cyclin-d1 and Esophageal-Neoplasms

The association between the Cyclin D1 gene (CCND1) G870A polymorphism and esophageal cancer has been widely evaluated, with conflicting results. As meta-analysis is a reliable approach to resolving discrepancies, we aimed to evaluate this association. Data were available from 9 study populations incorporating 1898 cases and 3046 controls. Overall, the allelic/genotypic association between the G870A polymorphism and esophageal cancer was nonsignificant [for allele: odds ratio (OR) = 1.14, 95% confidence interval (95%CI) = 0.94-1.38, P = 0.184; for genotype homozygous comparison: OR = 1.36, 95%CI = 0.90-2.06, P = 0.140; for dominant model: OR = 1.24, 95%CI = 0.88-1.75, P = 0.222; for recessive model: OR = 1.13, 95%CI = 0.90-1.43, P = 0.292]. Moreover, subgroup analyses according to study designs, geographic areas, types of esophageal cancer, genotyping methods, and ethnicities failed to demonstrate a significant association between this polymorphism and esophageal cancer. In addition, there was significant publication bias as reflected by funnel plots and the Egger test (P = 0.042). Taken together, our results suggest that the CCND1 G870A polymorphism might not be a potential candidate for predicting esophageal cancer risk.

Topics: Adenocarcinoma; Alleles; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Genetic Predisposition to Disease; Humans; Polymorphism, Single Nucleotide

Cyclin D1 is one of the most commonly over-expressed oncogenes; however, its role in esophageal squamous cell carcinoma (ESCC) remains controversial. We conducted a meta-analysis of 20 studies, comprising 2,041 patients to clarify this issue. In all studies, paraffin-embedded surgical specimens were collected and the status of cyclin D1 was determined by immunohistochemistry (IHC). The combined odds ratios (Ors) for cyclin D1 expression were 0.74 (95% confidence interval [CI]: 0.58-0.93) for well and moderately differentiated versus poorly differentiated tumors, 0.65 (95% CI: 0.45-0.94) for T1/T2 versus T3/ T4 tumors, 0.59 (95% CI: 0.39-0.90) for N0 versus N1 tumors, and 0.48 (95% CI: 0.33-0.71) for stage I/II versus stage III/IV diseases, respectively. The association between cyclin D1 expression and prognosis was examined in 10 studies, and the combined hazard ratio was 1.78 (95% CI: 1.49-2.12). Cyclin D1 expression level detected by IHC is associated with worst clinicopathological features and prognosis for ESCC.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Humans; Immunohistochemistry; Odds Ratio; Prognosis

Investigations concerning the association of Cyclin D1 (CCND1) G870A polymorphism with esophageal cancer risk have generated conflicting results. Thus, meta-analyses were conducted. The overall data suggest that CCND1 G870A variation might have an association with increased esophageal cancer susceptibility. In subgroup analyses on ethnicity, homozygous AA alleles might elevate esophageal cancer risk among Asians but not Caucasians. In subgroup analysis on histological types, no association was found in either the adenocarcinoma or the squamous cell carcinoma subgroup. Collectively, results suggest that CCND1 G870A polymorphism might be a low-penetrant risk factor for esophageal carcinoma, particularly among Asians.

Topics: Asia; Asian People; Cyclin D1; Esophageal Neoplasms; Genetic Predisposition to Disease; Humans; Polymorphism, Single Nucleotide; Risk Factors

Adenocarcinomas of the distal esophagus mainly develop from intestinal metaplasia (Barrett's esophagus) through intermediate steps of low-grade and high-grade intraepithelial neoplasia. Histopathological examination of endoscopic biopsies constitutes the gold standard for estimating the cancer risk of a patient with Barrett's esophagus. Several prospective biomarker phase IV studies have demonstrated the predictive value of e.g. allelic loss of TP53, tetraploidy and aneuploidy as well as cyclin D1 expression. Among the relevant biomarkers from retrospective phase III studies are polysomy and specific DNA gains and losses, markers of proliferation (Mib-1) and methylation markers. As there are conflicting results in the literature and these analyses are costly, their use in routine patient care cannot yet be recommended. However, immunostaining for several markers may assist in the classification of intraepithelial neoplasia in individual difficult cases.

Topics: Adenocarcinoma; Aneuploidy; Apoptosis; Barrett Esophagus; Biopsy; Carcinoma in Situ; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Esophageal Neoplasms; Esophagus; Gene Expression Regulation, Neoplastic; Genetic Markers; Immunohistochemistry; Loss of Heterozygosity; Prognosis; Tetraploidy; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer in Asia, characterized by high incidence and mortality rate. Although significant progress has been made in surgery and adjuvant chemoradiotherapy, the prognosis of the patients with this cancer still remains poor. Investigation into protein alterations that occurred in tumors can provide clues to discover new biomarkers for improving diagnosis and guiding targeted therapy. Hundreds of papers have appeared over the past several decades concerning protein alterations in ESCC. This review summarizes all the dysregulated proteins investigated in the disease from 187 published papers and analyzes their contributions to tumor development and progression. We document protein alterations associated with tumor metastasis and the transition from normal esophageal epithelia to dysplasia in order to reveal the most useful markers for prediction of clinical outcome, early detection, and identification of high-risk patients for targeted therapies. In particular, we discuss the largest and most rigorous studies on prognostic implications of proteins in ESCC, in which cyclin D1, p53, E-cadherin and VEGF appeared to have the strongest evidence as independent predictors of patient outcome.

Topics: Apoptosis; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin D1; Disease Progression; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neoplasm Proteins; Prognosis; Proportional Hazards Models; Signal Transduction; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

Barrett's esophagus (BE) represents the most serious histological consequence of gastroesophageal reflux disease (GERD) that develops in 5-10% of patients with GERD. Given that BE is the only known precursor to esophageal adenocarcinoma (EA), a systematic endoscopic biopsy protocol can detect EAs at an early stage. However, endoscopic and histopathological evaluation of BE are not adequate for effective screening of high risk patients. Therefore, molecular abnormalities associated with BE have been considered as surrogate markers and their use as such is proposed. Flow cytometry is the most useful adjunct to histology, and ploidy status of BE is an independent risk factor. Cyclin D1 overexpression is inversely correlated with survival in EA. C-erbB2 (+) patients have poorer prognosis. High plasma adenomatous polyposis coli levels correlate with reduced patient survival. p53 expression allows patient risk for EA stratification. Nuclear factor-kappaB overexpression inversely correlates with good response to adjuvant chemotherapy and radiotherapy in EA. Patients with cyclooxygenase-2 overexpression have reduced survival rates. Increased E-cadherin staining is associated with shorter survival in EA patients who received chemoradiotherapy. Finally, existing data cannot rule out a correlation between EA and colorectal tumors. Seventeen BE molecular alterations yielded noteworthy clinical implications. Apart from endoscopy and histology, these data allow for better risk stratification for patients with BE and for more efficient and timely therapeutic approaches.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli Protein; Barrett Esophagus; Biomarkers; Biomarkers, Tumor; Cadherins; Clinical Trials as Topic; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Endoscopy, Gastrointestinal; Esophageal Neoplasms; Gastroesophageal Reflux; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; NF-kappa B; Prostaglandin-Endoperoxide Synthases; Receptor, ErbB-2; Risk Factors; Survival Rate; Tumor Suppressor Protein p53; Up-Regulation

Barrett's esophagus is an acquired metaplastic change that occurs in the distal esophagus secondary to chronic gastroesophageal reflux. This premalignant condition forms the most important risk factor for developing esophageal adenocarcinoma, which is an extremely aggressive tumor with a 5-year survival rate of less than 25%. Carcinomas that arise in the setting of Barrett's esophagus are thought to develop as part of the metaplasia-dysplasia-carcinoma sequence.. To review the current knowledge on the genomic alterations involved in the development of Barrett's esophagus and its progression to dysplasia and/or cancer.. Several changes in gene structure, gene expression, and protein structure are associated with the progression of Barrett's esophagus to adenocarcinoma. Accumulation of these changes seems to be essential, rather than the exact sequence of these changes. Multiple molecular pathways are involved and interact with each other. Alterations in tumor suppressor genes, amongst which p53 and p16, are early events in the metaplasia-dysplasia-adenocarcinoma sequence, followed by loss of cell cycle checkpoints. Ongoing genomic instability leads to cumulative genetic errors and thereby the generation of multiple clones of transformed cells.. Within the multistep process of esophageal adenocarcinogenesis, to date no single molecular marker came forward able to predict who will and who will not develop cancer in the setting of Barrett's esophagus. Instead, panels of markers need to be developed in the future allowing to indicate disease progression. Identification of crucial molecular pathways involved in esophageal adenocarcinogenesis would ultimately improve therapy and facilitate development of new treatment strategies.

Topics: Adenocarcinoma; Apoptosis; Barrett Esophagus; Chromosome Aberrations; Cyclin D1; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gastroesophageal Reflux; Gene Expression Regulation, Neoplastic; Humans; Metaplasia; Microsatellite Repeats; Precancerous Conditions; Receptor, ErbB-2; Tumor Suppressor Protein p53

Over the past three decades, there has been a marked change in the epidemiology of esophageal malignancy, with an increasing incidence of esophageal adenocarcinoma. The reasons for this are largely unknown and remain controversial, but several lifestyle risk factors have been proposed, including gastroesophageal reflux disease (GERD). It is hypothesized that chronic GERD results in acute mucosal injury, promotes cellular proliferation, and induces specialized columnar metaplasia (Barrett esophagus). Progression of Barrett esophagus to invasive adenocarcinoma is reflected histologically by the metaplasia-dysplasia-carcinoma sequence. Dysplasia is widely regarded as the precursor of invasive cancer, and high-grade dysplasia in Barrett epithelium is frequently associated with esophageal adenocarcinoma. Although several molecular alterations have been described in Barrett esophagus, it is anticipated that relatively few will prove to be clinically useful. To date, biomarkers which currently appear to predict the progression of Barrett esophagus to invasive malignancy include aneuploidy, loss of heterozygosity of 17p (implicating the p53 tumor suppressor gene), and cyclin D1 protein overexpression, and with further validation, will most likely be incorporated into routine clinical practice. It is anticipated that models incorporating objective scores of sociodemographic and lifestyle risk factors (ie, age, gender, body mass index), severity of reflux symptoms, endoscopic and histologic findings, and an assessment of a panel of biomarkers will be developed to further define subsets of patients with Barrett esophagus at increased risk for malignant progression, thereby permitting the development of more rational endoscopic surveillance and screening programs.

Topics: Adenocarcinoma; Barrett Esophagus; Biomarkers, Tumor; Chronic Disease; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Epithelium; Esophageal Neoplasms; Gastroesophageal Reflux; Genes, p53; Humans; Ploidies

The rising incidence and poor prognosis of esophageal adenocarcinoma in the Western world have intensified research efforts into earlier methods of detection of this disease and its relationship to Barrett's esophagus. The progression of Barrett's esophagus to adenocarcinoma has been the focus of particular scrutiny, and a number of potential tissue and serum-based disease biomarkers have emerged. The epidemiology and pathogenesis of esophageal adenocarcinoma are outlined. Tissue biomarkers allowing risk stratification of Barrett's are reviewed as well as strategies currently being used to discover novel biomarkers that will facilitate the early detection of esophageal adenocarcinoma. Finally, the uses of biomarkers as predictive tests for targeted treatments and as surrogate endpoints in chemoprevention trials are considered.

Topics: Adenocarcinoma; Barrett Esophagus; Biomarkers; Biomarkers, Tumor; Cyclin D1; DNA, Neoplasm; Esophageal Neoplasms; Humans; Tumor Suppressor Protein p53

Topics: Animals; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclooxygenase 2; DNA Repair; ErbB Receptors; Esophageal Neoplasms; Humans; Isoenzymes; Membrane Proteins; Mice; Prostaglandin-Endoperoxide Synthases; Tumor Suppressor Protein p53

Carcinomas of the upper gastrointestinal tract have been intensively studied for decades in order to identify markers for a) design of simple blood tests to detect presence or recurrence of the disease, b) prediction of therapy response, c) identification of molecular targets for novel therapies. These aims have not yet been fully reached by analysing single genes. However, some genes, including E-cadherin that is altered in sporadic and familial cases of gastric cancer and interleukin 1-beta whose polymorphisms together with Helicobacter pylori infection are associated with an increased risk for gastric cancer, may have the potential for clinical use. In this review we summarize current data for the single gene approach and provide an overview for recent results from cDNA microarray studies.

Topics: Adenocarcinoma; Barrett Esophagus; Cadherins; Cyclin D1; Disease Progression; Esophageal Neoplasms; Humans; Mutation; Stomach Neoplasms

To evaluate the prognostic significance of gene amplification and overexpression of cyclin D1 in the patients of esophageal squamous cancer, slot blot hybridization and immunohistochemical staining were performed. The patients with gene amplification or overexpression of cyclin D1 were significantly poorly prognosis than patients these were negative. And to investigate abnormality of p16 and p15 genes in 12 squamous cell carcinoma of esophagus, PCR and SSCP analysis were performed. In only one of 12 tumors, complete deletion of p16 and p15 genes was detected. But in other 11 tumors, no abnormality could be detected. Besides to evaluate the prognostic significance of expression of p16 in patients of esophageal squamous cancer, immunohistochemical staining for p16 was performed. The patients with overexpression of p16 were significantly better prognosis than patients that was negative, and this result was opposite contrast with the result of cyclin D1.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Esophageal Neoplasms; Gene Amplification; Gene Deletion; Gene Expression; Humans; Oncogene Proteins; Prognosis

Topics: Cell Transformation, Neoplastic; Cyclin D1; Cyclins; Esophageal Neoplasms; Genes, Tumor Suppressor; Humans; Oncogene Proteins; Oncogenes; Proto-Oncogenes; Signal Transduction

Pin1 promotes oncogenesis by regulating multiple oncogenic signaling. In this study, we investigated the involvement of Pin1 in tumor progression and in the prognosis of human esophageal squamous cell carcinoma (ESCC).. We observed that proliferation, clonogenicity and tumorigenesis of CE81T cells were inhibited by Pin1 knockdown. We next analyzed Pin1 expression in clinical ESCC specimens. When compared to the corresponding non-tumor part, Pin1 protein and mRNA levels in tumor part were higher in 84% and 62% patients, respectively. By immunohistochemistry, we identified that high Pin1 expression was associated with higher primary tumor stage (p = 0.035), higher overall cancer stage (p = 0.047) and poor overall survival (p < 0.001). Furthermore, the association between expression of Pin1 and levels of β-catenin and cyclin D in cell line and clinical specimens was evaluated. β-catenin and cyclin D1 were decreased in CE81T cells with Pin1 knockdown. Cyclin D1 level correlated with Pin1 expression in clinical ESCC specimens.. Pin1 upregulation was associated with advanced stage and poor prognosis of ESCC. Pin1 knockdown inhibited aggressiveness of ESCC cells. β-catenin and cyclin D1 were positively regulated by Pin1. These results indicated that targeting Pin1 pathway could represent a potential modality for treating ESCC.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease-Free Survival; Esophageal Neoplasms; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Retrospective Studies; Survival Rate; Up-Regulation

Esophageal cancer is one of the leading causes of cancer death and the seventh most prevalent cancer worldwide. Considering the positive association of high selenium with the prevalence of esophageal cancer, we have investigated the effect of high doses of selenium on gene expression in the normal esophageal tissue of rats. Twenty male rats were randomly divided into four groups: control group, group 2 mg Se/L, 10 mg Se/L, and 20 mg Se/L rats fed with a basal basic diet and 2, 10, and 20 mg Se/L as sodium selenite in drinking water, respectively, for 20 weeks. Serum malondialdehyde and glutathione peroxidase activity were measured. Moreover, the expression and concentration of the cyclin D1, cyclin E, KRAS, p53, NF-kB, TGF-β, and MGMT in the esophageal tissue were analyzed and compared between the four groups. In normal esophageal tissue, selenium supplementations (2, 10, and 20 mg Se/L) increased the mRNA levels of cyclin D1, P53, KRAS, NF-κB p65, and MGMT and decreased the mRNA level of TGFß1. The concentrations of cyclin D1 and MGMT were also significantly increased by selenium supplementations. Selenium supplementations had no significant effect on serum MDA but significantly increased GPX activity. The present study suggests that selenium supplementation (2, 10, and 20 mg Se/L) affects gene expression related to inflammation, Cell proliferation, and apoptosis in the normal esophageal tissue. However, there were no observed abnormalities other than reduced growth with supplementation of 20 mg/L as Na2SeO3 in rats.

Topics: Animals; Cyclin D1; Dietary Supplements; Esophageal Neoplasms; Gene Expression; Glutathione Peroxidase; Male; Proto-Oncogene Proteins p21(ras); Rats; RNA, Messenger; Selenium; Tumor Suppressor Protein p53

We explored the mechanism by which miR-139 modulates radioresistance of esophageal cancer (EC). The radioresistant cell line KYSE150R was obtained from the parental KYSE150 cell line by fractionated irradiation (15×2 Gy; total dose of 30 Gy). The cell cycle was assessed by flow cytometry. A gene profiling study was conducted to detect the expression of genes related to the radioresistance of EC. In the KYSE150R line, flow cytometry revealed increased number of G1-phase cells and decreased number of G2-phase cells; the expression of miR-139 increased. Knockdown of miR-139 decreased radioresistance and changed the distribution of cell cycle phases in KYSE150R cells. Western blotting showed that miR-139 knockdown increased the expression levels of cyclin D1, p-AKT, and PDK1. However, PDK1 inhibitor GSK2334470 reversed this effect for p-AKT and cyclin D1 expression. A luciferase reporter assay indicated that miR-139 directly bound to the PDK1 mRNA 3'-UTR. Analysis of the clinical data from 110 patients with EC showed an association of miR-139 expression with the TNM stage and the effect of therapy. MiR-139 expression significantly correlated with EC and progression-free survival. In conclusion, miR-139 enhances the radiosensitivity of EC by regulating the cell cycle through the PDK1/Akt/Cyclin D1 signaling pathway.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Signal Transduction

The goal was to investigate the inhibitory effect of formononetin, an active component in Astragalus membranaceus, on the pathogenesis and development of esophageal cancer and the mechanism of action.. The expression of COX-2 in cancer tissue and paracancerous tissue of patients with esophageal cancer detected early. C57BL/6 mice were used to construct a 4-nitroquinoline 1-oxide (4-NQO)-induced esophageal cancer model to verify the inhibitory effect of formononetin on the pathogenesis of esophageal cancer. Additionally, human esophageal cancer cells were treated with formononetin, and the effects on the proliferation and cell cycle of esophageal cancer cells were assessed by the CCK-8 assay and flow cytometry. Changes in the expression levels of cyclin D1 and COX-2 mRNA in cells were detected by RT-qPCR and western blot (WB) analysis.. The expression level of COX-2 mRNA in esophageal cancer tissue was significantly higher than that in paracancerous tissue. In the mouse cancer model, the incidence of esophageal cancer in mice in the formononetin treatment group was significantly reduced at week 18 (0/15 vs. 2/15) and at week 24 (6/15 vs. 13/15) (all p < 0.05). Formononetin significantly inhibited the proliferation ability of KYSE170 and KYSE150 cells and inhibited the protein expression of COX-2 and cyclin D1 (both p < 0.05).. Formononetin, an active component of Astragalus membranaceus, can prevent the pathogenesis and progression of esophageal cancer by reducing the expression of the inflammatory proteins COX-2 and cyclin D1.

Topics: Animals; Astragalus propinquus; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Esophageal Neoplasms; Humans; Mice; Mice, Inbred C57BL

Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer, with 5-year survival rate less than 30%. In order to offer an individual therapeutic approach, it is necessary to identify novel prognostic factors to recognize high-risk patients. Given the high frequency of CCND1 abnormalities and the important biological effects of smoking in ESCC, we explored the potential relationship between CCND1 abnormalities and smoking in ESCC patients. CCND1 status was examined by fluorescence in situ hybridization and immunohistochemical staining in ESCC tissue microarrays (n = 519). CCND1 amplification and cyclinD1 overexpression were found in 53.2 and 34.1% ESCC, respectively. CCND1 amplification (P = 0.142 for DFS and P = 0.191 for OS) and cyclinD1 overexpression (P = 0.035 for DFS and P = 0.092 for OS) tended to be poorer prognostic factors in all patients. Among smoking patients, those with CCND1 amplification had significantly poorer prognosis, with a median DFS and OS of 25.0 and 30.0 months compared to not reached and 52.0 months for those without CCND1 amplification (P = 0.020 and 0.018). A similar trend was found in the 68 patients with cyclinD1 overexpression (P = 0.043 and 0.048). Further univariate and multivariate analysis revealed CCND1 amplification was independently poorer prognostic factor in smoking patients, which was not found in non-smoking patients. Smokers with CCND1 amplification or cyclinD1 overexpression have poorer survival, which help us to identify distinct groups of patients with apparently poorer outcome and would enable appropriate follow-up and treatment strategies.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; In Situ Hybridization, Fluorescence; Prognosis; Smoking

Esophageal cancer is one of the most common malignant tumors of the digestive system, with high incidence and mortality.. Immunohistochemical method was used to detect the expression of MACC1, c-Met, and cyclin D1 in ESCC and its adjacent tissues. Statistical analysis was done by SPSS 23.0.. The high expression of MACC1 and cyclin D1 was significantly correlated with tumor size. High c-Met expression was associated with patient ethnicity. MACC1 expression was positively correlated with both c-Met and cyclin D1. c-Met expression was also positively correlated with cyclin D1. Patients with high expression of MACC1 and c-Met had worse OS; patients with high c-Met expression also had worse PFS.. MACC1, c-Met, and cyclin D1 proteins are closely related to the occurrence and development of esophageal squamous cell carcinoma. MACC1 may affect the prognosis of ESCC by regulating the expression of the c-Met/cyclin D1 axis.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; Proto-Oncogene Proteins c-met; Trans-Activators

Myosin-related proteins play an important role in cancer progression. However, the clinical significance, biological functions, and mechanisms of myosin 1B (MYO1B), in esophageal squamous cell carcinoma (ESCC) remain unclear. The clinical relevance of MYO1B, SNAI2, and cyclin D1 in ESCC was determined by immunohistochemistry, Oncomine, and GEPIA databases. The oncogenic roles of MYO1B were determined by CCK8, colony formation assays, wound healing, and Transwell assay. MYO1B, SNAI2, and cyclin D1 at mRNA and protein levels in ESCC cells were detected by qPCR and Western blot analysis. In our study, we found that MYO1B expression was increased in ESCC tissue samples and correlated with tumor stage, TNM stage, and poor outcomes. Functional assays indicated that depletion of MYO1B impaired oncogenesis, and enhanced chemosensitivity in ESCC. Bioinformatic analysis and mechanistic studies illustrated that SNAI2 was a key downstream effector of MYO1B. Suppression of MYO1B downregulated expression of SNAI2, thereby inhibiting the SNAI2/cyclin D1 pathway. Furthermore, a selective inhibitor of cyclin D1 activation reversed siMYO1B cells overexpressing SNAI2-elicited aggressive phenotypes of ESCC cells. MYO1B positively correlated with SNAI2 and cyclin D1 in ESCC samples, and higher SNAI2 expression was also associated with poor prognosis in ESCC patients. Our finding demonstrated that MYO1B activates the SNAI2/cyclin D1 pathway to drive tumorigenesis and cisplatin cytotoxicity in ESCC, indicating that MYO1B is a potential therapeutic target for patients with ESCC.

Topics: Carcinogenesis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; Myosin Type I; Myosins; Snail Family Transcription Factors

As a neuronal transmembrane protein, leucine-rich repeat and fibronectin type-III domain-containing protein 2 (LRFN2) can recruit and combine with N-methyl-d-aspartate receptors (NMDARs) to promote nerve growth. Genetic studies suggest that mutations in LRFN2 are associated with various cancers. However, the role and mechanism of LRFN2 in the progression of ESCC have not been elucidated. In this study, we demonstrated that LRFN2 was significantly downregulated in ESCC tissues by qRT-PCR and immunohistochemistry. Low LRFN2 expression was an adverse prognostic factor in patients with ESCC. Overexpression of LRFN2 effectively suppressed the proliferation, migration, invasion, and epithelial-to-mesenchymal transition in vitro and tumor growth in vivo. Bioinformatics analysis indicated that Wnt/β-catenin signaling regulation was one of the most potential mechanisms and studies confirmed that overexpression of LFRN2 obviously downregulated the expression of β-catenin, c-Myc, and cyclin D1 in ESCC cells and tumor tissues. Further studies revealed that LRFN2 plays an anti-ESCC role by binding with NMDAR-GRIN2B and this effect can be weakened by NR2B-selective NMDA antagonist-NMDA-IN-1. Moreover, the bioinformatics analysis showed that the interaction of GRIN2B and GSK3β affects the NF-κB pathway, which was demonstrated by western blot experiments. Collectively, our results indicate that LRFN2 binding to NMDARs inhibits the progression of ESCC by regulating the Wnt/β-catenin and NF-κB pathway, which provides a new therapeutic target for improving the prognosis of patients with ESCC.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Fibronectins; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Membrane Glycoproteins; N-Methylaspartate; Nerve Tissue Proteins; NF-kappa B; Receptors, N-Methyl-D-Aspartate; Wnt Signaling Pathway

Bromodomain-containing protein 4 (BRD4) binds acetylated lysine residues on histones to facilitate the epigenetic regulation of many genes, and it plays a key role in many cancer types. Despite many prior reports that have explored the importance of BRD4 in oncogenesis and the regulation of epigenetic memory, its role in esophageal squamous cell carcinoma (ESCC) progression is poorly understood. Here, we investigated. BRD4 expression in ESCC tissues was measured

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Male; Middle Aged; Proto-Oncogene Proteins c-myc; Transcription Factors

Topics: Animals; Apoptosis; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; RNA, Long Noncoding; Transcriptional Activation; Up-Regulation; Xenograft Model Antitumor Assays

More than 40 000 patients worldwide die from esophageal cancer annually. The 5-year survival rate of patients is only ~ 15-20%, and thus, there is an ongoing need to improve diagnosis and treatment of esophageal cancer. Breast cancer type 1 susceptibility protein (BRCA1)-associated protein (BAP1) is a marker of poor prognosis in several cancers, including uveal melanoma, renal cell carcinoma, cholangiocarcinoma, non-small cell lung cancer, and colorectal cancer. BAP1 mutations are early and rare events in esophageal carcinoma, but the involvement of BAP1 in progression of esophageal carcinoma is unclear. Here, we report that cell proliferation and migration were significantly enhanced in esophageal carcinoma ECA109 cells overexpressing BAP1, while they were diminished upon BAP1 knockdown. In addition, the expression of Krüppel-like factor 5 (KLF5), CyclinD1, and FGF-BP1 was increased by BAP1 overexpression and decreased by BAP1 knockdown. Our data suggest that BAP1 promotes cell proliferation and migration, and enhances the expression of KLF5 and its downstream genes, including CyclinD1 and FGF-BP1, in the esophageal carcinoma cell line ECA109.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Kruppel-Like Transcription Factors; Prognosis; Transcription Factors; Tumor Suppressor Proteins; Ubiquitin Thiolesterase

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

Topics: Cell Cycle; Cyclin D1; Cycloheximide; DNA Damage; Down-Regulation; Epithelial Cells; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagus; F-Box Proteins; Gamma Rays; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteolysis; Tumor Suppressor Protein p53

Radiation therapy is a widely used treatment for esophageal cancer. However, radiation resistance might result in a poor prognosis. Overexpression of HER2 has been related to adaptive radiation resistance. Pyrotinib is a HER2 inhibitor that shows an anti-tumor effect in breast cancer. The present study aims to explore the influence of pyrotinib combined with radiotherapy on HER2-positive esophageal cancer cells and explore the underlying mechanism. We screened two cell lines (TE-1 and KYSE30) that highly express HER2 from several human esophageal cancer cell lines. Cells were treated with pyrotinib or/and radiation. Cell proliferation, cell cycle distribution, and cell migration were measured. The protein levels involved in cell cycle and DNA repair were measured by Western blot. Results showed that pyrotinib inhibited HER2 activation and exerted an anti-proliferative effect in TE-1 and KYSE30 cells. Furthermore, it enhanced the anti-proliferative effect of radiation in these two cell lines. These effects might be via inhibiting HER2 phosphorylation, inducing G0/G1 arrest, and reducing EMT and DNA repair. Our results indicated that pyrotinib sensitivitied HER2 positive esophageal cancer cells to radiation treatment through various mechanisms. These findings may provide a new therapeutic strategy for treating HER2 positive esophageal cancer.

Topics: Acrylamides; Aminoquinolines; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; DNA Repair; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Humans; Phosphorylation; Radiation Tolerance; Radiation-Sensitizing Agents; Receptor, ErbB-2; Signal Transduction

Isoliquiritigenin (ILQ) is a natural product isolated from licorice root which has served as traditional Chinese medicine for a long time. Recently, the antitumor effects of ILQ have been widely studied in various cancers, but the role and related mechanisms of ILQ in esophageal squamous carcinoma cells (ESCC) are still poorly understood. In our studies, ILQ showed profound antitumor activities in ESCC cells. In vitro, ILQ substantially inhibited cell proliferation and anchorage-independent growth in a panel of human ESCC cells. Mechanism studies showed that EGFR signaling pathway played an important role for ILQ to exert its antitumor activity in ESCC. Exposure to isoliquiritigenin substantially decreased EGF-induced EGFR activation and its downstream Akt and ERK1/2 signaling pathway. EGFR knockdown with shRNA in ESCC cell significantly reduced the sensitivity of cancer cells to ILQ. Moreover, it was found that ILQ had a significantly inhibitory effect on AP-1 family, the protein of Jun and Fos subfamilies was substantially downregulated, and the transcriptional activity of AP-1 family was dramatically suppressed by ILQ. By reducing the expression of cyclin D1, ESCC cells were induced G0/G1 arrest, and cell division was substantially blocked. Finally, the antitumor potency of ILQ was validated in xenograft models and the tumor growth was prominently restrained by ILQ. Briefly, our study showed that ILQ, or its analogue, appeared to be a promising new therapeutic agent for ESCC management.

Topics: Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chalcones; Cyclin D1; Down-Regulation; ErbB Receptors; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; G1 Phase; Gene Knockdown Techniques; Glycyrrhiza; Humans; Mice; Mice, Inbred BALB C; Plant Roots; Protein Kinase Inhibitors; Signal Transduction; Xenograft Model Antitumor Assays

The poor prognosis of esophagus cancer (EC) is mainly due to its high invasiveness and metastasis, so it is urgent to search effectively prognostic markers and explore their roles in the mechanism of metastasis.. Based on the TCGA database, we downloaded the RNA-Seq for analyzing the expression of ATP6V0D2. QRT-PCR was used to test the mRNA levels of ATP6V0D2 in cell lines. Chi-square tests were used to evaluate the correlation between ATP6V0D2 and clinical characteristics. Prognostic values were determined by Kaplan-Meier methods and cox's regression models. CCK-8 and clone formation assays were employed to evaluate the cell viability, and Transwell assay was implemented to determine the invasive and migratory abilities. Correlations between ATP6V0D2 and motion-related markers were analyzed by the GEPIA database and confirmed by western blot. Moreover, the relationship between ATP6V0D2 and molecules related to cell cycle and apoptosis was also determined by western blot.. A significant increase was observed in 3 EC-related cell lines compared to the normal cell line. ATP6V0D2 has a connection with the poor prognosis and can be considered as an independent prognosticator for patients with EC. Besides, ATP6V0D2 can improve cells viability as well as invasive and migratory abilities. What's more, downregulation of ATP6V0D2 notably enhanced E-cadherin expression, while decreased N-cadherin, Vimentin, and MMP9 expression, whereas overexpression of ATP6V0D2 presented the opposite outcomes. Furthermore, we found that silencing ATP6V0D2 led to a significant reduction on the protein expression of Cyclin D1, CDK4, Bcl-2, whereas resulted in a notable enhancement on the Bax level.. ATP6V0D2 might be an independent prognosticator for EC patients, and it possibly promotes tumorigenesis by regulating epithelial-mesenchymal transition, cell cycle and apoptosis-related markers, providing the possibility that ATP6V0D2 may be a novel biomarker for the therapeutic intervention of EC.

Topics: Apoptosis; Biomarkers, Tumor; Cadherins; Carcinogenesis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Female; Genes, bcl-2; Humans; Male; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Protons; RNA, Messenger; Sincalide; Vacuolar Proton-Translocating ATPases; Vimentin

Although radiotherapy is an important treatment mode for esophageal cancer (EC), the outcome remains unsatisfactory due to radioresistance, and the key cause of radiotherapy resistance is a change in the cell cycle. Zinc deficiency (ZD) has a significant influence on the cell cycle, and this effect is a common phenomenon in areas with a high incidence of esophageal cancer.. Radioresistant sub-cell lines were established by exposing esophageal cancer cells to nine rounds of X-ray irradiation at a dose of 2 Gy. The cells were treated with a range of different concentrations of zinc and overexpression of miR-193b. And proliferation, colony formation, cell cycle and apoptosis assays were then conducted. Luciferase reporter assays were performed to confirm direct interactions between miR-193b and ZRT/IRT-like protein 5 (ZIP5) and between miR-193b and Cyclin D1. Analysis of clinical and follow-up data was performed using data obtained from 75 patients from Cixian, a well-known high incidence area of esophageal cancer. All these patients are unable to tolerate surgery due to their advanced age or advanced stage, and serum specimens were obtained before the patients received therapy.. The cell cycle of radioresistant cells is blocked in G0/G1 phase (from 50% to 68%). The expression level of Cyclin D1 was decreased and miR-193b was increased in radioresistant cells (P < 0.001). ZD decreased the proportion of cells in G0/G1 phase both in EC (from 50% to 32%) and radioresistant (from 68% to 47%) cells. And the radioresistance of these two cells were decreased. ZD increased the expression of Cyclin D1 (P < 0.001) and inhibited the level of miR-193b (P < 0.001). Up-regulation of miR-193b recovered the proportion of cells in G0/G1 phase and the radioresistance, meanwhile, recovered the altered expression levels of Cyclin D1 and miR-193b of these two cells by ZD. ZD enhanced DNMT activity both in EC (32%) and radioresistant (26%). And miR-193b was hypermethylated both in EC and radioresistant cells. MiR-193b supp ressed Cyclin D1 expression by targeting the 3'UTR of Cyclin D1 mRNA. The expression level of miR-193b in the serum of patients was correlated with the disease control rate (DCR) and had a good diagnostic value for distinguishing DCR of EC patients (AUC = 0.710, 95%CI: 0.580-0.839, P = 0.003). And the level of miR-193b was correlated with overall survival (OS) (HR = 0.208, 95%CI: 0.094-0.464).. The methylation-mediated silencing of miR-193b in EC cells due to ZD increased the expression of ZIP5, and the overexpression of ZIP5 increased the intracellular zinc levels to maintain zinc homeostasis. Meanwhile, the silencing of miR-193b increased the sensitivity of radiotherapy by promoting the expression of Cyclin D1.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Methylation; MicroRNAs; Zinc

Compound Kushen injection (CKI), a medicine in widespread clinical use in China, has proven therapeutic effects on cancer. However, few molecular mechanism analyses have been carried out. To address this problem, bioinformatics approaches combining weighted gene co-expression network analysis with network pharmacology methods were undertaken to elucidate the underlying molecular mechanisms of CKI in the treatment of esophageal cancer (ESCA). First, the key gene modules related to the clinical traits of ESCA were analysed by WCGNA. Based on the results, the hub genes related to CKI treatment for ESCA were explored through network pharmacology. Molecular docking simulation was performed to recognize the binding activity of hub genes with CKI compounds. The results showed that the potential hub targets, including EGFR, ErbB2, CCND1 and IGF1R, are therapeutic targets of CKI for the treatment of ESCA. Moreover, these targets were significantly enriched in many pathways related to cancer and signalling pathways, such as the PI3K-Akt signalling pathway and ErbB signalling pathway. In conclusion, this research partially highlighted the molecular mechanism of CKI in the treatment of ESCA, offering great potential in the identification of the effective compounds in CKI and biomarkers for ESCA treatment.

Topics: Algorithms; Antineoplastic Agents; Computational Biology; Cyclin D1; Databases, Genetic; Drugs, Chinese Herbal; ErbB Receptors; Esophageal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kaplan-Meier Estimate; Models, Molecular; Molecular Docking Simulation; Receptor, ErbB-2; Receptor, IGF Type 1; Sequence Analysis, RNA

Epithelial ovarian carcinoma (EOC) is the most common cause of gynecological cancer mortality, and poses a threat to women. MicroRNA‑195 (miR‑195) has been reported to induce apoptosis of human OVCAR‑3 cells by inhibiting the VEGFR2/AKT pathway. However, the role of miR‑195 in EOC remains unknown. A previous study reported that cell division cycle 42 (CDC42) can serve as a target gene of miR‑195 and mediate malignant progression of esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the role of miR‑195 in EOC and the regulation in CDC42/CCND1 pathway. Tissues samples and clinical materials were collected from 78 enrolled patients with EOC to analyze the expression and clinical significance of miR‑195, CDC42 and cyclin D1 (CCND1). Human EOC cell lines OVCA420, OVCAR‑3, A2780 and SKOV3 cell lines were used to assess the expression and function of miR‑195, CDC42 and CCND1 in vitro. Cell proliferation, the cell cycle and apoptosis, as well as the cell migratory and invasive abilities were detected in vitro using BrdU incorporation, colony formation, wound healing and Transwell invasion assays, along with flow cytometry. miR‑195 was downregulated, while CDC42 and CCND1 were upregulated in human EOC tissues and cells, and the aberrant expression of both was associated with increased EOC malignancy. Moreover, miR‑195 expression was negatively correlated with CDC42 and CCND1 expression levels, and negatively regulated these expression levels. Thus, it was suggested that miR‑195 functions as a tumor suppressor, but CDC42 and CCND1 act as tumor promoters based their abilities to enhance cell proliferation, cell cycle entry, migration and invasion, as well as decrease apoptosis in OVCAR‑3 cells. the present results demonstrated that miR‑195 inhibited human EOC progression by downregulating CDC42 and CCND1 expression. Furthermore, it was identified that miR‑195, CDC42 and CCND1 may be effective biomarkers for EOC diagnosis and treatment.

Topics: Adult; Apoptosis; Carcinoma, Ovarian Epithelial; cdc42 GTP-Binding Protein; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; MicroRNAs; Middle Aged; Ovarian Neoplasms

Esophageal cancer (EC) recently has become a common malignancy of digestive system worldwide. RAB11A is a critical member of the small GTPases superfamily and was reported to affect a variety of cellular functions. However, its potential effects on EC progression and the specific regulatory mechanisms are still unclear. In this study, RAB11A was upregulated in human EC tissues and cells and predicted poor diagnosis. RAB11A expression was correlated with clinical-pathological features including pTNM stage (P=0.001*) and recurrence (P=0.000**) in patients with EC. Furthermore, RAB11A knockdown decreased the proliferation, migration, and invasion of EC cells via WNT pathway in vitro. Subsequently, the in vivo experiments confirmed that RAB11A contributed to EC tumor growth via WNT pathway. Therefore, these results provided evidence showing that RAB11A could promote the progression of EC via WNT pathway and might serve as a promising therapeutic target for EC treatment.

Topics: Aged; Animals; beta Catenin; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Ki-67 Antigen; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; rab GTP-Binding Proteins; RNA, Small Interfering; Survival Analysis; Tumor Burden; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

PDRG1, a short p53 and DNA damage-regulated gene, is overexpressed in many human tumors. This gene can promote proliferation and inhibit apoptosis in lung cancer. The aim of this study is to evaluate the effects of the PDRG1 gene on the Wnt signaling pathway in esophageal cancer cells.. In this study, 72 esophageal cancer tissues and adjacent normal tissues were collected, and the level of PDRG1 expression was assessed by quantitative real-time PCR (qRT-PCR). PDRG1 over-expression and knockdown cell lines (PDRG1o and PDRG1i) were established to analyze the effect of PDRG1 on esophageal cancer cells.. The expression level of PDRG1 in esophageal cancer gradually increased with advanced TNM grades. PDRG1 knockdown can potentially promote apoptosis and inhibit the proliferation of esophageal cancer cells. Meanwhile, cisplatin chemosensitivity was enhanced by PDRG1 knockdown, via the suppression of cyclinD1 and β-catenin expression within esophageal cancer cells. This indicates that PDRG1 silencing could inhibit the Wnt signaling pathway in esophageal cancer cells.. Our data provides convincing evidence that PDRG1 may promote proliferation while inhibiting apoptosis and chemotherapy sensitivity in esophageal cancer cells.

Topics: Adult; Aged; Antineoplastic Agents; Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; DNA-Binding Proteins; Drug Resistance, Neoplasm; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Wnt Signaling Pathway

Topics: Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Humans; Receptor, PAR-2

Esophageal cancer (EC) is one of the leading causes of death among malignancies. Radiotherapy for esophageal squamous cell carcinoma (ESCC) patients is limited by resistance to ionizing radiation (IR). An increasing body of evidence has demonstrated that aberrant expression of microRNA‑301a (miR‑301a) contributes to cancer progression and sensitivity to radiation. The aim of the present study was to investigate the exact functions and potential mechanisms of miR‑301a in ESCC radioresistance. Initially, the miR‑301a‑transfected radioresistant ESCC cells KYSE‑150R exhibited a decreased proliferation rate, and enhanced radiosensitivity and migration, whereas downregulation of miR‑301a in radiosensitive KYSE‑150 cells produced the opposite results. miR‑301a regulates WNT1 expression at both the mRNA and protein levels. Furthermore, dual‑luciferase reporter assays revealed that WNT1 was a target gene of miR‑301a. In addition, the expression of miR‑301a markedly affected the expression of Wnt/β‑catenin‑related proteins such as β‑catenin and cyclin D1. Finally, overexpression of miR‑301a inhibited epithelial‑mesenchymal transition (EMT) conversion by directly targeting Snail and vimentin in radioresistant‑ESCC cell lines; however, no inhibitory effects were exerted on Twist. Collectively, these results indicated that miR‑301a increased the radiosensitivity and inhibited the migration of radioresistant‑ESCC cells by targeting WNT1, thereby inactivating the Wnt/β‑catenin signaling pathway and EMT reversal. Thus, miR‑301a may be a potential therapeutic target for the treatment of EC radioresistance.

Topics: beta Catenin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Humans; MicroRNAs; Radiation Tolerance; Signal Transduction; Wnt1 Protein

An altered Wnt-signaling activation has been reported during Barrett's esophagus progression, but with rarely detected mutations in APC and β-catenin (CTNNB1) genes.. In this study, a robust in-depth expression pattern analysis of frizzled receptors, co-receptors, the Wnt-ligands Wnt3a and Wnt5a, the Wnt-signaling downstream targets Axin2, and CyclinD1, as well as the activation of the intracellular signaling kinases Akt and GSK3β was performed in an in vitro cell culture model of Barrett's esophagus. Representing the Barrett's sequence, we used normal esophageal squamous epithelium (EPC-1, EPC-2), metaplasia (CP-A) and dysplasia (CP-B) to esophageal adenocarcinoma (EAC) cell lines (OE33, OE19) and primary specimens of squamous epithelium, metaplasia and EAC.. A loss of Wnt3a expression was observed beginning from the metaplastic cell line CP-A towards dysplasia (CP-B) and EAC (OE33 and OE19), confirmed by a lower staining index of WNT3A in Barrett's metaplasia and EAC, than in squamous epithelium specimens. Frizzled 1-10 expression analysis revealed a distinct expression pattern, showing the highest expression for Fzd2, Fzd3, Fzd4, Fzd5, Fzd7, and the co-receptor LRP5/6 in EAC cells, while Fzd3 and Fzd7 were rarely expressed in primary specimens from squamous epithelium.. Despite the absence of an in-depth characterization of Wnt-signaling-associated receptors in Barrett's esophagus, by showing variations of the Fzd- and co-receptor profiles, we provide evidence to have a significant role during Barrett's progression and the underlying pathological mechanisms.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Apoptosis Regulatory Proteins; Axin Protein; Barrett Esophagus; beta Catenin; Cell Line; Cyclin D1; Disease Progression; Esophageal Neoplasms; Frizzled Receptors; Gene Expression; Humans; Low Density Lipoprotein Receptor-Related Protein-5; Low Density Lipoprotein Receptor-Related Protein-6; Repressor Proteins; Wnt Signaling Pathway; Wnt-5a Protein; Wnt3A Protein

The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.

Topics: Animals; Antineoplastic Agents; Benzeneacetamides; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Drug Synergism; Energy Metabolism; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; F-Box Proteins; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Humans; Hypoglycemic Agents; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Molecular Targeted Therapy; Phenformin; Protein Kinase Inhibitors; Retinoblastoma Protein; Signal Transduction; Thiadiazoles; Xenograft Model Antitumor Assays

Esophageal cancer is one of the most common cancers around the world, and it has high incidence and mortality rates. The conventional therapy for esophageal cancer is radiotherapy, although its effect is highly limited by the resistance of esophageal cancer cells. Thus, strong radiosensitizers can be very crucial during radiotherapy against esophageal cancer.. To evaluate the anti-cancer effect and mechanism of BJOE and explore the potential use of BJOE as a radiosensitizer during radiotherapy.. The inhibitory effect of BJOE and its enhancement function with radiation on cell viability were examined with the calculated half-maximal effective concentration and half-maximal lethal concentration. The influence of BJOE on cell migration and invasion were measured with EC109 and JAR cells by wound-healing and transwell assay. Clonogenesis and apoptotic rate, which was measured by Hoechst staining, were investigated to confirm its enhancement function with radiation. To investigate the molecular pathway underlying the effect of BJOE, the expressions of several apoptosis- and cycle-related proteins was detected by western blotting.. Our results demonstrated that BJOE inhibited the growth of esophageal cancer cell lines more than normal cell lines, and it markedly reduced migration and invasion in esophageal cancer cells (EC109 and JAR). Moreover, it promoted cell apoptosis and enhanced the effect of radiotherapy against esophageal cancerous cells. In the viability test, the values of half-maximal effective concentration and half-maximal lethal concentration were reduced. Compared to the control, only around 1/5 colonies formed when using BJOE and radiation together in the clonogenic assay. The apoptotic rate in EC109 was obviously promoted when BJOE was added during radiotherapy. Our study suggests that the expression of the apoptosis-proteins Bax and p21 were increased, while the expression of Bcl-2 was stable. Further detection of downstream proteins revealed that the expression of cyclin D1 and cyclin-dependent kinase 4/6 were significantly decreased.

Topics: Apoptosis; Brucea; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chemoradiotherapy; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Screening Assays, Antitumor; Emulsions; Esophageal Neoplasms; Humans; Neoplasm Invasiveness; Plant Oils; Radiation-Sensitizing Agents; Signal Transduction

α-Hederin has been shown promising anti-tumor potential against various cancer cell lines. However, reports about effects of α-hederin on esophageal squamous cell carcinoma (ESCC) are still unavailable.. To investigate the inhibitory effects of α-hederin on ESCC and explore the underlying mechanism.. Human esophageal carcinoma cell line (Eca-109) was used for the experiment. Cell Counting Kit-8, flow cytometry, Hoechst 33258 staining, enhanced ATP assay kit, 2',7'-dichlorofluorescin diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, cycle, apoptosis, cellular ATP content, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. Xenografted tumor model was constructed to evaluate the in vivo anti-tumor effects of α-hederin.. Compared with control group, α-hederin significantly inhibited the proliferation, induced apoptosis of ESCC, and arrested the cell cycle in G1 phase (P < 0.05). α-Hederin induced the accumulation of ROS, decrement of ATP levels, and disruption of MMP (P < 0.05). The detection of mitochondrial and cytosol proteins showed that AIF, Apaf-1, and Cyt C were released and increased in cytoplasm, and then, caspase-3, caspase-9, and Bax were involved and increased, while Bcl-2 level was decreased (P < 0.05). Furthermore, the above changes were amplified in the group pretreated with L-buthionine sulfoximine, while N-acetyl-L-cysteine plays an opposite role (P < 0.05). Meanwhile, α-hederin significantly inhibited the growth of xenografted tumors with favorable safety.. α-Hederin could inhibit the proliferation and induce apoptosis of ESCC via dissipation of the MMP with simultaneous ROS generation and activation of the mitochondrial pathway.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Flow Cytometry; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Mice, Nude; Mitochondria; Neoplasm Transplantation; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Saponins; Xenograft Model Antitumor Assays

Aberrant microRNA-449a (miR-449a-5p) expression has been demonstrated to be associated with the development of various cancer types. However, the effect of miR‑449a‑5p on esophageal squamous cell carcinoma (ESCC) cell proliferation remains unknown. The present study aimed to determine whether miR‑449a‑5p may regulate ESCC cell proliferation via negative regulation of cyclin D1. Reverse transcription quantitative‑polymerase chain reaction was used to measure the expression of miR‑449a‑5p in ESCC tissues and cells. Western blot was performed to analyze the protein level of cyclin D1. The proliferation of ESCC cells was determined by MTT and clone formation assay. Paired ESCC and adjacent normal esophageal squamous tissues were collected from patients with ESCC. It was demonstrated that miR‑449a‑5p expression was reduced, whereas cyclin D1 expression was increased in ESCC tissues compared with adjacent normal tissues. Proliferation was investigated in vivo using the ESCC cell line Eca‑190. miR‑449a‑5p inhibitor transfection facilitated the proliferation of Eca‑109 cells. By contrast, transfection with miR‑449a‑5p mimics inhibited Eca‑109 cell proliferation. Furthermore, it was confirmed that miR‑449a‑5p directly bound to the 3'‑untranslated region of cyclin D1. Transfection with cyclin D1 small interfering RNA reversed the effects of the miR‑449a‑5p inhibitor on Eca‑109 cell proliferation. In conclusion, miR‑449a‑5p may control ESCC proliferation through the negative regulation of cyclin D1 expression.

Topics: Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; RNA, Neoplasm

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is closely related to the occurrence and development of malignant tumors. This study discusses the expression of LETM1 in esophageal squamous cell carcinoma (ESCC) and its association with cancer stem-like cells (CSC). We used immunohistochemistry in 166 ESCC tissue samples, as well as Western blot and immunofluorescent methods in ESCC cell lines, to study the role of LETM1 and its association with CSC in ESCC. The expression of LETM1 was significantly higher in ESCC, and it was closely related to the primary tumor stage and clinical stage. LETM1 expression was significantly associated with lower overall survival and disease-free survival. In addition, the protein expression of LETM1 and CSC markers was higher in TE11 and ECG10 than in other ESCC cell lines. Moreover, the expression of LETM1 positively correlated with LSD1, CD44, and OCT4. Immunofluorescence revealed that LETM1 costained with CD44 and OCT4 in ECG10. The expression of LETM1 was associated with not only HIF-1α but also higher microvessel density and tumor-associated macrophage infiltration. Furthermore, LETM1 significantly correlated with cyclinD1 and pAkt. High expression of LETM1 indicates poor prognosis and may be a potential CSC marker in ESCC. Moreover, LETM1 may be a novel therapeutic target for the treatment of ESCC.

Topics: Aged; Calcium-Binding Proteins; Cell Line, Tumor; Cyclin D1; Disease-Free Survival; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Histone Demethylases; Humans; Hyaluronan Receptors; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Membrane Proteins; Middle Aged; Neoplasm Staging; Neoplastic Stem Cells; Neovascularization, Pathologic; Octamer Transcription Factor-3; Phenotype; Proto-Oncogene Proteins c-akt; Signal Transduction; Time Factors; Tumor Microenvironment

Topics: Animals; Apoptosis; Artemisia annua; Artesunate; Cell Proliferation; Cell Survival; Cells, Cultured; Cyclin D1; DNA Damage; DNA Repair; Esophageal Neoplasms; Female; Histones; Humans; Mice, Inbred BALB C; Radiation Tolerance; Stimulation, Chemical

Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. Histone deacetylase inhibitors (HDACIs) have been demonstrated as an emerging class of anticancer drugs for a range of haematological and solid tumours. However, the effect of HDACIs has not yet been investigated on ESCC cells. In this study, HDACIs were initially considered to have anticancer activity for ESCC, due to the high expression of HDAC genes in ESCC cell lines by analysing expression data of 27 ESCC cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia. Next, we used five ESCC cell lines and one normal immortalized esophageal epithelial cell line to screen three HDACIs, panobinostat (LBH589), vorinostat (SAHA), and trichostatin A (TSA), for the ability to inhibit growth. Here, we report that LBH589 more effectively suppressed cell proliferation of ESCC cell lines, in a dose-dependent manner, than TSA and SAHA, as well as had lower toxicity against the SHEE normal immortalized esophageal epithelial cell line. Further experiments indicated that LBH589 treatment significantly inhibited TP53 (mutated TP53) expression, both at the mRNA and protein level, and simultaneously increased p21 and decreased cyclin D1 expression. Taken together, we propose that LBH589 inhibits ESCC cell proliferation mainly through inducing cell cycle arrest by increasing p21 and decreasing cyclin D1 in a p53-independent manner. SIGNIFICANCE OF THE STUDY: In this study, the antitumor activity of HDACIs LBH589, SAHA, and TSA on ESCC was characterized, with LBH589 displaying the most potent anti-proliferative activity while not harming normal immortalized esophageal epithelial cells. Furthermore, we propose that LBH589 exerts its anti-proliferative effect by inducing cell cycle arrest. The ability to specifically target cancer cells indicates therapeutic potential for use of LBH589 in the treatment of ESCC.

Topics: Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Panobinostat; Tumor Suppressor Protein p53; Up-Regulation

In the present study, we aimed to evaluate the relationship between the survival rate of patients with esophageal squamous cell carcinoma (SCC) and expression of two biomarkers along with age, gender, tumor margin, depth of invasion, site of tumor, tumor diameter, tumor grade, number of involved nodes, and vascular invasion.. In this retrospective survey, medical records of patients referred to the Shohada-e Tajrish hospital during 2001 to 2005 were reviewed and subjects with definite diagnosis of SCC were included. Required data were extracted from the patients' records, and their prepared paraffin-embedded tissue blocks were collected under supervision of two pathologists. Immunohistochemistry (IHC) analysis was performed at the Firoozgar hospital in Tehran, Iran.. The studied population included 20 men (74%) and 7 women (26%). The mean age at diagnosis was 58 ± 22. Results showed significantly higher survival rates in women compared to men (85.7 vs. 40%) (p = 0.001) and in patients with well-differentiated tumors compared to poor-differentiated cases (20 vs. 5%) (p = 0.004). No significant relationship was found between p53 expression and prognostic factors like age, gender, the site, grade, and size of the tumor, depth of invasion, involvement of lymph nodes, and vascular invasion.. Positivity of p53 and cyclin D1 was not found to be predictive of survival in patients with esophageal SCC which might be due to the small sample size of the present survey. Further investigations with larger sample populations and longer follow-ups are required to evaluate this correlation.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Survival Rate; Tumor Suppressor Protein p53

The roles and mechanisms of mini-chromosome maintenance complex component 7 (MCM7) amplification and overexpression in esophageal carcinogenesis were investigated. By analyzing the TCGA datasets, we found that MCM7 was amplified in approximately 12% of esophageal squamous cell carcinomas (ESCCs), and in more than 4% of head and neck squamous cell carcinomas and stomach carcinomas. Overexpression of MCM7 was further verified in three independent GEO datasets of esophageal cancer. Knockdown of MCM7 using two siRNAs significantly inhibited cell proliferation, colony formation and migration of KYSE510 and EC9706 cells in vitro. Noteworthy, we further found that silencing of MCM7 suppressed the phosphorylation of AKT1 and mTOR both in KYSE510 and EC9706 cells, and reduced the cell cycle regulatory proteins cyclin D1, cyclin E2 and CDK2. Taken together, our findings suggested that MCM7 promoted tumor cell proliferation, colony formation and migration of ESCC cells via activating AKT1/mTOR signaling pathway.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Minichromosome Maintenance Complex Component 7; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Stem Cells; TOR Serine-Threonine Kinases

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in China, but the underlying molecular mechanism of ESCC is still unclear. Involvement of microRNAs has been demonstrated in cancer initiation and progression. Despite the reported function of miR-503 in several human cancers, its detailed anti-oncogenic role and clinical significance in ESCC remain undefined. In this study, we examined miR-503 expression by qPCR and found the downregulation of miR-503 expression in ESCC tissue relative to adjacent normal tissues. Further investigation in the effect of miR-503 on ESCC cell proliferation, migration, and invasion showed that enhanced expression of miR-503 inhibited ESCC aggressive phenotype and overexpression of CCND1 reversed the effect of miR-503-mediated ESCC cell aggressive phenotype. Our study further identified CCND1 as the target gene of miR-503. Thus, miR-503 functions as a tumor suppressor and has an important role in ESCC by targeting CCND1.

Topics: 3' Untranslated Regions; Aged; Antagomirs; Base Sequence; Binding Sites; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; G1 Phase Cell Cycle Checkpoints; Humans; Male; MicroRNAs; Middle Aged; Phenotype; RNA Interference; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; Sequence Alignment

Oesophageal cancer is one of the most common malignancies worldwide,and oesophageal squamous cell carcinoma (ESCC) is the predominant histological type both globally and in China. Collagen triple helix repeat containing 1 (CTHRC1) has been found to be upregulated in ESCC. However, its role in tumourigenesis and progression of ESCC remains unclear.. Using our previous ESCC mRNA profiling data, we screened upregulated genes to identify those required for proliferation. Immunohistochemistry was performed to determine the level of CTHRC1 protein expression in 204 ESCC patients. Correlations between CTHRC1 expression and clinicopathological characteristics were assessed. In addition, pyrosequencing and 5-aza-dC treatment were performed to evaluate methylation status of CTHRC1 promoter. In vitro and in vivo analyses were also conducted to determine the role of CTHRC1 in ESCC cell proliferation, migration and invasion, and RNA sequencing and molecular experiments were performed to study the underlying mechanisms.. Based on mRNA profiling data, CTHRC1 was identified as one of the most significantly upregulated genes in ESCC tissues (n = 119, fold change = 20.5, P = 2.12E-66). RNA interference screening also showed that CTHRC1 was required for cell proliferation. Immunohistochemistry confirmed markedly high CTHRC1 protein expression in tumour tissues, and high CTHRC1 expression was positively correlated with advanced T stage (P = 0.043), lymph node metastasis (P = 0.023), TNM stage (P = 0.024) and poor overall survival (P = 0.020). Promoter hypomethylation at cg07757887 may contribute to increased CTHRC1 expression in ESCC cells and tumours. Forced overexpression of CTHRC1 significantly enhanced cell proliferation, migration and invasion, whereas depletion of CTHRC1 suppressed these cellular functions in three ESCC cell lines and xenografts. CTHRC1 was found to activate FRA-1 (Fos-related antigen 1, also known as FOSL1) through the MAPK/MEK/ERK cascade, which led to upregulation of cyclin D1 and thus promoted cell proliferation. FRA-1 also induced snail1-mediated MMP14 (matrix metallopeptidase 14, also known as MT1-MMP) expression to facilitate ESCC cell invasion, migration, and metastasis.. Our data suggest that CTHRC1 may act as an oncogenic driver in progression and metastasis of ESCC, and may serve as a potential biomarker for prognosis and personalized therapy.

Topics: Adult; Aged; Animals; Biomarkers; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Disease Progression; DNA Methylation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Extracellular Matrix Proteins; Female; Gene Expression; Heterografts; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 14; Middle Aged; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Signal Transduction; Snail Family Transcription Factors; Tumor Burden

The authors' previous study demonstrated that Golgi phosphoprotein 3 (GOLPH3) was significantly overexpressed in esophageal squamous cell carcinoma (ESCC), correlating with poor patient survival. In the present study, GOLPH3 stable overexpression and knockdown KYSE‑140 cell lines were constructed. Cell proliferation, colony formation, cell cycle progression and tumorigenesis assays were performed. The results revealed that GOLPH3 promoted ESCC cell growth and proliferation. The effects of GOLPH3 on the mechanistic target of rapamycin (mTOR) and Wnt/β‑catenin signaling pathways were investigated using western blot analyis and dual‑luciferase reporter assays, and were observed to be activated in cells with GOLPH3 overexpression. Furthermore, overexpression of GOLPH3 resulted in the downregulation of p21 protein, upregulation of cyclin D1 and increased retinoblastoma‑associated protein phosphorylation, consequently leading to accelerated cell cycle progression. In addition, GOLPH3 knockdown resulted in reversed effects. The results of the current study suggest that GOLPH3 serves an important role in promoting tumorigenicity of ESCC via mTOR and Wnt/β‑catenin signaling pathway activation.

Topics: Animals; beta Catenin; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; RNA Interference; TOR Serine-Threonine Kinases; Up-Regulation; Wnt Proteins; Wnt Signaling Pathway

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers in Japan. Anticancer chemotherapy has been useful for ESCC treatment. However, therapeutic options are limited. Recently, bisphosphonates (BPs), which are osteoporosis drugs, have shown anticancer effects in several cancer cell lines, but the effects against ESCC cell lines are unknown. In this study, we examined the cytotoxic effects of BPs and their mechanisms of cytotoxicity in human ESCC cell lines. A first-generation BP (etidronate), two second-generation BPs (alendronate and pamidronate), and two third-generation BPs (risedronate and zoledronate) were used in this study. All BPs, except etidronate, were cytotoxic, as indicated by increased caspase-3/7 activity and numbers of Annexin-fluorescein isothiocyanate positive cells in ESCC cell lines. From cell cycle analysis, G0/G1-phase arrest was observed upon treatment with second- and third-generation BPs. In addition, Cyclin D1 protein expression levels were decreased by second- and third-generation BP treatment. Although squalene and trans, trans-farnesol minimally affected BP cytotoxicity, treatment with geranylgeraniol inhibited BP cytotoxicity almost completely. We concluded that second- and third-generation BPs are cytotoxic to ESCC cell lines as they induce apoptosis and inhibit the cell cycle through mevalonate pathway inhibition. Therefore, BP treatment may be a beneficial therapy in ESCC patients.

Topics: Annexins; Apoptosis; Bone Density Conservation Agents; Carcinoma, Squamous Cell; Caspase 3; Caspase 7; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cyclin D1; Diphosphonates; Diterpenes; Drug Screening Assays, Antitumor; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Farnesol; G1 Phase Cell Cycle Checkpoints; Humans; Squalene

Acquired radioresistance during radiotherapy has significantly affected the treatment efficacy in esophageal cancer. Many of radioresistant cancer cells demonstrated epithelial-mesenchymal transition (EMT).We found in previous study that a radioresistant cell line (KYSE-150R) possessed EMT characteristic with cyclin D1 overexpression. Cyclin D1 has been demonstrated to affect the radiation sensitivity in cancer cells. To elucidate the molecular functions of cyclin D1 on EMT phenotypes and esophageal cancer radiosensitivity, we treated the radioresistant esophageal cancer cells (KYSE-150R) and parental cells (KYSE-150) with cyclin D1 small interfering RNA (siRNA). The cell proliferation rate of KYSE-150R and the radiation survival fraction were significantly decreased in cyclin D1 siRNA treatment group. Knocking down cyclin D1 resulted in G0/G1 arrest in KYSE-150R cells. The average number of irradiation-induced γ-H2AX foci increased in the cells treated with cyclin D1 siRNA, indicating impaired DNA double-strand break (DSB) repair in KYSE-150R cells. Cyclin D1 also reversed EMT phenotypes with significantly increased expression of E-cadherin in KYSE-150R cells. However, cyclin D1 siRNA have no radiosensitizing effects on KYSE-150 cells, with no obvious change in EMT marker expression .Our work showed that EMT phenotypes can be reduced and the radiosensitivity of esophageal cancer cells can be enhanced by inhibiting cyclin D1.

Topics: Cadherins; Cell Line, Tumor; Cell Survival; Cyclin D1; DNA Breaks, Double-Stranded; DNA Repair; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Radiation Tolerance; Radiation-Sensitizing Agents; RNA, Small Interfering

Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs.

Topics: Carcinoma, Squamous Cell; Cell Line; Cyclin D1; DNA Copy Number Variations; ErbB Receptors; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Deletion; Gene Rearrangement; Genes, p16; Genetic Association Studies; Genetic Variation; Genome, Human; Genomics; Humans; In Situ Hybridization, Fluorescence; Receptor, ErbB-2; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Notch1; Reproducibility of Results; Sequence Analysis, RNA; Translocation, Genetic

Suprabasin is a recently identified oncoprotein that is upregulated in multiple cancers. However, the clinical significance and biological role of suprabasin in human esophageal squamous cell carcinoma (ESCC) remains unclear. In the current study, we reported that suprabasin was markedly overexpressed in ESCC cell lines and tissues at both mRNA and protein levels, and this was associated with advanced clinical stage, tumor-nodes-metastasis (TNM) classification, histological differentiation, tumor size and poorer survival. Furthermore, we found that both proliferation and tumorigenicity of ESCC cells were significantly induced by suprabasin overexpression, but inhibited by suprabasin knock-down. Moreover, we demonstrated that upregulation of suprabasin activated the Wnt/β-catenin signaling pathway and led to nuclear localization of β-catenin and upregulation of Cyclin D1 and c-Myc. Together, our results suggest that suprabasin plays an important oncogenic role in promoting proliferation and tumorigenesis of ESCC.

Topics: Antigens, Differentiation; beta Catenin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasm Proteins; Proto-Oncogene Proteins c-myc; Wnt Signaling Pathway

Tumorigenesis results from various types of dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis and senescence. The transcription factor Myc-interacting zinc finger protein 1 (Miz-1) can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein in several types of tumors. Known target genes of these complexes encode the cyclin‑dependent kinase inhibitors such as cdkn2b (p15) and cdkn1a (p21). In the present study, we showed that the silencing of Miz-1 expression, through shRNA in a lentiviral vector, influenced various biological processes in two types of esophageal carcinoma cell lines. Silencing of the expression of Miz-1 inhibited cell proliferation and promoted apoptosis in vitro. Loss of Miz-1 reduced the migration ability in esophageal carcinoma cells. High expression of p21 and downregulation of cyclin D1 accompanied the knockdown of Miz-1. Our data demonstrated that esophageal cancer has a cell cycle arrest pathway via Miz-1, p21 and cyclin D1.

Topics: Apoptosis; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Esophagus; Humans; Kruppel-Like Transcription Factors; Neoplasm Invasiveness; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; RNA Interference; RNA, Small Interfering

We aim to identify esophageal squamous cell carcinoma patients with increased risk of postoperative metastases.. A high level of cyclin D1 expression, together with poor tumor cell differentiation and advanced tumor stages, increased risk of postoperative metastasis and decreased distant metastasis-free survival in ESCC in both cohorts. A high level of cyclin D1 expression also decreased overall survival in the training cohort (p < 0.01) but not in the validation cohort (p = 0.415). However, when the two cohorts of patients were pooled to obtain a larger case number, a high level of cyclin D1 expression was again demonstrated as an independent predictor that decreased overall survival (p < 0.01).. We used data from two institutions to establish training (n = 319) and validation (n = 164) cohorts. Tissue microarrays were generated for immunohistochemical evaluation. The correlation among cyclin D1 expression, clinicopathologic variables, postoperative distant metastases, overall survival, and distant metastasis-free survival were analyzed. Multivariate analyses were used to test the independent factors impacting postoperative distant metastases and survival. The outcomes generated from the training cohort were then tested using the validation cohort and pooled dataset.. High level of cyclin D1 expression increased distant metastasis, decreased overall survival and distant metastasis-free survival in resectable ESCC. Using a combination of cyclin D1 expression, tumor cell differentiation grade, and tumor stages, identifying patients with increased risk of postoperative metastases becomes possible.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cohort Studies; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Postoperative Period; Predictive Value of Tests; Survival Analysis

Several aberrant microRNAs (miRNAs or miRs) have been implicated in esophageal cancer (EC), which is widely prevalent in China. However, their role in EC tumorigenesis has not yet been fully elucidated. In the present study, we determined that miR‑1 was downregulated in esophageal squamous cell carcinoma (ESCC) tissues compared with adjacent non-neoplastic tissues using RT-qPCR, and confirmed this using an ESCC cell line. Using a nude mouse xenograft model, we confirmed that the re-expression of miR‑1 significantly inhibited ESCC tumor growth. A tetrazolium assay and a trypan blue exclusion assay revealed that miR‑1 suppressed ESCC cell proliferation and increased apoptosis, whereas the silencing of miR‑1 promoted cell proliferation and decreased apoptosis, suggesting that miR‑1 is a novel tumor suppressor. To elucidate the molecular mechanisms of action of miR‑1 in ESCC, we investigated putative targets using bioinformatics tools. MET, cyclin D1 and cyclin-dependent kinase 4 (CDK4), which are involved in the hepatocyte growth factor (HGF)/MET signaling pathway, were found to be targets of miR‑1. miR‑1 expression inversely correlated with MET, cyclin D1 and CDK4 expression in ESCC cells. miR‑1 directly targeted MET, cyclin D1 and CDK4, suppressing ESCC cell growth. The newly identified miR‑1/MET/cyclin D1/CDK4 axis provides new insight into the molecular mechanisms of ESCC pathogenesis and indicates a novel strategy for the diagnosis and treatment of ESCC.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Base Sequence; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Luciferases; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Proto-Oncogene Proteins c-met; Transfection; Xenograft Model Antitumor Assays

Esophageal squamous cell carcinoma (ESCC) is one of the lethal cancers with a high incidence rate in Asia. Cyclin D1 is overexpressed and plays an important role in the carcinogenesis of ESCC; however the mechanism of the deregulation of Cyclin D1 in ESCC remains to be determined. In the study, we found that miR-18a promotes the expression Cyclin D1 by targeting PTEN in eophageal squamous cell carcinoma TE13 and Eca109 cells. Transfection of miR-18a mimetics increased cyclin D1, while transfection of miR-18a antagomir decreased D1. Moreover, miR-18a-mediated upregulation of cyclin D1 was accompanied with downregulation of PTEN, which is a direct target of miR-18a, and increase of the phosphorylation of AKT and S6K1. In addition, pharmacologic inhibition of AKT or mTOR kinases abolished the increase of cyclinD1 by miR-18a, which was accompanied with decreased phosphorylation of RbS780 and inhibition of cell proliferation. Our results demonstrated the upregulation of miR-18a promoted cell proliferation by increasing cylin D1 via regulating PTEN-PI3K-AKT-mTOR signaling axis, suggesting that small molecule inhibitors of AKT-mTOR signaling are potential agents for the treatment of ESCC patients with upregulation of miR-17-92 cluster.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Cisplatin; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; MicroRNAs; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; TOR Serine-Threonine Kinases

Esophageal Cancer (EC) is a common malignant tumor occurred in the digestive tract. In this study, we investigated the mechanism of Protease Activated Receptor 2 (PAR-2) on the proliferation of esophageal cancer cell.. Transfected esophageal cancer (EC) cell (PAR-2shRNA EC109) was established with low stable PAR-2 expression. EC109 cell was treated with PAR-2 agonist, PAR-2 anti-agonist and MAPK inhibitor respectively; Untreated EC109 cell (blank control) and PAR-2shRNA EC109 cell were used for analysis also. The mRNA expressions of PAR-2, ERK1, Cyclin D1, and c-fos in each group were detected by reverse transcript and polymerase chain reaction. Western blot was used to detect the protein expressions in each group. The cell growth curves were drawn to compare the cell growth.. Compared with the blank control, the mRNA and protein expressions of PAR-2, Cyclin D1, and c-fos in PAR-2 agonist group increased significantly (p < 0.05), while decreased significantly in PAR-2shRNA EC109 cell and MAPK inhibitor group (p < 0.05). The mRNA expression of ERK1 and protein expression of p-ERK1 increased in PAR-2 agonist group, decreased in PAR-2shRNA EC109 cell and MAPK inhibitor group when compared with blank control (p < 0.05). The growth of cells was upward in PAR-2 agonist group at cell growth phase when compared with blank control, while decreased in PAR-2 shRNA EC109 cell and MAPK inhibitor group with statistical difference (p < 0.05).. PAR-2 regulate cell proliferation through the MAPK pathway in esophageal carcinoma cell, and Cyclin D1, c-fos are involved in this process.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Receptor, PAR-2; RNA, Messenger

Cyclin D1 is frequently overexpressed in esophageal squamous cell carcinoma (ESCC) and is considered a key driver of this disease. Mutations in FBXO4, F-box specificity factor that directs SCF-mediated ubiquitylation of cyclin D1, occur in ESCC with concurrent overexpression of cyclin D1 suggesting a potential tumor suppressor role for FBXO4. To evaluate the contribution of FBXO4-dependent regulation cyclin D1 in esophageal squamous cell homeostasis, we exposed FBXO4 knockout mice to N-nitrosomethylbenzylamine (NMBA), an esophageal carcinogen. Our results revealed that loss of FBXO4 function facilitates NMBA induced papillomas in FBXO4 het (+/-) and null (-/-) mice both by numbers and sizes 11 months after single dose NMBA treatment at 2mg/kg by gavage when compared to that in wt (+/+) mice (P < 0.01). No significant difference was noted between heterozygous or nullizygous mice consistent with previous work. To assess cyclin D1/CDK4 dependence, mice were treated with the CDK4/6 specific inhibitor, PD0332991, for 4 weeks. PD0332991 treatment (150mg/kg daily), reduced tumor size and tumor number. Collectively, our data support a role for FBXO4 as a suppressor of esophageal tumorigenesis.

Topics: Animals; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; F-Box Proteins; Humans; Mice; Mutation

E-cadherin, β-catenin, epidermal growth factor receptor (EGFR), neuronal cadherin (N-cadherin) and Cyclin D1 are involved in epithelial to mesenchymal transition (EMT). However, the prognostic significance of EMT markers in oesophageal adenocarcinoma (OAC) is unknown. Aim of this study was to evaluate the prognostic value of, and the association between different EMT markers in OAC.. Tumour cores of 154 patients with OAC were included in a tissue microarray. Scoring criteria was based on immunohistochemical staining intensity.. EMT-associated markers were expressed in OAC: reduced membranous E-cadherin and β-catenin were seen in 11.4% and 51.7%, nuclear β-catenin in 19.1% and EGFR and Cyclin D1 overexpression in 56.5% and 27.4% of tumours. Mesenchymal marker N-cadherin was not expressed in OAC. A positive correlation was seen between membranous β-catenin and E-cadherin expression (R=0.209, p=0.001) and between EGFR and Cyclin D1 (R=0.257, p=0.002). In univariate analysis, EGFR overexpression and membranous β-catenin staining were significantly associated with a poor survival (HR 2.145; 95% CI 1.429 to 3.218, p<0.001 and HR 1.665; 95% CI 1.114 to 2.488; p=0.013). However, Cyclin D1 (HR 1.092; 95% CI 0.702 to 1.698; p=0.697), nuclear β-catenin (HR 1.322; 95% CI 0.799 to 2.189; p=0.277) and E-cadherin (HR 1.012; 95% CI 0.554 to 1.851; p=0.968) were not associated with survival. In multivariate analysis, EGFR overexpression was an independent prognostic factor for poor survival (HR 1.678; 95% CI 1.055 to 2.668; p=0.029) together with T stage (HR 2.759; 95% CI 1.356 to 5.576; p=0.005).. This study supports the presence of EMT in OAC. Moreover, EGFR overexpression was independently associated with a poor survival.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antigens, CD; beta Catenin; Biomarkers, Tumor; Cadherins; Chi-Square Distribution; Cyclin D1; Disease-Free Survival; Epithelial-Mesenchymal Transition; ErbB Receptors; Esophageal Neoplasms; Esophagectomy; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Predictive Value of Tests; Proportional Hazards Models; Time Factors; Tissue Array Analysis; Treatment Outcome

ZIP5 is a central player in mammalian zinc metabolism. Studies suggest that ZIP5 is differentially expressed during esophageal tumorigenesis, yet the role of ZIP5 in esophageal cancer cells has not yet been clarified. Immunohistochemistry, western blotting and qRT-PCR techniques were used to detect ZIP5 expression in esophageal squamous cell carcinoma (ESCC) tissues. We established a stable knockdown ZIP5 cell line (KYSE170K) derived from the ESCC cell line KYSE170. We conducted MTT and CCK-8 assays to determine the role of ZIP5 in cell proliferation, Transwell assays to detect migration and invasion, and flow cytometry (FCM) to detect apoptosis and cell cycle percentage using KYSE170K cells. We conducted a gene profiling study to detect the expression of genes related to tumor progression. The results demonstrated that ZIP5 protein and mRNA expression was highest in ESCC, intermediate in para-carcinoma and lowest in normal tissue. ZIP5 knockdown decreased proliferation by 28 and 38%, respectively, according to the MTT and CCK-8 assays. Migration and invasion decreased by 54 and 68%, respectively, according to the Transwell assays. COX2 expression was decreased by 68 and 75% at the mRNA and protein level, respectively, and cyclin D1 mRNA and protein expression was decreased following 62 and 60%, respectively, by knockdown of ZIP5, which upregulated the mRNA and protein expression of E-cadherin by 80 and 60%, respectively. ZIP5 knockdown inhibited the proliferation, migration and invasion of ESCC and suppressed COX2, cyclin D1 and E-cadherin expression, which led to the inhibition of cell progression in ESCC.

Topics: Adult; Aged; Apoptosis; Cadherins; Carcinoma, Squamous Cell; Cation Transport Proteins; Cell Movement; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Middle Aged; Neoplasm Invasiveness; RNA, Messenger

Rhizoma Paridis Saponins (RPS), a natural compound purified from Rhizoma Paridis, has been found to inhibit cancer growth in vitro and in animal models of cancer. However, its effects on esophageal cancer remain unexplored. The purpose of this study was to investigate the effects of RPS on tumor growth in a rat model of esophageal cancer and the molecular mechanism underlying the effects. A rat model of esophageal cancer was established by subcutaneous injection of N-nitrosomethylbenzylamine (NMBA, 1 mg/kg) for 10 weeks. RPS (350 mg/kg or 100 mg/kg) was administered by oral gavage once daily for 24 weeks starting at the first NMBA injection. RPS significantly reduced the size and number of tumors in the esophagus of rats exposed to NMBA and inhibited the viability, migration, and invasion of esophageal cancer cells EC9706 and KYSE150 in a dose dependent manner (all P < 0.01). Flow cytometry revealed that RPS induced apoptosis and cell cycle G2/M arrest in the esophageal cancer cells. The expression of cyclooxygenases-2 (COX-2) and Cyclin D1 in rat esophageal tissues and the esophageal cancer cells were also significantly reduced by RPS (all P < 0.01). Consistently, RPS also significantly decreased the release of prostaglandin E2, a downstream molecule of COX-2, in a dose-dependent manner (P < 0.01). Our study suggests that RPS inhibit esophageal cancer development by promoting apoptosis and cell cycle arrest and inhibiting the COX-2 pathway. RPS might be a promising therapeutic agent for esophageal cancer.

Topics: Animals; Cell Cycle Checkpoints; Cell Movement; Cell Survival; Cyclin D1; Cyclooxygenase 2; Dimethylnitrosamine; Dinoprostone; Esophageal Neoplasms; Esophagus; G2 Phase Cell Cycle Checkpoints; Male; Plant Extracts; Rats; Rats, Inbred F344; Rhizome; Saponins

Esophageal squamous cell carcinomas (ESCC) have become a severe threat to health and the current treatments for ESCC are frequently not effective. Recent epidemiological studies suggest that the anti-hyperglycemic agent metformin may reduce the risk of developing cancer, including ESCC, among diabetic patients. However, the antitumor effects of metformin on ESCC and the mechanisms underlying its cell cycle regulation remain elusive. The findings reported herein show that the anti-proliferative action of metformin on ESCC cell lines is partially mediated by AMPK. Moreover, we observed that metformin induced G0/G1 phase arrest accompanied by the up-regulation of p21CIP1 and p27KIP1. In vivo experiments further showed that metformin inhibited tumor growth in a ESCC xenograft model. Most importantly, the up-regulation of AMPK, p53, p21CIP1, p27KIP1 and the down-regulation of cyclinD1 are involved in the anti-tumor action of metformin in vivo. In conclusion, metformin inhibits the growth of ESCC cells both in cell cultures and in an animal model. AMPK, p53, p21CIP1, p27KIP1 and cyclinD1 are involved in the inhibition of tumor growth that is induced by metformin and cell cycle arrest in ESCC. These findings indicate that metformin has the potential for use in the treatment of ESCC.

Topics: AMP-Activated Protein Kinases; Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; Esophageal Neoplasms; G1 Phase Cell Cycle Checkpoints; Humans; Hypoglycemic Agents; Male; Metformin; Mice, Nude; Resting Phase, Cell Cycle; Tumor Burden; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate. To determine the molecular basis of ESCC development, this study sought to identify characteristic genome-wide alterations in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic alterations were also analyzed. Exome sequencing and verification were performed for nine pairs of ESCC and the matched blood samples, followed by validation with additional samples using Sanger sequencing. Whole-genome SNP arrays were employed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) in 55 cases, including the nine ESCC samples subjected to exome sequencing. A total of 108 non-synonymous somatic mutations (NSSMs) in 102 genes were verified in nine patients. The chromatin modification process was found to be enriched in our gene ontology (GO) analysis. Tumor genomes with TP53 mutations were significantly more unstable than those without TP53 mutations. In terms of the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A/2B (30.9%), amplification of 11q13.3 covering CCND1 (30.9%), and TP53 point mutation (50.9%) occurred in two-thirds of the cases. These results suggest that the deregulation of the G1 phase during the cell cycle is a key event in ESCC. Furthermore, six minimal common regions were found to be significantly altered in ESCC samples and three of them, 9p21.3, 7p11.2, and 3p12.1, were associated with lymph node metastasis. With the high correlation of TP53 mutation and genomic instability in ESCC, the amplification of CCND1, the deletion of CDKN2A/2B, and the somatic mutation of TP53 appear to play pivotal roles via G1 deregulation and therefore helps to classify this cancer into different genomic subtypes. These findings provide clinical significance that could be useful in future molecular diagnoses and therapeutic targeting.

Topics: Adult; Carcinoma, Squamous Cell; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagus; Exome; Female; G1 Phase Cell Cycle Checkpoints; Gene Dosage; Genomic Instability; Genomics; Humans; Loss of Heterozygosity; Male; Middle Aged; Mutation; Tumor Suppressor Protein p53

Aquaporins (AQPs) are water channel proteins that facilitate transcellular water movements. Recent studies have shown that AQP5 is expressed in various cancers, and plays a role in tumor progression. However, its expression and role in esophageal squamous cell carcinoma (ESCC) have not been investigated. We examined the pathophysiologic role of AQP5 in cell proliferation and survival, and also investigated its expression and effects on the prognosis of ESCC patients.. AQP5 expression in human ESCC cell lines was analyzed by Western blot testing. Knockdown experiments with AQP5 siRNA were conducted, and the effects on cell proliferation, cell cycle progression, and cell survival were analyzed. The cells' gene expression profiles were analyzed by microarray analysis. Immunohistochemistry of AQP5 for 68 primary tumor samples obtained from ESCC patients undergoing esophagectomy was performed.. AQP5 expression was high in TE2 and TE5 cells. In these cells, the knockdown of AQP5 using siRNA inhibited cell proliferation and G1-S phase progression, and induced apoptosis. The AQP5 siRNA transfected TE5 cells showed significant increase in p21 and decrease in CCND1 mRNA expression, respectively. The expression pattern of AQP5 and p21 protein was sharply contrasted, but AQP5 and CCND1 protein expression showed a similar pattern in ESCC tissue. These findings agree with the microarray results. Immunohistochemical staining of 68 ESCC patients showed the AQP5 expression is associated with tumor size, histological type, and tumor recurrence.. The AQP5 expression in ESCC cells may affect cell proliferation and survival, and impact on the prognosis of ESCC patients.

Topics: Aged; Apoptosis; Aquaporin 5; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Profiling; Humans; Male; Microarray Analysis; Middle Aged; Neoplasm Recurrence, Local; Prognosis; RNA, Messenger; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; Survival Rate

The tumor suppressor LKB1 gene encodes a serine-threonine kinase that regulates cell proliferation and polarity. Inactivation of LKB1 by mutations in LKB1 or loss of its expression is highly correlated with lung, ovarian, and pancreatic cancers, and WNT/β-catenin pathway is also known to be involved in many human malignancies. However, the relationship between LKB1 and WNT signaling pathway in esophageal carcinoma remains unknown. The expression of LKB1 in 62 cases of esophageal cancer patients was determined by quantitative real-time PCR. It was found that LKB1 mRNA level was significantly lower than the adjacent normal epithelium and that the LKB1 downregulation was correlating with TNM stages. Moreover, the expression of WNT target genes such as Cyclin D1, C-MYC, MMP2, and FZD2 was significantly upregulated in esophageal cancer tissues. LKB1 overexpression in TE10 cells inhibited TOPFlash luciferase reporter activity and WNT target gene expression even in the presence of WNT3A. Conversely, LKB1 knockdown enhanced WNT signaling activity in esophageal cancer cells. It was also found that LKB1 antagonized WNT signaling pathway through interaction with GSK3β to downregulate β-catenin expression level. Functional investigation revealed that LKB1 suppressed the promotion effects of WNT3A on the cell growth of TE10 cells. The LKB1 functions in regulating cell growth and WNT target genes expression were impaired by GSK3β inhibition, suggesting that LKB1 antagonized WNT-induced cell proliferation through enhancement of GSK3β activity. Together, the interaction between LKB1 and GSK3β upregulates GSK3β activity to suppress WNT-induced cell proliferation in esophageal carcinoma cells. Loss of LKB1 expression may result in the deregulation of WNT/β-catenin pathway to promote malignant progression of esophageal cancer.

Topics: AMP-Activated Protein Kinase Kinases; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Protein Serine-Threonine Kinases; Wnt Signaling Pathway

Global DNA hypomethylation plays a crucial role in genomic instability and carcinogenesis. DNA methylation of the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level, and is attracting interest as a useful marker for predicting cancer prognosis. Our previous study using more than 200 esophageal squamous cell carcinoma (ESCC) specimens demonstrated the significant relationship between LINE-1 hypomethylation and poor prognosis. However, the mechanism by which LINE-1 hypomethylation affects aggressive tumor behavior has yet to be revealed.. To examine the relationship between LINE-1 hypomethylation and DNA copy number variations, we investigated LINE-1-hypomethylated and LINE-1-hypermethylated ESCC tumors by comparative genomic hybridization array.. LINE-1-hypomethylated tumors showed highly frequent genomic gains at various loci containing candidate oncogenes such as CDK6. LINE-1 methylation levels were significantly associated with CDK6 mRNA and CDK6 protein expression levels in ESCC specimens. In our cohort of 129 patients with ESCC, cases with CDK6-positive expression experienced worse clinical outcome compared with those with CDK6-negative expression, supporting the oncogenic role of CDK6 in ESCC. In addition, we found that the prognostic impact of LINE-1 hypomethylation might be attenuated by CDK6 expression.. LINE-1 hypomethylation (i.e., global DNA hypomethylation) in ESCC might contribute to the acquisition of aggressive tumor behavior through genomic gains of oncogenes such as CDK6.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Chromosome Aberrations; Cyclin D1; Cyclin-Dependent Kinase 6; DNA Copy Number Variations; DNA Methylation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Amplification; Gene Expression; Humans; Long Interspersed Nucleotide Elements; Male; Prognosis

This study was designed to evaluate the clinical benefit of predicting the cyclin D1 (CCND1) status using cell-free plasma DNA in superficial esophageal squamous cell carcinoma (ESCC) patients.. The ratio of the CCND1 (11q13) dosage to the DRD2 (11q22-23) dosage (C/D ratio) as the CCND1 copy number was evaluated. This study was divided into three steps: (1) demonstration of the feasibility, (2) evaluation of whether the plasma C/D ratio assay could monitor tumor dynamics, and (3) a validation study in 63 consecutive superficial ESCC (pTis-T1) patients and 40 healthy volunteers.. (1) The plasma C/D ratio was significantly higher (p = 0.0369) in superficial ESCC patients than in the controls in a preliminary test. (2) The high plasma C/D ratio appeared to reflect the tumor levels of the CCND1 status and was reduced in postoperative plasma samples (p = 0.1154) and samples following endoscopic resection (p = 0.0845). (3) Validation analysis revealed that the plasma C/D ratio was significantly higher in superficial ESCC patients than in controls (p < 0.0001). The frequency of recurrence was significantly higher (p = 0.0198), and recurrence-free survival was significantly shorter (p = 0.0075) in patients with a high plasma C/D ratio. Moreover, a high C/D ratio was shown to be an independent risk factor for recurrence on multivariate analysis [p = 0.0334; odds ratio 10.58 (range 1.203-93.23)].. The prediction of CCND1 amplification by plasma DNA may be a new complementary clinical biomarker for recurrence in patients with superficial ESCC.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; DNA; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged

To explore the effect of downregulation of Tiam1 by siRNA on the esophageal squamous cell carcinoma (ESCC) EC9706 cells, and provide theoretical basis for gene therapy of ESCC using Tiam1 as a molecular target.. Tiam1 siRNA was transfected into EC9706 cells, and expression changes of Tiam1 mRNA and protein after transfection were detected by quantitative real-time PCR and Western blotting. Cell proliferation was analyzed using CCK-8 kit. Cell cycle and apoptosis of the EC9706 cells were assessed by flow cytometry. Cell cycle-related proteins and cell apoptosis-associated proteins were analyzed by Western blotting.. Compared with the untreated group and control siRNA group, the relative expression levels of Tiam1 mRNA (1.00 and 0.11 ± 0.02) were not significantly different (P > 0.05). The relative expression levels of Tiam1 mRNA in the Tiam1 siRNA group at 24, 48 and 72 h after transfection were 0.30 ± 0.04, 0.09 ± 0.01 and 0.09 ± 0.006, respectively, significantly lower than that of the untreated group (P < 0.05 for all). The expression level of Tiam1 protein at 24 h after Tiam1 siRNA transfection in the EC9706 cells was 0.11 ± 0.02, significantly lower than that in the un-treated group (0.44 ± 0.05) and control siRNA group (0.44 ± 0.04, P < 0.05 for all). The percentages of G0/G1 cells in the Tiam1 siRNA group, untreated group and control siRNA group were (54.48 ± 2.14)%, (40.69 ± 1.85)% and (41.78 ± 1.31)%, respectively (P < 0.01). The percentages of S phase cells in the Tiam1 siRNA group, untreated group and control siRNA group were (27.18 ± 1.65)%, (32.32 ± 1.15)% and (30.35 ± 1.09)%, respectively (P < 0.01). The expression levels of cyclin D1 protein in the untreated group, control siRNA group and Tiam1 siRNA group were 0.43 ± 0.02, 0.41 ± 0.01 and 0.11 ± 0.02, respectively (P < 0.05). The expression levels of p27 protein in the untreated group, control siRNA group and Tiam1 siRNA group were 0.10 ± 0.01, 0.09 ± 0.02 and 0.20 ± 0.02, respectively (P < 0.05). The ratios of early apoptotic cells in the untreated group, control siRNA group and Tiam1 siRNA group were (10 ± 0.9)%, (10 ± 0.5)% and (27 ± 0.7)%, respectively (P < 0.01). The expression levels of Mcl-1 protein in EC9706 cells of untreated group, control siRNA group and Tiam1 siRNA group were 0.47 ± 0.12, 0.48 ± 0.13 and 0.16 ± 0.02, respectively (P < 0.05). The expression levels of Bcl-2 protein in EC9706 cells of the untreated group, control siRNA group and Tiam1 siRNA group were 0.49 ± 0.08, 0.50 ± 0.05 and 0.04 ± 0.03, respectively (P < 0.05). The caspase-3 activities in the untreated group, control siRNA group and Tiam1 siRNA group were 2.3 ± 0.09, 2.3 ± 0.10 and 16.0 ± 1.50, respectively; and that of caspase-9 were 2.3 ± 0.08, 2.3 ± 0.11 and 14.5 ± 0.9, respectively (P < 0.05 for all).. Tiam1 siRNA can significantly inhibit the proliferation of esophageal cancer EC9706 cells, induce cell cycle arrest and cell apoptosis. These effects are related to the regulation of the expressions of cell cycle-related genes (cyclin D1 and p27) and cell apoptosis-related genes (Mcl-1, Bcl-1, caspase-3 and caspase-9) by Tiam1 siRNA.

Topics: Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Guanine Nucleotide Exchange Factors; Humans; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Messenger; RNA, Small Interfering; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Transfection

Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a poor prognosis. The phospholipase C? gene (PLCE1) encodes a novel ras-related protein effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion. However, molecular mechanisms pertinent to ESCC are unclear. We therefore designed PLCE1-special small interfering RNA and transfected to esophageal squamous cell (EC) 9706 cells to investigate the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC. Esophageal cancer tissue specimens and normal esophageal mucosa were obtained and assayed by immunohistochemical staining to confirm overexpression of PLCE1 in neoplasias. Fluorescence microscopy was used to examine transfection efficiency, while the result of PLCE1 silencing was examined by reverse transcription (RT-PCR). Flow cytometry and annexin V apoptosis assays were used to assess the cell cycle and apoptosis, respectively. Expression of cyclin D1 and caspase-3 was detected by Western-blotting. The level of PLCE1 protein in esophageal cancer tissue was significantly higher than that in normal tissue. After transfection, the expression of PLCE1 mRNA in EC 9706 was significantly reduced, compared with the control group. Furthermore, flow cytometry results suggested that the PLCE1 gene silencing arrested the cell cycle in the G0/G1 phase; apoptosis was significantly higher than in the negative control group and mock group. PLCE1 gene silencing by RNAi resulted in decreased expression of cyclin D1 and increased expression of caspase-3. Our study suggests that PLCE1 may be an oncogene and play an important role in esophageal carcinogenesis through regulating proteins which control cell cycling and apoptosis.

Topics: Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; G1 Phase; Gene Silencing; Humans; Phosphoinositide Phospholipase C; Resting Phase, Cell Cycle; RNA Interference; RNA, Messenger; RNA, Small Interfering

The CCND1 gene is overexpressed in esophageal cancer and accelerates cell cycle progression. However, the mechanism whereby the upstream genes or factors directly regulate CCND1 expression remains unknown. By analyzing the 5'-UTR region of the CCND1 gene, we found that this region contains an octamer motif (ATTTTGCAT), which suggests that the expression of CCND1 might be directly associated with octamer-binding transcription factor 4 (OCT4). In this study, the wild-type and the octamer motif-mutanted CCND1 promoters were cloned, and their corresponding luciferase reporter vectors were then constructed to study the molecular mechanism by which OCT4 regulates the expression of CCND1 and influences the biological behaviors of esophageal cancer cells. The results indicated that suppressing the expression of CCND1 and OCT4 in esophageal cancer cells reduced cell proliferative and invasive abilities, induced cell cycle G1-phase arrest, and slowed the growth of xenografts in nude mice. Suppression of OCT4 expression significantly decreased the wild-type CCND1 promoter activity and down-regulated the expression of CCND1, but did not affect the activity of the mutant promoter. Whereas, suppression of CCND1 did not affect OCT4 expression, suggesting that OCT4 regulates CCND1 expression by activating the CCND1 promoter and subsequently promoting cell cycle progression. The results revealed and confirmed that OCT4 is the upstream factor that directly binds to the CCND1 promoter to regulate CCND1 expression, then to promote cell cycle progression and accelerate the proliferation and invasion of esophageal cancer cells. This finding may significantly contribute to elucidating the regulatory mechanism involved in the cell cycle progression of esophageal cancer cells and may aid in screening potential gene targets for the biological therapy of esophageal cancer.

Topics: Amino Acid Motifs; Animals; Base Sequence; Carcinoma; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Transplantation; Octamer Transcription Factor-3; Promoter Regions, Genetic

CD133 was recently reported to be a cancer stem cell and prognostic marker. Quercetin is considered as a potential chemopreventive agent due to its involvement in suppression of oxidative stress, proliferation and metastasis. In this study, the expression of CD133/CD44 in esophageal carcinomas and Eca109/9706 cells was explored. In immunoflurorescence the locations of CD133+ and multidrug resistance 1 (MDR 1)+ in the same E-cancer cells were coincident, mainly in cytomembranes. In esophageal squamous cell carcinomas detected by double/single immunocytochemistry, small CD133+ cells were located in the basal layer of stratified squamous epithelium, determined as CSLC (cancer stem like cells); CD44+ surrounding the cells appeared in diffuse pattern, and the larger CD44+ (hi) cells were mainly located in the prickle cell layer of the epithelium, as progenitor cells. In E-cancer cells exposed to nanoliposomal quercetin (nLQ with cytomembrane permeability), down-regulation of NF-κBp65, histone deacetylase 1 (HDAC1) and cyclin D1 and up-regulation of caspase-3 were shown by immunoblotting, and attenuated HDAC1 with nuclear translocation and promoted E-cadherin expression were demonstrated by immunocytochemistry. In particular, enhanced E-cadherin expression reflected the reversed epithelial mesenchymal transition (EMT) capacity of nLQ, acting as cancer attenuator/preventive agent. nLQ acting as an HDAC inhibitor induced apoptotic cells detected by TUNEL assay mediated via HDAC-NF-κB signaling. Apoptotic effects of liposomal quercetin (LQ, with cytomembrane-philia) combined with CD133 antiserum were also detected by CD133 immunocytochemistry combined with TUNEL assay. The combination could induce greater apoptotic effects than nLQ induced alone, suggesting a novel anti-CSC treatment strategy.

Topics: AC133 Antigen; Antigens, CD; Antioxidants; Apoptosis; Cadherins; Carcinoma, Squamous Cell; Caspase 3; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Carriers; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Glycoproteins; Histone Deacetylase 1; Humans; Immune Sera; Liposomes; Nanoparticles; Neoplastic Stem Cells; NF-kappa B; Peptides; Quercetin; Sensitivity and Specificity; Tumor Cells, Cultured

To investigate esophageal Helicobacter pylori (H. pylori) colonization on esophageal injury caused by reflux and the related mechanisms.. An esophagitis model, with acid and bile reflux, was surgically produced in male rats. The rats were randomly divided into either: (1) an esophagogastroduodenal anastomosis (EGDA) group; (2) an EGDA with H. pylori infection group; (3) a pseudo-operation with H. pylori infection group; or (4) a pseudo-operation group. All rats were kept for 36 wk. Based on the location of H. pylori colonization, the EGDA rats with H. pylori infection were subdivided into those with concomitant esophageal H. pylori colonization or those with only gastric H. pylori colonization. The esophageal injuries were evaluated grossly and microscopically. The expressions of CDX2 and MUC2 were determined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Ki-67 antigen expression was determined by immunohistochemistry. The mRNA levels of cyclin D1, c-Myc, Bax and Bcl-2 were determined by RT-PCR. Cell apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method.. Esophagitis, Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC) developed in rats that underwent EGDA. When comparing rats with EGDA and concomitant esophageal H. pylori colonization to EGDA-only rats, the severity of injury (87.9 ± 5.2 vs 77.2 ± 8.6, macroscopically, 92.5 ± 8.0 vs 83.8 ± 5.5, microscopically, both P < 0.05) and the incidences of BE (80.0% vs 33.3%, P = 0.055) and EAC (60.0% vs 11.1%, P < 0.05) were increased. These increases were associated with upregulation of CDX2 and MUC2 mRNA (10.1 ± 5.4 vs 3.0 ± 2.9, 8.4 ± 4.6 vs 2.0 ± 3.2, respectively, Ps < 0.01) and protein (8.1 ± 2.3 vs 3.3 ± 3.1, 7.3 ± 4.0 vs 1.8 ± 2.7, respectively, all P < 0.05). The expression of Ki-67 (8.9 ± 0.7 vs 6.0 ± 1.7, P < 0.01) and the presence of apoptotic cells (8.3 ± 1.1 vs 5.3 ± 1.7, P < 0.01) were also increased significantly in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. The mRNA levels of cyclin D1 (5.8 ± 1.9 vs 3.4 ± 1.3, P < 0.01), c-Myc (6.4 ± 1.7 vs 3.7 ± 1.2, P < 0.01), and Bax (8.6 ± 1.6 vs 5.1 ± 1.3, P < 0.01) were significantly increased, whereas the mRNA level of Bcl-2 (0.6 ± 0.3 vs 0.8 ± 0.3, P < 0.01) was significantly reduced in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only.. Esophageal H. pylori colonization increases esophagitis severity, and facilitates the development of BE and EAC with the augmentation of cell proliferation and apoptosis in esophageal mucosa.

Topics: Adenocarcinoma; Animals; Apoptosis; Barrett Esophagus; bcl-2-Associated X Protein; CDX2 Transcription Factor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Esophageal Neoplasms; Esophagitis, Peptic; Esophagus; Gastroesophageal Reflux; Gene Expression Regulation; Helicobacter Infections; Helicobacter pylori; Homeodomain Proteins; Ki-67 Antigen; Male; Mucin-2; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Rats, Sprague-Dawley; RNA, Messenger; Severity of Illness Index; Transcription Factors

The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Drug Resistance; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Heterografts; Male; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Paclitaxel; ras Proteins; RNA, Small Interfering; Signal Transduction

Signal transducer of activator of transcription 3 (STAT3) and cyclinD1 are overexpressed in various human cancers, and their overexpression positively correlates to tumor progression and poor prognosis. However, the clinical significance of dual high expression of these two proteins in esophageal squamous cell carcinoma (ESCC) has yet to be determined.. The expression of STAT3 and cyclinD1 was analyzed in tissue microarrays containing tumor and adjacent tissue samples from 82 patients who had undergone curative resection for histologically proven ESCC. Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the prognostic value of STAT3 and cyclinD1 expression.. We discovered that expressions of STAT3 and cyclinD1 in cancer tissues were significantly higher than that in adjacent tissues. High expression of STAT3 and cyclinD1 was associated with malignant behaviors. Moreover, the expression of STAT3 was positively associated with the expression of cyclinD1. High STAT3 or cyclinD1 expression alone was associated with lower overall survival (OS) rates. Furthermore, dual high expression of STAT3 and cyclinD1 expression predict even worse survival outcome in both univariate and multivariate analysis.. STAT3 and cyclinD1 correlate with more aggressive tumor behavior in ESCC. When STAT3 and cyclinD1 are considered together, they serve as effective prognostic markers in patients with surgically resected ESCC.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; STAT3 Transcription Factor; Survival Rate

Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy. To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma treated with radiotherapy followed by surgery, we evaluated the expression of cell cycle checkpoint-related proteins Chk2, Cdc25C, and Cyclin D1. A total of 56 patients with locally advanced esophageal squamous cell carcinoma were treated with radiotherapy followed by surgery. Pretreatment tumor biopsy specimens were analyzed for Chk2, Cdc25C, and Cyclin D1 expression by immunohistochemistry. High expression of Chk2, Cyclin D1, and Cdc25C was observed in 44 (78.6%), 15 (26.8%), and 27 (48.2%) patients, respectively. The median survival was 16 months (range, 3-154 months), with a 5-year overall survival rate of 19.6%. Overexpression of Chk2 was associated with smoking (P = 0.021), overexpression of Cdc25C was associated with patient age (P = 0.033) and tumor length (P = 0.001), and overexpression of Cdc25C was associated with pathologic complete response (P = 0.038). Univariate analysis demonstrated that overexpression of Cdc25C and pathologic complete response was associated with better survival. In multivariate analysis, Cdc25C was the most significant independent predictor of better survival (P = 0.014) for patients treated with radiotherapy followed by surgery. Overexpression of Cdc25C was significantly associated with pathologic complete response and better survival of patients with locally advanced esophageal cancer treated with radiotherapy followed by surgery. These results suggest that Cdc25C may be a biomarker of treatment response and good prognosis for esophageal carcinoma patients. Thus, immunohistochemical staining of Cdc25C in a pretreatment specimen may be a useful method of identifying optimal treatment for patients with esophageal carcinoma.

Topics: Adult; Aged; Carcinoma, Squamous Cell; cdc25 Phosphatases; Checkpoint Kinase 2; Combined Modality Therapy; Cyclin D1; Esophageal Neoplasms; Female; Follow-Up Studies; Humans; Male; Middle Aged; Neoplasm Staging; Particle Accelerators; Proportional Hazards Models; Smoking; Survival Rate

STAT is the backward position of cytokine and growth factor receptors in the nucleus, STAT dimers could bind to DNA and induce transcription of specific target genes. Several lines of evidence support the important roles of STAT, especially STAT5, in carcinogenesis. The overexpression of STAT 5 is related to the differentiation and apoptosis of tumor cells. However, the role of STAT5 in esophageal squamous cell carcinoma remains unclear.. The siRNA vectors aiming to STAT5 gene were constructed. STAT5 siRNA was transfected into Eca-109 cells by Lipofectamine™2000. Expression of STAT5、Bcl-2 and Cyclin D1 were analyzed by Western blot and RT-PCR. Eca-109 cells proliferation was determined by MTT. Eca-109 cell cycle and apoptosis were detected by the flow cytometry. Boyden chamber was used to evaluate the invasion and metastasis capabilities of Eca-109 cells.. The double strands oligonucleotide of siRNA aiming to STAT5 was successfully cloned into the pRNAT-U6.1 vector, and the target sequence coincided with the design. RT-PCR and Western blotting detection demonstrated that the expression levels of STAT5、Bcl-2 and Cyclin D1 gene were obviously decreased in Eca-109 cells transfected with STAT5 siRNA. STAT5 siRNA could suppress the proliferation of Eca-109 cells. The proportion of S and G2/M period frequency was significantly decreased (p<0.05). The proportion of G0/G1 period frequency was significantly increased (p<0.05). The average amount of cells penetrating Matrigel was significantly decreased (p<0.05).. STAT5 silenced by siRNA could induce the apoptosis and suppress the proliferation、invasion and metastasis of esophageal carcinoma cell line Eca-109, which indicated STAT5 might be a novel therapeutic strategy for the human ESCC.. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1351913072103000.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; STAT5 Transcription Factor; Time Factors; Transfection

To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells.. EC9706 cells were treated in the following six groups: blank group (no transfection), negative control group (transfection no-carrier), transfection group (transfected with pSilencer-siK-ras), taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and western blotting were used to analyze the expression of MAPK1 (mitogen-activated protein kinases 1) and cyclin D1 in response to siRNA (small interfering RNA) transfection and taxol treatment.. K-Ras (K-Ras gene) siRNA transfection of EC9706 esophageal squamous carcinoma cells decreased the expression of K-Ras, MAPK1 and cyclin D1 at the mRNA and protein level. Reverse transcription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group (P < 0.05). Western blotting showed that 72 h after EC9706 cell transfection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the expression levels in the transfection group were significantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as compared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe.. Apoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-mediated inhibition of Ras signaling, with the most obvious increase observed in the transfection plus taxol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeutic agent, taxol. Ras knockdown also down-regulated the expression of the downstream genes, MAPK1 and cyclin D1, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Therapy; Humans; ras Proteins; RNA, Small Interfering; Signal Transduction

Although p21 is an important component that regulates cell-cycle progression, no consensus has been reached about its clinicopathological significance in esophageal squamous cell carcinoma (ESCC). In the present study, we investigated its prognostic significance and correlation with cyclin-D1 (CCND1) expression in ESCC.. The p21 labeling index (LI) was calculated by immunohistochemistry for 69 primary tumor samples obtained from patients with ESCC who had undergone curative esophagectomy, and correlations between p21 LI and various clinicopathological features, prognosis, and CCND1 LI were studied.. The p21 LI of these tumors ranged from 2.0% to 57.0% (median=28.4%, mean±SD=27.3% ± 13.0). p21 LI was positively correlated with CCND1 LI. When patients were divided into two groups using a p21 LI cut-off value of 30%, the 5-year survival rate of patients with p21 LI of ≥ 30% was 80.0%, which was significantly higher than that of patients with p21 LI of <30% (55.5%). Furthermore, when patients were divided into four groups according to p21 and CCND1 expression, the 5-year survival rate of patients with p21 LI of <30% and CCND1 LI of ≥ 45% was the lowest (44.4%). Multivariate analysis demonstrated that venous invasion, lymphatic invasion, and p21 LI were independent prognostic factors.. Our results indicate that p21 LI is correlated with CCND1 LI and can be used as an independent prognostic factor for patients with ESCC following selection of a suitable cut-off value.

Topics: Aged; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Esophagus; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Mucous Membrane; Multivariate Analysis; Prognosis; Proportional Hazards Models; Retrospective Studies

Recent population studies suggest that the use of metformin is associated with reduced incidence and improved prognosis of certain cancers.. In the current study, we assessed the effect of metformin on esophagus cancer cells using two cell lines (Eca-109 and TE-1 cells).. We found that metformin inhibited growth and decreased expression of cell-cycle regulators in these cells. Treatment with metformin was also associated with activation of AMP kinase and inhibition of mTOR/p70S6K/pS6 signaling in both cells. However, inhibition of AMPK signaling has little impact on the anti-proliferative roles of metformin. In addition, we found USP7, a positive regulator of tumor suppressor p53, as a new molecular target of metformin. Esophagus cancer cells can be protected against metformin-induced growth inhibition by small interfering RNA against USP7.. These results provide evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent population studies and justify further work to explore potential roles for it in esophagus cancer prevention and treatment.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Esophageal Neoplasms; Humans; Metformin; Protein Stability; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Ubiquitin Thiolesterase; Ubiquitin-Specific Peptidase 7; Up-Regulation

Cyclin D1 (CCND1) plays a significant role in G1-S transition of cell cycle, and phosphatase and a tensin homologue (PTEN) negatively regulate cell cycle through phosphatidylinositol 3-kinase (PI3K)/AKT signaling. CCND1 and PTEN genetic polymorphisms might induce susceptibility to the occurrence of esophageal squamous cell carcinoma (ESCC). Three hundred and four ESCC patients and 413 healthy controls from Anyang, China, were enrolled in this study. All genotyping at CCND1 (807 G/A) and PTEN (rs701848 T/C and rs2735343 C/G) were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Unconditional logistic regression model was used to analyze the correlation between the polymorphisms and the susceptibility to develop ESCC. Statistically significant differences were observed between cases and controls in distribution of genotypes or alleles at PTEN rs701848 T/C and rs2735343 C/G, with either haplotype TG or CG possessing notably higher proportion in cases than in the controls. However, such difference could not be found in the distribution of the polymorphisms at CCND1 807 G/A. In summary, the polymorphisms of PTEN rs701848 T/C and rs2735343 C/G might represent crucial modifying factors for development of ESCC.

Topics: Carcinoma, Squamous Cell; Case-Control Studies; Cyclin D1; Disease Progression; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Haplotypes; Humans; Male; Middle Aged; Phosphatidylinositol 3-Kinase; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction

Mammalian target of rapamycin (mTOR) has emerged as a key regulator of cell metabolism, growth, and proliferation. Despite the increasing significance of mTOR signaling in cancer cell cycle and proliferation, the clinical significance of activated mTOR in esophageal squamous cell carcinoma and its role in esophageal cancer cell proliferation and invasion remain unclear. Here, we show that both high levels of phosphorylated-mTOR and an increased ratio of phosphorylated-mTOR/mTOR (ratio ≥0.2) were significantly associated with shortened disease-specific survival in 165 patients with esophageal squamous cell carcinoma in univariate analysis (P = .047 for phosphorylated-mTOR, P = .021 for phosphorylated-mTOR/mTOR); phosphorylated-mTOR and phosphorylated-mTOR/mTOR remained independent prognostic factors after adjusting for age, TNM stage, chemotherapy, and radiation therapy in multivariate analysis (hazard ratio, 1.67, P = .025 for phosphorylated-mTOR; hazard ratio, 1.95, P = .006 for phosphorylated-mTOR/mTOR). Moreover, down-regulation of mTOR or mTOR complex components led to attenuation of proliferation, migration, and invasion of esophageal squamous cell carcinoma cell lines through suppression of cyclin D1 expression. Collectively, our findings suggest that phosphorylated-mTOR and the ratio of phosphorylated-mTOR/mTOR are closely linked to tumor progression and represent independent prognostic factors in esophageal squamous cell carcinoma, thereby providing a potential therapeutic target for this malignancy.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Cyclin D1; Disease Progression; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Phosphorylation; Prognosis; Signal Transduction; TOR Serine-Threonine Kinases; Young Adult

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal tumors in the world, and the development of new therapeutic targets is needed. Recent studies have shown that aerobic glycolysis, also known as the Warburg effect, mediated the anti-apoptotic effects in cancer cells. Lactate dehydrogenase A (LDHA) which executed the final step of aerobic lactate production has been reported to be involved in the tumor progression. However, the function of LDHA in ESCC has not been investigated. In this study, it was found that LDHA was up-regulated in ESCC clinical samples. Knockdown of the expression of LDHA inhibited cell growth and cell migration in vitro as well as tumorigenesis in vivo. With regard to the molecular mechanism, silencing the expression of LDHA was related to decreased AKT activation and cyclin D1 expression and increased cleavage of PARP and caspase 8. Taken together, our findings suggest that LDHA plays an important role in the progression of ESCC by modulating cell growth, and LDHA might be a potential therapeutic target in ESCC.

Topics: Adult; Apoptosis; Carcinoma, Squamous Cell; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Enzyme Activation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Glycolysis; Humans; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Male; Middle Aged; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Up-Regulation

Ribosomal protein S6 is a key regulator of 40S ribosome biogenesis, and its phosphorylation is closely related to cell growth capacity. However, as a downstream target of S6 kinases, the clinical significance and the roles of S6 and S6 phosphorylation in cell viability and motility of esophageal squamous cell carcinoma remain unclear. Here, we show that high level of phosphorylated-ribosomal protein S6 (p-S6) (immunohistochemistry score ≥5) and an increased ratio of p-S6/S6 (immunohistochemistry score ≥0.75) were significantly associated with shortened disease-free survival in patients with esophageal squamous cell carcinoma in univariate analysis (P=0.049 and P<0.001, respectively). After adjusting for age, tumor-nodes-metastasis stage, chemotherapy, and radiation therapy in multivariate analysis, both p-S6 (hazard ratio 2.21, P=0.005) and p-S6/S6 (hazard ratio 2.40, P<0.001) remained independent adverse prognostic factors. In addition, S6 and S6 kinase 1 knockdown resulted in attenuation of viability by suppressing cyclin D1 expression in esophageal cancer cells. Furthermore, depletion of S6 and S6 kinase 1 resulted in a reduction in esophageal cancer cell migration and invasion. This was paralleled by a reduction in focal adhesion and by suppression of extracellular signal-regulated kinase and c-jun N-terminal kinase phosphorylation, which control cell motility. Collectively, these findings suggest that p-S6 and the ratio of p-S6/S6 are closely relevant to tumor progression and have prognostic significance in esophageal squamous cell carcinoma.

Topics: Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Disease-Free Survival; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Female; Focal Adhesions; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Phosphorylation; Prognosis; Proportional Hazards Models; Ribosomal Protein S6; Ribosomal Protein S6 Kinases; Risk Assessment; Risk Factors; RNA Interference; Time Factors; Transfection

Emerging evidence has demonstrated that high-temperature requirement protein A1 (HtrA1) appears to be involved in several important biological processes in mammals such as growth, apoptosis, embryogenesis, invasion, metastasis, and cancer and has been verified to be reduced in a variety of human tumors. However, its precise functions and molecular mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. Here, we detected HtrA1 level in ESCC tissues and cells and investigated the biological roles of HtrA1 in ESCC. We found that expressions of HtrA1 mRNA and protein in ESCC tissues and cells were significantly lower than those in normal esophageal epithelial tissues and cells (P < 0.05). Expressions of HtrA1 mRNA and protein were closely associated with TNM staging and lymph node metastasis (P < 0.05). Additionally, the survival rate of patients with low HtrA1 level was lower than those patients with high HtrA1 level (P < 0.05). Elevated HtrA1 level markedly inhibited cell proliferation in vitro and in vivo, reduced cell invasion in vitro, and induced cell apoptosis. Notably, HtrA1 overexpression inhibited phosphorylation levels of IκBα and p65 subunit of the NF-κB signaling pathway, but increased total IκBα level, coupled with decreases of Ki-67, Bcl-2, Bcl-xL, cyclin D1, and MMP-9 proteins and increase of caspase-3 activity. Overall, these data suggest that HtrA1 may play critical roles in the tumorgenesis and progression of ESCC, and HtrA1 overexpression exerts its anti-tumor effect by blocking the NF-κB signaling pathway; thus, manipulation of HtrA1 may be an effective molecular target for ESCC treatment.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; High-Temperature Requirement A Serine Peptidase 1; Humans; I-kappa B Proteins; Ki-67 Antigen; Lymphatic Metastasis; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Transplantation; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; RNA, Messenger; Serine Endopeptidases; Signal Transduction; Survival Rate; Transcription Factor RelA; Transplantation, Heterologous

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal tumors in the world. Thus, it is very urgent to develop new therapeutic targets against this disease. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase, is required for the generation of several fundamental end products including cholesterol and isoprenoids. The function of the MVA pathway in ESCC has not been investigated. In this study, it was found that the MVA pathway was upregulated in ESCC clinical samples. Statin, the inhibitor of the MVA pathway, exerted potent cytotoxicity against human ESCC cells by inhibiting cell growth and proliferation, while it exerted lesser effects on non-tumorigenic SHEE cells. Further study revealed that statin could potently induce cell apoptosis and cell cycle arrest and also dose-dependently inhibit the growth of xenograft tumors in nude mice. With regard to the molecular mechanism, statin treatment was related to decreased extracellular signal-regulated kinase activation and proliferating cell nuclear antigen, cyclin D1 expression, and increased cleavage of poly(ADP-ribose) polymerase. Taken together, our findings suggest that the MVA pathway plays an important role in the progression of ESCC by modulating cell growth and statin might be a potential therapeutic agent in ESCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Extracellular Signal-Regulated MAP Kinases; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; MAP Kinase Signaling System; Mevalonic Acid; Mice; Mice, Nude; Neoplasm Transplantation; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Transplantation, Heterologous

Endoscopic resection is a less invasive treatment than esophagectomy for superficial esophageal squamous cell carcinoma, but patients with lymph node metastasis need additional treatment after endoscopic resection. The purpose of this study was to establish a set of indicators to identify superficial esophageal squamous cell carcinoma patients at a high risk of metastasis. In all, 271 superficial esophageal squamous cell carcinoma esophagectomy cases were reviewed retrospectively. The relationships between clinicopathological parameters and immunohistochemical findings (p53, cyclin D1, EGFR and VEGF) on tissue microarrays, on the one hand, and lymph node metastasis were assessed by univariate and multivariate logistic regression analyses. Patients with intraluminal masses and ulcerated masses had a high risk of lymph node metastasis. Patients with superficial esophageal squamous cell carcinoma (1) thinner than 1200 μm; (2) confined to the mucosa; (3) with submucosal invasion <250 μm; (4) with submucosal invasion ≥250 μm but with negative VEGF expression and well/moderately differentiated or basaloid histology; or (5) with submucosal invasion ≥250 μm but with weak VEGF expression and well-differentiated histology had almost no risk of lymph node metastasis. We recommend endoscopic resection for all erosive, papillary and plaque-like superficial esophageal squamous cell carcinomas where endoscopic resection is clinically feasible, and esophagectomy for all other erosive, papillary and plaque-like cases and all intraluminal masses and ulcerated tumors. No additional treatment is needed for endoscopic resection cases with superficial esophageal squamous cell carcinoma (1) thinner than 1200 μm; (2) confined to the mucosa; (3) with submucosal invasion <250 μm; (4) with submucosal invasion ≥250 μm but with negative VEGF expression and well/moderately differentiated or basaloid histology; or (5) with submucosal invasion ≥250 μm but with weak VEGF expression and well-differentiated histology. These clinical and pathological criteria should enable more accurate selection of patients for these procedures.

Topics: Algorithms; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Disease Progression; ErbB Receptors; Esophageal Neoplasms; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Mucous Membrane; Prognosis; Tissue Array Analysis; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

To determine the functional significance of TC21 in esophageal squamous cell carcinoma (ESCC).. TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and transcripts were analyzed by reverse transcription-polymerase chain reaction and protein by Western blotting. We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assessing the phosphorylation status of its downstream targets, phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), protein kinase B (pAkt), nuclear factor-κB (NF-κB) and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treatment was carried out by cell viability assay and cell cycle analysis using flow cytometry.. TC21 knockdown in human ESCC cell line TE13 cells, showed only a marginal increase (14.2%) in cell death compared with control cells. The expressions of the signaling proteins PI3K and pAkt, transcription factor NF-κB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregulation, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death (38.4%) was observed in cells treated with cisplatin after TC21 knockdown compared with cells which were treated with cisplatin alone (20% cell death).. Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-κB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Gene Knockdown Techniques; Humans; Membrane Proteins; Monomeric GTP-Binding Proteins; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Transfection

The aim of the present study was to explore mechanisms underlying the effects of down-regulating β-catenin expression on esophageal carcinoma (EC) cells.. Cell cycle distribution and apoptosis were determined using flow cytometry and annexin V apoptosis assay, respectively. Transmission electron microscopy (TEM) was used to examine changes in ultrastructure, while expression of cyclin D1 protein and mRNA was detected by western blot and real-time PCR. Proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated kinase (ERK) 1/2 were evaluated by Western blot analysis. PCNA labeling index (LI) was determined by immunocytochemistry.. Compared with pGen-3-con transfected and Eca-109 cells, the percentage of G0/G1-phase pGen-3-CTNNB1 transfected cells was obviously increased (P<0.05), with no significant difference among the three groups with regard to apoptosis (P>0.05). pGen-3-CTNNB1 transfected cells exhibited obvious decrease in cyclin D1 mRNA and protein expression (P<0.05) and the ultrastructure of Eca-109 cells underwent a significant change after being transfected with pGen-3-CTNNB1, suggesting that down-regulating β-catenin expression can promote the differentiation and maturation. The expression of PCNA and the ERKI/2 phosphorylation state were also down-regulated in pGen-3-CTNNB1 transfected cells (P<0.05). At the same time, the PCNA labeling index was decreased accordingly (P<0.05).. Inhibition of EC Eca-109 cellproliferation by down-regulating β-catenin expression could improve cell ultrastructure by mediating blockade in G0/G1 through inhibiting cyclin D1, PCNA and the MAPK pathway (p-ERK1/2).

Topics: Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Proliferating Cell Nuclear Antigen; RNA Interference; RNA, Messenger; RNA, Small Interfering

Esophageal carcinoma (EC) is one of the most aggressive cancers with a poor prognosis. Understanding the molecular mechanisms underlying esophageal cancer progression is a high priority for improved EC diagnosis and prognosis. Recently, MSP58 was shown to behave as an oncogene in colorectal carcinomas and gliomas. However, little is known about its function in esophageal carcinomas. We therefore examined the effects of MSP58 knockdown on the growth of esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo in order to gain a better understanding of its potential as a tumor therapeutic target. We employed lentiviral-mediated small hairpin RNA (shRNA) to knock down the expression of MSP58 in the ESCC cell lines Eca-109 and EC9706 and demonstrated inhibition of ESCC cell proliferation and colony formation in vitro. Furthermore, flow cytometry and western blot analyses revealed that MSP58 depletion induced cell cycle arrest by regulating the expression of P21, CDK4 and cyclin D1. Notably, the downregulation of MSP58 significantly inhibited the growth of ESCC xenografts in nude mice. Our results suggest that MSP58 may play an important role in ESCC progression.

Topics: Animals; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; HEK293 Cells; Humans; Mice; Mice, Nude; Nuclear Proteins; Prognosis; RNA-Binding Proteins

Although constitutive activation of nuclear factor-kappaB (NF-κB) signaling pathway has been reported in multiple different human tumors, the role of NF-κB pathway in esophageal squamous cell carcinoma (ESCC) remains ill-defined. Abundant sources have provided interesting insights into the multiple mechanisms by which curcumin may mediate chemotherapy and chemopreventive effects on cancer. In this study, we first analyzed the status of NF-κB pathway in the two ESCC cell lines Eca109 and EC9706, and then further investigated whether curcumin alone or in combination with 5-fluorouracil (5-FU) could modulate NF-κB pathway in vitro and in vivo. The results showed that NF-κB signaling pathway was constitutively activated in the ESCC cell lines. Curcumin suppressed the activation of NF-κB via the inhibition of IκBα phosphorylation, and downregulated the expressions of Bcl-2 and CyclinD1 in ESCC cell lines. Curcumin combined with 5-FU led to the lower cell viability and higher apoptosis than 5-FU treated alone. In a human ESCC xenograft model, curcumin or 5-FU alone reduced the tumor volume, but their combination had the strongest anticancer effects. Besides, curcumin could also inhibit NF-κB signaling pathway through downregulation of the IκBα phosphorylation and induction of cell apoptosis in vivo. Overall, our results indicated that constitutively activated NF-κB signaling pathway exists in the two ESCC cells and the chemopreventive effects of curcumin were associated with downregulation of NF-κB signaling pathway and its downstream genes.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Curcumin; Cyclin D1; Down-Regulation; Drug Synergism; Esophageal Neoplasms; Fluorouracil; Humans; I-kappa B Proteins; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Nude; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed malignant tumors in North China. We have identified that Wnt2/β-catenin pathway is activated in ESCC cells and sodium nitroprusside (SNP) and siRNA against β-catenin not only inhibit the expressions of β-catenin and its major downstream effectors including c-myc and cyclin D1 but induce cell cycle arrest and apoptosis. The purpose of the present study was to analyze the relationship between pathological parameters including invasion depth and lymph node metastasis and the expressions of β-catenin, c-myc, and cyclin D1 in order to evaluate their values of prognosis in patients with ESCC. The expressions of β-catenin, c-myc, and cyclin D1 were detected immunohistochemically in the resected cancer tissues from 40 patients with ESCC. The β-catenin expression was reduced in 22 (55.0%) patients, which was closely correlated with invasion depth (P = 0.023) and lymph node metastasis (P = 0.003). There was the positive c-myc expression in 21 (52.5%), which was significantly correlated with invasion depth (P = 0.009) and lymph node metastasis (P = 0.001). Furthermore, the results of survival rates analyzed by Kaplan-Meier curve revealed that patients with the reduced expression of β-catenin had a poorer prognosis than those with the preserved expression (P = 0.031), and patients with the positive expression of c-myc also had a significantly poorer prognosis than those with the negative expression (P = 0.008). These findings demonstrate that β-catenin pathway plays a crucial role in the progression of ESCC, suggesting that both β-catenin and c-myc may be used as markers for predicting the prognosis of patients with ESCC.

Topics: beta Catenin; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Prognosis; Proto-Oncogene Proteins c-myc; Survival Rate

Investigation of potential association of SNPs (G870A, rs9344; G1722C, rs678653) of cyclin D1 gene (CCND1) with susceptibility to esophageal squamous cell carcinoma (ESCC) in Kashmir valley (India). The study included 302 subjects comprising 151 ESCC cases and 151 controls. PCR-RFLP and direct sequencing were employed for genotyping. The G870A polymorphism, the individuals carrying GA + AA genotype was having 2.80-fold increased risk for development of ESCC (OR 2.8, 95% CI = 1.77-4.4; P = 0.0001) compared to GG genotype. Further a significantly higher risk was observed in individuals who consume >3 cups per day of salted tea (OR = 5.1; 95% CI = 1.6-16.7; P = 0.0016) and had smoking habits (OR = 6.3; 95% CI = 2.9-13.9; P = 0.0005). We also demonstrate for the first time in CCND1 1722 locus, the CC genotype was strongly associated with increased risk of developing ESCC (OR = 2.58; 95% CI = 1.61-4.15; P = 0.0001). In addition, the frequency of polymorphic C allele was also found to be higher in cases (OR = 1.92; 95% CI = 1.37-2.69; P = 0.0002). There appears to be an influence of CCND1 G870A/G1772C genotypes on genetic susceptibility to ESCC.

Topics: Adult; Carcinoma, Squamous Cell; Cyclin D1; Electrophoresis, Polyacrylamide Gel; Esophageal Neoplasms; Female; Genetic Predisposition to Disease; Genotype; Humans; India; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk Factors

The correlation of biomarker expression between primary tumors and corresponding metastases has not yet been well reported in esophageal squamous cell carcinoma (ESCC). This study was to confirm whether primary ESCC tumors differ from their regional metastatic lymph nodes (RMLN) in CyclinD1, p53, E-cadherin, and vascular endothelial growth factor (VEGF) expression and determine prognostic value of their alteration.. There were 134 patients with stage T3N1-3M0 ESCC recruited for the research. Expression of CyclinD1, p53, E-cadherin, and VEGF was evaluated in primary ESCC tumors and their paired RMLN assembled on tissue microarrays by immunohistochemistry (IHC). The comparison of expression in different lesion and their correlation with prognosis was analyzed.. E-cadherin was discordant expression in 55.2% cases and appeared to be more frequently positive in metastatic lymph nodes (P < 0.001). The VEGF expression level was significantly higher in primary tumors (P < 0.001). Combined analysis of VEGF expressions in paired lesions (P = 0.003) and its decreased expression (P = 0.006) were both predictive.. Biomarker expression was discordant between the primary tumor and its paired lymphatic metastasis in over 25% of patient with ESCC. VEGF discordant expression was a new prognostic factor and combined analysis of expression in paired lesions was useful to predict. Analysis of protein expression only in primary tumors would be inadequate to judge prognosis.

Topics: Adult; Aged; Aged, 80 and over; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Rate; Tissue Array Analysis; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

Cyclin D1 plays important roles in esophageal squamous cell carcinoma (ESCC) cases by amplification of the 11q13.3 locus. FBXO31 is a subunit of the SCF ubiquitin ligase, which targets cyclin D1 for degradation. In this study, we clarified the clinical significance of FBXO31 and characterized the association between cyclin D1 and FBXO31 in ESCC cases. Total RNA was extracted from tumor tissues obtained from 68 ESCC patients who underwent surgical resection. FBXO31 expression levels were determined by quantitative RT-PCR, and both FBXO31 and cyclin D1 protein expression and localization were evaluated by immunohistochemistry (IHC). Furthermore, using CGH and gene expression array data of another subset, we validated the association between cyclin D1 genomic amplification and FBXO31 expression levels. Higher FBXO31 expression levels significantly correlated with depth of tumor invasion and clinical stage (P<0.05). In addition, the FBXO31 high expression group showed a significantly poorer prognosis than the low expression group (P<0.001). Multivariate analysis indicated that FBXO31 expression was an independent prognostic factor [relative risk (RR): 1.79, confidence interval (CI): 1.14-3.01, P=0.01]. Using IHC, concordant expression was observed between cyclin D1 and FBXO31 in the nucleus and cytoplasm, respectively. CGH array indicated that cases having cyclin D1 with increased copy number were significantly associated with elevated FBXO31 expression levels (P<0.05). FBXO31 could be a novel and robust prognostic marker for ESCC.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; F-Box Proteins; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Prognosis; RNA, Messenger; Survival Analysis; Tumor Suppressor Proteins

Zinc deficiency was implicated in the etiologies of human esophageal squamous cell carcinoma (ESCC). Wild-type p53-induced gene 1 (WIG-1), a kind of zinc finger protein, was cloned from the human 3q26.3 region and encoded a putative polypeptide of 289 amino acids. Our previous studies have demonstrated that the expression of WIG-1 was downregulated in ESCC tissues. Herein, we investigated the effect of zinc on cell proliferation, apoptosis, as well as expression of WIG-1 in EC109 cells. Meanwhile, an RNAi vector of WIG-1 was transfected into EC109 cells and the effect of zinc on WIG-1 expression was investigated. We found that zinc could suppress cell proliferation and induce G0/G1 cell cycle arrest and apoptosis of EC109, and this efficacy might result from the expression altering of several apoptosis-related genes, such as Bax, p21 ( WAF ), and cyclin D1. In particular, upregulation of WIG-1 was observed after zinc supplementation, indicating that WIG-1 might be involved in the zinc-induced cell cycle arrest and apoptosis of EC109 cells by regulating the expression of Bax, p21 ( WAF ), and cyclin D1.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Squamous Cell; Carrier Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Nuclear Proteins; RNA-Binding Proteins; Transfection; Tumor Suppressor Protein p53; Up-Regulation; Zinc

Esophageal cancer is one of the most common malignancies and is associated with a dismal prognosis. Although treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We found that Baohuoside-I, a flavonoid extracted from a Chinese medicinal plant, exhibits anticancer activity. Here, we demonstrated that Baohuoside-I significantly inhibited Eca109 human esophageal squamous carcinoma cell proliferation and induced Eca109 cell apoptosis in vitro and in vivo. The growth inhibitory effect of Baohuoside-I on the Eca109 tumor cell line was examined by MTT assay; the induction of apoptosis was analyzed by flow cytometry. Eca109-luc cells were injected into the subcutaneous tissue of nude mice to establish xenograft tumors. Our results revealed that Baohuoside-I caused a dose- and time-dependent inhibition of cell growth and an induction of apoptosis. Furthermore, Baohuoside-I-treated cells were characterized by decreased expression of the β-catenin gene and protein in the total cell lysates. Thus, the gene and protein expression of the downstream elements survivin and cyclin D1 was downregulated. To determine the precise inhibitory mechanisms involved, further in-depth in vivo studies of Baohuoside-I are warranted. Our study provides the first evidence that Baohuoside-I inhibits tumor growth and induces apoptosis by inhibiting β-catenin-dependent signaling pathways. Thus, Baohuoside-I is a potential candidate in ESCC disease therapy.

Topics: Animals; beta Catenin; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; Drugs, Chinese Herbal; Esophageal Neoplasms; Female; Flavonoids; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Signal Transduction; Survivin; Xenograft Model Antitumor Assays

Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRAS(G12V) and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma.

Topics: Animals; Carcinoma; Cell Line, Tumor; Cell Survival; Cyclin D1; Cytotoxicity, Immunologic; Esophageal Neoplasms; Female; Genes, ras; Genetic Therapy; Genetic Vectors; Humans; Immunotherapy; Mice; Mice, Inbred C57BL; Neoadjuvant Therapy; Neoplasm Recurrence, Local; Neoplasms, Experimental; Simplexvirus; Thymidine Kinase; Treatment Outcome; Tumor Suppressor Protein p53

p53 Arg72Pro, MDM2 T309G, and CCND1 G870A are functional single-nucleotide polymorphisms (SNPs) in key genes that regulate apoptosis and cell cycle. Variant genotypes of these SNPs have been associated with increased risk and earlier age of onset in some cancers. We investigated the association of these SNPs with susceptibility to esophageal adenocarcinoma in a large, North American case-control study. Three hundred and twelve cases and 454 cancer-free controls recruited in Boston, USA were genotyped for each of the three SNPs, and demographic and clinical data were collected. Genotype frequencies for each of the three SNPs did not deviate from the Hardy-Weinberg equilibrium, and did not differ between cases and controls. Odds ratios (OR), adjusted for clinical risk factors, for the homozygous variant genotypes were 0.99 (95% confidence interval [CI] 0.57-1.72) for p53 Pro/Pro, 0.81 (95% CI 0.52-1.28) for MDM2 G/G, and 0.97 (95% CI 0.64-1.49) for CCND1 A/A. The analysis was adequately powered (80%) to detect ORs of 1.37, 1.35, and 1.34 for each SNP, respectively. In contrast to the results of smaller published studies, no association between p53 Arg72Pro, MDM2 T309G, and CCND1 G870A SNPs and susceptibility to esophageal adenocarcinoma, age of onset, or stage of disease at diagnosis was detected.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Case-Control Studies; Cyclin D1; Esophageal Neoplasms; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

We aimed to develop a new biomarker to predict cyclin D1 (CCND1) status using plasma DNA in oesophageal squamous cell carcinoma (ESCC) patients.. We evaluated the ratio of the CCND1 (11q13) dosage to the dopamine receptor D2 (DRD2; 11q22-23) dosage (C/D ratio) as CCND1 copy number. This study was divided into three steps: (1) Determination of a cutoff value for the C/D ratio in test scale; (2) Comparison of the C/D ratio in between plasma samples and cancer tissues in ESCC patients showing high plasma C/D ratio; (3) Validation study of the clinical application of the plasma C/D ratio as a diagnostic and prognostic marker, by comparing with clinicopathologic factors in 96 ESCC patients.. The plasma C/D ratio was significantly higher in the ESCC group than the controls (P=0.0134). A high plasma C/D ratio reflected the tumour C/D ratio, and significantly correlated with a poorer prognosis (P=0.0186). Moreover, the high C/D ratio was found to be an independent prognostic factor on multivariate analysis (P=0.0266; hazard ratio 5.988).. Prediction of CCND1 amplification using plasma DNA is thought to be a promising prognostic biomarker in ESCC patients.

Topics: Carcinoma, Squamous Cell; Cyclin D1; DNA, Neoplasm; Esophageal Neoplasms; Gene Amplification; Gene Dosage; Humans; Polymerase Chain Reaction; Prognosis; Receptors, Dopamine D2; Recurrence; Survival Analysis; Survival Rate

Dysregulation of cell cycle control, especially G1/S phase transition, is implicated in the pathogenesis of most human cancers, including esophageal squamous cell carcinoma (ESCC). However, the clinicopathological significance of aberrant expression of G1/S phase-related proteins in ESCC remains unclear. In the present study, cyclin D1, p21WAF1/Cip1 and p53 protein expression were examined in 148 ESCC cases using immunohistochemistry combined with tissue microarray. We then analyzed the correlations between their expression and clinicopathological parameters and found that overexpression of p53 was associated with lymph node metastasis (P=0.001). p21WAF1/Cip1 down-regulation was an independent prognostic factor by multivariate analysis (RR=0.418, P<0.001). Further more, array-CGH results revealed that cyclin D1 gene was amplified in 45.4% of ESCC patients. This study confirms that dysregulation of G1/S related genes is common in ESCC and reduced expression of p21WAF1/Cip1 protein can predict shorter overall survival time of patients with ESCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Comparative Genomic Hybridization; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Prognosis; S Phase; Tissue Array Analysis; Tumor Suppressor Protein p53

The purpose of this study was to evaluate the use of thymidilate synthetase (TS), thymidilate phosphorylase (TP), dihydropyrimidin dehydrogenase (DPD), Her-2/neu, and cyclin D1 as predictors of therapy response, survival, and recurrence in patients with esophageal squamous cell carcinoma (ESCC) following radiochemotherapy.. Twenty-six patients with histologically proven intrathoracic, locally advanced ESCC (cT3, cN0/+, cM0) underwent preoperative, combined simultaneous radiochemotherapy followed by R0-transthoracic esophagectomy. Because R0 resection is the strongest known independent prognostic factor in this tumor entity, only R0-resected patients were included in this study. Pre-therapeutically taken, formalin-fixed, and paraffin-embedded tumor biopsies were used for laser-assisted microdissection of tumor cells and RNA extraction and subjected to real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR).. No significant correlation between clinical or histopathological parameters and the relative gene expression of TS, TP, DPD, or Her-2/neu was observed. However, patients with relative cyclin D1 levels below the median gene expression did not reach median survival compared to the 19.9 months seen in patients with relative cyclin D1 gene expression above the median (P = 0.02). Patients with low cyclin D1 levels experienced significantly less frequent recurrence of the tumor (20% versus 63%; P = 0.006), and there was a significant difference in the recurrence-free interval (P = 0.003).. Despite the small number of investigated patients, our data seem to show that high levels of cyclin D1 measured by real-time Q-RT-PCR before neoadjuvant radiochemotherapy correlate significantly with patient survival, tumor recurrence, and recurrence-free-interval. Cyclin D1 might be useful in identifying patients at high risk of poor prognosis and suffering from recurrence after neoadjuvant radiochemotherapy treatment and R0 resection. Further investigations with a larger cohort are warranted.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Dihydrouracil Dehydrogenase (NADP); Esophageal Neoplasms; Esophagectomy; Female; Gene Expression; Humans; Male; Middle Aged; Neoadjuvant Therapy; Neoplasm Recurrence, Local; Prognosis; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; Thymidine Phosphorylase; Thymidylate Synthase

The relationship between smoking and esophageal squamous cell carcinoma (ESCC) has been confirmed by epidemiology. Cyclin D(1) plays a critical role in regulating the cell cycle; it is an important regulator of cell cycle and can function as a transcriptional co-regulator. The importance of cyclin D(1) makes it an attractive target for anticancer therapy. Human ESCC cell line EC109 was cultured with aspirin and cigarette smoke extract (CSE) at different concentrations for 48 h. Cell growth was tested with 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide reduction assay; cyclin D(1) mRNA level was detected by reverse transcription-polymerase chain reaction assays; protein level of cyclin D(1) was detected by Western blot; the cell cycle change was monitored by flow cytometry detection assays. CSE stimulated cell proliferation, increased the protein level of cyclin D(1) in a dose-dependent manner (P < 0.01), and decreased the proportion of G(0)/G(1) phase cell of cell cycle. However, aspirin can inhibit the cell growth and suppress the protein level of cyclin D(1) after CSE affected the EC109 cell line in a dose-dependent manner (P < 0.01). Meanwhile, aspirin increased the proportion of G(0)/G(1) phase cell, while that of S and G(2)/M phases decreased. Aspirin can inhibit the cell growth and suppress the protein level of cyclin D(1) after CSE affected EC109 cell line. The probable mechanism is through decreasing the expression of cyclin D(1), thus stopping the transition of cell cycle from G(0)/G(1) to S phase.

Topics: Analysis of Variance; Aspirin; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Esophageal Neoplasms; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Nicotiana; Probability; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Smoke

Cyclin D1 plays a pivotal role in cell-cycle transition through G1 phase. In this article, we found that Degenerative Spermatocyte Homolog 1 (DEGS1) up-regulated the expression of cyclin D1 and the activation of transcription factor NF-kappaB was essential for DEGS1-induced cyclin D1 production. Forced expression of DEGS1 in Esophageal carcinoma cell line Eca109 cells increased their ability of cell migration and significantly induced tumor metastasis in nude mice, whereas RNA interference-mediated knockdown of DEGS1 cells significantly inhibited cell migration in vitro, as well as tumor metastasis in vivo. Our results demonstrated that expression of DEGS1 up-regulated the expression of cyclin D1 and enhanced the efficiency of tumor metastasis.

Topics: Animals; Blotting, Western; Cadherins; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Fatty Acid Desaturases; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Luciferases; Mice; Mice, Nude; Neoplasm Metastasis; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transfection; Tumor Cells, Cultured; Up-Regulation

Cell cycle regulators, such as cyclinD1 and p53, play major roles in the tumor response to radiation and chemotherapy in esophageal squamous cell carcinoma (SCC). Pin1-mediated prolyl-isomerization potentiates cell cycle progression and cell proliferation, including the regulation of cyclinD1 and p53. Herein, we investigated the effect of Pin1 in association with cyclinD1 and p53 on the sensitivity of esophageal SCC to chemoradiotherapy (CRT). The expression levels of Pin1, cyclinD1 and p53 were examined immunohistochemically in endoscopic biopsy specimens from 68 advanced esophageal SCC patients before CRT to determine whether their expression levels predicted the clinical effectiveness of CRT in individual cancers. Forty-six of the 68 patients (67.6%) had an effective response to CRT, whereas 22 patients (32.4%) had an ineffective response. There was no significant correlation between clinical responses and expression levels of cyclinD1 or p53. However, the clinical response of the high Pin1 expression group was significantly higher than that of the low expression group (P=0.0200). Moreover, our data indicate that the combined immunohistochemical evaluation of Pin1, cyclinD1 and p53 expression in pretreatment biopsy samples is a useful indicator of sensitivity to CRT in advanced esophageal SCC. Thus, Pin1 may influence cyclinD1 and p53 functions and predict CRT sensitivity.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclin D1; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Tumor Suppressor Protein p53

Neoadjuvant chemotherapy may improve the outcome of esophageal cancer after esophagectomy, but is accompanied by considerable toxicity by collateral destruction of normal cells. Such side effects may be avoided by developing therapies that specifically target molecular characteristics of tumors. The aim of the present study was to determine the proportion of esophageal squamous cell carcinoma (ESCC) patients that could possibly benefit from (a combination of) currently available targeted therapies, by assessing the frequency of immunohistochemical expression of their target molecular markers in ESCC tissues. Sections from a validated tissue microarray comprising 108 ESCCs were immunohistochemically stained for Bcl-2, c-KIT, cyclo-oxygenase-2 (COX-2), cyclin D1, estrogen receptor (ER), epidermal growth factor receptor (EGFR), Her-2/neu, progesterone receptor (PR), and vascular endothelial growth factor (VEGF). VEGF, cyclin D1, EGFR, and COX-2 could be detected in 55, 42, 40, and 40%, respectively. Her-2/neu, Bcl-2, and c-KIT were detected in 12, 11, and 10% of the tumors, respectively. No nuclear expression of ER or PR was noticed. Concurrent expression of two markers was noticed in 28% of ESCCs, whereas 25% of ESCCs showed concurrent expression of three markers. The concurrent expression of two of the most frequently expressed markers (VEGF, cyclin D1, EGFR, and COX-2) ranged from 11 (COX-2 and EGFR) to 26% (cyclin D1 and VEGF). The expression of all of these four markers was seen in 5% of ESCCs. Promising targets for molecular therapy in ESCC appear to be COX-2, VEGF, EGFR, and cyclin D1, as they are frequently overexpressed. Phase II clinical studies on these molecular markers may therefore be warranted. The role for targeted therapy against ER, PR, Her-2/neu, c-KIT, or Bcl-2 in ESCC seems limited.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Cyclooxygenase 2; ErbB Receptors; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Neoplasm Staging; Protein Array Analysis; Receptors, Estrogen; Vascular Endothelial Growth Factors

The genesis of esophageal squamous cell carcinoma(ESCC)is a multifactor and multistage process, in which Wnt signaling transduction pathway plays an important role in tumorigenesis and tumor progression. This study was to investigate the roles of four proteins in the Wnt pathway in tumorigenesis of ESCC, and their significances in the early diagnosis of ESCC.. The expression of adenomatous polyposis coli (APC), beta-catenin, E-cadherin and cyclinD1 was detected by immunohistochemistry using tissue microarrays consisting of 199 specimens of ESCC, 164 specimens of normal mucosa, 34 specimens of basal cell hyperplasia and 30 specimens of dysplasia adjacent to cancer tissues.. The positive rates of APC and E-cadherin in ESCC were lower than those in the normal group (69.6% vs. 98.0%, p < 0.01; 19.6% vs. 96.3%, p < 0.01). The abnormal expression rates of beta-catenin and cyclin D1 in ESCC were higher than those in the normal group (65.5% vs. 1.2%,p < 0.01; 70.9% vs. 0.8%, p < 0.01). In accordance with the following order, normal epithelia --> basal cell hyperplasia --> dysplasia --> ESCC, hypoexpression of APC proteins occurred in ESCC, abnormalities of beta-catenin and E-cadherin started to appear in dysplasia, and overexpression of Cyclin D1 emerged from basal cell hyperplasia. From well to poorly differentiated ESCC, the expression of APC, E-cadherin and cyclin D1 were gradually reduced, while beta-catenin was increased. The expression of beta-catenin was not correlated with APC (r = -0.10, p > 0.05), was negatively correlated with E-cadherin (r = -0.31,p < 0.01) and positively correlated with cyclin D1(r = 0.49, p < 0.01).. APC, E-cadherin, beta-catenin and cyclin D1 may play important roles in tumorigenesis of ESCC. Therefore, detection of E-cadherin, beta-catenin and cyclin D1 proteins may be helpful for the early diagnosis of ESCC.

Topics: Adenomatous Polyposis Coli Protein; Adult; Aged; Aged, 80 and over; beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Genes, APC; Humans; Male; Middle Aged; Signal Transduction; Tissue Array Analysis; Wnt Proteins

Current therapies offer scant benefit to patients with advanced esophageal adenocarcinoma. We investigated the effects of Sorafenib, a multifunctional kinase inhibitor, on several growth regulatory pathways that control cell growth and survival in SEG-1 cells derived from Barrett's adenocarcinoma.. SEG-1 cells were exposed to acidified medium or taurocholic acid, with and without pre-incubation with Sorafenib. Cyclin D1 and E, c-Myc, and Bcl-2 expression levels as well as STAT3 activations were determined by Western blotting. Cyclin D1 mRNA was measured by real-time PCR. Apoptosis was assessed by TUNEL assay.. Sorafenib significantly inhibited SEG-1 cell proliferation stimulated by acid or bile acid treatments and reduced cell survival. This drug significantly reduced the up-regulations of cyclin D1, cyclin E, c-Myc, and Bcl-2 as well as the activation of STAT3 in SEG-1 cells.. These results support a rational basis for future clinical studies to assess the therapeutic benefit of Sorafenib in esophageal adenocarcinoma.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Benzenesulfonates; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Humans; Niacinamide; p38 Mitogen-Activated Protein Kinases; Phenylurea Compounds; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Pyridines; Sorafenib; STAT3 Transcription Factor

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as a growth factor for gastrointestinal (GI) tumours. The tumourigenic effects of GHRH appear to be mediated by the splice variant 1 (SV-1) of GHRH receptor as well as the full length pituitary type receptor for GHRH (GHRH-R). We examined the protein and mRNA expression of GHRH-R and SV-1 in normal human tissues and tumours of the gastrointestinal (GI-) tract by immunohistochemical staining and reverse transcriptase (RT)-PCR. Squamous cells and squamous cell carcinoma of the oesophagus were negative for GHRH-R and SV-1, while Barrett's mucosa and adenocarcinomas of the oesophagus showed a strong expression of both receptors. The expression of GHRH-R was absent in normal colonic mucosa other than neuroendocrine cells (NE) and lining epithelium (LE) but strong in tubular adenomas of the colon, while the staining for SV-1 was absent in cells other than NE. However, the expression of both receptors was significantly increased in tubulovillous adenomas and colorectal cancers. No differences were seen in protein levels for both receptors between normal and neoplastic tissues of the stomach, pancreas and liver. Because of low mRNA levels for both receptors in all samples tested, only a qualitative assessment could be made. However, mRNA for GHRH-R and SV-1 showed a near-perfect correlation with the assessment of receptor proteins by immunostaining. Our study shows that in contrast to normal mucosa, transformed mucosa of the oesophagus and the colon expresses GHRH-R and SV-1. This aberrant expression of GHRH-R and SV-1 in oesophageal and colorectal malignancies may provide a molecular target for a therapeutic approach based on GHRH antagonists.

Topics: Colon; Colonic Neoplasms; Cyclin D1; Esophageal Neoplasms; Esophagus; Growth Hormone-Releasing Hormone; Humans; Immunohistochemistry; Intestinal Mucosa; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; RNA Splicing; RNA, Messenger

Barrett esophagus (BE) is an established precursor of esophageal adenocarcinoma (AdenoCa). One hundred and one cases of BE diagnosed by esophageal biopsy and resections were examined morphologically for dysplasia. These were categorized as BE without dysplasia (n=25), indefinite for dysplasia (IND, n=17), low-grade dysplasia (LGD, n=18), high-grade dysplasia (HGD, n=15), and AdenoCa (n=26). Immunostaining for p16 (INK4A/CDKN2A), Cyclin D1 (CCND1), Ki-67, and alpha-methylacyl-CoA racemase (AMACR) was employed to assess their potential as diagnostic discriminators. Abnormal p16 expression (negative, cytoplasmic, or combined cytoplasmic and nuclear staining) was present in all categories, rising from 68% in BE without dysplasia to 100% in AdenoCa, with cytoplasmic staining only showing a significant correlation with the severity of dysplasia. Cyclin D1 expression was present in almost all cases, but high expression (>50% cells positive) was displayed mostly in HGD and AdenoCa (46.7% and 42.3%, respectively). Ki-67 index increased with the severity of dysplasia and labeling extended from the lower third of the crypts to the superficial epithelium. The frequency of AMACR-positivity was 12% in BE, 47.1% in IND, 44.4% in LGD, 93.3% in HGD, and 96.2% in AdenoCa. The intensity and extent of AMACR staining also increased with the severity of dysplasia. Aberrant p16 and high-cyclin D1 expression may reflect early genetic events during the progression of Barrett-associated carcinogenesis. Cytoplasmic staining of p16 is specific. It may represent a different pathway of p16 dysfunction. The pattern and extent of Ki-67 staining and AMACR overexpression are useful additional parameters for identifying dysplasia in BE.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Barrett Esophagus; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Proteins; Racemases and Epimerases

Cyclin D1 is found on 11q13, which is a region frequently amplified in several tumor types. The CCND1 locus gives rise to at least two protein isoforms of D1 (D1a and D1b). A common G/A polymorphism (G/A870) is thought to influence the expression levels of D1a and D1b. D1b has been suggested to be increased in the presence of the A allele and more oncogenic than D1a. Furthermore, the A allele has been reported to correlate with increased risk of carcinoma in several tumor types, suggesting that this polymorphism and D1b are important in tumor progression. However, contradictory data about the polymorphism, D1 variant expression, and correlation with survival have been reported. We explored the relationship between gene amplification, G/A870 genotype, D1a and D1b expression, and overall survival in esophageal adenocarcinoma and non-small cell lung cancer.. DNA and RNA were isolated from 54 esophageal adenocarcinoma samples and 89 non-small cell lung cancer samples and were analyzed for gene amplification, genotype at the polymorphism, gene expression, and association with overall survival.. The D1 variant expression did not correlate with amplification, genotype, or overall survival in either tumor type. The total D1 expression correlated with decreased patient survival. Several other genes on 11q13 also seem to be overexpressed and correlated with decreased survival.. We report that the G/A870 polymorphism does not correlate with patient survival, or with D1a or D1b expression. However, the total D1 expression and the expression of several other genes on 11q13 seem to be associated with esophageal adenocarcinoma patient survival.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Alleles; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Esophageal Neoplasms; Female; Gene Amplification; Gene Expression; Genotype; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction

To rapidly detect molecular alterations in different malignancies and investigate the possible role of Tp53, C-myc, and CCND1 genes in development of tumors in human organs and their adjacent normal tissues, as well as the possible relation between well- and poorly-differentiated tumors.. A tissue array consisting of seven different tumors was generated. The tissue array included 120 points of esophagus, 120 points of stomach, 80 points of rectum, 60 points of thyroid gland, 100 points of mammary gland, 80 points of liver, and 80 points of colon. Expressions of Tp53, C-myc, and CCND1 were determined by RNA in situ hybridization. 3' terminal digoxin-labeled anti-sense single stranded oligonucleotide and locked nucleic acid modifying probe were used.. The expression level of Tp53 gene was higher in six different carcinoma tissue samples than in paracancerous tissue samples with the exception in colon carcinoma tissue samples (P < 0.05). The expression level of CCND1 gene was significantly different in different carcinoma tissue samples with the exception in esophagus and colon carcinoma tissue samples. The expression level of C-myc gene was different in esophagus carcinoma tissue samples (chi2 = 18.495, P = 0.000), stomach carcinoma tissue samples (chi2 = 23.750, P = 0.000), and thyroid gland tissue samples (chi2 = 10.999, P = 0.004). The intensity of signals was also different in different carcinoma tissue samples and paracancerous tissue samples.. Over-expression of the Tp53, CCND1, and C-myc genes appears to play a role in development of human cancer by regulating the expression of mRNA. Tp53, CCND1 and C-myc genes are significantly correlated with the development of different carcinomas.

Topics: Breast Neoplasms; China; Colonic Neoplasms; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-myc; Rectal Neoplasms; RNA, Messenger; Stomach Neoplasms; Thyroid Neoplasms; Tissue Array Analysis; Tumor Suppressor Protein p53

Altered cyclin D1 (CD1), a cell cycle regulator, may play an important role in imparting aggressive nature to esophageal adenocarcinoma (EAC). CD1 gene single nucleotide polymorphism G/A870 results in two alternatively spliced transcripts, CD1a and CD1b. CD1b, preferentially encoded by the A870 allele, is putatively oncogenic. We hypothesized that CD1 A870 allele would be associated with higher CD1 protein expression, and increased genomic instability during EAC evolution, leading to more aggressive phenotype.. One hundred twenty-four archival specimens of EAC, and 39 associated Barrett's esophagus (BE) specimens were examined for CD1 genotype, CD1 protein expression, and chromosome 9 polysomy (representing genomic instability). We correlated CD1 genotypes with CD1 protein expression, genomic instability, age at diagnosis of EAC, and overall survival (OS).. The A870 allele was associated with higher levels of CD1 protein expression in EAC (P = .032); in BE (P = .01) where it was associated with concomitant increased chromosome 9 polysomy (P = .002); and with a younger age at diagnosis (P < .001) and poor OS (P = .0003) of EAC patients.. Our data suggest that CD1 A870 background may be imparting aggressive phenotype to EAC. It provides a molecular basis to explain the clinical biology associated with CD1 polymorphism whereas aberrant nuclear accumulation of CD1 protein enhances the acquisition of genomic instability (ie, clonal diversity), thus leading to early age of EAC diagnosis and poor OS. CD1 genotyping with other biomarkers may help create a biomarker-based prognostic model for EAC and CD1 may also serve as a therapeutic target.

Topics: Adenine; Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Biopsy, Needle; Cohort Studies; Cyclin D1; Disease Progression; Esophageal Neoplasms; Esophagectomy; Esophagoscopy; Female; Gene Expression Regulation, Neoplastic; Genomic Instability; Guanine; Humans; Immunohistochemistry; Male; Middle Aged; Polymorphism, Genetic; Probability; Proportional Hazards Models; Retrospective Studies; Risk Assessment; Sensitivity and Specificity; Survival Analysis

The dramatic increase in the incidence and poor overall survival rates of esophageal/gastroesophageal junction adenocarcinoma underscore the necessity to discover molecular markers that can be used for risk assessment, early diagnosis, and targeted therapeutic intervention. Barrett's esophagus (BE) is proposed to represent a precursor of esophageal/gastroesophageal junction adenocarcinoma. BE progression to invasive cancer is defined by a metaplasia-dysplasia-carcinoma progression characterized by an increasing accumulation of genetic changes associated with alterations in molecular gatekeepers of cell circuitries and tissue homeostasis. Using a combination of in situ tissue-based and high-throughput analyses, we investigated alterations of cell-cycle regulators and inflammation-associated molecular effectors. Our data suggest a potential synergistic effect of these alterations for the BE progression to cancer, and underscore the potential use of these markers: (1) in molecular panels assessing cancer risk in BE patients; and (2) as potential therapeutic targets for chemopreventive interventions and to enhance response to anti-neoplastic therapies.

Topics: Adenocarcinoma; Barrett Esophagus; Biomarkers, Tumor; Cell Transformation, Neoplastic; Chemoprevention; Cyclin D1; Disease Progression; Early Diagnosis; Esophageal Neoplasms; Humans; Metaplasia; NF-kappa B; Precancerous Conditions; Risk Assessment; Signal Transduction; Treatment Outcome

Recently, attention has been directed to concurrent chemoradiotherapy (CRT) for the treatment of squamous cell carcinoma of the esophagus with regard to efficacy, quality of life and functional preservation, and survival periods comparable to those after standard surgical therapy have been reported in responders to CRT. However, there are some non-responders to CRT, and the prediction of the outcome after CRT is an important subject for future studies. In this study, using biopsy specimens obtained before CRT, we evaluated the relationships between biological markers and the outcome after CRT in order to determine the prognostic factors of CRT.. The subjects were 51 patients (42 males and nine females: median age 68 years). who were histologically confirmed to have squamous cell carcinoma of the esophagus at stage II or III (UICC). Concurrent CRT consisting of chemotherapy using 5FU and CDDP and radiation therapy (60 Gy) was performed as the initial treatment, and the relationships of overexpression of EGFR, p53, VEGF, PCNA and CyclinD1 were examined immunohistochemically in biopsy specimens collected before treatment. Overall survival was estimated by multivariate analysis.. The percentages of patients overexpressing p53, VEGF, PCNA, CyclinD1, and EGFR were 33, 31, 37, 31 and 29%, respectively. On multivariate analysis, T stage (P = 0.0393) and PCNA (P = 0.0302) were found to be significant prognostic factors.. PCNA overexpression appears to be a prognostic factor for squamous cell carcinoma of the esophagus after CRT.

Topics: Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclin D1; Cyclophosphamide; Doxorubicin; ErbB Receptors; Esophageal Neoplasms; Etoposide; Female; Fluorouracil; Humans; Male; Prognosis; Proliferating Cell Nuclear Antigen; Tumor Suppressor Protein p53

The purpose of the study was to explore the effects and molecular mechanisms of lutein on the differentiation of esophagus cancer EC9706 cell.. EC9706 cells were seeded in 1640 medium before the addition of test compounds. The respective test compound was added in fresh medium and the control cell received the vehicle (DMSO) or Fluorouracil. The proliferation and cell cycle of EC9706 were determined by MTT assay and flow cytometry, respectively. The change in cytomorphology was investigated by using HE staining. Proliferation and differentiation cells were checked and observed by methyl green-pyronine staining. The protein expression of cyclin D1 was detected by immunohistochemistry.. Compared with the DMSO control group, the proliferation of the EC9706 cells treated with lutein (100 microg/mL and 150 microg/mL) could markedly be decreased and the cell cycle was blocked at G0/G1 phage which caused significant changes in the cytomorphology of EC9706 cell line, and the cell malignant degree tended to drop down, the protein expression of cyclin D1 was also down-regulated significantly.. Lutein can inhibit the proliferation of EC9706 cell, and promote the cancer cell differentiation. cyclin D1 may be involved in cell proliferation and differentiation events in esophageal cancer EC9706 cell, which is regulated by lutein.

Topics: Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Lutein

The study aimed at investigating whether genetic polymorphisms in BCL2, FAS, CCND1, EGF and EGFR genes influence the outcome of patients of esophageal squamous cell cancer treated with radiotherapy, with or without chemotherapy. Sixty nine histologically confirmed, previously untreated, patients with a squamous cell esophageal cancer were inducted into this study. Genotyping of BCL2 (ala43thr), FAS (A-670G), CCND1 (G870A), EGF (+61A/G) and EGFR (G497A) polymorphisms were determined using the polymerase chain reaction followed by restriction fragment length polymorphism methodology. Genotyped data was analyzed using univariate and multivariate logistic regression statistical tests for predicting the survival outcome. Genotypes of BCL2, FAS, CCND1 and EGFR polymorphisms independently did not influence outcome significantly. However, patients with EGF +61AG genotype had median survival of 25.5 months (95% CI = 5.2-45.5), whereas those with EGF +61GG genotype had survival of only 3.7 months (95% CI = 0.0-9.8, p = 0.006). In univariate cox-regression analysis, interaction of genotypes EGF+61GG*radiotherapy tumor dose (< or =50 Gy) and EGF +61GG *upper third tumor location showed high hazard of death, 6.6 (95% CI = 2.0-21.5, p = 0.002) and 26.8 (95% CI = 3.7-194.2, p = 0.001) while EGF+61AG*middle third tumor location had reduced hazard 0.20 (95%CI = 0.06-0.60, p = 0.004). The pilot study suggests that EGF +61AG and +61GG genotypes may predict clinical outcome in esophageal cancer patients treated with radiotherapy with or without chemotherapy. EGF +61AG genotype was associated with improved survival, however +61GG genotype adversely affected the outcome in patients particularly with upper third location of tumor and lower dose (< or =50) of radiotherapy.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; fas Receptor; Female; Humans; Male; Middle Aged; Polymorphism, Genetic; Proto-Oncogene Proteins c-bcl-2; Survival Analysis; Treatment Outcome

It is known that cyclooxygenase (COX)-2 expression is increased in Barrett's esophagus and esophageal adenocarcinomas. We studied COX-2 expression and the effect sulindac has on the genesis of Barrett's esophagus and adenocarcinoma in rats undergoing esophagogastroduodenal anastomosis (EGDA).. Fifty-one rats were divided into a control group (n=27), a 500 ppm sulindac-treated group (n=15) and 1000 ppm sulindac-treated group (n=9). Randomly selected rats were killed by diethyl ether inhalation at 20 and 40 weeks after surgery.. At 40 weeks, rats treated with 1000 ppm sulindac showed narrower esophageal diameter and milder inflammation than the control rats. At 40 weeks, the incidence of Barrett's esophagus was similar between control and sulindac-treated groups, but the incidence of adenocarcinoma was significantly lower in the 1000 ppm sulindac-treated group than either the control or 500 ppm sulindac-treated groups. COX-2 was significantly increased in the lower esophagus of control rats killed at 40 weeks. Cyclin D1 expression was negligible in the sulindac- treated group compared with the control group.. We suggest that the chemopreventive effect of sulindac is related to decreased COX-2 and cyclin D1 expression, which may be influenced by reduced inflammation.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Barrett Esophagus; Blotting, Western; Cyclin D1; Cyclooxygenase 2; Duodenogastric Reflux; Esophageal Neoplasms; Immunohistochemistry; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Sulindac

Little reports showed cyclin D1 changes in esophageal cancer from southern China. In this study, we detected cyclin D1 expression in esophageal carcinomas from southern China 61% and 35% cases showed increased expression of cyclin D1 in esophageal carcinomas and the adjacent epithelia, respectively. Significant difference for cyclin D1 expression was found between esophageal carcinomas and the adjacent epithelia. Comparing cyclin D1 expression in the carcinomas at different stages, we found significant alterations. The results suggested that cyclin D1 was involved in the earlier event and accumulated as the cancer evolved to a later stage in some esophageal carcinomas.

Topics: Carcinoma, Squamous Cell; China; Cyclin D1; Esophageal Neoplasms; Gene Expression Profiling; Humans; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction

We evaluated the relationship of amplification and polysomy of both the CCND1 and the ERBB2 (alias HER-2/NEU) genes to the overexpression of their proteins in esophageal and gastric cancers and also their association with clinicopathological features. CCND1 gene amplification (45%) was more prevalent than polysomy (25%) in esophageal carcinoma, but the pattern observed was similar in gastric adenocarcinoma (10% amplification, 15% polysomy). For ERBB2, polysomy was a more frequent mechanism than amplification in both esophageal (32.5 vs. 7.5%) and gastric (15 vs. 5%) cancers. Overexpression of cyclin D1 protein was identified in 37.5% of the specimens of esophageal tumors and 35% of gastric tumors, and overexpression of Her-2/neu protein in 12.5 and 7.5%, respectively. The kappa-statistics revealed a fair agreement in both types of tumors only in overexpression and amplification of the CCND1 gene; the ERBB2 gene showed a fair agreement in amplification and polysomy and the level of protein expression in gastric adenocarcinoma. Thus, polysomy 17 could contribute to a high Her-2/neu protein level, at least in gastric cancer. Our data indicated an association with alcohol consumption and the CCND1 gene or protein levels, in both esophageal and gastric cancers.

Topics: Adenocarcinoma; Aged; Cyclin D1; Esophageal Neoplasms; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Helicobacter Infections; Helicobacter pylori; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Middle Aged; Receptor, ErbB-2; Stomach Neoplasms; Survival Analysis

Oesophageal adenocarcinoma frequently develops on a background of metaplastic Barrett's epithelium. The development of malignancy is accompanied by genetic alterations, which may be promising biomarkers of disease progression.. A case control study was conducted nested within a large unselected population based cohort of Barrett's patients. Incident oesophageal malignancies and high grade dysplasias were identified. For each case up to five controls were matched on age, sex, and year of diagnosis. Biopsies from the time of diagnosis of Barrett's epithelium were stained immunohistochemically for TP53, cyclin D1, cyclooxygenase 2 (COX-2), and beta-catenin proteins.. Twenty nine incident oesophageal malignancies and six cases of high grade dysplasia were identified. The odds of diffuse or intense TP53 staining were substantially elevated in biopsies from patients who developed oesophageal adenocarcinoma compared with controls (odds ratio (OR) 11.7 (95% confidence interval (CI) 1.93, 71.4)). This difference was also present when all cases were considered (OR 8.42 (95% CI 2.37, 30.0). Despite the association with TP53 staining, only 32.4% of cases had an initial biopsy showing diffuse/intense TP53 staining. There were no significant associations between cyclin D1, COX-2, or beta-catenin staining and case control status. The OR for positive staining for both TP53 and COX-2 was markedly increased in cases compared with controls (OR 27.3 (95% CI 2.89, 257.0)) although only 15% of cases had positive staining for both markers.. Immunohistochemical detection of TP53 expression is a biomarker of malignant progression in Barrett's oesophagus but sensitivity is too low to act as a criterion to inform endoscopic surveillance strategies. Additional biomarkers are required which when combined with TP53 will identify, with adequate sensitivity and specificity, Barrett's patients who are at risk of developing cancer.

Topics: Adenocarcinoma; Aged; Barrett Esophagus; beta Catenin; Biopsy; Case-Control Studies; Cohort Studies; Cyclin D1; Cyclooxygenase 2; Disease Progression; Esophageal Neoplasms; Esophagus; Female; Humans; Immunohistochemistry; Male; Metaplasia; Tumor Suppressor Protein p53

To correlate immunohistochemical expression patterns and prognosis in oesophageal adenocarcinoma.. The expression of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor (EGFR) was studied in a series of 137 primarily resected oesophageal adenocarcinoma samples. The expression analysis on protein level was performed on routine paraffin wax-embedded material, with immunohistochemical staining of the samples, assembled on a tissue microarray. The results were correlated with clinicopathological features (pT, pN and G) and survival.. 22 (16%) tumours showed an overexpression of the c-erbB-2 oncoprotein. Expression of EGFR was observed in 72 (55%) cases, accumulation of p53 in 68 (52%) cases and of cyclin D1 in 102 (77%) cases. Loss of p16INK4A expression was observed in 101 (76%) cases and low expression of p27KIP1 in 91 (71%) cases. Expression of these proteins did not correlate with tumour stage, grade, Lauren's or World Health Organization classification or lymph node status. On univariate survival analysis, more advanced tumour stage (p = 0.002), lymph node involvement (p = 0.003), high tumour grade (p = 0.017) and lack of EGFR expression (p = 0.034) were found to be associated with poorer survival. On multiple regression analysis, only tumour stage (p = 0.03) and lymph node involvement (p = 0.004) were shown to have an association with the survival of the patient.. The immunohistochemical expression of c-erbB-2 oncoprotein, cylin D1, p16INK4A, p27KIP1, p53 and EGFR in most oesophageal adenocarcinomas suggests their implication in the pathogenesis of this entity. None of the molecular markers assessed, however, was of prognostic value. Identification of any marker superior to or even approaching the prognostic value of conventional histopathological markers (pT and pN) was therefore not possible.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Epidemiologic Methods; ErbB Receptors; Esophageal Neoplasms; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prognosis; Protein Array Analysis; Receptor, ErbB-2; Tumor Suppressor Protein p53

Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.

Topics: Alleles; Amino Acid Substitution; Animals; Carcinoma; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Transformation, Neoplastic; Cyclin D; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclins; DNA Mutational Analysis; DNA, Neoplasm; Esophageal Neoplasms; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mutation, Missense; Neoplasm Proteins; NIH 3T3 Cells; Phosphorylation; Phosphothreonine; Point Mutation; Protein Processing, Post-Translational; Protein Transport; Recombinant Fusion Proteins; Sequence Deletion; Spodoptera

Esophageal carcinoma is one of the most lethal tumors, and identification of prognostic factors for patients with this disease is important. Propyl isomerase Pin1 is overexpressed in some human cancers and thought to be an important regulator of cyclinD1. However, the relationships between Pin1 expression and clinicopathologic features in patients with esophageal squamous cell carcinoma (SCC) have not been explored. Here, we investigated the role of Pin1 in association with cyclinD1 in esophageal SCC progression and its clinicopathological significance. The expressions of Pin1 and cyclinD1 were examined immunohistochemically in surgical specimens from 119 esophageal SCC patients. The expression levels of Pin1 and cyclinD1 in 6 esophageal SCC-derived cell lines were compared with those in an immortalized human esophageal cell line by western blotting. Pin1 overexpression was correlated with lymph node metastasis (P=0.0384), and its expression was related to cyclinD1 expression. Pin1 expression was correlated with poor prognosis in esophageal SCC patients (P=0.0044), and found to be an independent prognostic factor (P=0.0277). Pin1 was overexpressed in 5 of 6 esophageal SCC-derived cell lines compared with immortalized esophageal keratinocytes. Moreover, the Pin1 level was correlated with the cyclinD1 level in 4 of the 6 cell lines. In conclusion, Pin1 expression is correlated with cyclinD1 expression and may be a useful prognostic factor for esophageal SCC.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Lymphatic Metastasis; Male; Middle Aged; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Prognosis; Treatment Outcome

Esophageal squamous cell carcinoma (ESCC) shows a high frequency of lymphatic and/or systemic metastasis, even when the tumor invades only the submucosa. To investigate the genetic alterations in circulating esophageal tumor cells, we performed array-based comparative genomic hybridization (CGH) analysis of 8 DNA samples of xenografts, which were previously established from the thoracic duct lymph of 13 ESCC patients. A total of 5 loci (or genes), 10q21.3 (EGR2), 11q13.3 (CCND1/CyclinD1, FGF4, and EMS1), 11q14 (PAK1), and 22qtel (ARSA) were found to be candidate amplified loci in the xenograft. In contrast, a total of 24 loci including 9p21 (p16 and MTAP) were found to be homozygously deleted candidates in the xenograft. Both p16 homozygous deletion and CCND1 amplification were detected in 6 (75%) and 5 (62.5%) of the 8 xenografts. Furthermore, by quantitative Southern blot analysis, we found p16 homozygous deletion in 30.8% (8/26) of the primary tumors and in 50% (4/8) of the metastasized lymph nodes. The frequency of CCND1 amplification and p16 homozygous deletion is suggested to be associated with ESCC progression. Matrigel invasion assays of p16-deleted ESCC cells showed that restoring wild-type p16 activity into the cells significantly inhibits tumor-cell invasion, suggesting that p16 inactivation could be involved in ESCC invasion. This is the first report showing the genetic alteration of concealed tumor cells in the thoracic duct lymph. The present gene list should be helpful for identifying new amplified and deleted genes in primary ESCCs as well as in metastasized lymph nodes.

Topics: Adenoviridae; Animals; Blotting, Southern; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Gene Amplification; Gene Deletion; Gene Transfer Techniques; Genes, p16; Humans; Lymphatic Metastasis; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Cells, Circulating; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Thoracic Duct; Transplantation, Heterologous

p53 is known to play a central role in sensing and signaling for the growth arrest and apoptosis in cells with DNA damage. Mutation of p53 is a frequent event in esophageal squamous cell carcinoma (ESCC). p16 protein binds to cyclin dependent kinase 4 (CDK4) inhibiting the ability of CDK4 to interact with cyclin D1, and stimulates the passage through the G1 phase of cell cycle. We observed the expression patterns and frequencies of p53, p16, and cyclin D1 in esophageal dysplasia and in esophageal squamous cell carcinomas.. In 15 patients of ESCC, 5 patients of esophageal dysplasia and 5 volunteers with normal esophagus, tissue specimens were taken from esophageal lesions during the operation or endoscopic examination. We used specific monoclonal antibodies for p53 protein, p16(INK4 ) protein and cyclin D1. Immunoreactivity was scored.. Mean age of all groups was 66 years old (range 47-93) and men to women ratio was 19:1. p53 mutation was observed in 87% (13/15) of ESCC, in 80% (4/5) of esophageal dysplasia, in 0% (0/5) of normal mucosa (p=0.001). p16 expression was seen in 40% (2/5) of esophageal dysplasia, 27% (4/15) of ESCC and 100% (5/5) of normal mucosa (p=0.016). Cyclin D1 expression was not significantly different among 20% (1/5) of esophageal dysplasia, 53% (8/15) of ESCC and 20% (1/5) of normal mucosa. Either the expression of p53 mutation or the loss of p16 occurred in 80% (4/5) of esophageal dysplasia and in 93% (14/15) of ESCC.. The expression of p53 mutation and the loss of p16 might play a central role in the pathogenesis of esophageal squamous cell carcinoma (ESCC), and contribute to the development of precancerous lesion such as dysplasia. In addition, there is a possibility that the mutations of p53 and p16 silencing would be the early events in ESCC development.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Esophagus; Female; G1 Phase; Humans; Immunohistochemistry; Male; Middle Aged; Tumor Suppressor Protein p53

Alvocidib (Flavopiridol, HMR1275) is a potent inhibitor of multiple cyclin-dependent kinases and has been identified recently as an antitumor agent in several cancers. Previous studies have shown that alvocidib could potentially treat esophageal cancer in vitro. This study evaluates alvocidib for its ability to suppress tumor growth in severe combined immunodeficiency (SCID) mice bearing TE8 human esophageal squamous cell carcinoma (SCC) xenografts. Alvocidib treatment of 10mg/kg body weight reduced tumor volume significantly. Immunohistochemistry analysis of alvocidib-treated tumor sections showed significant reductions in cyclin D1, VEGF, and Rb levels. Alvocidib treatment did not cause a marked increase in apoptotic tumor cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, yet hematoxylin and eosin staining revealed tumor necrosis. In vivo investigation of alvocidib treatment confirmed antitumor activity in TE8 esophageal xenografts. These findings suggest that alvocidib could be a useful anti-cancer agent for esophageal cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyclin D1; Esophageal Neoplasms; Female; Flavonoids; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, SCID; Necrosis; Neoplasm Transplantation; Piperidines; Retinoblastoma Protein; Vascular Endothelial Growth Factor A

To assess the status of EGFR, HER-2, and CCND1 at the gene and protein levels in esophageal squamous cell carcinoma.. Dual-color FISH assays were performed using DNA probes for EGFR/CEP 7, HER-2/CEP 17, and CCND1/CEP 11. The respective proteins, furthermore, was assessed in IHC assays and correlated with patient and tumor characteristics.. From 55 ESCCs, 8 (15%) tumors showed gene amplification and 20 (36%) had gene overrepresentation (balanced gene and chromosome 7 polysomy) for EGFR. High-level protein expression was frequent (49%), positively correlated with gene copy numbers (kappa=0.4), and associated with well-differentiated histology (p=0.02). For HER-2, gene amplification was detected in a single tumor (2%) and protein overexpression was rare (9%). CCND1 gene was amplified in 23 (42%) tumors; likewise, CCND1 protein overexpression was common (58%) and prevailed in gene overrepresentation or amplification. Only 1 patient showed gene amplification for both EGFR and CCND1. Survival was not associated with EGFR or CCND1 gene/protein status, whereas negative patients for HER-2 protein had a better survival than positive patients (p=0.04).. Frequent overexpression and gene amplification of EGFR and CCND1 make these molecules and their pathways potential therapeutic targets for ESCC. In addition, EGFR and CCND1 appeared to be independently altered suggesting alternative mechanisms for pathway activation. Therapeutic agents targeting these molecules are urged to be tested in clinical trials and comprehensive biological analyses should be included to properly interpret the outcome.

Topics: Aged; Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Esophageal Neoplasms; Female; Gene Amplification; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Middle Aged; Polymerase Chain Reaction; Receptor, ErbB-2; Up-Regulation

The cyclin-dependent kinase (cdk) inhibitor p27 preferentially inactivates cdk complexes required for progression through the G1/S transition. Loss of p27 is associated with aggressive behavior in a variety of tumors, including Barrett's associated adenocarcinoma (BAA). We have previously shown that gastroduodenal-esophageal reflux (GDER) together with N-methyl-N-benzylnitrosamine (MBN) induces Barrett's esophagus (BE) and malignant transformation of the esophageal mucosa in mice. This process is enhanced in a p27 null background. Here, we show that chronic flavopiridol administration sharply reduced the prevalence of BE in GDER/MBN-treated p27 knockout mice when compared to animals treated with diluent only (7 vs 26%, P=0.0079). Similarly, flavopiridol reduced the prevalence of BAA (11 vs 32%, P=0.0098) and overall cancer prevalence (15 vs 60%, P<0.0001). In addition, appropriate molecular targeting by flavopiridol in tumor cells was confirmed by downregulation of cyclin D1, a known target of this pan-cdk inhibitor. The results of this study represent the experimental basis for chemoprevention with cdk inhibitors in human BE and BAA.

Topics: Adenocarcinoma; Animals; Anticarcinogenic Agents; Barrett Esophagus; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Esophageal Neoplasms; Flavonoids; Mice; Phosphorylation; Piperidines; Retinoblastoma Protein; Tumor Suppressor Proteins

Squamous cancers of the oral cavity and esophagus are common worldwide. A number of environmental factors as well as genetic alterations have been identified. However, the specific combination of genetic events and their interplay with environmental carcinogens are largely un-known. Furthermore, no good animal model existed to study the molecular changes important in the induction and progression of the disease. Here we summarize the efforts made to establish a mouse model of oral-esophageal carcinogenesis. Cyclin D1 overexpressing(L2D1+) mice were generated using an EBV promoter to specifically target the oral cavity and the esophageal squamous epithelium. Besides analyzing different environmental factors, such as nitrosamines and zinc deficiency, cyclin D1 transgenic mice were crossbred with p53-deficient mice. While L2D1+ mice exhibited a phenotype of dysplasia, different combinations of mice result-ed in invasive oral-esophageal cancer. This mouse model provides a well-defined and reproducible model of oral-esophageal cancer that should be useful for testing chemopreventive, diagnostic, and therapeutic strategies.

Topics: Animals; Carcinogenicity Tests; Cyclin D1; Disease Models, Animal; Esophageal Neoplasms; Genetic Engineering; Humans; Mice; Mice, Transgenic; Mouth Neoplasms; Transfection

To investigate individual susceptibility to gastroesophageal reflux disease, Barrett esophagus, and esophageal adenocarcinoma, the authors studied the frequency of the common G870A polymorphism of CCND1, which encodes cyclin D1, a key cell cycle regulatory protein.. The study population included 307 patients who were enrolled in a prospective case-control study to evaluate lifestyle risk factors and molecular alterations in gastroesophageal reflux disease (n = 126 patients), Barrett esophagus (n = 125 patients), and esophageal adenocarcinoma (n = 56 patients). A control group included 95 strictly asymptomatic individuals. Genomic DNA was extracted from cases and controls, and polymerase chain reaction was used to amplify exon 4 of CCND1. After digestion with BsrI, acrylamide gel electrophoresis was used to identify the wild type and common G870A polymorphic alleles. The frequency of alleles (G/G, G/A, A/A) was compared between cases and controls. Immunohistochemistry was used to study cyclin D1 distribution in among patients in the case group.. Compared with the asymptomatic control group, and adjusted for age and gender, increasing frequencies were seen for the A/A genotype in patients with gastroesophageal reflux disease (odds ratio [OR], 2.83; 95% confidence interval [95% CI], 1.09-7.34), Barrett esophagus (OR, 3.69; 95% CI, 1.46-9.29), and esophageal adenocarcinoma (OR, 5.99; 95% CI, 1.86-18.96). No association was seen between genotype and cyclin D1 overexpression.. The CCND1 A/A genotype was associated with increased risk for gastroesophageal reflux disease, Barrett esophagus, and esophageal adenocarcinoma. The contribution of this polymorphism to susceptibility of defined stages of progression to esophageal adenocarcinoma suggested potential application in endoscopic Barrett surveillance programs.

Topics: Adenocarcinoma; Barrett Esophagus; Cyclin D1; Esophageal Neoplasms; Gastroesophageal Reflux; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Immunohistochemistry; Polymerase Chain Reaction; Polymorphism, Genetic; Precancerous Conditions; Risk Factors

The aim of the present study is to use immunohistochemical methods to investigate the clinical implications of tumor markers in esophageal squamous cell carcinoma and evaluate their impact on prognosis.. From November 1990 to December 1996, 47 patients were treated with preoperative radiation followed by radical esophagectomy. All patients were confirmed pathologically as suffering from squamous cell carcinoma. Immunohistochemical stain was done for PCNA, cyclinD1 protein expression and DNA content analyzed by image cytometry. Kaplan-Meier method for single prognostic factor and log-rank test was used to test the significant difference. Cox stepwise regression model and prognosis index model were used for survival analysis with multiple prognostic factors.. Radio-pathological change, T stage and N stage, as the traditional prognostic factors had statistical difference in 3-, 5- and 10-year survival rates. While, tumor cell proliferating marked PCNA, cyclinD1 and DNA content served as independent prognostic factors of esophageal carcinoma. There was definitely an identity between the single and multiple factor analyses. PI was more accurate to evaluate the prognosis of esophageal carcinoma.. It is possible that tumor cell proliferating marked PCNA, cyclinD1 and DNA content would become the endpoints for evaluating the prognosis of esophageal carcinoma.

Topics: Adult; Aged; Cyclin D1; DNA, Neoplasm; Esophageal Neoplasms; Female; Humans; Male; Middle Aged; Neoplasm Staging; Postoperative Period; Preoperative Care; Prognosis; Proliferating Cell Nuclear Antigen

SUMMARY. We performed a multi-institutional analysis of E2F1 and cyclin D1 expression in cases of esophageal squamous cell carcinoma (ESCC). Cyclin D1 and E2F1 are involved in the transition of cell cycle phases and associated with tumor progression. However, no previous studies have concurrently analyzed combined E2F1 and cyclin D1 expression. The purpose of this study was to clarify the relationship of E2F1 and cyclin D1 in ESCC. We studied 122 patients with primary ESCC who underwent surgical tumor resection. Immunohistochemical analyses were performed for E2F1 and cyclin D1. A statistical analysis of immunohistochemistry results, clinicopathological features, and prognosis was performed. E2F1/cyclin D1 (-/-) tumors were present in 31 patients (25.4%) and correlated with reduced tumor progression. In these patients, pT (P=0.0001), pN (P<0.0001), p-Stage (P=0.0019), and survival rates were better than in patients who were positive for either E2F1 or cyclin D1 (P=0.0232). The expression of E2F1 and cyclin D1 is an indicator of tumor progression and prognosis in patients with ESCC. Combined analysis of E2F1 and cyclin D1 expression helps to determine the characteristics and prognosis of ESCC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin D1; Disease Progression; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Esophageal Neoplasms; Female; Humans; Male; Middle Aged; Prognosis; Retrospective Studies; Transcription Factors

We investigated the prevalence of single nucleotide polymorphisms in the p16 gene (C540G) and the cyclin D1 gene (G870A), both known to regulate function in G1 arrest and therefore, may play an important role in carcinogenesis.. Using PCR based restriction fragment length polymorphism and single strand conformational polymorphism, we determined single nucleotide exchanges in the p16 and cyclin D1 genes among 56 esophageal adenocarcinomas (ADC) arising in Barrett's esophagus, 95 cardiac gastric ADC, and in 191 distal gastric ADC. The allelic frequencies were compared to a control group of 253 healthy blood donors.. The C/G genotype of p16 was identified in 10.4% of esophageal carcinomas, 13.3% of cardiac carcinomas, and in 14.1% of gastric carcinomas, compared to 17.4% in the healthy control group. All other cases showed the C/C wildtype, as no homozygous G/G nucleotide exchange was detected in the group of cancer patients or in the control group. In esophageal cancer, cyclin D1 G/G genotype was found 28.6%, A/G in 46.4%, and A/A in 25.0%. In cardiac carcinoma, frequency of cyclin D1 genotype was 27.4% for G/G, 57.9% for A/G, and 14.7% for AA. In distal gastric carcinoma, both homozygous genotypes (G/G and A/A) had a frequency of 15.2% each, while the heterozygous A/G genotype occurred in 69.6% of patients. The control group displayed 24.9% G/G, 53.8% A/G, and 21.3% A/A genotype.. Our results show that frequencies of p16 or cyclin D1 polymorphisms in gastric and esophageal ADC do not differ significantly from the healthy control group. Therefore, these polymorphisms are unlikely to be associated with risk of ADC of the upper gastrointestinal tract.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Alanine; Barrett Esophagus; Cardia; Case-Control Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cysteine; DNA, Neoplasm; Esophageal Neoplasms; Female; Gene Frequency; Glycine; Homozygote; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Stomach Neoplasms

Immortalization and malignant transformation are important steps in tumor development. The ability to induce these processes from normal human epithelial cells with genetic alterations frequently found in the corresponding human cancer would significantly enhance our understanding of tumor development. Alterations in several key intracellular regulatory pathways (the pRB, p53, and mitogenic signaling pathways and the telomere maintenance system) appear to be sufficient for the neoplastic transformation of normal human cells. Nevertheless, in vitro transformation models to date depend on viral oncogenes, most prominently the simian virus 40 early region, to induce immortalization and malignant transformation of normal human epithelial cells. Here, we demonstrate a transformation model creating oral-esophageal cancer cells by using a limited set of genetic alterations frequently observed in the corresponding human cancer. In a stepwise model, cyclin D1 overexpression and p53 inactivation led to immortalization of oral keratinocytes. Additional ectopic epithelial growth factor receptor overexpression followed by c-myc overexpression as well as consecutive reactivation of telomerase induced by epithelial growth factor receptor sufficed to transform oral epithelial cells, truly recapitulating the development of the corresponding human disease.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; ErbB Receptors; Esophageal Neoplasms; Humans; Keratinocytes; Mouth Mucosa; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Telomere; Tumor Suppressor Protein p53

Many molecular alterations occur in esophageal carcinogenesis; however, little is known about the molecular genetic events responsible for the development of carcinoma. We investigated the expression of ki67, p53, cyclin D1 and pRB in 105 biopsy specimens using immunohistochemistry from iodine unstained lesions as indicators of carcinogenesis of the esophagus. Also, the genetic alternation of esophageal dysplasia from patients with accompanying esophageal squamous cell carcinoma (ESCC) was examined to study the evidence for field carcinogenesis in the esophagus. The expression of p53, cyclin D1 and pRB was detected in 31, 0 and 51.7% respectively of mild dysplasia; 40, 0 and 70% of moderate dysplasia; 40, 20 and 70% of severe dysplasia; and 48, 32 and 80% of carcinoma specimens. p53 expression was significantly increased in mild dysplasia, whereas cyclin D1 and pRB expression were significantly increased in carcinoma as compared to both normal epithelium and esophagitis. The ki67 LI and the rate of p53 expression were significantly higher in dysplasia with ESCC than in dysplasia without ESCC. Ki67, p53, cyclin D1 and pRB expression may be useful biomarkers for assessing the risk of developing esophageal cancer. Dysplasia observed at screening for secondary lesions has a highly malignant potential and careful follow-up studies are required.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophagitis; Esophagus; Humans; Immunohistochemistry; Neoplasm Proteins; Precancerous Conditions; Retinoblastoma Protein; Tumor Suppressor Protein p53

The dramatically increased incidence and poor survival rates of esophageal adenocarcinoma (EAC) underscore the need for novel targets useful for risk assessment and therapeutic intervention. Altered expression of cyclin D1 has been proposed as an early predictor for malignant transformation in EAC; however, the mechanisms underlying cyclin D1 deregulation have not been identified. A single nucleotide polymorphism, A870G, of the cyclin D1 gene has been associated with the preferential encoding of a protein with an extended half-life. We investigated the association of the cyclin D1 A870G polymorphism with cyclin D1 protein expression and clinical characteristics and outcome in 124 patients treated at our institution for EAC. Our results indicate that the cyclin D1 AA/AG genotype is associated with earlier age of cancer onset, cyclin D1 protein deregulation in the primary tumors, and increased frequency of distant metastasis. Our findings suggest that cyclin D1 status could be useful to assess risk of progression to EAC, and strategies directed to modulate cyclin D1 expression may prove useful for interventions to slow or interrupt the EAC tumorigenesis process.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cyclin D1; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Polymorphism, Genetic; Retrospective Studies

Cytogenetic analyses of four squamous cell carcinomas (SCC) of the esophagus showed complex numerical and structural abnormalities. Chromosomal bands or regions preferentially involved were 11q13, 8q10, 21q10, 3p10 approximately p11, 1p11 approximately q11, 5p11 approximately q11, and 14p11 approximately q11. For the first time, to our knowledge, recurrent aberrations were identified in esophageal SCC, including homogeneous staining region (hsr), isochromosomes i(3q) and i(21q), and ring chromosome. Losses of chromosomal material dominated over gains. Recurrent imbalances included under-representation of 4p13 approximately pter, 5q14 approximately qter, 9p22 approximately pter, 10p, 11p13 approximately pter, 12p13 approximately pter, 17p10 approximately pter, 18p11 approximately pter, 21p, and 22p, as well as over-representation of 1q25 approximately qter, 3q, 7q, and 8q. Interestingly, hsr at different chromosomal regions occurred in three of four cases. With the application of fluorescence in situ hybridization (FISH) and multicolor combined binary ratio labeling-FISH with specific DNA probes, it could be shown that in two cases the hsr was derived from chromosome 11 material and that the amplicon included CCND1. Our results, together with previous molecular genetic findings, indicate that CCND1might be a prime target in 11q13 amplification, and that amplification of this gene might be crucial in the tumorigenesis of esophageal SCC. These observed chromosomal aberrations and imbalances thus provide important information for further molecular genetic investigation of esophageal SCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Chromosome Aberrations; Cyclin D1; Esophageal Neoplasms; Female; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Male; Middle Aged

This study was undertaken to determine whether selected risk factors for esophageal and gastric cancer are associated with tumors that overexpress cyclin D1. Archived tumor tissue was available for 630 esophageal and gastric cancer patients who participated in a population-based case-control study. Patients were categorized into case groups based on whether protein overexpression of the cyclin D1 gene, as assessed by immunohistochemistry, was present (cyclin D1+, n = 285) or not (cyclin D1-, n = 345) in the tumor. The distribution of risk factors in each of these case groups was then compared with the distribution among the 695 controls. Multivariate-adjusted odds ratios (OR) for esophageal adenocarcinoma were reduced in relation to use of aspirin and other nonsteroidal anti-inflammatory drug (NSAID) use but only among patients with cyclin D1+ tumors (0.45, 95% confidence interval [CI] = 0.26, 0.79) and not among those with cyclin D1- tumors (1.12, 95% CI = 0.67, 1.86). A similar pattern was observed for gastric cardia adenocarcinomas. In contrast, ORs for esophageal squamous cell carcinoma and noncardia gastric adenocarcinomas in relation to NSAID use were reduced, regardless of cyclin D1 status. ORs did not vary with cyclin D1 status in relation to alcohol, body size, or cigarette smoking, with the following exception; for noncardia gastric adenocarcinomas the cyclin D1- tumors showed a 2-fold elevation in the OR with ever smoking. These data suggest that the reduction in risk associated with NSAID use may be restricted to those esophageal and gastric cardia adenocarcinomas that overexpress cyclin D1.

Topics: Adenocarcinoma; Aged; Anti-Inflammatory Agents, Non-Steroidal; Confidence Intervals; Cyclin D1; Esophageal Neoplasms; Female; Humans; Life Style; Male; Middle Aged; Population Surveillance; Prevalence; Risk Factors; Stomach Neoplasms; Surveys and Questionnaires; United States

To examine the expression of Egr-1, c-fos and cyclin D1 at both transcript and protein levels in esophageal carcinoma and to correlate the level of their expressions with precancerous and paracancerous esophageal lesions and esophageal carcinoma.. In situ hybridization and immunohistochemistry were used respectively to detect the expression of mRNA and proteins of Egr-1, c-fos and cyclin D1 in 70 cases of esophageal squamous cell carcinoma and their corresponding para-cancerous mucosa and upper cut edge mucosa.. In situ hybridization and immunohistochemistry showed positive staining of all three mRNAs in the cytoplasm and those of the proteins in nuclei. Overexpression of Egr-1, c-fos and cyclin D1 mRNAs and their proteins was found in dysplasia and squamous carcinomas. The expression level of Egr-1 and c-fos was high, and cyclin D1 was low in dysplasia mucosa, whereas the expression of Egr-1 was decreased, c-fos was maintained and cyclin D1 was increased in the cancers. The expression of both c-fos and cyclinD1 was consistent between the mRNA and protein in their corresponding high expression lesions.. The expression of Egr-1, c-fos and cyclin D1 varies in esophageal precancerous lesions and cancer tissues, suggesting an involvement of these genes in the development of esophageal carcinoma.

Topics: Cyclin D1; DNA-Binding Proteins; Early Growth Response Protein 1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Lymphatic Metastasis; Precancerous Conditions; Proto-Oncogene Proteins c-fos; RNA, Messenger; Transcription Factors

Patients with chagasic achalasia (megaesophagus) are liable to have an additional 1.7-20% possibility of developing esophageal squamous cell carcinoma (ESCC). We applied a fluorescence in situ hybridization technique in 20 such patients and found aneuploidies of chromosomes 7, 11, and 17 in 60% (12 of 20 specimens) and deletion of the TP53 gene in 54.5% (6 of 11 specimens; it was only possible to obtain data by FISH technique from 11 of the 20 achalasia patients). The main aneuploidies detected were chromosome 7 monosomy or trisomy (35%) in mid-third megaesophagus cases, and chromosome 17 monosomy or trisomy (25%) in distal-third cases. TP53 gene deletion was more frequent in mid-third (62.5%) than in distal-third megaesophagus cases (40%). In chagasic megaesophagus, no amplification of the cyclin D1 gene (CCND1) was observed. Comparing chagasic megaesophagus to ESCC, we found a higher frequency of aneuploidies in all 10 tumors. The main alterations were trisomy or tetrasomy of chromosomes 17 (90%), 11 (70%), and 7 (70%). Amplification of CCND1 was evidenced as a cluster in 70% of the tumors (22-99% of nuclei), while TP53 gene deletion occurred in 100%. To our knowledge, this is the first cytogenetic analysis of chagasic megaesophagus to show that aneuploidies of chromosomes 7, 11, and 17, and TP53 gene deletion might be related to increased risk for malignancy.

Topics: Adult; Aged; Aneuploidy; Carcinoma, Squamous Cell; Chagas Disease; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 7; Cyclin D1; Esophageal Achalasia; Esophageal Neoplasms; Female; Gene Deletion; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Monosomy; Trisomy; Tumor Suppressor Protein p53

Squamous cell carcinoma (ESCC) is the most frequent histological subtype in esophageal cancer, although the incidence of esophageal adenocarcinoma (EAC) is increasing faster than any other malignancy in the western world. New developments in the understanding of molecular mechanisms in esophageal cancer comprise analysis of the genetic tumor profiles by CGH (comparative genomic hybridization), the detection of tumor suppressor gene inactivation, and the analysis of proto-oncogenes. Especially the inactivation of the p53 gene proved to be of particular importance for the development of esophageal cancer. Also p15 and p16 have been identified to be involved in the pathogenesis of esophageal cancer by influencing the cyclin kinase inhibitor cascade and DNA mismatch repair processes. Amplification of cyclin D1 results in growth advantage for tumor cells and enhances tumorigenesis; gene amplification and overexpression of cyclin D1 were frequently demonstrated especially in ESCC. Regarding the dysplasia-metaplasia-carcinoma sequence of Barrett's esophagus, inhibition of apoptosis by overexpression of bcl-2 proteins occurs as an early event.

Topics: Adenocarcinoma; Barrett Esophagus; Biomarkers, Tumor; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Molecular Biology; Neoplasms, Squamous Cell; Risk Factors; Smoking; Tumor Suppressor Proteins

To investigate the expression of E-cadherin, alpha-catenin, beta-catenin, gamma-catenin and cyclin D(1) in patients with esophageal squamous cell carcinoma (ESCC), and analyze their interrelationship with clinicopathological variables and their effects on prognosis.. Expression of E-cadherin, alpha-catenin, beta-catenin, gamma-catenin and cyclin D(1) was determined by EnVision or SABC immunohistochemical technique in patients with ESCC consecutively, their correlation with clinical characteristics was evaluated and analyzed by univariate analysis.. The reduced expression rate of E-cadherin, alpha-catenin, beta-catenin and gamma-catenin was 88.7%, 69.4%, 35.5% and 53.2%, respectively. Cyclin D1 positive expression rate was 56.5%. Expression of gamma-catenin was inversely correlated with the degree of tumor differentiation and lymph node metastasis (chi(2) = 4.183 and chi(2) = 5.035, respectively, P<0.05), whereas the expression of E-cadherin was correlated only with the degree of differentiation (chi(2) = 5.769, P<0.05). Reduced expression of E-cadherin and gamma-catenin was associated with poor differentiation of tumor, reduced expression of gamma-catenin was also associated with lymph node metastasis. There obviously existed an inverse correlation between level of E-cadherin and gamma-catenin protein and survival. The 3-year survival rates were 100% and 56% in E-cadherin preserved expression group and in reduced expression one and were 78% and 48% in gamma-catenin preserved expression group and in reduced expression one, respectively. The differences were both statistically significant. Correlation analysis showed the expression level of alpha-catenin correlated with that of E-cadherin and beta-catenin (P<0.05).. The reduced expression of E-cadherin and gamma-catenin, but not alpha-catenin, beta-catenin and cyclin D1, implies more aggressive malignant behaviors of esophageal carcinoma cells and predicts the poor prognosis of patients.

Topics: alpha Catenin; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; Cytoskeletal Proteins; Desmoplakins; Esophageal Neoplasms; gamma Catenin; Humans; Immunohistochemistry; Prognosis; Survival Rate; Trans-Activators

Esophageal cancer has been reported to be frequently associated with cancer of the head and neck. The iodine dye method is reportedly useful to detect early esophageal cancer. The aim of this study was to clarify clinicopathological and biological characteristics of esophageal squamous cell carcinoma associated with head and neck cancer (HN group).. Thirty-seven patients of the HN group who underwent esophagectomy were examined clinicopathologically compared to 42 patients with esophageal cancer alone (SE group). All resected specimens were histologically studied after iodine dye staining, and p53 and cyclin D1 (CD1) expression were immunohistochemically examined in esophageal cancer.. The HN group had more multiple iodine-unstained lesions and multiple primary cancers within the esophagus compared with the SE group (p = 0.0027, p = 0.067, respectively). There was no significant difference in smoking, drinking, family history and the other clinicopathological factors between the HN and SE groups. Coexpression of p53 and CD1 was found to be significant in the HN group (p = 0.03).. The coexistence of multiple iodine-unstained lesions, multiple cancers in the esophagus and overexpression of p53 and CD1 is suggested as a risk factor for the HN group.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophagectomy; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasms, Multiple Primary; Predictive Value of Tests; Risk Factors; Tumor Suppressor Protein p53; Up-Regulation

Chemoradiotherapy is a multimodal therapy routinely used as a primary treatment for advanced esophageal cancer. However, it is beneficial only to patients who respond. To identify pretreatment markers predicting response and survival, we examined the expression of cell cycle regulatory molecules, p53, p21(Waf1/Cip1) cyclin D1, and CDC25B, in biopsy specimens from 76 patients with stage III and stage IV squamous cell carcinoma. Overexpression of p53, p21, cyclin D1 and CDC25B was observed in 58%, 30%, 28%, and 32% of patients, respectively. The expression of p21 correlated significantly with response to chemoradiotherapy (P = 0.0001). Survival of patients with p21-expressing tumors was better than that of patients with p21-negative tumors (P = 0.013). Expression of other genes was not significantly correlated with treatment response and survival. In patients with p53-negative tumors, survival of those patients with p21-positive tumors was significantly higher than that of those with p21-negative tumors (P = 0.0452), but no significant difference was found in patients with p53-positive tumors. Multivariate analysis revealed that p21 expression was an independent variable among pretreatment parameters in predicting survival. These results suggest that p21 expression is potentially useful for predicting the response to chemoradiotherapy and survival of patients with advanced esophageal squamous cell cancer.

Topics: Biopsy; Carcinoma, Squamous Cell; cdc25 Phosphatases; Cell Cycle Proteins; Chemotherapy, Adjuvant; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Multivariate Analysis; Prognosis; Proportional Hazards Models; Radiotherapy, Adjuvant; Survival Rate; Treatment Outcome; Tumor Suppressor Protein p53

Flavopiridol is a synthetic flavone that has shown an antitumor effect against several cancers. Here, we investigated the in vitro effect of flavopiridol alone and the combined effect of low-dose flavopiridol plus radiation on esophageal squamous cell carcinoma cell lines. Esophageal squamous cell carcinoma cell lines (TE8, TE9 and KE4) were exposed to flavopiridol (0.05-400 nmol/L) for 48 h. Growth inhibition was evaluated by MTT assay, cell cycle distribution was determined by flow cytometry, and cyclin D1, Bcl-2 and Rb protein expression was detected by Western blotting. The effect of 0.05 nmol/L flavopiridol as a radio-sensitizer was determined by clonogenic assay. The IC50 was approximately 110-250 nmol/L. Exposure to 0.05 nmol/L flavopiridol for 48 h increased the G2/M population, while 300 nmol/L increased the G1 population. At a concentration of 300 nmol/L, nuclear fragmentation and chromatin condensation were observed in all three cell lines. Exposure to 300 nmol/L flavopiridol decreased the levels of cyclin D1 and Rb protein in all three cell lines and Bcl-2 protein was also decreased in TE8 and KE4 cells. Moreover, exposure to 0.05 nmol/L flavopiridol slightly decreased the levels of cyclin D1, Rb and Bcl-2 protein in KE4 cells. Flavopiridol treatment (0.05 nmol/L) enhanced the radio-sensitivity in all three cell lines. Low-dose flavopiridol augmented the response of esophageal squamous cell carcinoma cell lines to radiation. Administration of a low dose of flavopiridol could be a potent new therapeutic approach for improving the efficacy of radiotherapy against esophageal cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Flavonoids; Flow Cytometry; Formazans; Genes, bcl-2; Growth Inhibitors; Humans; Piperidines; Radiation Tolerance; Radiation-Sensitizing Agents; Retinoblastoma Protein; Tetrazolium Salts; Treatment Outcome

To study the expression and significance of cyclin E, cyclin D1, CDK4 and p27 protein in esophageal squamous cell cancer (ESCC) and their correlation with tumor differentiation and lymph node metastasis.. Expressions of cyclin E, cyclin D1, CDK4 and p27 protein in 65 patients with ESCC were quantitatively detected by flow cytometry.. The expressions of cyclin E, cyclin D1, CDK4 in poorly-differentiated ESCC were higher than those in well-differentiated ESCC (P = 0.0275, 0.0001, 0.0174). The expression of p27 in poorly-differentiated ESCC was lower than that in well-differentiated ESCC (P = 0.0042). There was positive correlation between cyclin E and cyclin D1, cyclin D1 and CDK4, but negative correlation between cyclin D1 and p27. The expressions of all four proteins were not correlated with lymph node metastasis.. The expressions of cyclin E, cyclin D1, CDK4 and p27 are closely related to tumor differentiation of ESCC. An imbalance between positive and negative control of cell cycling might be critical in the carcinogenesis of esophageal squamous cell cancer.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Differentiation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Esophageal Neoplasms; Female; Flow Cytometry; Humans; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged

Deregulation of cell-cycle G(1)-restriction point control by disruption of Rb-pathway components is a frequent event in cancer. In concert with the inactivation of cell death pathways, such events not only contribute to tumor development but also determine the intrinsic and acquired resistance to cancer therapy and, ultimately, disease prognosis. We previously observed that the cyclin-dependent kinase inhibitor p16(INK4a) and the proapoptotic Bcl-2 homolog Bax are positive prognostic factors and identify patients with good prognosis in esophageal squamous cell carcinoma (SCC). In the present study, we therefore extend our analysis to additional genes controlling the G(1) restriction point and apoptosis, respectively. This retrospective analysis was performed in a cohort of 53 patients undergoing surgery for esophageal SCC with curative intent, i.e., R0 resection. Protein expression profiles of cyclin D1, p16(INK4a), Rb, p21(CIP/WAF-1), p53, Bax and Bcl-2 were analyzed by immunohistochemistry and compared to p53 mutational status, as determined by SSCP-PCR of exons 5-8. Loss of p16(INK4a), Rb, p21(CIP/WAF-1) or Bax and overexpression of cyclin D1 were associated individually with shorter overall survival, while Bcl-2 expression and p53 mutation were not of prognostic relevance. The longest survival was observed in a subgroup of patients whose tumors bore a combination of favorite genotypes, i.e., low cyclin D1 and high Rb, p21(CIP/WAF-1), p16(INK4a) and Bax protein expression. These results show that multigene analyses based on limited sets of functionally linked genes reliably identify patients with good vs. poor prognosis.

Topics: Adult; Aged; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Mutational Analysis; Esophageal Neoplasms; Female; G1 Phase; Genes, p53; Genetic Markers; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Mutation; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Regression Analysis; Retinoblastoma Protein; Retrospective Studies; Time Factors

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; China; Cyclin D1; Esophageal Neoplasms; Female; Gene Frequency; Genetic Markers; Genetic Predisposition to Disease; Genotype; Humans; Male; Polymorphism, Genetic; Risk Factors; Statistics as Topic

Our aim was to investigate the association of cyclin D1 (G870A) single nucleotide polymorphism with susceptibility to esophageal and cardiac carcinoma in a northern Chinese population. By polymerase chain reaction-single strand conformation polymorphism analysis, cyclin D1 (G870A) genotyping was carried out among 120 patients with esophageal squamous cell carcinoma (ESCC), 87 patients with gastric cardiac adenocarcinoma (CAC), and 183 age- and gender-matched controls. The cyclin D1 genotype distribution among ESCC patients was significantly different from that among healthy controls (chi(2) = 7.372, p = 0.025). The G/G genotype was significantly less frequent among ESCC patients (9.2%) than among healthy controls (20.8%) (chi(2) = 7.192, p = 0.007). The G/G genotype significantly reduced risk for the development of ESCC compared to the combination of G/A and A/A genotypes (adjusted odds ratio [OR] = 0.37, 95% confidence interval [CI] = 0.16-0.83). After stratification according to smoking status, the A/A frequency among smoking ESCC (34.3%) and CAC patients (35.7%) was significantly higher than that among smoking healthy controls (18.6%) (chi(2) = 5.426 and 5.599, p = 0.020 and 0.018, respectively). Smokers with the A/A genotype had an about 2-fold increased risk for both of ESCC and CAC compared to the G/A and G/G genotypes, with an adjusted OR of 2.26 in ESCC (95% CI = 1.14-4.49) and of 2.42 in CAC (95% CI = 1.17-4.98). No correlation between the cyclin D1 genotype and development of ESCC or CAC was found among nonsmokers. Determination of the cyclin D1 (G870A) single nucleotide polymorphism may be suitable to identify individuals with increased risk for ESCC or CAC in the northern Chinese population.

Topics: Adenocarcinoma; Asian People; Carcinoma, Squamous Cell; Cardia; Case-Control Studies; Cyclin D1; DNA; Esophageal Neoplasms; Esophagus; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Risk Factors; Smoking; Stomach Neoplasms

Overexpression of cyclin D1 and disruption of cell cycle control in G(1) occur frequently in human esophageal cancer. Transgenic (TG) mice with cyclin D1 overexpression targeted to the oral-esophageal tissue by the EBV ED-L2 promoter showed increased severity in esophageal dysplasia without cancer development, after multiple doses of N-nitrosomethylbenzylamine (NMBA). Dietary zinc deficiency (ZD) in mice enhances cellular proliferation in esophagus/forestomach and susceptibility to NMBA-induced carcinogenesis. We investigated whether cyclin D1 overexpression in TG mice, together with ZD, might lead to unchecked cell proliferation and accelerated NMBA-induced tumorigenesis. Five-week-old TG and wild-type (WT) mice were fed a ZD- or -sufficient (ZS) diet, forming four groups: ZD:TG; ZS:TG; ZD:WT; and ZS:WT. After 4 weeks, animals were given a single intragastric NMBA dose and were sacrificed 25 and 77 days later. Without NMBA, cell proliferation was greatest in ZD:TG esophagus/forestomach, followed by ZD:WT, and then ZS:TG>/=ZS:WT. The high rate of cell proliferation was accompanied by overexpression of cell cycle progression and tumorigenesis biomarkers, including proliferating cell nuclear antigen, cyclin D1, cyclin-dependent kinase 4, p53, cytokeratin 14, epidermal growth factor receptor, and by a reduced rate of apoptosis. ZD substantially increased forestomach tumor incidence in TG mice: 85% of ZD:TG versus 14% of ZS:TG mice had forestomach tumors (P < 0.001), with progression to malignancy occurring only in ZD:TG tumors. Additionally, 14% of ZD:TG mice developed esophageal tumors and esophageal intestinal metaplasia at 77 days. Thus, cyclin D1 overexpression, in cooperation with ZD, decontrols cell proliferation, ensuring cell expansion, a prerequisite for cancer development.

Topics: Animals; Apoptosis; Cell Cycle; Cell Division; Cyclin D1; Disease Progression; Esophageal Neoplasms; Female; Male; Mice; Mice, Transgenic; Stomach Neoplasms; Zinc

To investigate the association of Cyclin D1 (A870G) single nucleotide polymorphism (SNP) with the susceptibility to esophageal and cardiac cancer in northern Chinese population.. By polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP), the Cyclin D1 (A870G) genotyping was performed among 178 patients with esophageal or esophageal -gastric junction carcinoma (120 with esophageal squamous cell cancer and 58 with cardiac adenoma cancer) and 122 health controls.. There were no significant differences in Cyclin D1 (A870G) allele frequencies between cancer patients and health controls (P = 0.075). There is no genotype distribution difference was found between non-smoker patients and controls (P > 0.05). The risk for esophageal and cardiac cancer is 2.5 times higher in A/A genotype carried smokers than that in A/G or G/G genotype carried non-smokers, with the adjusted Odd Ratio of 2.57 (95% confidence interval is 1.19 approximately 5.57). The G/G genotype reduces the susceptibility to esophageal cancer, with the adjusted Odd Ratio of 0.39 and 95% confidence interval of 0.18 - 0.82. The A/A frequency in cardiac patients (34.48%) is higher than that in health controls (23.77%) but the difference does not reach significant (P = 0.212).. In northern Chinese population, the smoking individuals carrying Cyclin D1 (A870G) A/A genotype increase the susceptibility to esophageal and cardiac cancer. The G/G genotype probably plays a protecting role in the occurrence of esophageal cancer.

Topics: Adult; Aged; Cardia; Cyclin D1; Esophageal Neoplasms; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymorphism, Genetic; Stomach Neoplasms

We investigated whether aberrant p53 and p16 expression, the Ki-67 labeling index (LI), and int-2/cyclin D1 gene amplification predict the response to chemoradiotherapy (CRT) in patients with locally advanced esophageal squamous cell carcinoma (ESCC).. p53 and p16 expression status, the Ki-67 LI, and int-2/cyclin D1 amplification were assessed by immunohistochemical staining and slot blot analysis in pretreatment endoscopic biopsy specimens of 41 patients with T4 or M1 Lym (distant lymph node metastasis) ESCC. All patients received a course of chemotherapy (5-fluorouracil and cisplatin) with radiotherapy.. The CRT therapeutic response rate was 71%, and resection after CRT was successful in 15 of the cases in which the CRT effect was significant. The cumulative survival rate after CRT in the p53-negative patients was significantly higher than in the p53-positive patients (P =.037). The mean Ki-67 LI in the CRT response cases was significantly higher than in the CRT no-response cases (P =.023). Multivariate regression analysis revealed high Ki-67 LI to be an independent variable linked to a pathologic complete response to CRT (P =.033). The cumulative survival rate after CRT in the group that was p53-negative and int-2/cyclin D1 amplification-positive was significantly higher than in the other groups (P =.008).. Evaluating predictive factors in pretreatment endoscopic biopsy specimens may allow selection of more suitable multimodal treatment for ESCC patients and improve their quality of life.

Topics: Adenocarcinoma; Adult; Aged; Combined Modality Therapy; Cyclin D1; Esophageal Neoplasms; Female; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Proto-Oncogene Proteins; Survival Analysis; Tumor Suppressor Protein p53

To investigate the alteration of molecular events and the early carcinogenesis mechanism of esophageal epithelial cells in the high incidence area of esophageal cancer.. Esophageal epithelial cells of esophageal cancer patients were collected from the high incidence area in China. Content of DNA and telomerase as well as multi-gene expressions such as p53, p21 and cyclin D1 in esophageal precancer cells were quantitatively analysed by flow cytometry (FCM) with indirect immunofluorescence technique and DNA propidium iodide fluorescence staining.. FCM analysis results showed the DNA content increased significantly and the heteroploid rate was 87.9% in occurred carcinogenesis. P53 protein accumulation and ras p21 increase were seen in the early carcinogenesis of the esophagus. The positive rate of p53 and ras p21 was 100% (5/5, 4/4 respectively) in the cancer group. Telomerase and oncogene cyclin D1 were over- expressed in all of the cancer patients.. Increased DNA content and heteroploid rate, accumulation of p53 protein, and over-expression of p21, telomerase and cyclin D1 proteins were early molecular events during the development of esophageal cancer.

Topics: Carrier Proteins; Cyclin D1; DNA; Epithelial Cells; Esophageal Neoplasms; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins p21(ras); RNA-Binding Proteins; Telomerase; Tumor Suppressor Protein p53

Glycogen synthase kinase-3beta-dependent phosphorylation of cyclin D1 at a conserved COOH-terminal residue, Thr-286, promotes CRM1-dependent cyclin D1 nuclear export at the G(1)-S boundary. Mutations that perturb the phosphorylation of cyclin D1 at Thr-286 contribute to cell transformation, although to date, no such mutations have been found in human cancers. Cyclin D1 (CCND1) undergoes alternative splicing leading to the production of an mRNA predicted to encode a unique cyclin D1 isoform, cyclin D1b, which lacks Thr-286. We have cloned and expressed cyclin D1b, and find that it retains the ability to bind to and activate CDK4. Unlike canonical cyclin D1a, cyclin D1b remains nuclear through the cell cycle where its constitutive expression facilitates cellular transformation. Using antisera specific for cyclin D1b, the protein was detected in a high percentage of esophageal cancer-derived cell lines and in primary esophageal carcinomas. Therefore, alternative splicing leads to expression of a nuclear, oncogenic cyclin D1 isoform that is expressed in human cancer.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Esophageal Neoplasms; Humans; Male; Mice; Mice, SCID; Molecular Sequence Data; NIH 3T3 Cells; Oncogenes; Protein Isoforms; Proto-Oncogene Proteins; RNA, Messenger; Transfection

The incidence of oesophageal adenocarcinoma is increasing rapidly and this may be related to the presence of intestinal metaplasia (IM) at the gastro-oesophageal junction (GOJ). Recent studies have distinguished two subtypes of IM at the GOJ: short segment Barrett's oesophagus (SSBO) and IM at a normal squamo-columnar junction (IMNSCJ). Because abnormal expression of cell cycle regulators is common in cancer and precancerous states, cell cycle regulation was studied in patients with IM at the GOJ.. Biopsy samples and resected materials were identified from patients with SSBO (10), IMNSCJ (14), a normal SCJ with (14) and without (12) inflammation, conventional Barrett's oesophagus (BO) (12), and oesophageal adenocarcinoma (12). Sections were stained with antibodies to p21, p27, p53, Ki67, cyclin D1, and c-erbB2 and were assessed independently by two observers, using predetermined criteria.. Patients with oesophageal adenocarcinoma showed high expression of c-erbB2, p53, p27, and Ki67. Patients with BO showed expression of c-erbB2 but little expression of other markers. Greatly increased expression of cyclin D1 was seen in patients with IMNSCJ. The expression of all other markers was similar in patients with IMNSCJ and those with SSBO. Cyclin D1 and c-erbB-2 were coexpressed in patients with SSBO and IMNSCJ, and their expression was associated with the presence of p53 and p21.. Although the proposed aetiologies of SSBO (gastro-oesophageal reflux) and IMNSCJ (Helicobacter pylori infection) differ, the cell cycle response is similar and both may have malignant potential.

Topics: Adenocarcinoma; Adult; Aged; Barrett Esophagus; Biomarkers; Case-Control Studies; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Esophageal Neoplasms; Esophagogastric Junction; Female; Helicobacter Infections; Helicobacter pylori; Humans; Intestinal Mucosa; Ki-67 Antigen; Male; Metaplasia; Microfilament Proteins; Middle Aged; Muscle Proteins; Receptor, ErbB-2; Tumor Suppressor Protein p53

DNA amplifications at 11q13 are frequently observed in esophageal squamous cell carcinoma (ESC) and correlate with a malignant phenotype. Although this amplicon spans a region of several megabases and harbors numerous genes, CCND1 and EMS1 are thought to be the relevant candidates in ESC. We investigated whether the putative transforming gene MYEOV, mapping 360 kb centromeric to CCND1 and activated concomitantly with CCND1 in a subset of t(11;14)(q13;q32) positive multiple myeloma cell lines, represents a target of 11q13 amplification in ESC. To evaluate the role of MYEOV in ESC, we tested 31 ESC cell lines and 48 primary tumors for copy number levels of MYEOVand demonstrated that MYEOV was always coamplified with CCND1. However, MYEOV expression levels correlated only inconsistently with DNA amplification data. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored MYEOV expression in a subset of cell lines exhibiting DNA amplification without high MYEOV expression, suggesting that MYEOV is transcriptionally silenced by a DNA methylation mechanism in most of the latter cell lines. Our results indicate that MYEOV is a coamplified gene with CCND1 at 11q13, but its activation is sometimes inhibited by an epigenetic mechanism.

Topics: Azacitidine; Blotting, Northern; Blotting, Southern; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Cyclin D1; Decitabine; DNA Methylation; Esophageal Neoplasms; Gene Amplification; Gene Silencing; Humans; Neoplasm Proteins; Oncogene Proteins; Proto-Oncogene Proteins; Tumor Cells, Cultured

To clarify modes of silencing of p16 and their concerns to the development and progression of esophageal squamous cell carcinomas (ESCCs), we examined immunoreactivity of p16, loss of heterozygosity (LOH) at six microsatellite loci in 9p13-22, and the methylation status of the p16 promoter in 42 cases of the ESCC at various stages. The samples taken from step sections in and around the tumors were examined to map heterogeneity of those changes. Thereby at least one focus of dysplasia was detected in each case. No immunoexpression of p16 was detected in the ESCCs of 38 cases (90.5%) and in dysplasias of 34 cases (81%), whereas the histologically normal epithelia adjacent to the ESCC showed the p16 expression even in the presence of p16 methylation. Of the ESCCs/dysplasias without p16 expression (38/34), 16/0 showed both p16 methylation and LOH at the near-p16 loci (+/+), 14/30 did only methylation (+/-) and 8/4 did only LOH (-/+). The presence of LOH with/without homozygous deletion (HD) at the near-p16 loci correlated with the advanced tumor stages (P < 0.001). The mapping of +/+ cases indicated that the +/+ carcinomas were included in the +/- carcinomas, which were, in turn, surrounded by the +/- dysplasias and/or by the +/- normal-looking epithelia, whereas the -/+ dysplasias were always accompanied by the -/+ carcinomas. These results suggest that the mode of p16 silencing either through methylation or through LOH and possible mutation is determined in each patient before the occurrence of ESCCs and dysplasia. The mapping of HD and p16 expression suggest that the HD is a later event than the p16 silencing.

Topics: Aged; Alleles; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 9; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, p16; Humans; Loss of Heterozygosity; Male; Middle Aged; Neoplasm Staging; Promoter Regions, Genetic; Retinoblastoma Protein

Alterations of cell cycle-regulated genes play an important role in the process of carcinogenesis, and some of them are thought to be prognostic factors in esophageal cancer. The expressions of p53, p16, pRB and Cyclin D1 proteins were evaluated immunohistochemically in 144 patients who underwent curative esophagectomy without any adjuvant therapy before surgery. p53 overexpression was observed in 99 (69%) out of the 144 patients. No significant correlation was noted between p53 and any other gene expression. p16 expression was observed in 12 (8.3%) out of all cases. A negative correlation was recognized between p16 and Cyclin D1 expression (P=0.0004). pRB expression was observed in 130 (90.3%) out of all cases, whereas pRB expression was not observed in 11 out of the 12 patients with p16-positive tumors. A negative correlation was also found between p16 and pRB (P < 0.0001). A positive correlation was noted between pRB and Cyclin D1 expression (P=0.0009). The cumulative survival rate of patients without pRB expression was significantly lower than that of patients with positive expression (P=0.003). In the multivariate survival analysis, pRB expression was an independent prognostic factor. In 98% of all patients with esophageal cancer, impairment of the G1 checkpoint is due to a loss of function by p16, pRB or Cyclin D1, which showed a negative correlation in each factor. In addition, aberrant expression of pRB is useful as a prognostic factor in esophageal cancer.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Proportional Hazards Models; Tumor Suppressor Protein p53

Squamous cancers of the oral cavity and esophagus are common worldwide, but no good genetically based animal model exists. A number of environmental factors as well as genetic alterations have been identified in these cancers, yet the specific combination of genetic events required for cancer progression remains unknown. The Epstein-Barr virus ED-L2 promoter (L2) can be used to target genes in a specific fashion to the oral-esophageal squamous epithelium. To that end, we generated L2-cyclin D1 (L2D1(+)) mice and crossbred these with p53-deficient mice. Whereas L2D1(+) mice exhibit a histologic phenotype of oral-esophageal dysplasia, the combination of cyclin D1 expression and p53 deficiency results in invasive oral-esophageal cancer. The development of the precancerous lesions was significantly reversed by the application of sulindac in the drinking water of the L2D1(+)/p53(+/-) mice. Furthermore, cell lines derived from oral epithelia of L2D1(+)/p53(+/-) and L2D1(+)/p53(-/-) mice, but not control mice, formed tumors in athymic nude mice. These data demonstrate that L2D1(+)/p53(+/-) mice provide a well-defined, novel, and faithful model of oral-esophageal cancer, which allows for the testing of novel chemopreventive, diagnostic, and therapeutic approaches.

Topics: Animals; Antineoplastic Agents; Cyclin D1; Disease Models, Animal; ErbB Receptors; Esophageal Neoplasms; Genotype; Herpesvirus 4, Human; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Mouth Neoplasms; Neoplasms, Squamous Cell; Promoter Regions, Genetic; Sulindac; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The present study was conducted to assess the inhibitory effects of EGCG (epigallocatechin-3-gallate) on NMBA-induced rat esophageal tumorigenesis and to seek the potential mechanisms. In experiment I, 81 F344 rats were randomly divided into seven experimental groups according to the different regiments of NMBA 1 mg/kg subcutaneously (s.c.) and EGCG 4 mg/kg or 10 mg/kg orally or intraperitoneally (i.p.). The experiment was terminated at 24 weeks. In experiment II, 48 rats were allocated into two groups, each group contained 24 rats, in which the rats were injected with NMBA 1 mg/kg only or a combination of NMBA 1 mg/kg and EGCG 4 mg/kg i.p. Six rats from each group were sacrificed at the 12th, 16th, 20th and 24th week, respectively. The expression of cyclin D1 and cyclooxygenases (COX-2 and COX-1) was detected using semi-quantitative RT-PCR, and the production of prostaglandin E2 (PGE2) was measured by ELISA. In the groups which were treated with EGCG at a dose of 4 mg/kg i.p., or 10 mg/kg both orally and i.p., the mean number of tumors per rat was significantly reduced to 48, 56 and 61%, respectively (p<0.05). The incidence rate of esophageal carcinomas in the rats that were treated with EGCG 4 mg/kg i.p., was significantly lower than that in the rats which only received NMBA 1 mg/kg (p<0.05). The expression of cyclin D1 and COX-2, and the levels of PGE2 were also decreased by EGCG treatment. These results indicated that EGCG significantly inhibits the NMBA-induced rat esophageal carcinogenesis and it inhibitory effects may partly target cyclin D1 and COX-2 expression, and PGE2 production.

Topics: Animals; Anticarcinogenic Agents; Catechin; Cyclin D1; Cyclooxygenase 1; Cyclooxygenase 2; Dimethylnitrosamine; Dinoprostone; Disease Models, Animal; DNA Primers; Enzyme-Linked Immunosorbent Assay; Esophageal Neoplasms; Esophagus; Isoenzymes; Male; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Urokinase-Type Plasminogen Activator

The purpose of this study was to identify prognostic markers for chemoradiotherapy (CRT) in T(2-4)M(0) esophageal cancer.. We investigated clinicopathological and biological markers in biopsy specimens from 73 T(2-4)M(0) esophageal cancer patients treated with CRT (5-fluorouracil plus cisplatin and 60 Gy of radiation). Expressions of p53 gene product, Ki-67 labeling index, epidermal growth factor receptor, cyclin D1, vascular endothelial growth factor, microvessel density (MVD), thymidylate synthase, dihydropyrimidine dehydrogenase, and glutathione S-transferase pi in formalin-fixed biopsy samples of primary tumors before CRT were examined immunohistochemically. Clinicopathological and biological marker expressions were compared in terms of survival.. Univariate analysis revealed that performance status and T stage in clinicopathological features had a significant association with survival (P = 0.007 and 0.04, respectively) and that patients whose tumors showed high MVD [>median (19.7 vessels)] in biological markers had significantly better survival than those with low MVD (< or = median, P = 0.02). Also, there were weak associations of p53 and Ki-67 with survival (P = 0.08 and 0.07, respectively). Multivariate analysis, using both clinicopathological and biological markers, showed that MVD, T stage, and performance status became independent variables (P = 0.002, 0.02, and 0.02, respectively). Kaplan-Meier analysis showed that the patients with high MVD tumors survived longer than those with low MVD tumors (median survival time, not reached and 13 months, respectively; 3-year survival rate, 61% and 33%, respectively), with a significant difference of P = 0.02.. These results indicate that MVD using pretreatment biopsy specimens is a potentially useful prognostic marker for CRT in patients with T(2-4)M(0) esophageal cancer who are treated with CRT.

Topics: Adult; Aged; Biomarkers, Tumor; Cisplatin; Combined Modality Therapy; Cyclin D1; Dihydrouracil Dehydrogenase (NADP); Endothelial Growth Factors; ErbB Receptors; Esophageal Neoplasms; Female; Fluorouracil; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Immunoenzyme Techniques; Isoenzymes; Ki-67 Antigen; Lymphokines; Male; Middle Aged; Neoplasm Staging; Neovascularization, Pathologic; Oxidoreductases; Platelet Endothelial Cell Adhesion Molecule-1; Radiation Dosage; Survival Rate; Thymidylate Synthase; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Aiming to clarify and possibly extend indications for minimally invasive treatment, we characterized superficial esophageal cancers (SEC) with respect to biologic properties regulating malignant potential.. Surgical specimens obtained at eight cancer institutes from 222 Japanese patients with SEC (all squamous cell carcinomas) were investigated immunohistochemically for expression of cyclin D1 and E-cadherin.. Perturbations of cyclin D1 (overexpression) and E-cadherin (reduced expression) were observed in 37.6% (68 of 181) and 39.9% (71 of 178) of SEC patients. E-cadherin expression was more frequently reduced in cancers that invaded the submucosal layer, while cyclin D1 overexpression was constant, irrespective of depth of invasion. Overexpression of cyclin D1 was more frequent in poorly differentiated squamous cell carcinomas, while E-cadherin did not vary according to histologic differentiation. Lymph node metastasis, the only independent postoperative prognostic factor in these patients, occurred in only 4.8% of mucosal cancers (2 of 42), but in 51.1% of submucosal cancers (92 of 180). However, neither cyclin D1 nor E-cadherin status affected lymph node metastasis.. Both E-cadherin and cyclin D1 play important roles in esophageal carcinogenesis, but neither can be used to identify patients who do not require lymph node dissection and might be treated by endoscopic mucosal resection.

Topics: Cadherins; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Humans; Immunohistochemistry; Lymphatic Metastasis; Neoplasm Invasiveness; Prognosis

Lymph node metastasis is a major prognostic factor for esophageal squamous cell carcinoma (ESCC). In recent years, endoscopic mucosal resection (EMR) has been developed with excellent results for the treatment of the superficial ESCC. To make the EMR treatment successful, it is important to establish a good indicator to identify ESCC patients at a high risk of lymph node metastasis. In this study, we examined clinicopathological and immunohistochemical factors to investigate the factors involved in lymph node metastasis of ESCC invading to the submucosal layer (sm-ESCC). Surgical specimens from 84 sm-ESCC patients were examined. Among 84 sm-ESCC patients, 33 (39.3%) had lymph node metastases. Clinicopathologically, tumor depth, lymphatic invasion and blood vessel invasion showed significant correlations with lymph node metastasis by univariate analysis. Tumor depth and lymphatic invasion showed significant correlations by multivariate analysis of these factors. Immunohistochemically, P53 accumulation was observed in 45 cases (53.6%), cyclin D1 overexpression in 25 (29.8%), and pRB in 65 (77.4%). P53 accumulation, cyclin D1 overexpression and MIB-1 Labeling Index were significantly associated with lymph node metastasis by univariate analysis, and P53 accumulation showed a significant correlation with lymph node metastasis by multivariate analysis. Among tumor depth, lymphatic invasion and P53 accumulation, tumor depth and lymphatic invasion were significantly correlated with lymph node metastasis (P = 0.0023 and P = 0.0092, respectively) by multivariate analysis. These data suggest that tumor depth and lymphatic invasion can be considered as good indicators for lymph node metastasis among patients with sm-ESCC. In addition, P53 accumulation could be helpful to identify the patients who need additional treatment after EMR.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Nuclear; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Humans; Ki-67 Antigen; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Mucous Membrane; Neoplasm Invasiveness; Nuclear Proteins; Prognosis; Retinoblastoma; Survival Rate; Tumor Suppressor Protein p53

The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis. To evaluate which of these cell cycle regulatory proteins are related to patients' prognosis, a comprehensive analysis of alterations in these components was carried out in 100 ESCCs (oesophageal squamous cell carcinoma) using immunohistochemistry and correlated with clinicopathological parameters by univariate analysis. Overexpression of p53, MDM2 and cyclin D1 proteins was observed in 73, 42 and 67% of the cases, respectively, while loss of expression of p21, p16 and pRb was observed in 36, 45 and 75% of the cases, respectively. Multiple logistic regression analysis revealed that loss of p16 immunoreactivity was a significant risk factor for tumour stage (pT) (Odds Ratio (OR)=3.3), whereas the loss of pRb was a significant risk factor for nodal metastasis (pN) (OR=8.8). MDM2 overexpression emerged as the most significant risk factor for distant organ metastasis (pM) (OR=4.6). Of the ESCC patients who underwent oesophagectomy, 50 cases were followed-up for a maximum period of 44 months and median of 16 months. Survival analysis revealed that Cyclin D1 overexpression is an adverse prognosticator for disease-free survival, as well as overall survival, and tumour stage (pT) is an adverse prognosticator for disease-free survival. In conclusion, these data support a model of oesophageal cancer pathogenesis in which both the pRb and p53 pathways are inactivated and suggests an in-depth evaluation of the clinical utility of these putative markers is warranted.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Nuclear Proteins; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Retinoblastoma Protein; Risk Factors; Tumor Suppressor Protein p53

To explore the molecular events and mechanism in the carcinogenesis of esophageal epithelium in the high incidence area of esophageal carcinoma.. Epithelial cells collected from the high incidence area of esophageal carcinoma were used to detect DNA content and ploidy by propidium iodide(PI) stain. The expressions of p53, p16 and cyclin D1 were stained by indirect immunofluorescence of fluorescein isothiocyanate(FTTC), which were detected by flow cytometry (FCM).. During the process of carcinogenesis, DNA content increased significantly. The diploid cells decreased while heteroploid cells increased sharply, with a heteroploidy rate of 84.2%. At the same time, the p53 protein accumulated and p16 was deleted. The positive rates of p53 and oncogene cyclin D1 were both 100%(5/5, 6/6) in the cancer group.. In the early carcinogenesis of esophageal epithelium, DNA content and heteroploidy rates increase with tumor suppressor gene p16 deletion and p53 protein accumulation while oncogene cyclin D1 is overexpressed. Multiple molecular events have already occurred when esophageal carcinoma develops.

Topics: Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA, Neoplasm; Esophageal Neoplasms; Flow Cytometry; Gene Expression; Humans; Precancerous Conditions; Tumor Suppressor Protein p53

The clinicopathological characteristics of esophageal cancer have gradually been clarified using molecular biologic methods developed over the past 20 years. For example, amplification of the c-erb B gene is a prognostic factor and predictive of lymph node involvement, while the amplification of the cyclin D1 gene is also a prognostic factor and predictive of distant organ metastasis. Alteration of the p16 gene is also a prognostic factor and predicts lymph node involvement. As telomerase activity is almost a unique phenomenon of cancer cells, highly sensitive detection of esophageal cancer cells in the peripheral blood can be performed. Recently, such new methods as comparative genomic hybridization analysis and cDNA microarray analysis have been used to determine meaningful genetic changes. For therapeutic purposes, although tailor-made therapy has been proposed for several years, the validity of these approaches should be confirmed in a well-designed clinical trial. As molecular targeted therapies, tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR) and monoclonal antibodies against EGFR are being studied in clinical trials in Western countries. A clinical trial of p53 gene therapy against esophageal cancer is also promising.

Topics: Cyclin D1; DNA; Esophageal Neoplasms; Genes, DCC; Genes, MCC; Genes, p53; Genes, ras; Genes, Tumor Suppressor; Humans; Oncogene Proteins v-erbB; Ploidies

The cell cycle is controlled by protein complexes composed of cyclins and cyclin-dependent kinases. p27KIP1 (p27) is one of the Kip/Cip family cyclin-dependent kinase inhibitory proteins which negatively regulate cell cycle progression, and have been proposed as candidate tumor suppressor genes. To examine the role of p27 in the development of human esophageal squamous cell carcinoma (ESCC), we performed Western blot and immunoprecipitation analyses of the levels of expression of p27 protein in a series of ESCC cell lines. This protein was expressed at various levels in these cell lines during exponential growth. p27 level was significantly associated with that of cyclin D1, but not of cyclin E. Further cell cycle synchronization studies demonstrated that p27 was free or bound with affinity to cyclin E-CDK2 more than to cyclin D1-CDK4 or cyclin D1-CDK6. It is known that overexpression of cyclin D1 rather than cyclin E is involved in the pathogenesis of ESCC. Our findings indicated that high expression of p27 throughout the G1 to S phase may inhibit more likely cyclin E, than cyclin D1, which promotes tumor growth of esophageal squamous cell carcinoma.

Topics: Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Esophageal Neoplasms; G1 Phase; Gene Expression; Humans; Microtubule-Associated Proteins; S Phase; Tumor Cells, Cultured; Tumor Suppressor Proteins

Previous studies in our laboratory have shown that 6-phenylhexyl isothiocyanate (PHITC), enhances N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats while the shorter chain analogs, phenylethyl isothiocyanate (PEITC), and 3-phenylpropyl isothiocyanate (PPITC), inhibit NMBA-induced esophageal tumorigenesis. To test the hypothesis that PHITC influences the promotional stage of esophageal tumorigenesis, groups of 22-27 rats were dosed with vehicle or NMBA three times a week for 5 week, and fed a modified AIN-76A diet containing PHITC at concentrations of 0.0, 1.0, and 2.5 micromol/g. At the 25th week, the rats were killed, esophagi harvested and tumors counted. In the groups that received NMBA+PHITC, apparent but statistically insignificant increases in tumor multiplicity of 32 and 42% were found in comparison to rats treated with NMBA alone. A higher frequency of dysplastic lesions was found in rats treated with NMBA+2.5 micromol/g PHITC (71%) when compared to rats treated with NMBA only (12%). To test whether PHITC increased cellular proliferation, we evaluated proliferating cell nuclear antigen (PCNA) expression by immunohistochemistry. While there were no significant increases in PCNA staining in rats treated with NMBA+PHITC compared to rats treated with NMBA only, rats treated with PHITC only had a significantly higher PCNA index compared to untreated controls. Expression of cyclin D1, another biomarker of proliferation, was analyzed by semi-quantitative reverse transcription-polymerase chain reaction. There were no significant increases in cyclin D1 expression in groups treated with NMBA+PHITC compared to the group treated with NMBA only. Thus, while the data suggest a promotional effect by PHITC as manifested by a significant increase in dysplastic leukoplakia by the high dose of PHITC and an increase in the PCNA index by PHITC alone, PHITC does not appear to have a significant effect on esophageal cell proliferation.

Topics: Animals; Carcinogens; Cyclin D1; Dimethylnitrosamine; Esophageal Neoplasms; Isothiocyanates; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344

Alterations in expression of the p53 and cyclin D1 genes have been implicated in the development of esophageal carcinomas in both humans and animal models. We hypothesize that altered expression of cyclin D1 and p53 may be involved in the sequential development of esophageal carcinomas with glandular differentiation induced by the carcinogen, 2,6-dimethylnitrosomorpholine (DMNM) in rats with duodenal content reflux esophagitis. In the present study Sprague-Dawley rats were given DMNM 15 days after performing an esophago-jejunostomy in order to induce chronic duodenal content reflux esophagitis. Expression and localization of p53, cyclin D1 and Ki-67 were examined by immunohistochemical analyses. Twenty of 24 animals developed different types of esophageal carcinomas, including pure squamous carcinoma, adenosquamous carcinoma and pure adenocarcinoma. Undifferentiated basaloid areas were frequently observed in these tumors. Cyclin D1 overexpression was observed in hyperplastic lesions and increased through dysplasia and in undifferentiated areas of infiltrating carcinoma. Cyclin D1 expression coincided with increased Ki-67 expression and decreased along with cell differentiation. The p53 immunohistochemical pattern was parallel to that of cyclin D1, although the percentage of positive cells was usually smaller in all lesions and increased p53 expression started at the dysplastic stage. These findings suggest that overexpression of cyclin D1 may be an early event in DMNM-induced rat esophageal tumorigenesis, causing increased proliferation of esophageal stem cells. Abnormal p53 expression may then be required to promote the development of neoplastic transformation from dysplastic epithelium through invasive phenotype, being more evident in cancer cells with squamous differentiation.

Topics: Adenocarcinoma; Animals; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Cell Division; Cyclin D1; Duodenogastric Reflux; Esophageal Neoplasms; Esophagectomy; Female; Immunoenzyme Techniques; Jejunostomy; Ki-67 Antigen; Male; Quinoxalines; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53

The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alterations in expression of tumour suppressor gene products, p16 and pRb in esophageal cancer. Knowledge of alterations in cyclin D1, a vital component of this pathway in esophageal carcinomas from the Indian subcontinent, where the etiology and pathogenesis may be confounded by various unique dietary and environmental factors, is presently scanty. In order to bridge the gap between the accentuating incidence of esophageal cancer and aberrations in the components of this vital pathway, we analysed cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population.. Immunohistochemical analysis of cyclin D1 expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinomas (ESCC) (70 patients) and matched with histopathologically normal esophageal tissues from a distant site. The findings were correlated with clinicopathological parameters.. Overexpression of cyclin D1 was observed in the tumour nuclei in 41 out of 70 (59%) patients. We found concomitant alterations in 16 and cyclin D1 (p16-/CycD1+ phenotype) in 16 of the 70 patients (23%), while alterations of pRb and cyclin D1 (pRb-/CycD1+) were observed in 36 of the 70 (51%) patients of ESCCs. Cyclin D1 overexpression was significantly associated with the loss of p16 immunoreactivity (P = 0.005). The pRb- and p16-/pRb-/Cyc D+ phenotypes showed significant association with differentiation of the tumour (P = 0.005, 0.05, respectively). Kaplan-Meier analysis for disease recurrence showed increased disease recurrence in cyclin D1 overexpressed patients. Median time to disease recurrence in the cyclin D1+ group was 15 months as against 18 months observed in the cyclin D1- patients (P = 0.067; log-rank test).. Alterations in at least one of the components of the p16/cyclin D1/pRb pathway in majority of the 70 patients analysed herein, and concomitant alterations in all the three proteins in 19 patients (35%) underscore the critical role of this pathway in esophageal tumourigenesis. The results of the present study taken together with our previous findings on p16 and pRb alterations in ESCCs suggest that these alterations are not mutually exclusive and may cooperatively provide greater tumour growth advantage. The prognostic significance of alterations in the expression of these components cyclin D1, p16, and pRb remains to be established in a larger cohort.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; India; Male; Middle Aged; Retinoblastoma Protein

Although many molecular biologic molecules have been analyzed for their prognostic influence on patients with esophageal cancer, previous studies have not been able to raise statistically significant prognostic factors.. Immunohistochemical analysis of CyclinD1 expression and E-Cadherin expression was performed retrospectively in 416 esophageal squamous cell cancer patients who underwent curative resection of esophageal cancer at 10 major surgical departments in Japan, where more than 30 esophagectomies are performed in a year. The prognostic impact of these molecules and their relationship to clinicopathologic data of the patients were evaluated.. Univariate analysis revealed that pN (pTNM), pT (pTNM), CyclinD1 expression, and E-Cadherin expression were significant prognostic factors, and multivariate analysis revealed that pN (risk ratio (RR) 2.19), pT (RR 3.35), CyclinD1 (RR 1.42), and E-Cadherin (RR 0.71) were significant prognostic factors. Combination analysis of these genes revealed that E-Cadherin-preserved and CyclinD1-negative patients had the best prognosis; E-Cadherin-reduced and CyclinD1-positive patients had the worst prognosis.. Increased CyclinD1 expression and reduced E-Cadherin expression were significant prognostic factors in patients with esophageal squamous cell carcinoma.

Topics: Analysis of Variance; Biopsy; Cadherins; Carcinoma, Squamous Cell; Chemotherapy, Adjuvant; Cyclin D1; Esophageal Neoplasms; Esophagectomy; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Radiotherapy, Adjuvant; Retrospective Studies; Survival Analysis; Treatment Outcome

Post-initiation ethanol modification on N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal carcinogenesis model was investigated in male, 6-week-old, F344 rats that received s.c. injections, 3 times per week, of 0.5 mg/kg NMBA for the first 5 weeks and then were treated with 0% (Group 1), 3.3% (Group 2), and 10% (Group 3) ethanol in the drinking water for up to 20 weeks. Group 4 received 10% ethanol without NMBA administration and Group 5 was maintained without any chemical treatment. There were no statistical differences in the incidence and multiplicity of esophageal tumors among Groups 1 to 3. However, the multiplicity of hyperplasias was statistically greater in Group 3 than in Groups 1 or 2. Esophageal epithelia of all rats in Groups 4 and 5 demonstrated a normal histology. BrdU labelling indices of tumors and hyperplasias in NMBA-treated groups were essentially similar, although cycline D1 was overexpressed to a greater extent in tumors and also hyperplasias of Group 3 than in Groups 1 or 2. The results indicated ethanol to exert weak promotion effects through cycline D1 overexpression on rat esophageal tumorigenesis initiated with NMBA.

Topics: Animals; Bromodeoxyuridine; Carcinogens; Cyclin D1; Dimethylnitrosamine; Disease Progression; Epithelium; Esophageal Neoplasms; Esophagus; Ethanol; Hyperplasia; Immunohistochemistry; Male; Rats; Rats, Inbred F344

N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis is an important model for squamous cell carcinoma of the human esophagus. In this model, previous studies have shown that the GGA-->GAA Ha-ras codon 12 mutation is present in the majority of papillomas. No other Ha-ras mutation has been identified. Studies using other models of chemical carcinogenesis suggest that Ha-ras activation has a critical role during tumor initiation. We have used laser-capture microdissection and polymerase chain reaction-restriction fragment length polymorphism analysis to study the role of codon 12 Ha-ras mutation at various stages of tumor development in the rat esophagus. Our results indicate that Ha-ras mutation was present infrequently (4.3%) in premalignant lesions. The incidence of Ha-ras mutation was high in papillomas (57.1%), however, and 50% of papillomas expressed mutant Ha-ras RNA message. Additionally, there was a linear trend correlating increased incidence of Ha-ras mutation with later papilloma stage. These data suggest the role of ras activation later in neoplastic development. To evaluate the potential mechanism of action by which Ha-ras contributes to promotion and progression in this model, we compared mRNA expression of cyclin D1 and p27 in Ha-ras mutant and Ha-ras normal papillomas. We found no differences in mRNA expression of either cyclin D1 or p27 between these two papilloma populations. Our data suggest an important paradigm shift for the role of ras mutations in this model of chemical carcinogenesis, indicating a functional role of Ha-ras activation in promotion/progression and not in the initiation phase of NMBA-induced papillomagenesis.

Topics: Animals; Carcinogens; Cell Cycle Proteins; Codon; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dimethylnitrosamine; Disease Progression; DNA Primers; Esophageal Neoplasms; Genes, ras; Hypoxanthine Phosphoribosyltransferase; Incidence; Male; Papilloma; Point Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Precancerous Conditions; Rats; Rats, Inbred F344; RNA, Messenger; Tumor Suppressor Proteins

Alterations in the cell cycle regulatory cyclin/retinoblastoma protein (pRB) pathway play a important role in tumor progression in esophageal squamous cell carcinoma (ESCC). In the present study, we evaluated the prognostic significance of the combined analysis of cyclin D1 and pRB in ESCC retrospectively.. Immunoreactivities of cyclin D1 and pRB were evaluated in 148 surgically resected ESCC by use of monoclonal antibodies. Disease-free survival of patients was compared among the four subgroups according to the phenotypes of cyclin D1 and pRB expressions.. High immunoreactivities of pRB and cyclin D1 were detected in 64.2% and 40.5% of tumors, respectively. The loss of pRB expression and overexpression of cyclin D1 correlated with short survival. However, these factors were not detected as independently prognostic in multivariate analysis. In 107 surviving patients who underwent curative operation, co-expressed pRB and cyclin D1 (pRB+/cyclin D1 +: 29 patients) were correlated with unfavorable prognosis (disease-free 5-year survival rate: 42.7%) and high cancer recurrence rate (44.8%) compared with that of 40 patients with pRB +/cyclin D1- tumors (70.5% and 27.5%). The disease-free 5-year survival rate of patients with pRB+/cyclin D1- tumors was significantly better than that of other groups (P=0.001). However, the disease-free 5-year survival rate of 29 patients with pRB+/cyclin D1 + tumors was equivalent to that of 29 patients with pRB-/cyclin D1tumors (48.3%), and that of nine patients with pRB-/cyclin D1+ tumors (22.2%, P=0.237).. Our results suggest that overexpression of cyclin D1 may suppress pRB function, and that combined analysis of pRB and cyclin D1 may be a useful parameter of patient prognosis in ESCC.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Disease-Free Survival; Esophageal Neoplasms; Female; Humans; Male; Middle Aged; Multivariate Analysis; Phosphorylation; Retinoblastoma Protein; Survival Rate

Cyclin D1 is known to play important roles in the G1/S check-point of the cell cycle. We investigated the correlation between cyclin D1 overexpression and clinical characteristics to clarify its prognostic significance in patients with esophageal cancer.. From 1991 to 1998, cyclin D1 was investigated in esophageal cancers from 86 patients who underwent esophagectomy. Overexpression of cyclin D1 was demonstrated using an immunohistochemical method.. Overexpression of cyclin D1 was found in 23 (26.7%) of 86 cases. Overexpression of cyclin D1 correlated with lymph node metastasis (P = 0.0083) and lymphatic vessel invasion (P = 0.018). Cyclin D1 overexpression may indicate resistance to chemotherapy. The patients with cyclin D1 overexpression had a significantly lower survival rate than those without overexpression (P = 0.013). The multivariate analysis revealed cyclin D1 overexpression to be an important prognostic factor in patients with esophageal cancer.. Immunohistochemical examination of cyclin D1 expression may provide important prognostic information in univariate and multivariate analysis and may be necessary for determining therapeutic strategies for esophageal cancer.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cyclin D1; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Survival Analysis

Cell cycle regulatory proteins were analysed by immunohistochemistry in order to clarify how their expression changes with the degree of atypia as oesophageal surface squamous epithelium progresses from normal mucosa, through reactive change, low-grade dysplasia, and high-grade dysplasia to mucosal invasive carcinoma.. Immunostaining for cyclin D1, cyclin E, p21, p27, p53 and Ki67 proteins was performed using 22 normal mucosa, 17 reactive change, 22 low-grade dysplasia, 15 high-grade dysplasia and 22 mucosal invasive carcinoma specimens. Normal mucosa, low-grade dysplasia and high-grade dysplasia samples were taken from patients without any oesophageal invasive carcinoma by endoscopic biopsy or endoscopic mucosal resection, and reactive change and mucosal invasive carcinoma were obtained from oesophagectomy material. Stepwise over-expression of cyclin E (P < 0.0001) and p53 (P < 0.0001), reduction of p21 (P=0.0189) and dysregulation of cyclin D1 and p27 were observed in the multistep process of oesophageal carcinogenesis. Significant differences in expression of p27 (P < 0.0001), p53 (P=0.0299) and Ki67 (P=0.0101) were observed between reactive change and low-grade dysplasia. Furthermore, expression of cyclin D1, cyclin E, p27 and p53 in mucosal invasive carcinoma were significantly different from those in high-grade dysplasia (P=0.0079, P=0.0237, P=0.0042 and P= 0.0299, respectively).. Cell cycle regulatory proteins, cyclin E, p53 and p21 show stepwise over-expression or reduction with progression of oesophageal carcinogenesis, correlating with the increased cell proliferation observed with Ki67 labelling. We conclude that immunohistochemical analysis for p27, p53 and Ki67 is practically useful for the discrimination between low-grade dysplasia and reactive change. Cyclin D1, cyclin E, p27 and p53 help to distinguish high-grade dysplasia from mucosal invasive carcinoma.

Topics: Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Esophageal Neoplasms; Humans; Immunohistochemistry; Ki-67 Antigen; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

There is controversy as to whether esophageal squamous dysplasia is a pre-cancerous lesion or a non-cancerous lesion. In this study, we conducted an immunohistochemical investigation of cyclin D1, retinoblastoma (Rb), p16INK4 and p27KIP1 expression in 36 squamous dysplasias and 34 early squamous cell carcinomas of the esophagus. The frequency of cyclin D1 overexpression was similar in dysplasias and early cancers (30% vs. 35%). Loss of p16INK4 and p27KIP1 expression was less frequent in dysplasias than in early cancers (p=0.005 and 0.001, respectively). Loss of Rb protein expression was not detected in dysplasia and rarely observed in early cancer (7%). The proliferation cell nuclear antigen index increased from moderate dysplasia to mucosal invasive carcinoma and was correlated significantly with the expression of cyclin D1, p16INK4 and p27KIP1 (p=0.0001, 0.003, and 0.007, respectively). Thus, this study found that cyclin D1 overexpression starts early in dysplasia and could be a useful marker for its malignant potentiality while reduction of p16INK4 and p27KIP1 occurs during the transformation from dysplasia to cancer. These findings suggest that esophageal dysplasia should be treated as a precancerous lesion.

Topics: Biomarkers, Tumor; Carcinoma in Situ; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Humans; Proliferating Cell Nuclear Antigen

The lymph nodes of 59 patients with pN0 esophageal squamous cell carcinomas were examined immunohistochemically using cytokeratin (CK) antibody. Primary tumors were immunostained with cyclin D1 (CD1) and E-cadherin (E-cad) antibody. Lymph node micrometastasis (MM) was found in 39 (55.5%) patients. Tumor recurrence was found in 17 patients and all but one of them had MM. The 5-year survival rate was significantly poorer in patients with MM than in those without MM. Almost all patients with positive CD1 and negative E-cad expression had MM. The examination of CD1 and E-cad expression in primary tumors may be useful for predicting MM.

Topics: Adult; Aged; Aged, 80 and over; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; Epithelium; Esophageal Neoplasms; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis

The purpose of the present study was to define the overexpression of cyclin D1 in superficial and advanced esophageal carcinomas and to investigate whether the expression of this molecule indicates a poor prognosis. This study included 41 patients with superficial esophageal carcinomas (Tis and T1) and 48 patients with advanced esophageal carcinomas (T2, T3, and T4). The expression of cyclin D1 in surgically resected specimens was evaluated immunohistochemically with a monoclonal antibody. Positive immunoreactivity was found in 31 of 89 cases (35%). Overexpression of cyclin D1 did not correlate with TNM classification or histologic type. Of the 48 patients with advanced esophageal carcinomas, 32 patients with cyclin D1-negative tumors survived longer than did 16 patients with cyclin D1-positive tumors (P = 0.0017). In contrast, we observed no survival difference between patients with cyclin D1-positive and -negative superficial esophageal carcinoma. These results suggest that cyclin D1 indicates a poor prognosis in cases of advanced esophageal carcinoma but not in cases of superficial esophageal carcinoma.

Topics: Biomarkers, Tumor; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Carcinosarcoma; Cyclin D1; Esophageal Neoplasms; Esophagus; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis

The aim of this study was to evaluate whether cisplatin sensitivity relates to patient's prognosis in oesophageal squamous cell carcinoma and to find a useful chemosensitivity molecular marker. 59 oesophageal squamous cell carcinoma (SCC) patients received cisplatin 30 mg/m2/week treatment of two to five cycles, followed by oesophagectomy. We analysed retrospectively whether the histological effect was related to patient's prognosis. Furthermore, to evaluate the relationship between the effect of preoperative cisplatin treatment and p53 and cyclin D1 expression, we investigated p53 and cyclin D1 expression in pretreatment biopsy samples using an immunohistochemical analysis and compared the results with the histological effect to cisplatin in the resected oesophagus. The cases that showed immunohistochemical p53 staining in the pretreatment biopsy samples were resistant to cisplatin (P = 0.032). However, there was no relationship between cyclin D1 expression and histological effect (P = 0.230). Nevertheless, combined analysis of p53 and cyclin D1 can predict histological effect (P = 0.032). The prognosis of cisplatin-sensitive cases was significantly better than that of cisplatin-resistant cases (P = 0.041). Cox's multivariate analysis revealed that the histological effect was an independent prognostic factor. In contrast, p53 protein accumulation and cyclin D1 were not. Histological response after neoadjuvant cisplatin treatment is a prognostic factor for oesophageal SCC and cisplatin chemotherapy may be selected according to the findings of p53 and cyclin D1 expression.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cisplatin; Cyclin D1; Esophageal Neoplasms; Esophagectomy; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoadjuvant Therapy; Neoplasm Staging; Prognosis; Survival Rate; Tumor Suppressor Protein p53

Topics: Adenocarcinoma; Bias; Biomarkers, Tumor; Case-Control Studies; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Research Design; Up-Regulation

Esophageal adenocarcinoma commonly arises from a precancerous condition, Barrett's esophagus, in which the normal squamous epithelium is replaced by a columnar cell-lined epithelium. Genetic alterations occurring in this process could serve as biomarkers for the risk of malignant progression, improve surveillance, and contribute to early diagnosis. We examined two potential biomarkers, cyclin D1 and p53, in a prospective cohort of Barrett's esophagus patients.. A total of 307 patients were enrolled in an endoscopic surveillance cohort, and esophageal biopsy specimens were collected at each endoscopy. Incident cases of adenocarcinoma were matched to control patients within the cohort by duration of follow-up, age, sex, and length of columnar cell-lined epithelium at recruitment. Biopsy specimens were analyzed for cyclin D1 and p53 protein levels by immunohistochemistry. Statistical tests were two-sided.. A total of 12 cases of adenocarcinoma occurred within the follow-up period, and tumor biopsy specimens from 11 cases stained positive for cyclin D1. Biopsy specimens from eight of these patients taken at recruitment also stained positive for cyclin D1. A case-control analysis of biopsy specimens obtained at recruitment revealed a statistically significantly increased risk of progression to adenocarcinoma in Barrett's esophagus patients whose biopsy specimens were cyclin D1 positive (odds ratio [OR] = 6. 85; 95% confidence interval [CI] = 1.57-29.91; P =.0106) but not in patients whose biopsy specimens were p53 positive (OR = 2.99; 95% CI = 0.57-15.76; P =.197).. Cyclin D1-positive staining could be a useful biomarker in identifying Barrett's esophagus patients at high risk of esophageal adenocarcinoma. Given the complexity of genetic alterations in the natural history of this cancer, additional biomarkers will be required to increase the sensitivity and specificity of molecular diagnosis.

Topics: Adenocarcinoma; Aged; Barrett Esophagus; Case-Control Studies; Cell Transformation, Neoplastic; Cyclin D1; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Odds Ratio; Prospective Studies; Risk; Tumor Suppressor Protein p53; Up-Regulation

The p16ink4a-cyclin D1/cyclin-dependent kinase 4 (Cdk4)-retinoblastoma (Rb) pathway has emerged as a critical target in oncogenesis. The zinc-deficient (ZD), N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer model provides a tool to study cell proliferation and cell cycle control in cancer initiation. Weanling rats were fed a ZD or zinc-sufficient (ZS) diet for 5 weeks, and then given a dose of NMBA. After 14 weeks, esophageal tumor incidence was 88% in ZD rats with highly proliferative esophagi versus 0% in ZS rats. Expression of p16ink4a, cyclin D1, Cdk4, and Rb in relation to that of proliferating cell nuclear antigen was characterized in esophagi by immunohistochemistry at 0, 24, and 48 h, and 1, 3, 7, 10, and 14 weeks after NMBA treatment. As early as 24 h, proliferating cell nuclear antigen-positive focal hyperplastic lesions were detected in the suprabasal layers of ZD esophagi. At the same time, overexpression of cyclin D1, Cdk4, and Rb was found in the corresponding lesion in adjacent esophageal sections. By contrast, p16ink4a expression was reduced or absent. At all time points, p16ink4a showed reduced nuclear staining in ZD esophagi compared with that in ZS esophagi. In addition, increased expression of the hyperphosphorylated forms of Rb was detected in ZD esophagi by immunoblotting. Importantly, tumors were consistently observed in ZD esophagi at very early time points. These data, obtained using a unique in vivo model for esophageal cancer with rapid tumor induction, provide strong evidence for a link between deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and the initiation of esophageal tumors.

Topics: Animals; Carcinogens; Carrier Proteins; Cell Division; Cocarcinogenesis; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Dimethylnitrosamine; Esophageal Neoplasms; Esophagus; G1 Phase; Immunohistochemistry; Male; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; S Phase; Signal Transduction; Zinc

Ionized radiation leads to G1 arrest and apoptosis by a p53-dependent pathway and G2-M arrest through a p53-independent pathway. In this study, we evaluated the role of cell cycle-regulating molecules in the sensitivity of cancer cells for radiation therapy. Forty-seven patients with squamous cell carcinomas of the esophagus had undergone radiation therapy, followed by surgical resection. They were classified as sensitive to radiation (SR, 14 cases) with no residual tumor in the surgical specimen or as resistant to radiation (RR, 33 cases) with viable residual tumors. Their preradiation biopsy samples were immunohistochemically investigated for the expressions of cell cycle-related molecules, including p53, CDC25A, CDC25B, cyclin D1, cyclin B1, and Ki-67. p53 expression was negative in 71% (10 of 14) of SR and positive in 91% (30 of 33) of RR. The association was strong between high radiation sensitivity and negative p53 expression (P < 0.0001). CDC25B, which is not expressed in normal epithelium but is in the cytoplasm of esophageal cancers, was strongly expressed (2+) in 46% (6 of 14) of SR and in 6% (2 of 23) of RR. Thus, the sensitivity for radiation therapy was significantly correlated with CDC25B overexpression. With respect to CDC25A, cyclin D1, cyclin B1, and Ki-67, no statistically significant differences were found in their expressions between SR and RR tumors. p53 and CDC25B expressions showed no significant associations, and multivariate analysis revealed that both p53 and CDC25B are significant independent markers for predicting radiation sensitivity. CDC25B was revealed to be a novel predictor of radiation sensitivity in esophageal cancers. Because CDC25B is an oncogene, which affects G2-M progression, these results suggest the importance of a p53-independent G2-M checkpoint in radiation therapy.

Topics: Antimetabolites, Antineoplastic; Biopsy; Carcinoma, Squamous Cell; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cyclin B; Cyclin B1; Cyclin D1; Cytoplasm; Dose-Response Relationship, Radiation; Esophageal Neoplasms; Fluorouracil; Humans; Immunoblotting; Immunohistochemistry; Ki-67 Antigen; Radiation Tolerance; Tumor Suppressor Protein p53

To investigate the effects of different refluxant on esophageal carcinogenesis in rats.. The animal models of gastroesophageal reflux (G), duodenoesophageal reflux (D) and duodenogastroesophageal reflux (DG) and no reflux as control (C) were made by operations. The rats in all of the groups were given carcinogen (methyl-n-amyl nitrosamine, MANA). They were killed at 4, 20, 26, 40-week, then their esophagi were taken to the morphologic study and the expression of p53, cyclinD(1), CDK(4) studied with immunohistochemical studies.. At 4-week without carcinogen, most of rats in reflux groups displayed evidence of esophageal mucosal injury. The degree of mucosal injury in D group was greater than that in DG which was greater than that in G; and became severity with the time. At 40 weeks, the incidences of papillomas in group D, DG, G were 94.7%, 95.2%, 70.5%, respectively. All of them compared to the group C (38.5%) were significant difference. Only in both D and DG group, there were 24 with esophageal columnar metaplasia, 20 with dysplasia and 11 with cancer. The overexpression of p53, CDK(4), cyclin D(1) was seen in D and DG groups, while in G group, the overexpression of p53 and cyclin D(1) were seen only.. Both of gastric juice and duodenal contents reflux can promote tumourgenesis of MANA on esophagi by changing the expression of cell cycle-related protein. The effect of duodenal contents is stronger. It may play an important role in tumourgenesis of gastroesophageal reflux disease.

Topics: Animals; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Esophageal Neoplasms; Female; Gastroesophageal Reflux; Male; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53

We previously described the oral-esophageal tissue-specific expression of cyclin D1 with the Epstein-Barr virus ED-L2 promoter in transgenic mice, and resulting dysplasia. Given the evidence for an interplay between environmental and genetic factors in esophageal squamous carcinogenesis, the aim of this study was to determine the potential cooperation of the nitrosamine compound N-nitrosomethylbenzylamine (NMBA), an esophageal specific carcinogen, in the cyclin D1 transgenic mice. NMBA was first demonstrated to induce dysplasia in two strains of inbred mice, C57BL/6 and FVB/N. Subcutaneous NMBA was then administrated to wild type and transgenic mice beginning at 4 weeks of age. Mice were monitored for the duration of the study for general appearance, activity and weight, and were euthanized at 12 and 15 months. Histopathologic analysis revealed increased severity of dysplasia in cyclin D1 mice treated with NMBA compared with treated age-matched wild-type mice and untreated mice. There was also increased proliferating cell nuclear antigen (PCNA) expression in the esophagi of NMBA treated cyclin D1 mice. Taken together, these findings suggest that a genetic alteration, specifically cyclin D1 overexpression and a chemical carinogen, NMBA, may cooperate to increase the severity of esophageal squamous dysplasia, a prominent precursor to carcinoma.

Topics: Animals; Carcinogens; Cell Division; Cyclin D1; Dimethylnitrosamine; Epithelium; Esophageal Neoplasms; Gene Expression; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms, Squamous Cell

In the present study, the expression of cyclin D1, as detected by immunohistochemistry, was compared with other prognostic variables and its prognostic impact was evaluated in a group of 172 patients with squamous cell carcinoma (SCC) of the esophagus who underwent potentially curative resection therapy and in a second group of 38 patients with SCC of the esophagus who were treated by combined modality therapy (radiochemotherapy +/- surgery). Expression of cyclin D1 in surgically treated carcinomas correlated negatively with tumor differentiation (p = 0.026) but positively with mitotic activity (p = 0.0199) and nodal status (p = 0.040). There were no significant correlations with pT category. Patients with cyclin D1-positive carcinomas showed significantly worse overall survival than patients with cyclin D1-negative carcinomas, both in univariate (p = 0.0016) and in multivariate survival analyses (p = 0.0038). Expression of cyclin D1 in carcinomas with multimodal treatment was correlated with poor response to chemotherapy (p = 0.026) but not with overall survival. We thus consider expression of cyclin D1 to be an important parameter, predicting an unfavorable overall survival of surgically treated esophageal cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Cyclin D1; Epirubicin; Esophageal Neoplasms; Esophagus; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Mucous Membrane; Prognosis; Survival Rate

Esophageal adenocarcinoma often arises in association with metaplastic and dysplastic mucosa in Barrett's esophagus. Derangements in cell cycle control and apoptosis regulation might be responsible for the progression from metaplasia to dysplasia and adenocarcinoma We tested this hypothesis by performing cell cycle analysis, in situ detection of apoptosis, and evaluation for the immunohistochemical expression of proteins involved in proliferation (Ki-67), the control of apoptosis (bcl-2, bcl-x and bax), and cell cycle regulation (retinoblastoma and cyclin D1). We studied 17 randomly selected paraffin-embedded esophagectomy specimens that contained intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma Compared with gastric controls and intestinal metaplasia without dysplasia, high-grade dysplasia and esophageal adenocarcinoma demonstrated greater numbers of cells in S phase and G2 phase. Comparison of the proliferation index and the apoptotic rate in intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma showed a statistically significant trend that linked an increasing proliferation index and apoptotic rate with increasing histologic severity (P = .006 and P = .0002, respectively). A statistically significant linear association was found between bcl-x expression, bax expression, and the bcl-2-to-bax expression ratio versus increasing histologic severity (P = .0004, P = .007, and P = .03, respectively). These data support the hypothesis that neoplastic transformation of intestinal metaplastic epithelium in the esophagus might result from sequential changes in the expression of proteins involved in the control of apoptosis and the cell cycle. Furthermore, suppression of apoptosis does not seem to foster neoplastic growth in Barrett's esophagus. These observations will lead to a better understanding of the pathogenesis of esophageal adenocarcinoma and might contribute to enhancing the diagnostic accuracy when presented with dysplastic lesions.

Topics: Adenocarcinoma; Aged; Apoptosis; Barrett Esophagus; bcl-2-Associated X Protein; bcl-X Protein; Cell Cycle; Cell Division; Cyclin D1; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Parietal Cells, Gastric; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein

Recent developments in molecular biology have revealed that several oncogenes, suppressor genes and adhesion molecules are involved in the development of oesophageal cancer; however, the role of these genes is still unknown. To evaluate which molecular biological factors are related to patients' prognosis and recurrence, we checked p53, p16, p21/Waf1, cyclin D1, Ki-67, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), Mdm2, Bcl2, E-cadherin and MRP1/CD9 by means of immunohistochemical analysis in 116 cases of oesophageal cancer (R0). We also checked the regrowth capability of the primary cultures of the resected tumours and the effect of post-operative treatment. Although univariate analysis revealed that pN (pTNM), pT (pTNM), sex, cyclin D1, Ki-67, VEGF, E-cadherin and cell regrowth capability were prognostic factors, multivariate analysis revealed that pN (risk ratio (RR) 3.17), sex (RR 8.13), cell regrowth capability (RR 3.03) and E-cadherin (RR 0.30) were prognostic factors. Interestingly, step-wise analysis revealed that the following five factors were prognostic factors: pN (RR 5.74), sex (RR 3.14), cyclin D1 (RR 2.29), E-cadherin (RR 0.26) and cell regrowth capability (RR 1.94). Logistic regression analysis revealed that the risk factors of haematogenous recurrence were pN (odds ratio (OR) 8.97), cyclin D1 (OR 4.52) and EGFR (OR 0.18). On the other hand, the risk factor of lymph node recurrence was pN (OR 5.16). With regard to the effect of postoperative treatment, post-operative radiotherapy was a favourable risk factor (RR 0.43) and reduced the haematogenous recurrence (OR 0.18). Our data indicate that combination analysis using pN, sex, cyclin D1, E-cadherin, EGFR and cell regrowth capability may be useful for the prediction of patient survival and recurrence.

Topics: Aged; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Risk Factors; Survival Analysis; Treatment Outcome

The expressions of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4), and CDK2 were immunohistochemically examined in 90 patients with human oesophageal squamous cell carcinoma (SCC) to determine their relationship to the tumour behaviour and patient prognosis. Nuclear immunostaining of cyclin D1 and cyclin E was observed in 28 (31.1%) and 27 tumours (30.0%) respectively. Thirty-nine tumours (43.3%) and 31 tumours (34.4%) exhibited both cytoplasmic and nuclear positivity for CDK4 and CDK2 respectively. Of 28 cyclin D1-positive and 27 cyclin E-positive tumours, CDK4 was overexpressed in 12 (42.8%) tumours and CDK2 in seven (25.9%) tumours respectively. There was no significant relationship in immunopositivity between cyclin D1 and CDK4 or between cyclin E and CDK2. Simultaneous immunoreactivity for both cyclin D1 and CDK4 was significantly associated with venous invasion (P < 0.05). In a univariate analysis, the prognosis of patients with tumours that were both cyclin D1- and CDK4-positive was significantly poorer than that of patients with cyclin D1-negative tumours (P < 0.05). In a multivariate analysis, both cyclin D1 and CDK4 immunoreactivities (P < 0.01) and tumour stage (P < 0.001) were recognized as independent risk factors. In this analysis, the hazard ratio for cyclin D1-positive and CDK4-negative cases compared with cyclin D1-negative cases was significant (hazard ratio = 3.128, 95% confidence interval = 1.418-6.899, P = 0.0047). No significant prognostic relevance was detected in both cyclin E and CDK2 immunoreactivity. Our in vivo findings suggest that in human oesophageal SCC, cyclin D1 and cyclin E and their functional partners, CDK4 and CDK2, often exhibit dysregulated overexpression in many cases, and that tumours with simultaneous expression of cyclin D1 and CDK4 are frequently associated with venous invasion and have a worse prognosis, statistically. Moreover, overexpression of cyclin D1 alone may also contribute to tumour progression independent of CDK4 overexpression.

Topics: Aged; Carcinoma, Squamous Cell; CDC2-CDC28 Kinases; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Survival Analysis

This study investigated amplification of the cyclin D1 and MDM-2 genes, and overexpression of the cyclin D1 gene product, in oesophageal carcinoma.. Paired tumour and normal DNA samples from 26 oesophageal adenocarcinomas and 19 squamous cell carcinomas were analysed by Southern blotting with specific DNA probes for cyclin D1 and MDM-2, and for a control gene (alpha-lactalbumin). The cyclin D1 and MDM-2 gene copy numbers were calculated for each tumour. Expression of the cyclin D1 gene was assessed by immunohistochemical analysis of its protein product.. Cyclin D1 gene amplification (by a factor of between two and six) was identified in seven tumours (16%). MDM-2 gene amplification (by a factor of between two and 11) was identified in 10 tumours (22%). Overexpression of cyclin D1 protein was identified in eight tumours and was significantly associated with gene amplification (P=0.04; Fisher's exact test), and with early T stage (P=0.01; Fisher's exact test).. Cyclin D1 and MDM-2 amplification and cyclin D1 overexpression occur, although infrequently, in the development of oesophageal carcinoma. Cyclin D1 overexpression may influence tumour behaviour, causing the disease to present at an earlier T stage. The mechanism for this effect is unclear, and warrants further investigation.

Topics: Adenocarcinoma; Blotting, Southern; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Gene Amplification; Humans; Immunohistochemistry; Neoplasm Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Up-Regulation

Recently, various cell cycle regulators have been investigated as biological markers of malignant potential. These regulators might influence the survival rate and the effect of adjuvant therapies. In this study, we analyzed p53, p21(Waf1/Cip1) and cyclin D1 expression in 64 esophageal cancer patients and the relationship between clinicopathologic parameters and patient survival. The positive expression rate was 48.4%, 42.2% and 43.8% in the p53, p21 and cyclin D1 groups respectively. Multivariant analysis revealed that tumor depth, chemotherapy, p53, p21 and cyclin D1 expression showed significant values. p53- and cyclin D1-negative patients had a worse prognosis. p21-positive patients had a better prognosis. In stage 0, I and II patients, there was a significant difference between p53-positive and -negative, p21-positive and -negative, and cyclin D1-positive and -negative groups. In stage III and IV patients, there was no significant difference between any two groups. However, a significant difference was seen in the p21 group: among patients who received adjuvant chemotherapy, the p21-positive group had a 5-year survival rate of 50% compared with 13.4% in the p21-negative group (not significant).

Topics: Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Esophageal Neoplasms; Female; Humans; Male; Prognosis; Proportional Hazards Models; Survival Rate; Tumor Suppressor Protein p53

Increased expression of the cyclin D1 gene frequently occurs in human squamous carcinomas of the esophagus. However, the expression of cyclin D1 has not been previously examined in detail in adenocarcinomas of the esophagus or stomach. Therefore, we examined, in parallel, the expression of cyclin D1 in both squamous and adenocarcinomas of the esophagus and in adenocarcinomas of the stomach. The level of expression of the cyclin D1 protein was assessed by immunohistochemistry in 39 esophageal and 34 gastric carcinomas and correlated with clinical and pathology parameters. Within the esophagus, 71% of the squamous carcinomas and 64% of the adenocarcinomas were positive for increased cyclin D1 nuclear staining. For adenocarcinomas of the stomach, the overall positive rate was 47%; in the gastric cardia, the rate was 44%, and in other regions of the stomach, it was 50%. In esophageal and gastric adenocarcinomas of the intestinal type, increased expression of cyclin D1 was seen in 70% of the samples, whereas with the diffuse type only 13% were positive (P < .01). Tumors from patients older than the median age of 67 years were more frequently positive than tumors from patients younger than 67 years (74% v 42%, respectively) (P < .01). Positive staining was also seen more frequently in well and moderately differentiated tumors than in poorly differentiated tumors (74% v 49%, respectively) (P < .05). Cytoplasmic staining for cyclin D1 was noted in 22% of the tumors, of various types. Therefore, increased expression of cyclin D1 frequently occurs in both adenocarcinomas and squamous carcinomas of the esophagus, and in adenocarcinomas of the stomach. The increased expression in adenocarcinomas is especially frequent in the intestinal-type lesions.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Stomach Neoplasms

Esophageal carcinoma is one of the most lethal tumors. Therefore, it is important to identify prognostic factors for patients with this disease. The objective of this study was to clarify the relation between clinicopathologic and biologic factors in esophageal carcinoma and to determine the prognostic significance of different biologic factors.. DNA ploidy pattern, Ki-67 labeling index (LI), and cyclin D1 and p53 protein expression were examined and detailed pathologic examinations were conducted on tumors from 53 patients (46 males and 7 females with a mean age of 66 years [range, 47-85 years]) with surgically resected esophageal squamous cell carcinoma and the prognostic value of these factors was evaluated.. Of the 53 esophagus carcinomas examined, 26 (49%) were classified as DNA diploid. The mean Ki-67 LI was 45 +/- 4. 9% (range, 10.5-86.1%). p53 expression was detected in 38 of the carcinomas (71.7%) and cyclin D1 expression was detected in 35 (66%). Various prognostic factors were examined using the Cox stepwise regression model, four of which were found to correlate with overall survival: tumor size (P = 0.0346), lymph node status (P = 0.0384), Ki-67 LI (P = 0.0161), and p53 expression (P = 0.001). Lower Ki-67 LI and a lower rate of p53 expression were detected in the long term survival group (> 3 years) compared with the short term survival group (P = 0.00045 and P = 0.0023, respectively).. The biologic factors of Ki-67 LI and p53 expression, as well as clinicopathologic factors, may be used as independent prognostic factors for patients with esophageal carcinoma. However, the results of the current study do not support cyclin D1 expression as a prognostic factor.

Topics: Aged; Aged, 80 and over; Biomarkers; Carcinoma, Squamous Cell; Cyclin D1; DNA, Neoplasm; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Ploidies; Prognosis; Survival Analysis; Survival Rate; Tumor Suppressor Protein p53

Amplification and expression status of the CCND1 gene in 12 cell lines derived from esophageal cancer was evaluated and the differences in the cell proliferation and morphology were investigated. Amplification was observed in 6 cell lines, all of which consequently overexpressed the mRNA. Although the amplification of the gene did not contribute to an increase of cell proliferation, an in vitro morphological transformation was observed in the colonies with an amplification of the CCND1 gene. An amplification/overexpression of the CCND1 gene plays an important role in the enhancement of cell transformation in esophageal squamous cell carcinoma cells.

Topics: Blotting, Northern; Blotting, Southern; Cell Division; Cyclin D1; DNA, Neoplasm; Esophageal Neoplasms; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Proliferating Cell Nuclear Antigen; RNA, Neoplasm; Tumor Cells, Cultured

It has recently been reported that the reduced expression of p27(Kip1) is a negative prognostic marker in several carcinomas. In this study, we examined the expression of p27(Kip1) in esophageal squamous cell carcinomas in order to understand its prognostic role. We also examined the expression of cyclinD1, which is believed to be correlated with the prognosis. Of the 128 cases, 64 cases (50.0%) showed low grade p27(Kip1) immunostaining and 64 cases (50.0%) high grade immunostaining; there was no significant difference in survival (p = 0.0915) between the two groups. On the other hand, 51 of the 156 cases (32.7%) were classified as the high cyclinD1 group, and 105 of the 156 cases (67.3%) as the low group, thus representing prognostic significance with regard to survival (p = 0.0161). Multivariate analysis indicated that gender, lymph node metastasis and positive cyclinD1 were independent prognostic factors. Our results revealed that cyclinD1 was a significant prognostic predictor of esophageal squamous cell carcinomas, whereas p27(Kip1) was not a significant prognostic factor.

Topics: Aged; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Microtubule-Associated Proteins; Middle Aged; Multivariate Analysis; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Risk Factors; Survival Analysis; Tumor Suppressor Proteins

Multiple primary upper aerodigestive tract carcinomas can occur in up to 15% of patients. We have shown previously that half of the patients with multiple upper aerodigestive tract squamous cell carcinomas are initially seen with synchronous tumors. Most metachronous squamous cell carcinomas become manifest within 3 years.. To examine the expression of 2 proteins-cyclin D1 and p53--in an attempt to predict the occurrence of multiple primary malignant neoplasms (MPs).. Monoclonal antibodies to cyclin D1 (DCS-6 [dilution, 1:50]. Novocastra Laboratories Ltd, Newcastle, England) and p53 (DO-7 [dilution, 1-100], Dako Corp, Carpinteria, Calif) proteins were used. Resection specimens from a total of 47 patients, 12 patients with MP and 35 patients with nonmultiple primary malignant neoplasms, were analyzed. Those in the nonmultiple primary malignant neoplasm group had longer than 3 years' follow-up to ascertain the absence of MP.. Tumor overexpression of cyclin D1 was significantly associated with the development of MP (P<.01). Tumor overexpression of p53 was also frequent in patients with MP although statistical significance was not achieved. The combination of these 2 parameters was an even greater predictor of MP (P<.001).. Overexpression of cyclin D1 and p53 proteins was highly correlated with the development of MP. Additional studies are necessary to confirm this finding. Immunohistochemical evaluation of primary squamous cell carcinomas for cyclin D1 and p53 overexpression may become an important fact of surgical pathologic reporting for primary upper aerodigestive tract squamous cell carcinomas.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Hypopharyngeal Neoplasms; Neoplasms, Multiple Primary; Predictive Value of Tests; Prognosis; Tumor Suppressor Protein p53

The relationship between aberrant expression of cyclin D1 and retinoblastoma (RB) protein and clinicopathological factors was investigated in 80 patients with oesophageal SCC using immunohistochemical analyses. Heterogeneous staining of cancer cell nuclei with antibody to cyclin D1 was found in 31.3% of patients (25 out of 80 patients). Nuclear staining of cancer cells with anti-RB antibody was homogeneous in 10.0% (8 out of 80 patients) and heterogeneous in 58.8% (47 out of 80 patients). Among cases with homogeneous staining for RB protein, 75% (six out of eight patients) exhibited simultaneous positivity for cyclin D1 (P < 0.05). No significant relationship was found between cyclin D1 or RB protein expression and various clinicopathological parameters. The prognosis of patients with cyclin D1-positive tumours was significantly poorer than that of the other patients (P < 0.01). In addition, when patients with cyclin D1-positive and -negative tumours were stratified according to presence or absence of lymph node metastasis and RB status, the cumulative survival rates in the cyclin D1-positive groups were significantly lower for patients without lymph node metastasis (P < 0.01) and for patients whose tumours were positive for RB (P< 0.0001). These findings suggest the possibility that cyclin D1 positivity is a useful prognostic marker related to lymph node metastasis and RB protein expression in human oesophageal SCC, in addition to clinicopathological factors.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Retinoblastoma Protein; Survival Analysis

We studied cyclin D1 gene amplification in four esophageal carcinosarcomas using the differential polymerase chain reaction. Three cases showed amplification in the sarcomatous components, and one of these also showed amplification in the carcinomatous component. No amplification was detected in the other case. We also examined p53 expression in these four tumors by immunostaining. Both components of all four cases showed diffuse overexpression of p53 protein in the nuclei. Our results showed that the cyclin D1 gene is frequently amplified in esophageal carcinosarcoma, especially in the sarcomatous component. Two cases showed amplification only in the sarcomatous component, suggesting that amplification may have a role in the pathogenesis of that component of this malignancy. The diffuse overexpression of p53 observed in both components suggests that such overexpression, which could be the result of somatic mutation, might be an early event in the pathogenesis of esophageal carcinosarcoma.

Topics: Blotting, Southern; Carcinosarcoma; Cyclin D1; DNA Primers; DNA, Neoplasm; Esophageal Neoplasms; Gene Amplification; HeLa Cells; Humans; Immunoenzyme Techniques; Polymerase Chain Reaction; Receptors, Dopamine D2; Tumor Suppressor Protein p53

Cyclin D1, which functionally competes with the tumor suppressor genes retinoblastoma (Rb) and p16INK4, is widely recognized as an oncogene. P27KIP1, which inhibits the cyclin D1-CDK4 complex, is also a putative tumor suppressor gene. In order to evaluate the regulatory interaction of these molecules, a retrospective series of tissues from 66 patients with esophageal squamous cell carcinoma was evaluated immunohistochemically for the expressions of cyclin D1, Rb, p16INK4 and p27KIP1. The expressions of these molecules were correlated with the proliferation cell nuclear antigen (PCNA) index as an indicator of cell proliferation. Cyclin D1 was overexpressed (++) in 28 cases (42%), Rb was lost (-) in 19 cases (24%), p16INK4 was lost (-) in 37 cases (56%) and p27KIP1 was lost (-) in 27 cases (41%). Taken together, disorder of at least one or more of these molecules was observed in 62 cases (92%). Expression of cyclin D1 and p16INK4 was negatively correlated (p<0.03), while expression of cyclin D1 and p27KIP1 was positively correlated (p<0.0004). We found strong overall correlation between expression of cyclin D1 and the PCNA index (p<0.0001), however expression of p16INK4 and p27KIP1 was significantly correlated with the PCNA index in tumors devoid of cyclin D1 overexpression (p<0.03 and p<0.02 respectively). Thus, it was found that cyclin D1 plays a major role and closely related to abnormal cell proliferation in esophageal cancer, however assessment of p16INK4 and p27KIP1 status, particularly in tumors devoid of cyclin D1 overexpression, is necessary for comprehensive evaluation of cancer cell proliferation. Furthermore, expression of cyclin D1 is correlated with that of p16INK4 and p27KIP1 in squamous cell carcinoma of the esophagus.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Esophageal Neoplasms; Female; Humans; Male; Microtubule-Associated Proteins; Middle Aged; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; Retinoblastoma Protein; Retrospective Studies; Tumor Suppressor Proteins

In esophageal carcinoma, individual genetic alterations of cyclins, cyclin-dependent kinase inhibitors, and final effectors of the G1-to-S transition have been documented. Our aim was to design a comprehensive analysis of the role and clinical significance of some critical genes, namely cyclin D1, MTS1, and Rb. To this end, cyclin D1 gene amplification and protein accumulation, Rb gene allelic loss and protein expression, and MTS1 gene mutation and DNA methylation were investigated in a series of 74 esophageal carcinomas. Cyclin D1 amplification was documented in 17 of 55 (31 %) cases, being a feature of squamous cell type (14 of 17 amplified cases). Cyclin D1 accumulation significantly correlated with lymph node metastasis (p < 0.02), advanced tumor stage (p < 0.05), and a reduced overall survival rate (p < 0.03). Rb gene loss of heterozygosity occurred in 14 of 39 (36%) informative cases and was associated with an unfavorable survival rate (p < 0.01). MTS1 gene mutations were detected in 2 adenocarcinomas only; gene methylation was observed in 17 of 72 cases (24%) without any correlations with the variables investigated. A direct association between cyclin D1 and Rb gene accumulation (p < 0.0005) and an inverse one between RB loss of heterozygosity and MTS1 abnormalities (p < 0.05) emerged from this study. These results have important clinical implications because both cyclin D1 and Rb gene deregulation are significantly related to an unfavorable survival rate. In addition, cyclin D1 amplification is associated with esophageal carcinoma of squamous cell type, being totally absent in adenocarcinomas (p < 0.01). The combined evaluation of these genes also demonstrates that molecular abnormalities of genes belonging to the same pathway are mutually exclusive and unnecessary for the neoplastic transformation and tumor progression.

Topics: Carcinoma; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Gene Amplification; Gene Expression; Genes, Retinoblastoma; Humans; Loss of Heterozygosity; Methylation; Mutation; Retinoblastoma Protein

A number of molecules involved in the process of invasion and metastasis of cancer cells have been demonstrated as a biological prognostic parameter. In esophageal cancer, overexpression of the oncogenes (c-erbB, int-2/hst-1/cyclin D1, MDM2), altered expression of suppressor genes (p 16, DCC), and abnormal expression of adhesion molecules (E-cadherin, alpha-catenin) has been reported as markers of high malignant potential. Proliferation markers (Ki-67, AgNORs, PCNA) and angiogenetic factors (intratumoral microvessel density, VEGF) are also related to the prognosis of the patients with various cancers including esophageal cancer. Prognostic significance of p53 is still controversial. In addition to the clinicopathological parameters, combination of these biological markers would be important to predict the clinical outcome of the cases and to establish an individualized strategy of the treatment of each case according to the biological behavior of the cancer cells.

Topics: Cell Division; Cyclin D1; Endothelial Growth Factors; Epidermal Growth Factor; Esophageal Neoplasms; Genes, Tumor Suppressor; Humans; Lymphatic Metastasis; Lymphokines; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Proliferating Cell Nuclear Antigen; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Cyclin D1 (CD1) gene amplification is frequently observed in esophageal carcinosarcoma by differential polymerase chain reaction (DPCR). In this study, fluorescence in situ hybridization (FISH) was performed to show more direct evidence of CD1 gene amplification in patients with esophageal carcinosarcoma. FISH results were also compared with DPCR results studied previously. FISH analysis revealed CD1 gene amplification in all four patients with esophageal carcinosarcoma. CD1 gene amplification occurred with a high incidence in both components of esophageal carcinosarcoma, suggesting that CD1 gene amplification could have an important role in malignant transformation processes of esophageal carcinosarcoma. The results of the current study also suggest that FISH is a more sensitive method than DPCR. Because inactivation of p16 gene (which is a putative tumor suppressor gene) is thought to have similar oncogenic effects with CD1 gene amplification, DPCR was used to examine whether p16 homozygous deletion occurs in esophageal carcinosarcoma. These results suggest that homozygous deletion of the p16 gene occurs less frequently than CD1 gene amplification in esophageal carcinosarcoma. It does not seem to be an alternative event to CD1 gene amplification, though the number of studied cases was small.

Topics: Aged; Aged, 80 and over; Carcinosarcoma; Cyclin D1; DNA Primers; Esophageal Neoplasms; Gene Amplification; Gene Deletion; Genes, p16; Homozygote; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction

To study the effect of cyclin D1 protein expression on the prognosis of esophageal carcinoma treated with preoperative radiotherapy.. From Nov. 1979 to Dec. 1996, 164 patients were treated with preoperative radiation followed by radical esophagectomy in our hospital. All cases were confirmed by pathology as squamous-cell carcinoma. Two vertical radiation fields were used to deliver a total tumor dose of 30-40 Gy, with 15-20 fractions in 3-4 weeks. Immunohistochemical staining of cyclin D1 was performed using a streptavidin-biotin staining technique.. Cyclin D1 protein expression was positive in 62.5%(65/104) and 60.8%(93/153) for pre- and post-radiation tumor specimens, respectively. The expression level did not change following radiation. Post-radiation cyclin D1 expression influenced the survival (P = 0.055), and the survival rate differed significantly between patients who expressed cyclin D1 and those who did not (P = 0.02).. Cyclin D1 expression is of prognostic value for esophageal carcinoma. It may provide a reference criterion for the clinical staging of esophageal cancer.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclin D1; Esophageal Neoplasms; Esophagectomy; Female; Humans; Male; Middle Aged; Prognosis; Radiotherapy, High-Energy

Cyclin D1 is a cell-cycle regulator and candidate proto-oncogene implicated in the pathogenesis of numerous tumor types. Amplification of the cyclin D1 gene occurs commonly in esophageal squamous cell carcinomas. However, no studies have examined the role of cyclin D1 in anal carcinogenesis. We examined 20 esophageal squamous cell carcinomas and 24 anal carcinomas for cyclin D1 alterations. Protein expression was evaluated by immunohistochemistry using the cyclin DIGM antibody (Novocastra, Newcastle upon Tyne, UK). Cyclin D1 amplification was examined by fluorescent in situ hybridization (FISH), using a cyclin D1 probe obtained from Toshiya Inaba at St. Jude Children's Research Hospital, Memphis, TN. The FISH sections were analyzed using a Leica (Deerfield, IL) confocal microscope. By immunohistochemistry, 75% of esophageal carcinomas showed evidence of cyclin D1 expression. Cyclin D1 amplification was detected by FISH in 65% of esophageal cancers. There was good correlation between cyclin D1 protein expression and gene amplification, although some tumors showed protein overexpression in the absence of gene amplification. Among the 24 anal carcinomas studied, 8% showed weak cyclin D1 immunoreactivity in rare tumor cells. None of the anal tumors showed cyclin D1 amplification. We conclude that cyclin D1 alterations are common in esophageal carcinomas but do not appear to be important in anal carcinogenesis. Immunohistochemical detection of cyclin D1 protein overexpression is a good predictor of cyclin D1 amplification.

Topics: Anus Neoplasms; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Esophageal Neoplasms; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Oncogene Proteins; Proto-Oncogene Mas

Overexpression of cyclin D1 in human esophageal carcinomas has been well documented. The aim of the present study was to assess the expression of cyclin D1 in different types of esophageal epithelial lesions induced by N-nitrosomethylbenzylamine (NMBA) in rats. A total of 30 rats received s.c.-injections, five times/week, of 1.0 mg/kg NMBA for a period of 5 weeks followed by the same dose once per week for another 10 weeks. An additional 15 rats were given saline and used as controls to provide normal epithelium. The tumor incidence was 100% at the termination point of 21 weeks. Seventeen rats (57%) showed nuclear staining for cyclin D1, with a great variation in the intensity, as demonstrated by using an immunohistochemical technique. The cyclin D1 positive indices were in the range of 0% to 60% of the individual cells. Negligible staining was observed for normal esophageal epithelium, with a minimal increase in hyperplastic and dysplastic lesions. A significant elevation of cyclin D1 levels was observed in tumors. However, no significant differences were found between papillomas and carcinomas. The immunohistochemical results were confirmed by western blotting analysis. Tumors, papillomas and carcinomas overexpressing cyclin D1 had elevated proliferating cell nuclear antigen (PCNA) indices (P < 0.05). The correlation coefficient of overexpressions of PCNA and cyclin D1 was r = 0.7 for papillomas, but only r=0.3 for carcinomas. The study thus provides strong evidence of relatively early overexpression of cyclin D1 during tumorigenesis in the present rat esophageal model. Cyclin D1 expression is not simply a direct consequence of increase cell proliferation.

Topics: Animals; Cyclin D1; Cyclins; Dimethylnitrosamine; Esophageal Neoplasms; Immunohistochemistry; Male; Oncogene Proteins; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344

Cyclin D1 in cooperation with its major catalytic partners, cyclin-dependent kinases cdk4 and cdk6, facilitates progression through the G1 phase of the eukaryotic cell cycle, in part through phosphorylation of the retinoblastoma protein. Cyclin D1's oncogenic properties have been suggested by its cooperation with ras or adenovirus E1a to transform cultured cells, as well its overexpression in transgenic mice that leads to breast cancer. Activated by a number of different mechanisms in human cancers, the cyclin D1 gene is frequently amplified in squamous epithelial cancers derived from the head/neck and esophageal regions. In order to study the functional consequences of cyclin D1 overexpression in these squamous epithelial specific sites, we have linked the Epstein-Barr virus ED-L2 promoter to the human cyclin D1 cDNA and utilized this transgene to generate founder lines. This transgene is transcribed specifically in the tongue, esophagus and forestomach, all sharing a stratified squamous epithelium. The transgene protein product localizes to the basal and suprabasal compartments of these squamous epithelial tissues, and mice from different lines develop dysplasia, a prominent precursor to carcinoma, by 16 months of age in contrast to age-matched wild-type mice. This transgenic model is useful in demonstrating cyclin D1 may be a tumor initiating event in aero-upper digestive squamous epithelial tissues.

Topics: Animals; Blotting, Southern; Cell Transformation, Neoplastic; Cyclin D1; Cyclins; DNA, Complementary; Esophageal Neoplasms; Esophagus; Gastric Mucosa; Herpesvirus 4, Human; Humans; Mice; Mice, Transgenic; Oncogene Proteins; Phenotype; Precancerous Conditions; Promoter Regions, Genetic; Stomach; Stomach Neoplasms; Tongue; Tongue Neoplasms

In the present study we have characterized eight human esophageal squamous carcinoma cell lines for levels of expression of cyclins D1, E, A and B1; CDKs 1, 2 and 4; the CDK inhibitors p16INK4, p21WAF1 and p27KIP1; the retinoblastoma (Rb) protein; and in vitro CDK2- and CDK4-associated kinase activity; and also compared the growth properties of these cell lines. The level of the cyclin D1 protein varied by over 30-fold amongst the eight cell lines. The high level in two cell lines was associated with amplification of this gene, but in three cell lines it was due to post-transcriptional events. Amongst the eight cell lines there was a significant correlation between the levels of cyclin D1, Rb and p27KIP1 proteins, and CDK4-associated kinase activity. Furthermore, when an exogenous cyclin D1 cDNA was over-expressed in the EC109 cell line by transfection, this led to increased expression of both Rb and p27KIP1. There was, however, no correlation between the level of cyclin D1 expression and the cell doubling times, duration of the G1 phase, or colony-forming efficiency in agar. Two of the cell lines displayed a high level of the cyclin E protein, low levels of cyclin D1, lacked expression of the Rb protein and expressed high levels of the p16INK4 protein. One of these cell lines displayed amplification of the latter gene. There was no correlation between the levels of cyclins E or A and in vitro CDK2 kinase activity, but CDK2 kinase activity was inversely correlated with the duration of the G1 phase of the cell cycle. Taken together, these studies indicate marked heterogeneity in the expression of cell cycle-related proteins amongst a series of esophageal carcinoma cell lines. The correlation between the levels of the cyclin D1, Rb and p27Kip1 proteins suggest the existence of a homeostatic feedback loop between positive and negative acting components of the cell cycle machinery.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Squamous Cell; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Esophageal Neoplasms; Humans; Microtubule-Associated Proteins; Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured; Tumor Suppressor Proteins

To assess the immunoexpression of cyclin D1 and retinoblastoma in a cohort of oesophageal squamous cell carcinoma cases from South Africa to see whether there is a relation between these two proteins. In addition, protein expression was correlated with clinicopathological features.. Fifty biopsies and 30 oesophagectomy specimens were immunostained with commercially available antibodies to cyclin D1 and retinoblastoma proteins, following microwave antigen retrieval.. Twenty three of the 80 cases (29%) showed cyclin D1 protein expression. However, only five cases had > 50% of the tumour cells displaying immunopositivity. Three of the four cases with lymph node spread were cyclin D1 positive in the primary tumour and the metastasis. Fifty three cases were immunoreactive with the antiretinoblastoma antibody; 29 of these cases showing > 50% of cells with immunolabelling. Of the 23 cyclin D1 positive cases, 18 were also retinoblastoma positive. No correlation was observed between cyclin D1 and retinoblastoma protein expression and age, sex, race, or histological grade.. Cyclin D1 is expressed in a minority of cases of oesophageal squamous carcinomas from South Africa. However, three of four cases with lymph node spread were cyclin D1 positive, thus indicating that cyclin D1 positive tumours may have a greater propensity for spread. In addition, 18 of 23 cyclin D1 positive cases also expressed retinoblastoma protein. These findings suggest a possible relation between cyclin D1 and retinoblastoma proteins in a proportion of cases of oesophageal squamous.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Gene Expression; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Proteins; Retinoblastoma Protein

The p16/MTS1/CDKN2 gene and the cyclin D1/PRAD-1 gene cooperatively regulate cyclin-dependent kinase 4-mediated phosphorylation of pRB in the cell cycle of normal cells. p16/CDKN2 gene and cyclin D1/PRAD-1 gene alterations have been detected in squamous cell carcinoma cell lines and in several primary squamous cell carcinomas of the esophagus. We immunohistochemically assessed p16 and cyclin D1 expression in 111 squamous cell carcinomas of the esophagus after evaluation of the antibodies against p16 and cyclin D1 protein using four squamous cell carcinoma cell lines. Loss of p16 expression was detected in 56 of 111 cases (50%). The mean number of metastatic lymph nodes without p16 expression was significantly higher than the number of nodes with p16 expression (P = 0.04). The postoperative survival rate for patients without p16 expression was significantly lower than that of patients with p16 expression (P = 0.04). Cyclin D1 overexpression was found in 28 of the 111 cases (25%) and correlated with distant organ metastasis after curative surgery (P = 0.05). The survival rate of patients with cyclin D1 overexpression was significantly lower than that of patients without cyclin D1 overexpression (P = 0.01). A positive correlation between the loss of p16 expression and cyclin D1 overexpression was observed (P = 0. 03). The loss of p16 expression and overexpression of cyclin D1 may be useful prognostic indicators in patients with squamous cell carcinomas of the esophagus. It may be possible to select more suitable treatment for patients with squamous cell carcinomas of the esophagus by evaluating the status of p16 and cyclin D1 expression.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, p16; Humans; Male; Middle Aged; Prognosis; Survival Analysis; Time Factors; Tumor Cells, Cultured

Dysregulation of G1 cyclins has been implicated in several human malignancies. To further investigate the role of G1 cyclins in chemical carcinogenesis, the expression of cyclin D1 and cyclin E was analyzed by RT-PCR and immunohistochemical studies in N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. Cyclin D1 mRNA levels were increased 2.8-fold in 25 week (P < 0.05) and 6.8-fold in 45 week (P < 0.01) papillomas induced by NMBA, when compared with normal rat esophageal epithelium. Cyclin E mRNA levels were increased 6.2-fold in 25 week (P < 0.01) and 6.9-fold in 45 week (P < 0.01) papillomas. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cyclin E. Furthermore, there was a sequential increase in cyclin D1- and cyclin E-positive cells from normal epithelium, to preneoplastic lesions, to papillomas. These findings suggest that overexpression of cyclin D1 and cyclin E occur relatively early in rat esophageal tumorigenesis and participate in tumor progression in this model.

Topics: Animals; Base Sequence; Carcinogens; Cyclin D1; Cyclins; Dimethylnitrosamine; DNA Primers; Esophageal Neoplasms; Immunohistochemistry; Male; Molecular Sequence Data; Oncogene Proteins; Papilloma; Polymerase Chain Reaction; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; RNA, Messenger

Previous studies have found a 3-10-fold amplification and overexpression of the cyclin D1 gene in about 32% of human esophageal squamous cell carcinoma. The purpose of this study was to evaluate the prevalence of increased expression of the cyclin D1 protein in Barrett's esophagus. Using 69 formalin-fixed and paraffin-embedded human esophageal specimens, which had been removed endoscopically or obtained at surgery during 1993 and 1994, all immunohistochemical analyses were performed using an avidin-biotin complex immunoperoxidase technique. Increased nuclear expression of the cyclin D1 protein was noted in 32 of 69 samples (46%; 44% of the samples from males and 50% of the samples from females). Positive nuclear staining for the cyclin D1 protein in Barrett's disease with intestinal metaplasia was found in 38% of the male cases and 25% of the female cases, whereas in gastric metaplasia it was positive in 33% of men and 48% of women. Nuclear accumulation of the cyclin D1 protein was also found in both dysplastic and nondysplastic lesions, and it was not associated with sex, age, or cigarette or alcohol consumption. Samples from patients taking proton pump inhibitors tended to be less frequently positive (32%) for cyclin D1 nuclear staining when compared to patients taking H2 antagonists (45%) or antacids (55%). These studies suggest that increased expression of cyclin D1 is an early event in the tumorigenic process of esophageal adenocarcinomas and that the increased expression of this gene might predispose the epithelium to malignant transformation.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Barrett Esophagus; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cyclin D1; Cyclins; Esophageal Neoplasms; Esophagus; Female; Humans; Immunoenzyme Techniques; Male; Metaplasia; Middle Aged; Oncogene Proteins; Risk Factors

The products of both CDKN2 and cyclin D1 genes are negative and positive regulators respectively in cell cycle. To study the involvement of the CDKN2 tumor suppressor gene and cyclin D1 oncogene in esophageal cancer development, we examined the situation of both genes in 21 pairs of primary human esophageal cancers and the mucosa adjacent to the cancers and also in four esophageal cancer cell lines by means of molecular biological and immunohistochemical techniques. Homozygous deletion was observed in 6 out of the 21 primary cancers and with lymph node metastasis in 5 out of the 6 cases. Loss of expression of p16 protein was identified immunohistochemically in 8 out of 21 primary cancers. Amplification of cyclin D1 was found in 12 out of 21 primary cancers and in 5 esophageal mucosa adjacent to the tumors, accompanying with overexpression of cyclin D1 protein. Homozygous deletion was observed in one (EC8712) out of the four cell lines, while by Northern and immunohisto chemistry analysis, loss of transcription of CDKN2 mRNA and loss of p16 protein expression were observed in two cell lines (EC8712 and EC8501). Amplification and overexpression of cyclin D1 were found in two cell lines (EC8733 and EC8501). These findings suggest that loss of CDKN2 gene and amplification of cyclin D1 gene are involved in esophageal cancers and that cyclin D1 alteration may be a earlier molecular event while CDKN2 to be a later one.

Topics: Carcinoma, Squamous Cell; Carrier Proteins; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p18; Enzyme Inhibitors; Esophageal Neoplasms; Gene Amplification; Gene Deletion; Gene Expression; Genes, Tumor Suppressor; Humans; Lymphatic Metastasis; Tumor Cells, Cultured; Tumor Suppressor Proteins

D-type cyclins being considered as oncogenes promote progression of the cell through the G1 phase of the cell cycle by CDK4 mediated phosphorylation of the retinoblastoma protein. The activities of CDK4 is constrained by inhibitors such as p16, the product of the CDKN2 in tumor cells and primary tumors suggests that p16 acts as a tumor suppressor. We examined these proteins and genes by immunohistochemistry and in situ hybridization techniques in 41 primary esophageal cancers. Overexpression of cyclin D1 was revealed in 26/41 samples (63.4%) and also in the mucosa adjacent to the cancers in 10 of 26 cyclin D1 overexpression samples, which also have high levels of cyclin D1. P16 was undetectable in 13 of 41 samples. Interestingly, 17 of 24 Rb positive cancers had no or low p16, while 9 Rb-negative cancers showed high levels of p16. These results suggest that the overexpression of cyclin D1 may be a common molecular abnormality and an early molecular event in esophageal cancer, followed either by Rb loss, as occurred in Rb negative samples, or by loss of p16, as occurred in p16 negative samples. Cyclin D1 overexpression and Rb inactivation can coexist in esophageal cancer. However, there is a reciprocity between Rb inactivation and p16 expression in esophageal cancer. Thus, abnormality in the negative feedback regulatory pathway of cyclin D1/CDK4, Rb and p16 may be involved in the molecular mechanism of esophageal cancer.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Esophageal Neoplasms; Humans; Immunohistochemistry; In Situ Hybridization; Retinoblastoma Protein

p16MTS1/INK4A negatively regulates cell cycle progression by inhibiting the cyclin D/CDK4 complex that phosphorylates pRb. Frequent homozygous deletions of the p16 gene were recently found in various tumor cell lines. We examined the relationship between the genetic status of p16 and the expression of the cell cycle regulating molecules in human esophageal carcinoma cell lines. Out of eight human esophageal carcinoma cell lines, seven (67.5%) and six (75%) cell lines showed homozygous deletions of the p16 and p15 genes, respectively. All the p16-negative cell lines expressed high levels of cyclin D1, CDK4 and p27KIP1 proteins. Interestingly, the expression level of cyclin D1 was closely correlated to the levels of not only CDK4 but also p27KIP1 protein in p16-negative cell lines. Furthermore, all the p16-negative cell lines expressed Rb protein of approx 110 kDa which corresponds to the phosphorylated form, whereas the cell line with intact p15 and p16 genes did not express pRb. These results suggest that the expression of cyclin D1, CDK4, Rb and p27 is associated with the p16 gene status in esophageal carcinoma cell lines. Alternatively, loss of the p16 gene and subsequent over-expression of cyclin D1 and CDK4 might be involved in autonomous growth of esophageal carcinoma cells.

Topics: Adenocarcinoma; Blotting, Northern; Blotting, Southern; Carcinoma, Squamous Cell; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; DNA Probes; Esophageal Neoplasms; Humans; Proto-Oncogene Proteins; Tumor Cells, Cultured; Tumor Suppressor Proteins

The cyclin D1, referred to as PRAD-1, has been mapped to the 11q13 region, and its expression has been detected in squamous cell lines and several primary esophageal carcinomas. We assessed cyclin D1 amplification in 122 squamous cell carcinomas of the esophagus. Samples for DNA extraction were obtained from formalin-fixed paraffin-embedded specimens, and 10 microgram of each DNA sample were subjected to slot blot analysis. The presence of more than three gene copies was considered evidence of gene amplification. Amplification of cyclin D1 was detected in 28 (23%) of 122 cases of squamous cell carcinoma of the esophagus. There were no significant differences between the clinicopathological background factors in groups positive and negative for cyclin D1 amplification, but the survival rate of patients exhibiting amplification was significantly lower (P < 0.001). The groups were stratified according to the pN (pathological N category) factor and pT (pathological T category) factor in the TNM classification, and the cumulative survival rates in the amplification groups were always significantly lower. Amplification of cyclin D1 was correlated with distant organ metastasis after curative operations, but there was no significant difference in lymph node recurrence rates of patients with or without amplification. Cyclin D1 amplification had the second highest partial regression coefficient in the multivariate analysis, after the pN factor. Amplification of cyclin D1 was independent of the TNM classification as a prognostic factor, and was a useful marker for predicting outcome and distant organ metastasis in patients with squamous cell carcinoma of the esophagus. It appears that appropriate treatment can be selected by evaluating both TNM factors and cyclin D1 amplification.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Gene Amplification; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Rate

The cyclin D1 gene is amplified and overexpressed in a significant fraction of human esophageal tumors, and several other types of human cancer, but the functional significance of this overexpression has not been established. To further address the roles of cyclin D1 in growth control and tumorigenesis, we have overexpressed an antisense cyclin D1 cDNA construct, either constitutively or inducibly, in the HCE7 human esophageal cancer cell line in which cyclin D1 is amplified and expressed at high levels. The expression of antisense cyclin D1 led to decreased expression of cyclin D1 at both mRNA and protein levels, and this was associated with a marked inhibition of cell proliferation. Antisense cyclin D1 expressing cells displayed a decreased plating efficiency, increased doubling time, decreased saturation density, increased cell size, decreased cyclin D1-associated in vitro kinase activity, decreased anchorage-independent growth, and a loss of tumorigenicity in nude mice. These findings provide direct evidence that the overexpression of cyclin D1 in certain tumor cells contributes to their abnormal growth and tumorigenicity. The ability to revert the transformed phenotype of these cells with antisense cyclin D1 suggests that cyclin D1 may be a useful target in cancer therapy.

Topics: Cell Adhesion; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Cyclins; DNA, Antisense; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Oncogene Proteins; Phosphorylation; Protein Kinases; RNA, Messenger; RNA, Neoplasm; Transfection; Tumor Cells, Cultured

We examined the expression of cyclin D1 mRNA in two human carcinoma cell lines (A431 and TT) and 17 specimens of esophageal cancer with in situ hybridization. Cyclin D1 mRNA was overexpressed in the cytoplasm of cancer cells that showed cyclin D1 gene amplification by Southern blot hybridization. Cyclin D1 antigen was overexpressed in the nucleus of these cancer cells. The distribution of cyclin D1 mRNA-positive cells was similar to that of cyclin D1 antigen-positive cells in the cancer tissues. We then attempted to correlate overexpression of cyclin D1 antigen and prognosis, by using 55 formalin-fixed, paraffin-embedded specimens of esophageal cancer. The overall 5-year survival of patients with strongly staining tumors was significantly lower than that of patients with weakly or nonstaining tumors (7 versus 59%; P < 0.01). There was no significant correlation between cyclin D1 expression and other clinicopathological factors. These results suggest that cyclin D1 may play an important role in carcinogenesis and that cyclin D1 overexpression may be a useful prognostic factor in esophageal cancer.

Topics: Base Sequence; Blotting, Southern; Carcinoma; Cyclin D1; Cyclins; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; In Situ Hybridization; Male; Molecular Sequence Data; Oncogene Proteins; RNA, Messenger; Tumor Cells, Cultured

Oncogene activation and tumor suppressor gene inactivation have been implicated in the genetic basis of esophageal squamous cell carcinoma (ESCC). Cyclin D1, an oncogene that has a critical role in G1 progression of the cell cycle, has been observed to be amplified in carcinomas of the breast and head and neck, and translocated in parathyroid adenomas and centrocytic lymphomas.. Established ESCC cell lines were assayed for cyclin D1 amplification and overexpression by Southern, Northern, and Western blot analyses. In addition, cyclin D1 overexpression was determined in primary tumors and adjacent normal mucosa by differential polymerase chain reaction (PCR) and immunohistochemical staining.. The authors observed that approximately 50% of ESCC cell lines with cyclin D1 DNA amplification also had RNA and protein overexpression. Related genes, cyclin D2 and D3, were not amplified or overexpressed in these cell lines with rare exception. The cyclin D1 protein was able to associate with the cell-cycle-dependent kinases, cdk4 and cdk6, but not always with proliferating cell nuclear antigen in selected cell lines tested, representing a novel finding. In addition, approximately 50% of primary tumors had cyclin D1 overexpression that was not present in adjacent normal mucosa. Cyclin D1 overexpression based on PCR correlated with enhanced cyclin D1 protein nuclear staining in malignant cells.. Cyclin D1 is amplified and overexpressed in ESCC and may be important in its molecular pathogenesis.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; DNA Primers; Esophageal Neoplasms; Gene Amplification; Gene Expression; Humans; Molecular Sequence Data; Oncogene Proteins; RNA, Messenger; Tumor Cells, Cultured

The cyclin D1 gene, located on chromosome 11q13, is frequently rearranged in parathyroid neoplasms and amplified in some carcinomas of other organs. Recent studies have detected amplification of cyclin D1 and other markers on chromosome 11q13 (evaluated by Southern or slot blot assays) in approximately 25-50% of squamous cell carcinomas of the esophagus and noted that amplification was associated with lessened survival time. We applied the technique of differential polymerase chain reaction to the evaluation of cyclin D1 gene amplification in squamous cell carcinomas of the esophagus. Cyclin D1 was found to be amplified in 10 of 45 (22%) primary tumors and three of 12 (25%) lymph node metastases. Lymph node metastases tended to be more common in patients with cyclin D1 amplification (70%) than in those without amplification (37%). In 36 patients with follow-up, cyclin D1 amplification was associated with decreased 1 year survival (28% vs. 59%). Cyclin D1 gene amplification in esophageal carcinomas can be evaluated by differential polymerase chain reaction and may provide useful prognostic information.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Electrophoresis, Polyacrylamide Gel; Esophageal Neoplasms; Gene Amplification; Humans; Lymphatic Metastasis; Molecular Sequence Data; Oncogene Proteins; Paraffin Embedding; Polymerase Chain Reaction; Receptors, Dopamine D2; Tissue Fixation

The objective of this study was to quantify the changes in p53 and cyclin D1 protein levels in different stages of human esophageal and gastric cardia carcinogenesis in a high-risk population in Henan, China. Immunoreactivity of p53, cyclin D1 and proliferating-cell nuclear antigen (PCNA) was observed in the cell nuclei of esophageal and gastric cardia biopsies. The number of p53-immunostaining-positive cells was low in normal epithelia, slightly increased in basal-cell hyperplasia (BCH), markedly increased in dysplasia (DYS) (10-fold), and further increased in squamous-cell carcinoma (SCC) (40-fold). This pattern of change was similar to that of cell proliferation as indicated by PCNA immunostaining. On the other hand, the number of cyclin D1-immunostaining-positive cells did not increase from BCH to DYS, although a slight increase from DYS to SCC was noted. In the gastric cardia, again, the pattern of change of p53-positive cells in different stages of lesions paralleled the pattern of cell proliferation. The number of p53-positive cells was very low, much lower than that of PCNA-positive cells, in normal, chronic superficial gastritis (CSG) and chronic atrophic gastritis (CAG); therefore, the increase of p53-positive cells from CAG to DYS was more dramatic (100-fold). From DYS to adenocarcinoma (AC), the p53-positive and the PCNA-positive cells increased 4-fold. On the other hand, the number of cyclin D1-positive cells did not increase in pre-cancerous lesions, but increased slightly in AC. This study demonstrates that p53 protein accumulation increased with the progression of pre-cancerous lesions, especially in the genesis of dysplasia, both in the esophagus and in the gastric cardia. Our approach of quantitative immunohistochemistry sheds light on the mechanisms of genesis of esophageal and gastric-cardia cancers, which frequently occur together in many high-incidence areas.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cardia; Cell Division; Cell Transformation, Neoplastic; Chi-Square Distribution; Cyclin D1; Cyclins; Esophageal Neoplasms; Female; Humans; Hyperplasia; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Oncogene Proteins; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Stomach Neoplasms; Tumor Suppressor Protein p53

To study the involvement of the p16 tumor suppressor gene in esophageal cancer development, we examined homozygous deletion of the p16 gene in 13 human esophageal cancer cell lines and 9 gastric cancer cell lines, in which some of genetic alterations have already been characterized. By Southern blot analysis, homozygous deletion was observed in 12 out of the 13 esophageal cancer cell lines (92%), in 2 out of a total of 9 gastric cancer cell lines (22%). It was also found that the p16 gene loss, cyclin D1 amplification and p53 gene mutations occurred independently in these cell lines. These findings suggest that loss or mutations of the p16 gene are involved in most esophageal cancers and that mutation of this gene plays a critical role in the development of esophageal cancer.

Topics: Blotting, Southern; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclins; Esophageal Neoplasms; Gene Deletion; Genes, p53; Homozygote; Humans; Mutation; Oncogene Proteins; Protein Kinase Inhibitors; Stomach Neoplasms; Tumor Cells, Cultured

We previously identified two genes, EXP1 and EXP2/Cyclin D1, about 120 kb apart from HST1 and INT2 on human chromosome 11q13. Here we report the frequent amplification and overexpression of these genes in surgically resected esophageal carcinomas. Amplification was observed in 8 out of 22 cases. In the tumors with the amplification, these genes were overexpressed, while in the tumors without the amplification, expression levels of these genes were almost the same as those with normal mucosa. These results suggest that EXP1 and/or EXP2/Cyclin D1 may play an important role in the progression of human esophageal carcinomas.

Topics: Adult; Aged; Base Sequence; Chromosome Mapping; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; DNA Primers; Esophageal Neoplasms; Esophagus; Female; Gene Amplification; Gene Expression; Humans; Male; Middle Aged; Molecular Sequence Data; Mucous Membrane; Oligonucleotide Probes; Oncogene Proteins; Open Reading Frames; Polymerase Chain Reaction; Protein Biosynthesis; Proteins

To study the involvement of cyclins in cell-cycle progression, changes of mRNA levels for three G1 cyclins (cyclin C, D1 and E) and cyclin A were studied in a leukemia cell line, HEL cells, before and after incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, the cyclin D1 mRNA level markedly increased in the HEL cells when the cells were growth-arrested by TPA, while the amounts of cyclin E and A mRNAs decreased to an almost undetectable level in HEL cells after incubation with TPA. The similar marked increment of the cyclin D1 mRNA level was observed in other leukemia cell lines, CMK and HL-60 cells, after incubation with TPA. The cyclin C mRNA was not detected in HEL cells before and after incubation with TPA.

Topics: Base Sequence; Blotting, Northern; Cell Cycle; Cell Division; Cloning, Molecular; Cyclin D1; Cyclins; Dimethyl Sulfoxide; Esophageal Neoplasms; Humans; Kinetics; Leukemia, Myeloid; Molecular Sequence Data; Molecular Weight; Oligonucleotide Probes; Oncogene Proteins; Open Reading Frames; Polymerase Chain Reaction; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.

Topics: Blotting, Southern; Blotting, Western; Cell Line; Cyclin D1; Cyclins; DNA, Neoplasm; Esophageal Neoplasms; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Humans; Immunohistochemistry; Molecular Weight; Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured

Amplification of the hst-1 and int-2 genes on chromosome 11q13 has previously been found in over 20% of human primary esophageal cancers. However, these two genes do not appear to be transcribed in appreciable amounts. Recently, the human cyclin D gene (also referred to as prad1) has been mapped to the 11q13 locus. Here, we report coamplification of the cyclin D and hst-1 genes in 5 of 20 (25%) human squamous esophageal tumors. We also detected significant levels of cyclin D transcription in two esophageal carcinoma cell lines, even though they did not express detectable amounts of hst-1 transcription. These findings provide the first evidence for the amplification of a cyclin gene in human esophageal cancer and suggest that an increase in cyclin D gene dosage could be an important factor in the pathogenesis of esophageal cancer. Additionally, because the 11q13 locus is found to be amplified in many types of human tumors, cyclin gene amplification could also play an important role in the development of other forms of human cancer.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; DNA Probes; DNA, Neoplasm; Esophageal Neoplasms; Gene Amplification; Gene Expression; Humans; Molecular Sequence Data; Oncogene Proteins; Transcription, Genetic