Combined CCNE1 high-level amplification and overexpression is associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma.

CCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high-level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut-off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high-level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut-off for IHC to predict CCNE1 high-level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high-level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high-level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38-2.26, p < 0.0001). CCNE1 high-level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high-level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors.

The Prognostic Value of G1 Cyclins, p21 and Rb Protein in Patients With Colon Cancer.

BACKGROUND/AIM: Cyclins D1 and E play different roles in the cell cycle. Cyclin E promotes chromosome instability, whereas cyclin D1 regulates apoptosis of cells. This study evaluated the prognostic significance of G1 cyclins, p21 and pRb in tumor proliferation. PATIENTS AND METHODS: A total of 102 patients with colon cancer were operated on and staged according to TNM. Follow-up was 2 to 68 months (mean 38.3±16.7 months). Expression of cyclin E and D1 were evaluated using immunohistochemistry. RESULTS: Levels of cyclin E expression were correlated with cyclin D1 expression (p=0.038), p21 expression (p=0.047), and pRb expression (p=0.004). The 5-year survival rate along with prognosis of patients with advanced stage (III, IV) colon cancer and cyclin D1 positive tumors, were significantly worse (p=0.009). Statistically significant association was observed between tumor proliferative capacity Ki-67, cyclin D1 (p=0.009), pRb (p=0.031) and p21 (p=0.050). CONCLUSION: Cyclin D1 is highly expressed in advanced stage colon cancer patients, implying a potential prognostic value.

Anticancer Mechanism of Lobaplatin as Monotherapy and in Combination with Paclitaxel in Human Gastric Cancer.

OBJECTIVE: To explore the mechanism by which lobaplatin, as monotherapy and in combination with paclitaxel, inhibits the proliferation of human gastric cancer SGC-7901 cells. METHODS: After treatment, the MTT assay was used to assess cell viability; cell cycle distribution was evaluated flow-cytometrically. Western blot was used to quantitate cyclin D1, E1, B1, and Cdk2/4 protein levels. RESULTS: Lobaplatin and paclitaxel inhibited SGC-7901 cell growth in a concentration and timedependent manner, with IC25 values at 48h of 1.97±0.17µg/ml and 1.98±0.19 ng/ml, respectively. Interestingly, both drugs synergistically inhibited SGC-7901 cells (combination index [CI]<0.95). Lobaplatin did not affect cyclin D1 and CDK4 protein expression, while cyclin E1 and CDK2 levels were significantly increased, with cyclin B1 amounts markedly decreased (p<0.05). More S phase cells were observed after lobaplatin treatment compared with controls (60.03±1.25 vs. 18.69±0.96%; p<0.05). After treatment with paclitaxel, cyclin D1 and CDK4 protein levels were similar to control values; meanwhile, cylinE1 and CDK2 protein amounts were reduced, with increased cyclin B1 levels, compared with control values (p<0.05). More G2/M cells were obtained after treatment with paclitaxel compared with control values (74.54±0.92 vs. 18.62±0.44% (p<0.05). Lobaplatin and paclitaxel combination did not affect cyclin D1 and CDK4 protein levels (p>0.05); meanwhile, cyclin E1 and CDK2 levels were increased, with reduced cyclin B1 amounts, compared with control values (p<0.05). Notably, more S (43.23±0.81 vs. 22.32±0.86%) and G2/M (31.22±0.96 vs. 25.81±2.08%) phase cells were obtained after combined treatment compared with control values. CONCLUSION: Lobaplatin and paclitaxel synergistically inhibit SGC-7901 cells.

Protein expression patterns of cell cycle regulators in operable breast cancer.

BACKGROUND-AIM: To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes. METHODS: Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis. RESULTS: Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively. CONCLUSIONS: It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer.

Prognostic value of cyclin E expression in patients with ovarian cancer: a Meta-analysis.

PURPOSE: Cell cycle is mainly mediated by cyclins, cyclin- dependent kinases (CDK), and CDK inhibitors. Cyclin E is the main regulator for transition from G1 to S phase, and is involved in cancer pathogenesis, progression and metastasis. Nevertheless, there is still a controversy of the prognostic value of cyclin E overexpression in ovarian cancer patients. This meta-analysis is the first study aimed at analyzing the effect of cyclin E overexpression on the prognosis of ovarian cancer. METHODS: By systematically searching the PUBMED, EMBASE and MEDLINE databases for relevant articles with publication dates up to January 2016 and selection following inclusion and exclusion criteria, 8 studies with 1470 patients were enrolled in our meta-analysis. The overall survival (OS) of patients with cyclin E overexpression was calculated using hazard ratio (HR) with 95% confidence intervals (CIs). The studies were categorized according to the author and year, demographic data in each study, ovarian cancer related information, and cyclin E cut-off value. RESULTS: Cyclin E overexpression in ovarian cancer was a poor prognostic factor with statistical significance for OS (HR=1.48, 95%CI: 1.12,1.85). Using confunnel, we found no publication bias in our analysis. CONCLUSION: Cyclin E might be considered as a prognostic factor for ovarian cancer, as supported by our meta-analysis. However, more high-quality studies should be conducted to find better clinical use of cyclin E in ovarian cancer.

MCM4 and MCM7, potential novel proliferation markers, significantly correlated with Ki-67, Bmi1, and cyclin E expression in esophageal adenocarcinoma, squamous cell carcinoma, and precancerous lesions.

Minichromosomal maintenance (MCM) proteins are participants of DNA replication and may represent more accurate markers in determining the proliferative fraction within a tumor than proliferative marker Ki-67. Our study investigated the correlation between MCM4 and MCM7 expression and Ki-67, Bmi1, and cyclin E expression in esophageal adenocarcinoma, squamous cell carcinoma, and precancerous lesions. MCM4 and MCM7 expression had similar distribution as Ki-67 and Bmi1 expression in esophageal carcinoma and precancerous lesions. The mean percentage of MCM4, MCM7, and Ki-67 expression increased from squamous epithelium (5.5%, 7.3%, and 5.9%, respectively), to columnar cell metaplasia (11.2, 13.5%, and 3.4%), Barrett's esophagus (27.7%, 35.3%, and 8.3%), low-grade dysplasia (42.6%, 52.2%, and 12.9%), high-grade dysplasia (63.2%, 77.7%, and 29.6%), adenocarcinoma (61.3%, 75.5%, and 24.5%), and squamous cell carcinoma (74.1, 85.4%, and 36.3%). The percentages of MCM4 and MCM7 expression were significantly higher than Ki-67 expression. Using univariate analysis we found a high percentage of MCM4 expression (>70%) to be significantly associated with lymph node metastasis and shorter survival in the adenocarcinoma group. We also demonstrated the percentage of MCM4 and MCM7 expression to be significantly correlated with Ki-67, Bmi1, and cyclin E expression in esophageal carcinoma and precancerous lesions. MCM4 and MCM7 may serve as more sensitive proliferative markers for the evaluation of esophageal lesions.

Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib.

OBJECTIVE: Homologous recombination (HR) proficient ovarian cancers, including CCNE1 (cyclin E)-amplified tumors, are resistant to poly (ADP-ribose) polymerase inhibitors (PARPi). Histone deacetylase inhibitors (HDACi) are effective in overcoming tumor resistance to DNA damaging drugs. Our goal was to determine whether panobinostat, a newly FDA-approved HDACi, can sensitize cyclin E, HR-proficient ovarian cancer cells to the PARPi olaparib. METHODS: Expression levels of CCNE1 (cyclin E), BRCA1, RAD51 and E2F1 in ovarian tumors and cell lines were extracted from The Cancer Genome Atlas (TCGA) and Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). In HR-proficient ovarian cancer cell line models (OVCAR-3, OVCAR-4, SKOV-3, and UWB1.289+BRCA1 wild-type), cell growth and viability were assessed by sulforhodamine B and xenograft assays. DNA damage and repair (pH2AX and RAD51 co-localization and DRGFP reporter activity) and apoptosis (cleaved PARP and cleaved caspase-3) were assessed by immunofluorescence and Western blot assays. RESULTS: TCGA and CCLE data revealed positive correlations (Spearman) between cyclin E E2F1, and E2F1 gene targets related to DNA repair (BRCA1 and RAD51). Panobinostat downregulated cyclin E and HR repair pathway genes, and reduced HR efficiency in cyclin E-amplified OVCAR-3 cells. Further, panobinostat synergized with olaparib in reducing cell growth and viability in HR-proficient cells. Similar co-operative effects were observed in xenografts, and on pharmacodynamic markers of HR repair, DNA damage and apoptosis. CONCLUSIONS: These results provide preclinical rationale for using HDACi to reduce HR in cyclin E-overexpressing and other types of HR-proficient ovarian cancer as a means of enhancing PARPi activity.

miRNA-34a promotes proliferation of human pulmonary artery smooth muscle cells by targeting PDGFRA.

OBJECTIVES: Pulmonary arterial hypertension (PAH) is a fast progressing vascular disease characterized by uncontrolled cell proliferation of pulmonary artery smooth muscle cells (PASMCs). Some studies have suggested that PAH and cancers share an apoptosis-resistant state, featuring excessive cell proliferation. The miR-34 family consists of tumour-suppressive miRNAs, and its reduced expression has been reported in numerous cancers; however, its role in hypoxia-induced PAH has not been previously studied. MATERIALS AND METHODS: miR-34 family expression was evaluated in a rat model with hypoxia and in cultured hypoxic PASMCs, using real-time quantitative PCR (RT-qPCR). Function of miR-34 family was assessed by transfecting miR-34 mimics and inhibitors. Dual luciferase reporter gene assays, RT-qPCR and Western blotting were performed to validate target genes of miR-34. RESULTS: Significant down-regulation of miR-34a in hypoxic lung tissue, pulmonary artery and PASMCs was identified and then effects of miR-34a in modulating cell proliferation in human pulmonary artery smooth muscle cells (hPASMCs) was investigated in vitro. Reduction of miR-34a levels in hPASMCs caused increased proliferation and these effects were reversed by overexpression of miR-34a. miR-34a overexpression down-regulated platelet-derived growth factor receptor alpha (PDGFRA) expression, which is a key factor in PAH development. These results suggest that miR-34a is a potential regulator of proliferation in PASMCs, and that it could be used as a novel treatment strategy in PAH.

Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer.

Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.

Cyclin E is overexpressed by clear cell carcinomas of the endometrium and is a prognostic indicator of survival.

OBJECTIVE: Upregulation of cyclin E and cyclin D1-6 accelerates the transition from G1 to S phase. The objective of this study was to determine if cyclin D1 and E are prognostic indicators in endometrial cancer. MATERIALS AND METHODS: Surgically-treated patients with endometrial carcinoma had their tumors stained for nuclear expression of cyclin D1 and E. Quantification of staining and measurement of growth phase fraction were performed using image analysis. FIGO stage, grade, and histology were also analyzed. RESULTS: Cyclin D1 and E expression was unrelated to DNA index (p = 0.93). While cyclin D1 expression did not correlate with S+G2M phase fraction (p = 0.69), increased cyclin E expression was directly correlated with increased S+G2M phase fraction (p = 0.002). Cyclin E expression was highest in clear cell carcinomas (p = 0.042) while cyclin D1 expression was highest in adenosquamous carcinomas (p = 0.028). Patients dying from cancer had significantly higher expression of cyclin D1 (p = 0.042) and E (p = 0.02) as compared to patients surviving their disease. Multivariate logistic regression revealed FIGO stage, grade, and lack of cyclin E overexpression to be independent prognostic indicators of survival. CONCLUSION: Cyclin E expression is related to increased growth fraction, clear cell histology, and decreased survival in patients with endometrial cancer.

Synergistic effects of AKAP95, Cyclin D1, Cyclin E1, and Cx43 in the development of rectal cancer.

OBJECTIVE: To explore the expression of A-kinase anchor protein 95 (AKAP95), Cyclin D1, Cyclin E1, and Connexin43 (Cx43) in rectal cancer tissues and assess the associations between each of the proteins and pathological parameters, as well as their inter-relationships. METHODS: AKAP95, Cyclin D1, Cyclin E1, and Cx43 protein expression rates were evaluated by immunohistochemistry in 50 rectal cancer specimens and 16 pericarcinoma tissues. RESULTS: The positive rates of AKAP95, Cyclin E1, and Cyclin D1 proteins were 54.00 vs. 18.75%, 62.00 vs. 6.25%, and 72.00 vs. 31.25% in rectal cancer specimens and pericarcinoma tissues, respectively, representing statistically significant differences (P < 0.05). The positive rate of Cx43 protein expression in rectal cancer tissues was 44.00% and 62.50% in pericarcinoma tissues, and the difference between them was not significant (P > 0.05). No significant associations were found between protein expression of AKAP95, Cyclin E1, Cyclin D1, and Cx43, and the degree of differentiation, histological type, and lymph node metastasis of rectal cancer (P > 0.05). However, significant correlations were obtained between the expression rates of AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43, respectively (P < 0.05). CONCLUSION: AKAP95, Cyclin E1, and Cyclin D1 protein expression rates were significantly higher in rectal cancer tissues compared with pericarcinoma samples, suggesting an association between these proteins and the development and progression of rectal cancer. In addition, the significant correlations between the proteins (AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43) indicate the possible synergistic effects of these factors in the development and progression of rectal cancer.

Quantum dot assisted tracking of the intracellular protein Cyclin E in Xenopus laevis embryos.

BACKGROUND: Luminescent semiconductor nanocrystals, also known as quantum dots (QD), possess highly desirable optical properties that account for development of a variety of exciting biomedical techniques. These properties include long-term stability, brightness, narrow emission spectra, size tunable properties and resistance to photobleaching. QD have many promising applications in biology and the list is constantly growing. These applications include DNA or protein tagging for in vitro assays, deep-tissue imaging, fluorescence resonance energy transfer (FRET), and studying dynamics of cell surface receptors, among others. Here we explored the potential of QD-mediated labeling for the purpose of tracking an intracellular protein inside live cells. RESULTS: We manufactured dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QD, not available commercially, and coupled them to the cell cycle regulatory protein Cyclin E. We then utilized the QD fluorescence capabilities for visualization of Cyclin E trafficking within cells of Xenopus laevis embryos in real time. CONCLUSIONS: These studies provide "proof-of-concept" for this approach by tracking QD-tagged Cyclin E within cells of developing embryos, before and during an important developmental period, the midblastula transition. Importantly, we show that the attachment of QD to Cyclin E did not disrupt its proper intracellular distribution prior to and during the midblastula transition. The fate of the QD after cyclin E degradation following the midblastula transition remains unknown.

MUC4 regulates cellular senescence in head and neck squamous cell carcinoma through p16/Rb pathway.

The limited effectiveness of therapy for patients with advanced stage head and neck squamous cell carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. MUC4, a high molecular weight glycoprotein, is differentially overexpressed in many human cancers and implicated in cancer progression and resistance to several chemotherapies. However, its clinical relevance and the molecular mechanisms through which it mediates HNSCC progression are not well understood. This study revealed a significant upregulation of MUC4 in 78% (68/87) of HNSCC tissues compared with 10% positivity (1/10) in benign samples (P=0.006, odds ratio (95% confidence interval)=10.74 (2.0-57.56). MUC4 knockdown (KD) in SCC1 and SCC10B HNSCC cell lines resulted in significant inhibition of growth in vitro and in vivo, increased senescence as indicated by an increase in the number of flat, enlarged and senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. Decreased cellular proliferation was associated with G0/G1 cell cycle arrest and decrease expression of cell cycle regulatory proteins like cyclin E, cyclin D1 and decrease in BrdU incorporation. Mechanistic studies revealed upregulation of p16, pRb dephosphorylation and its interaction with histone deacetylase 1/2. This resulted in decreased histone acetylation (H3K9) at cyclin E promoter leading to its downregulation. Orthotopic implantation of MUC4 KD SCC1 cells into the floor of the mouth in nude mice resulted in the formation of significantly smaller tumors (170±18.30 mg) compared to those (375±17.29 mg) formed by control cells (P=0.00007). In conclusion, our findings showed that MUC4 overexpression has a critical role by regulating proliferation and cellular senescence of HNSCC cells. Downregulation of MUC4 may be a promising therapeutic approach for treating HNSCC patients.

Roles of Cx43 and AKAP95 in ovarian cancer tissues in G1/S phase.

OBJECTIVE: The purpose of this study was to investigate the expression of A-kinase anchor protein 95 (AKAP95), cell cycle protein E1 (cyclinE1) and D1 (cyclinD1), and gap junction protein connexin 43 (Cx43) in ovarian cancer tissues, the relationship between four proteins and clinicopathologic parameters, and the correlation between these proteins. METHODS: The expression of proteins in 54 cases of ovarian cancer tissues was detected by immunohistochemical method. RESULTS: The positive expression rates of AKAP95, cyclinD1 and cyclinE1 in ovarian cancer tissues were 72.22%, 66.67% and 79.63%, respectively, which were higher than that of ovarian pericarcinoma tissues expressing as 33.33%, 25% and 8.30% (P<0.05). The positive expression rate of Cx43 in ovarian cancer tissues was 40.74%, which was lower than that of ovarian pericarcinoma tissues expressing as 75%; respectively, and the difference was statistically significant between groups (P<0.05). The expression of cyclinD1 in ovarian cancer tissues was related to the histologic type (P<0.05) while it showed no correlation with the degree of differentiation (P>0.05). Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues showed no correlation with the degree of differentiation or the histologic type (P>0.05). Protein expressions of AKAP95, Cx43 and cyclinE1 were correlated with each other (P<0.05), and the expressions of cyclinD1, cyclinE1 and Cx43 were also correlated with each other (P<0.05). However, AKAP95 and cyclinD1 showed no correlation (P>0.05). CONCLUSION: AKAP95, cyclinD1 and cyclinE1 play an important role in promoting the process of ovarian cancer formation. The tumor inhibitory effects of Cx43 protein on the pathogenesis of ovarian cancer were weakened. The expression of cyclinD1 in ovarian cancer tissues is related to the histologic type while it shows no correlation with the degree of differentiation. Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues shows no correlation with the degree of differentiation or the histologic type. AKAP95 expression is correlated with Cx43 and cyclinE1 expression; Cx43 expression is correlated with AKAP95, cyclinD1 and cyclinE1 expression; cyclinE1 expression is correlated with AKAP95, Cx43, cyclinD1 expression; cyclinD1 expression is correlated with Cx43 and cyclinE1 expression, while AKAP95 and cyclinD1 show no correlation.

Prognostic importance of cyclin E1 expression in neuroblastic tumors in children.

A number of studies have indicated that cyclin E plays an important role in a variety of neoplastic processes. In our study we evaluated cyclin E1 expression and the possible prognostic value of this protein in neuroblastic tumors in children. Cyclin E1 expression was investigated by means of immunohistochemical analysis of 25 neuroblastic tumor tissue samples. We found a significant correlation between high cyclin E1 expression and deaths due to neoplastic disease. The mean values of cyclin E1 indexes in fatal cases were twice as high as in other cases. The results indicate that high cyclin E1 expression may have prognostic importance in neuroblastic tumors in children.

The role of estrogen in the survival of ovarian tumors--a study of the human ovarian adenocarcinoma cell lines OC-117-VGH and OVCAR3.

BACKGROUND: The role of estrogen in the growth and survival of ovarian cancer cells is controversial. In this study, we investigated the changes in cell-cycle regulatory proteins in ovarian cancer cell lines after estrogen treatment to explore the role of estrogen in ovarian cancers. METHODS: Two ovarian adenocarcinoma cell lines were used for the study: the first, OC-117-VGH, was deficient in estrogen receptors (ER)α and ERß, and the second, OVCAR3, was positive for ERα and ERß. Serial concentrations of estrogen were used to evaluate the effects of estrogen on the survival of ovarian cancer cells. The cell-cycle regulatory proteins, including cyclin D1, cyclin E, p16/INK4a, and p27/KIP1, were used to check the possible mechanism of an estrogen effect on survival of the cancer cell line. RESULTS: Estrogen 0.01-1.0 µM inhibited the growth of both cell lines. There were no differences in cyclin D1 and E expression between the two cell lines after estrogen treatment, but the expression of p16/INK4a and p27/KIP1 was significantly higher in the OC-1170-VGH cell line than in the OVCAR3 cell line. CONCLUSION: Although the ER-positive and ER-negative ovarian cancer cell lines were inhibited by estrogen, the influence of cell-cycle regulatory proteins was different between the two, suggesting that the inhibitory effect of estrogen on ovarian cancer cell lines might be mediated through different pathways.

Immunohistochemical expression of cell-cycle regulators in pediatric embryonal brain tumors.

Embryonal tumors constitute the most common malignant brain tumor group in children. Experimental results indicate that genes involved in cell cycle and signal transduction are deregulated in medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT). The cell cycle is regulated by protein complexes composed of a regulatory subunit called Cyclin and a catalytic domain named Cyclin-dependent kinase (CDK). Cyclins and CDKs are in turn regulated by cyclin-dependent kinase inhibitors (CDKI) which inhibit cell-cycle progression. Cyclins D and Cyclin E are important for the passage of cells through G1 to S phase. P-27, a member of the universal CDKI family, is important in regulating the G1/S transition. Thus, the purpose of this study was to investigate the expression of p-27, Cyclin D3, and Cyclin E, and their possible prognostic significance in pediatric embryonal brain tumors. We retrospectively evaluated 51 children with embryonal tumors that were treated surgically in our institute. All patients had regular follow up examinations. The streptavidin-biotin HRP method was performed on paraffin sections for detection of p-27, Cyclin D3, and Cyclin E. There were 42 cases of MB and nine cases of AT/RT. Cyclin D3 expression was detected in 11/42 MB and 3/9 AT/RT patients. Cyclin E expression was detected in 28/42 MB and 8/9 AT/RT patients. High expression of Ki-67 (>50 %) and p-27 (>50 %) was observed in 23.8-73.8 % of MB patients. Combined high Ki-67 and p-27 expression was observed in 21.4 % cases of MB. In these cases there was expression of Cyclin E in 88.8 % and Cyclin D3 in 22.2 % of MB. No significant correlation was found between Ki-67 and p-27, Cyclin D3, and E. No correlation was found between Cyclin D3, Cyclin E, p-27, and overall survival. Increased p-27 and Cyclin E expression was detected in a substantial number of MB patients and in nearly all AT/RT patients. Further studies on a larger number of patients are needed to clarify a possible correlation of p-27 and Cyclin E with tumor behavior.

Metformin suppresses growth of human head and neck squamous cell carcinoma via global inhibition of protein translation.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world; the main risk factors are alcohol and tobacco use. Advancements in therapies have yet to improve the prognosis of HNSCC. The connection between diabetes and cancer is being recognized, and metformin has been shown to decrease cancer incidence in diabetic patients. Accordingly, here, for the first time, we investigated metformin's efficacy on the growth and viability of human HNSCC FaDU and Detroit cells. Our results show that metformin treatment (5-20 mM) dose-dependently inhibits the growth of both cell lines. In FaDU cells, metformin caused 18-57% and 35-81% growth inhibition after 48 and 72 h treatments, respectively. Similarly, in Detroit 562 cells, 48 and 72 h metformin treatment resulted in 20-57% and 33-82% inhibition, respectively. Mechanistically, metformin caused G 1 arrest, which coincided with a decrease in the protein levels of CDKs (2, 4 and 6), cyclins (D1 and E) and CDK inhibitors (p15, p16, p18 and p27), but no change in p19 and p21. Metformin also decreased the levels of oncogenic proteins Skp2 and ß-Trcp. In other studies, metformin decreased the phosphorylation of 4E-BP1 at Ser65, Thr37/46 and Thr70 sites, but drastically increased the phosphorylation of EF2 at Thr56 and AMPK at Thr172, which results in global translational inhibition. In summary, the observed wide spectrum of mechanistic effects of metformin on HNSCC cells provides support for the anticancer capability of the drug and its potential use in future therapies.

Cyclin E1 is a common target of BMI1 and MYCN and a prognostic marker for neuroblastoma progression.

The Polycomb transcription repressor BMI1 is highly expressed in human neuroblastomas and is required for the clonogenic self-renewal and tumorigenicity of human neuroblastoma cell lines. The molecular basis of BMI1 action in neuroblastoma cells is not well understood. Here we report that BMI1 has a critical role in stabilizing cyclin E1 by repressing the expression of FBXW7, a substrate-recognition subunit of the SCF E3 ubiquitin ligase that targets cyclin E1 for degradation. BMI1 binds to the FBXW7 locus in vivo and represses its mRNA expression. Overexpression of cyclin E1 or abrogation of FBXW7 induction rescues the cell-death phenotype of BMI1 knockdown. Moreover, MYCN, an oncoprotein in the pathogenesis of high-risk neuroblastomas, is able to counteract the death-inducing effect of BMI1 knockdown by activating CCNE1 transcription. We further show that high cyclin E1 expression is associated with Stage 4 neuroblastomas and poor prognosis in patients. These findings suggest a molecular mechanism for the oncogenic activity of BMI1 and MYCN in neuroblastoma pathogenesis and progression by maintaining cyclin E1 levels.