方法分析其表达情况以及与肿瘤分期、病理分型、淋巴结转移、侵袭深度之间的相互关系.结果 E2F-1在癌组织与癌旁正常黏膜中的阳性表达率分别为79.6%(39/49)和26.5%(13/49),差异有显著性(P<0.001).癌组织、癌旁正常黏膜中E2F-1的表达在不同的临床病理特征之间差异无显著性(P>0.05).结论胃癌中E2F-1超表达和反常表达与胃癌的发生有关.     

Clinical significance of Skp2 expression, alone and combined with Jab1 and p27 in epithelial ovarian tumors

We have previously demonstrated the inverse correlation of Jab1 and p27 proteins, as well as prognostic significance in epithelial ovarian carcinomas. In order to investigate Skp2 protein and its correlation with Jab1, p27, and clinical outcome, we evaluated Skp2 expression in a group of epithelial ovarian tumors. Immunohistochemical analysis was performed on 80 cases of ovarian tumors (33 benign and 47 malignant), and 26 of the 80 cases were evaluated by Western blot analysis. Immunofluorescence was carried out in the human ovarian adenocarcinoma cell line OVCAR-3. Skp2 expression was detected in 53.2% of malignant tumors and 18.2% of benign tumors. The positive ratio of Skp2 expression was increased from benign to malignant ovarian tumors (p=0.002). A negative correlation between Skp2 and p27 was found in benign and malignant ovarian tumors (p=0.006 and p<0.0001, respectively). Skp2 expression was significantly associated with high tumor grade (p=0.001), lymph node metastasis (p=0.01), and residual disease (p=0.012). Kaplan-Meier survival analysis showed that Skp2 expression was significantly associated with poor prognosis (p=0.013), and patients with Skp2(+)/Jab1(+)p27(-) expression had the worst prognosis among all phenotypes of Skp2/Jab1/p27 expression (p=0.0007). Our results suggest that Skp2 expression was significantly associated with malignancy, and the Skp2 protein level may be a valuable prognostic factor for epithelial ovarian carcinomas. Furthermore, the combined evaluation of Skp2/Jab1/p27 proteins provides important prognostic information on patients with epithelial ovarian carcinoma.     

Correlation between FDG uptake and glucose transporter type 1 expression in neuroendocrine tumors of the lung

Neuroendocrine (NE) lung tumors are subdivided into the following types; typical (TC) and atypical carcinoids (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC). Moreover, the determinants of the FDG uptakes of NE lung tumors have not been elucidated. Thus, the aim of the present study was to investigate the relationships between FDG uptake and glucose transporter type 1 (Glut-1) expression in these NE tumors. Tissue-proven NE lung tumor patients (n=32; age, mean+/-S.D.=67.8+/-10 years; male:female=28:4) who had undergone F-18 FDG-PET before treatment were enrolled in this study. There were 1 TC, 3 AC, 5 LCNEC, and 23 SCLC patients. FDG uptakes were quantified using maximum standardized uptake values (maxSUV). Paraffin sections of tumor tissues were immunostained using anti-Glut-1 antibody (Neomarkers, 1:50). Levels of Glut-1 expression are presented as percentages of tumor cells positively immunostained (%Glut-1). Relations between FDG uptakes and Glut-1 expression were assessed using Pearson correlation analysis. The maxSUVs of all NE lung tumors ranged from 0.6 to 29.5 (mean+/-S.D.=7.7+/-5.4) and %Glut-1 expression ranged from 0 to 100% (18+/-24%). The maxSUVs of all NE lung tumors were found to be significantly correlated with %Glut-1 expression (r=0.6471, p=0.0001). By subgroup analysis, maxSUV was also found to be significantly correlated with %Glut-1 expression in SCLC (n=23, r=0.6189, p=0.0016). FDG uptake was found to be highly correlated with Glut-1 expression in NE lung tumors. This result suggests that Glut-1 plays a crucial role in determining FDG uptake in these tumors.     

Cyclin B and E2F-1 expression in prostate carcinoma cells treated with the novel retinoid CD437 are regulated by the ubiquitin-mediated pathway

E2F-1 and cyclin B are important regulators of the cell cycle, and their expressionand degradation are tightly regulated. Proteolysis of both molecules is mediated by the ubiquitin degradation pathway involving the activation of specific E3 ubiquitin ligases. Treatment of prostate carcinoma cells with the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437/AHPN) results in the enhanced expression of E2F-1 and rapid degradation of cyclin B in the absence of the modulation of mRNA levels; this is accompanied by the S phase arrest of the cells and subsequent apoptosis. The elevated level of E2F-1 is because of the enhanced stability of the molecule, as indicated by pulse-labeling studies, demonstrating a prolonged half-life. The enhanced E2F-1 stability is associated with the concomitant acetylation of E2F-1, the disassociation of E2F-1 from the E2F-1 E3 ligase p45(SKP2), and decreased E2F-1 ubiquitination, suggesting CD437 inhibition of E-3 E2F-1 ligase activity. Exposure of the cells to CD437 also results in the enhanced association of the cyclin B E3 ligase APC with cyclin B and the rapid proteolysis of cyclin B. The CD437-enhanced proteolysis of cyclin B is blocked in the presence of the ubiquitin proteolysis inhibitor N-acetyl-leu-leu-norleu-al. Thus, CD437 modulates the expression of E2F-1 and cyclin B through the simultaneous stimulation and inhibition of the cyclin B and E2F-1 E3 ligases, respectively.     

Isoenzyme pattern of enolase in the diagnosis of neuroendocrine bronchopulmonary tumors

Electrophoretic separation of enolase isoenzymes and the measurement of enolase activity were performed in 25 lung tumor extracts. In 13 neuroendocrine (NE) tumors (nine small cell lung carcinoma [SCLC], three atypical NE tumors, and one carcinoid tumor), the NE differentiation was assessed by ultrastructural determination of neurosecretory granule (NSG) density. Twelve non-NE lung tumors also were studied (three adenocarcinomas, four epidermoid, two composite, two large cell undifferentiated carcinomas, and one lymphoma). Four normal lung tissues and 1 human brain were used as controls. The gamma gamma isoenzyme was present at a high level (mean +/- SE, 12 +/- 3%) in all NE carcinomas and consistently absent in all non-NE tumors as well as in normal lung. The alpha gamma isoenzyme was found in significantly higher proportion in NE carcinomas (mean +/- SE, 29 +/- 2%) than in non-NE tumors (mean +/- SE, 8 +/- 1%) (P less than 0.0001), despite an equally high level of total enolase activity in both groups of tumor. The separation of alpha gamma and gamma gamma isoenzymes of enolase allows for the accurate diagnosis of NE tumors and NE components of atypical NE carcinomas, and the gamma gamma isoenzyme, in contrast to gamma chain detection by immunoassay, can be considered to be a specific marker in itself of NE differentiation in lung neoplasms.     

Expression levels of eIF4E, VEGF, and cyclin D1, and correlation of eIF4E with VEGF and cyclin D1 in multi-tumor tissue microarray

The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is a rate-limiting factor of cap-dependent translation initiation. When elevated, eIF4E greatly facilitates translation of a selected spectrum of mRNAs coding for proteins critical to angiogenesis and growth such as vascular endothelial growth factor (VEGF) and cyclin D1. Expression levels of eIF4E, VEGF, and cyclin D1 were examined in multi-tumor tissue microarray by immunohistochemistry and analyzed quantitatively. eIF4E, VEGF and cyclin D1 protein were elevated in tumors of the breast (62, 78, or 40%), colon (72, 77, or 12%), glioblastoma multiforme (48, 68, or 52%), lymphoma (66, 74, or 38%), melanoma (59, 73, or 58%), NSCLC (81, 82, or 29%), ovary (50, 39, or 13%), and prostate (78, 97, or 21%), respectively. eIF4E levels were strongly correlated with VEGF and cyclin D1 in melanoma (Spearman's r=0.97 and 0.77; all P<0.0001); moderately in tumors of the breast (r=0.55 and 0.41; all P<0.0005), colon (0.63 and 0.56; all P<0.0001), lung (0.53 and 0.53; all P<0.005), lymphoma (0.50 and 0.61; all P<0.0005), prostate (0.46 and 0.54; all P<0.005), or ovary (0.56 and 0.46; all P<0.005); and weakly in tumors of glioblastoma multiforme (r=0.20 and 0.31; all P>0.15). The significant association of eIF4E with VEGF and cyclin D1 in multiple tumors supports a role for eIF4E in translational regulation of proteins related to angiogenesis and growth.     

Skp2 在卵巢上皮性肿瘤中的表达及临床意义

 目的 检测S期激酶相关蛋白2(S-phase kinase associated protein2, Skp2)在卵巢上皮性肿瘤中的表达,探讨其表达与上皮性卵巢癌临床病理参数之间的关系,以及了解其表达在卵巢上皮性肿瘤发生、发展及上皮性卵巢癌预后估计中的意义. 方法 收集我院81例卵巢上皮性肿瘤的外科手术石蜡标本,用免疫组化的方法检测Skp2的表达水平. 结果 Skp2在卵巢上皮性良性肿瘤及交界性肿瘤中表达均为阴性,而在上皮性卵巢癌中表达率为46.43%(26/56),半定量分析表明Skp2在不同年龄及组织类型的卵巢癌中表达无显著性差异(P>0.05),而在中低度分化组、Ⅲ～Ⅳ期组及有淋巴结转移组分别明显高于高分化组(P<0.01)、Ⅰ～Ⅱ期组(P<0.01)及无淋巴结转移组(P<0.01). 结论 Skp2特异性表达于上皮性卵巢癌,其表达水平越高提示病人预后越差.     

Inhibition of cyclin A kinase activity in E2F-1 chemogene therapy of colon cancer.

Adenoviral-mediated gene transfer of the apoptotic gene E2F-1 has been shown to induce apoptosis in a variety of tumor cells and acts in an additive or cooperative fashion with several specific chemotherapeutic agents to induce tumor cell death. The apoptotic function of E2F-1 is dependent on its ability to bind DNA; cyclin A kinase activity has been shown to negatively regulate the DNA-binding capacity of E2F-1. In the present study, we sought to determine whether cyclin A kinase activity is involved in mediating the interaction between E2F-1 and chemotherapeutic agents in colon cancer cells. Therefore, human colon adenocarcinoma (SW620) cells were treated with an adenovirus expressing E2F-1 (Ad-E2F-1, multiplicity of infection 20). Immediately following infection, a panel of conventional chemotherapeutic agents with varying modes of cytotoxic action were administered at LD(25 )doses. Three days following treatment, viability and growth inhibition were determined by trypan blue exclusion assay. Apoptosis was confirmed using cellular morphology, poly (ADP-ribose) polymerase cleavage, and flow-cytometric analysis. E2F-1 overexpression and cyclin A protein expression were monitored by immunoblot, and cyclin A kinase activity was determined by kinase assay. Vincristine (VIN), camptothecin (CPT), and actinomycin D were found to have a cooperative (>38% over the additive single therapy values) effect on E2F-1-mediated apoptosis. Etoposide, cisplatin (CIS), and 5-fluorouracil (5-FU) showed the least cooperation ( 0.1) compared to Ad-E2F-1 treatment alone. Combination of Ad-LacZ/5-FU and Ad-LacZ/actinomycin D significantly inhibited cyclin A kinase activity compared to Ad-LacZ treatment alone (p < 0.005). No other Ad-LacZ/drug combinations significantly affected cyclin A kinase activity (p > 0.05). In conclusion, combinations of E2F-1 adenovirus and VIN, CPT, or actinomycin D at LD(25 )had significant cooperative effects on colon cancer apoptotic cell death in vitro. Although inhibition of cyclin A kinase activity was observed in most Ad-E2F-1/drug combination treatments compared to Ad-E2F-1 treatment alone, there was no consistent correlation between degree of inhibition of cyclin A kinase activity and the cooperative effect. Nonetheless, inhibition of cyclin A kinase activity may be an important mechanism by which the chemogene therapy effects involving E2F-1 are modulated.     

Cancer-testis antigen MAGE-C2 binds Rbx1 and inhibits ubiquitin ligase-mediated turnover of cyclin E

Cancer-testis antigen MAGE-C2 is normally expressed in testis but aberrantly expressed in various kinds of tumors. Its functions in tumor cells are mostly unknown. Here, we show that MAGE-C2 binds directly to the RING domain protein Rbx1, and participates in Skp1-Cullin1-F box protein (SCF) complex. Furthermore, MAGE-C2 can inhibit the E3 ubiquitin ligase activity of SCF complex. Ablation of endogenous MAGE-C2 decreases the level of cyclin E and accelerates cyclin E turnover by inhibiting ubiquitin-mediated proteasome degradation. Overexpression of MAGE-C2 increases the level of cyclin E and promotes G1-S transition and cell proliferation, and the results are further confirmed by knockdown of MAGE-C2. Overall, the study indicates that MAGE-C2 is involved in SCF complex and increases the stability of cyclin E in tumor cells.