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Original Research ARTICLE
Novel Potential Biomarkers Associated With Epithelial to Mesenchymal Transition and Bladder Cancer Prognosis Identified by Integrated Bioinformatic Analysis
- 1Department of Urology, The First Affiliated Hospital of China Medical University, Shenyang, China
- 2Department of Medical Oncology, The First Affiliated Hospital of China Medical University, Shenyang, China
Bladder cancer (BC) is one of the most common malignancies in terms of incidence and recurrence worldwide. The aim of this study was to identify novel prognostic biomarkers related to BC progression utilizing weighted gene co-expression network analysis (WGCNA) and further bioinformatic analysis. First, we constructed a co-expression network by using WGCNA among 274 TCGA-BLCA patients and preliminarily screened out four genes (CORO1C, TMPRSS4, PIK3C2B, and ZNF692) associated with advanced clinical traits. In support, GSE19915 and specimens from 124 patients were used to validate the genes selected by WGCNA; then, CORO1C and TMPRSS4 were confirmed as hub genes with strong prognostic values in BC. Moreover, the result of gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) indicated that CORO1C and TMPRSS4 might be involved in the process of epithelial to mesenchymal transition (EMT) reversely. In addition, high expression of CORO1C was found to be significantly correlated with tumor-infiltrating neutrophils (TINs), a negative regulatory component that facilitates tumor distant progression and induces poor clinical outcome. In conclusion, our study first identified CORO1C and TMPRSS4 as vital regulators in the process of tumor progression through influencing EMT and could be developed to effective prognostic and therapeutic targets in future BC treatment.
Bladder cancer is the 10th most common cancer in the world with an estimated 549,000 new cases and 200,000 deaths in 2018 (1). The median age of tumor diagnosis is 73 years old, which is the most elderly of diagnosis among all cancer sites (2), and the mortality rate increased by 1% per year from 2000 to 2016 in the oldest old men (3), which means the probability of most bladder cancer cases occurring with tumor progression and advanced disease before diagnosis still ranks high. Current recognized treatments are radical resection and transurethral resection of bladder tumor (TURBT) for muscle invasive bladder cancer (MIBC) and those treatments with or without intravesical therapy for non-muscle invasive bladder cancer (NMIBC). However, ~60% of NMIBC patients will undergo recurrence (4, 5) following the surgery due to rapid tumor invasive growth and high tendency of distant progression. Therefore, for such difficulties in early tumor diagnosis and dealing with the frequent recurrence rate, there remains an urgent need to explore novel effective prognostic biomarkers and potential therapeutic targets in bladder cancer.
The majority of researchers have just focused on the differential expression of genes associated with BC and have largely ignored the high degree of interconnectivity among genes, where genes with semblable expression patterns might be functionally correlated. Weighted gene co-expression network analysis (WGCNA) is a distinct approach in identifying modules with different categories of correlated genes, and candidate biomarkers or therapeutic targets for cancer management can be picked out according to the connectivity between the internal linkage of the gene sets in specific modules and by calculating the association between the gene sets and related clinical features (6–9). Furthermore, WGCNA has been successfully applied to identify biomarkers or potential therapeutic targets for BC (8, 10). Hence, we attempt to construct a network through a comprehensive bioinformatics method in the light of WGCNA and identified prognostic genes of BC.
In this study, we first extracted the level-three mRNA expression data and clinical information of 274 bladder cancer patients from The Cancer Genome Atlas (TCGA) database for subsequent weighted network construction. By WGCNA, four modules and their candidate hub genes were preliminarily identified through evaluating the significance between modules and clinical traits. Survival analysis of the candidate hub genes was then performed in three data sets (TCGA-BLCA, GSE19915 data set, and clinical specimens), and CORO1C and TMPRSS4 were recognized as hub genes due to the consistency of prognostic value in overall survival (OS). The diverse gene set analysis method was later used to depict the potential biological functions of the hub gene. Finally, protein–protein interaction (PPI) network analysis was performed to investigate the relationships between the hub genes and the associated genes' network in corresponding modules.
Co-expression Network Construction and Module Identification
According to the flowchart shown in Figure 1, after data processing, a total of 274 TCGA-BLCA samples and their selected clinical information were imported to WGCNA. In the present study, β = 5 (scale-free R2 = 0.85) was chosen as the soft thresholding for further adjacency calculation (Figure 2A). Genes in the gray module were regarded as non-specific genes and removed from further data processing. After merging the similar modules by a clustering height cutoff of 0.25 (Figure 2B), 17 modules possessing high credibility were finally generated in total with the initial modules and merged modules displaying under the clustering tree (Figure 2C). The candidate hub gene and module size of all 17 modules calculated by the “chooseTopHubInEachModule” function are shown in Table 1. According to the network adjacency of expression data, some modules indicated high levels of similarity, and three major clusters were formed by the “average” clustering method (Figure 2D). The heat map that demonstrates the relationship between seven clinical traits and each module identified by WGCNA is shown in Figure 2A. The cyan, turquoise, light green, and green modules were significantly negatively correlated not only with the M stage, but also with the papillary subtypes, grade, and T stage of bladder cancer. Interestingly, these three modules share high similarity between each other (Figure 2D), which implies that genes in such modules might work together to be protective regulators during tumor progression. Other modules, except the brown and grey60 modules, reflect significant positive correlation with most of the clinical traits, indicating that genes in these modules are more likely to promote advanced disease.
Figure 2. Co-expression network construction based on TCGA-BLCA mRNA data. (A) The analysis of topology for soft threshold powers and β = 5 (scale-free R2 = 0.85) was set as the soft thresholding for further adjacency calculation. (B) Clustering dendrograms were cut at a height of 0.25 to identify the similar modules. (C) The original 20 modules and merged 17 modules were displayed at top and bottom under the clustering dendrogram. (D) An eigengene network heat map showed the correlation between each module, and modules with close connectivity were clustered together.
Selection of Candidate Hub Genes From Identified Modules
To determine the prognostic values of the candidate hub genes in modules found by WGCNA, which as least significantly associated with one clinical trait (Figure 3A), the GEPIA online tool was used to perform survival analysis among them in TCGA-BLCA patients, and finally, four of all 15 candidate hub genes were identified to be valuable predictors of overall survival. Only the significant results were presented in Figures 4A–D. These four candidate hub genes (CORO1C, TMPRSS4, PIK3C2B, and ZNF692) were retained for further validation. Then, we selected the corresponding purple, light green, cyan, and green modules in order to evaluate their relationship with the M stage because metastasis was the primary cause of bladder cancer progression and treatment failure. As shown in Figures 3B–E, the relationship between gene significance (GS) and module membership (MM) are plotted to demonstrate that characteristic genes in these four modules are significant contributors to tumor M stage, and hub genes representing light green, cyan, and green modules could reverse tumor progression, but in the purple module, it could promote tumor progression (Figure 3A).
Figure 3. Relationships between WGCNA identified modules and clinical traits. (A) Heat map of the correlation among all 17 modules and seven selected clinical traits including age, tumor subtype (papillary and non-papillary), tumor grade (low grade and high grade), tumor stage (stage II and stage III-IV), tumor T stage (T2 and T3-4), tumor N stage (non-metastasis and metastasis), and tumor M stage (non-metastasis and metastasis). (B) Scatterplot of the relationship between gene significance (GS) and module membership (MM) in the purple module, (C) in the light green module, (D) in the green module, and (E) in the cyan module.
Figure 4. Survival analysis of four candidate hub genes in TCGA-BLCA and GSE19915 data. (A–D) Survival analysis result of CORO1C (A), TMPRSS4 (B), PIK3C2B (C), and ZNF692 (D) in TCGA-BLCA downloaded from GEPIA. (E–G) Survival analysis of CORO1C (E), TMPRSS4 (F), and ZNF692 (G) in GSE19915.
Validation of the Candidate Hub Genes by Immunohistochemistry and GSE19915
Clinical specimens for immunohistochemical analysis were collected from a cohort of 124 bladder cancer patients to clarify the expression pattern of the candidate hub genes (CORO1C, TMPRSS4, PIK3C2B, and ZNF692) identified in the above modules, and the main findings were shown in Figures 5E–L. Immunohistochemical staining revealed that, compared with other subcellular localization, these candidate hub genes were more highly expressed in the cytoplasm of tumor cells: 58.06% cases of bladder cancer specimens (72/124) were scored as CORO1C overexpression (Figures 5E,F), 34.68% cases of bladder cancer specimens (43/124) were scored as TMPRSS4 overexpression (Figures 5G,H), and 43.55% (54/124) and 81.45% (101/124) were scored as PIK3C2B and ZNF692 overexpression. To explore whether the candidate hub genes play consistent prognostic roles in clinical outcomes, overall survival analyses were performed on this immunohistochemical cohort. In particular, CORO1C and TMPRSS4 were noted significant (Figures 5A–D). We downloaded another online data set, GSE19915, which included 144 bladder cancer patients to test the prognostic performance of these genes, but only three genes (CORO1C, TMPRSS4, and ZNF692) could match the probes in later survival analysis (Figures 4E–G). We also assessed the association between candidate hub genes and related clinicopathological features. The relationship between the expression level of hub genes and clinicopathological parameters, including age, gender, stage, TNM stages, and grade, were concluded in Tables 2A,B. Notably, among all 274 TCGA-BLCA patients, CORO1C were significantly differentially expressed between the groups of T stage category, M stage category, and grade. It's evident that CORO1C overexpression was highly associated with advanced disease and worse clinical status in bladder cancer. Contrary to CORO1C, the expression level of TMPRSS4 was significantly higher in the groups of young age (<60 years old), early T stage, well-differentiated tumor patients, and cases without lymph node or distant metastasis, which provided strong evidence of the role that TMPRSS4 played in better clinical outcome involvement. The expression of CORO1C and TMPRSS4 in normal bladder tissues and the relationship between two genes and tumor stage were explored via GEPIA online tool (Supplementary Figure 1). The results show that the higher TMPRSS4 expression indicates the lower tumor stage.
Figure 5. Validation of the prognostic value of four candidate hub genes in clinical specimens. (A–D) Survival analysis of CORO1C (A), TMPRSS4 (B), PIK3C2B (C), and ZNF692 (D) in 124 bladder cancer patients' follow-up in our center. (E–L) Representative image of 200× immunohistochemical experiment on CORO1C (E,F), TMPRSS4 (G,H), PIK3C2B (I,J), and ZNF692 (K,L) clinical specimens.
Table 2A. The correlations between the CORO1C and clinicopathological features based on TCGA-BLCA data.
Table 2B. The correlations between the TMPRSS4 and clinicopathological features based on TCGA-BLCA data.
In general, good consistency of CORO1C and TMPRSS4's prognostic values for overall survival outcome has been observed among three data sets, robustly indicating that CORO1C is a risk factor in bladder cancer development while TMPRSS4 is a protective factor, and as hub genes, their relationships with clinicopathological features are in accord with the purple and light green modular characterizations that have been described by WGCNA analysis (Figure 3A).
Pathway Analysis of the Hub Genes
To investigate the potential molecular functions of CORO1C and TMPRSS4 in BC, we performed GSEA and GSVA based on the processed TCGA-BLCA data. The GSEA results of hallmark gene sets showed genes that have a positive correlation with CORO1C are significantly enriched in the following pathways: epithelial to mesenchymal transition pathway, angiogenesis pathway, and hypoxia pathway (Figures 6A–C). Both the angiogenesis and hypoxia pathways are closely related to the occurrence and development of EMT-associated tumor metastasis. Accumulating research has reported that a hypoxic tumor microenvironment is a key regulator in affecting tumor EMT transformation and active angiogenesis would facilitate distant transportation of EMT-progressing tumor cells through the microvasculature while abnormal vasculatures in the tumor microenvironment also led to a hypoxic condition (11–13). Conversely, TMPRSS4-related downregulated genes were only significantly enriched in the epithelial to mesenchymal transition pathway (Figure 6D) while upregulated genes did not observe enrichment in any pathways. To further verify the relationship between EMT and hub genes, the GSVA method was utilized to evaluate the EMT pathway score variation. The Spearman correlation coefficient shows a significantly strong positive correlation (r = 0.76, p < 0.0001) between CORO1C expression and mesenchymal pathway (Figures 6E,F), and the correlation between CORO1C and epithelial pathway is significantly negative (r = −0.38, p < 0.0001). On the contrary, TMPRSS4 has a significantly strong positive correlation with genes in epithelial pathways (r = 0.61, p < 0.0001) but is opposite to the mesenchymal process in bladder cancer (r = −0.36, p < 0.0001) (Figures 6G,H). In summary, this result elucidates that there is a high possibility of the participation of CORO1C and TMPRSS4 in the bladder tumor cell EMT based on the opposite effects, which suggested their different roles during disease metastatic course.
Figure 6. Hallmark GSEA and EMT GSVA of hub genes based on TCGA-BLCA mRNA data. Some representative top enriched pathways of CORO1C were (A) epithelial to mesenchymal transition pathway, (B) angiogenesis pathway, and (C) hypoxia pathway. Only one downregulated GSEA result of TMPRSS4 enriched in (D) mesenchymal transition pathway. (E) GSVA pathway score of mesenchymal state gene set and (F) epithelial state gene set vs. normalized Log2(FPKM + 1) expression of CORO1C. (G) GSVA pathway score of mesenchymal state gene set and (H) epithelial state gene set vs. normalized Log2(FPKM + 1) expression of TMPRSS4.
CORO1C Expression and TME Evaluation
To better demonstrate the accordance between hub genes and corresponding modules, GO_BP enrichment analysis was performed in the purple and light green modules by R package “clusterProfiler.” Ontology analysis of the light green module did not find any significantly enriched pathways, and genes in the purple module were positively related to the top four enriched processes of neutrophil activation, neutrophil degranulation, neutrophil activation involved in the immune response, and neutrophil-mediated immunity (Figure 7A). The hallmark GSEA results of the hub gene CORO1C indicate that many pathways associated with the immune process were significantly enriched, such as interferon gamma response, inflammatory response, IL6-JAK-STAT3 signaling, IL2-STAT5 signaling, and TNFα signaling via NF-κB (Figure 7B). In addition, the TIMER online tool was exerted to evaluate the potential relationships between the expression of CORO1C and both tumor purity score as well as six types of tumor-infiltrating immune cells (Figure 7C). Based on the linear least square regression calculating method, the expression of CORO1C was shown to have a negative tendency with tumor purity (r = −0.475, p = 3.71e-22) and the level of infiltrating B cell (r = −0.193, p = 2.12e-04); meanwhile, CORO1C expression was positively correlated with the infiltrating level of CD8+ T cells (r = 0.487, p = 3.13e-23), CD4+ T cells (r = 0.145, p = 5.59e-03), macrophages (r = 0.19, p = 2.60e-04), neutrophils (r = 0.437, p = 2.20e-18), and dendritic cells (r = 0.563, p = 6.67e-32). The ssGSEA method was selected as another estimation tool to verify the probable relationship between CORO1C expression and 23 types of immune components with a heat map displaying the order ranked from high to low correlation appraised by Pearson correlation (Figure 7D). Interestingly, the relation between CORO1C and neutrophil share the strongest consistency among different means, which also confirms the hub role of CORO1C in a neutrophil mainly related immune infiltrating module.
Figure 7. Evaluation of CORO1C expression and tumor immune infiltration based on TCGA-BLCA data. (A) Biological process of gene ontology enrichment for WGCNA purple module. (B) Integrated plot presented some top GSEA enriched immune-related pathway associated with CORO1C expression. (C) Evaluation of CORO1C expression level and immune infiltration level in BC via TIMER tool. (D) Relationship between CORO1C and 23 types of immune cells via ssGSEA analysis. The Pearson correlative coefficients were marked following the name of each immune components (“*” represented statistical significance p < 0.01).
PPI and Genetic Alteration Characterizations
PPI construction was performed with the aim to explore the interaction network between hub genes and other genes in the corresponding modules. As shown in Figures 8A,B, 163 genes were obtained from the WGCNA purple module out of 394 genes, and 25 genes were obtained in the WGCNA light green module out of 44 genes that were generated by taking the intersection with the highly correlated genes identified from hub genes' Pearson analysis (r > 0.4). It means that these screened-out genes would form crosslink networks with the hub genes and could well represent the major functional modules. Because the number of genes in the light green module is rather small, the connectivity between TMPRSS4 and other genes in this module were evaluated by setting up the adjacency threshold as 0.02, and finally, a weighted network with highest strengths containing only four genes was structured to represent the module (Figure 8C). The total alteration frequency is included to illustrate that CORO1C and TMPRSS4 are altered in 4.2% cases, which looks relatively not much higher in bladder cancer (Figure 8D). We then separately assessed the gene alternation type and mutational rates of CORO1C and TMPRSS4 in TCGA-BLCA data. The two genes had almost identical mutational rates, both around 2%, but the major genetic changes in CORO1C are mutation and copy number amplification while deep depletion of TMPRSS4 gene is detectable in a small group of patients, which suggests a heterogeneous gene alteration pattern and exclusive molecular function of these two genes (Figures 8E,F).
Figure 8. PPI construction and genetic alteration analysis. (A) In WGCNA purple modules, genes highly related to CORO1C were established in a protein network. (B) In WGCNA light green modules, protein interaction of genes highly related to TMPRSS4. (C) The strongest weighted genes network in WGCNA light green module. (D) The overall genetic alternation status of two hub genes in TCGA-BLCA data. (E) The alternation type and frequency of CORO1C and (F) TMPRSS4, respectively, in TCGA-BLCA data.
Bladder cancer is the most common urinary malignancy with the characteristic of high incidence and mortality rate. Currently, radical cystectomy is effectively exerted on patients at an early stage, but in many cases, patients suffer tumor recurrence within 5 years (4, 14, 15), and those who are diagnosed with advanced disease could barely benefit from such therapeutic approaches, which suggests recurrence management and early diagnosis remain the major challenges in bladder cancer improvement. Therefore, there is an urgent need to find new biomarkers that accurately predict the tumor progressive status and clinical prognosis of bladder cancer.
EMT is a cellular process that is known to be essential for malignant progression, which could give tumor cells potential capability in further local invasion and distant metastasis. Moreover, cancer cells with stromal features are more resistant to traditional therapy, and this is the main reason accounting for death and treatment failure (16, 17). In the process of driving tumor progression, EMT has been proven to be a set of multiple dynamic transition states between the epithelial and mesenchymal phenotypes rather than a process involving a single binary decision, and different EMT stages have been identified in multiple tumor subpopulations: epithelial states, intermediate hybrid states, and completely mesenchymal states (18, 19). In bladder urothelial carcinoma, tumor metastasis caused by EMT is the main cause of death, and reversing EMT has been widely considered to be a method to combat the progression of bladder cancer in treatment (20, 21). The reprogramming of gene expression during EMT, as well as non-transcriptional changes, is initiated and controlled by signaling pathways that respond to extracellular cues (22). Interestingly, we use GSEA and GSVA to explore the function of TMPRSS4 and CORO1C, and we find that both TMPRSS4 and CORO1C are significantly enriched in pathways related to epithelial–mesenchymal transition but with a completely adverse direction. CORO1C was positively correlated with the process of EMT, and TMPRS4 is negatively correlated with the process of EMT. The correlation analysis between EMT gene sets and the hub genes also supports the discovery from GSEA. Therefore, we speculate that these two genes may play key roles in the pathogenesis of bladder cancer via influencing EMT.
Transmembrane protease serine 4 (TMPRSS4), a novel type II transmembrane serine protease, has been proven to have a crucial role in multiple tumors. In lung squamous cell carcinoma, overexpressed TMPRSS4 is associated with disease recurrence and poor survival by promoting invasion and metastasis (23–25). TMPRSS4 is overexpressed in breast cancer cells, and its overexpression promoted the proliferation, migration, and invasion of breast cancer cells and is related to poor prognosis (26). In colorectal cancer, the high expression state of TMPRSS4 is significantly correlated with advanced TNM stage and predicted poor prognosis (27). In thyroid cancer, TMPRSS4 is detected to be significantly associated with the promotion of proliferation in thyroid cancer cells (28). In gastric cancer, upregulation of TMPRSS4 facilitates cell migration and invasion through the activation of NF-κB/MMP-9 signaling (29). In pancreatic carcinoma, TMPRSS4 is identified as a candidate biomarker by affecting the clonability and invasiveness of pancreatic cancer cells, and overexpressed TMPRSS4 predicts poor prognosis (30, 31). In prostate cancer cells, overexpressed TMPRSS4 promotes migration via the progression of EMT (32). Interestingly, in contrast to most cancer types in published research, our study finds that high expression of TMPRSS4 predicts a better clinical outcome in bladder cancer, and this finding has been verified in three data sets (two online and one from our center). The relationship between TMPRSS4 expression level and clinical traits also supports the protective role of TMPRSS4 in bladder cancer, which might be related to its counteractive function in the EMT process.
Coronin-like actin binding protein 1C (CORO1C), a member of the WD repeat protein family, regulates the actin-dependent process by assembling F-actin. In triple-negative breast cancer (TNBC) cells, miR-206 inhibits cancer cell migration by directly targeting CORO1C, which regulates actin filaments (33). In breast cancer MDA-MB-231 cells, YBX1 could regulate invasion and migration by regulating its downstream target CORO1C (34). In lung squamous cell carcinoma, CORO1C was identified as a target of the miR-1/133a cluster, and silencing CORO1C inhibited cancer cell proliferation, migration, and invasion (35). In gastric cancer, overexpressed CORO1C is associated with poor prognosis and could promote metastasis by regulating cyclin D1 and vimentin (22). In our study, the high expression level of CORO1C also predicted poor survival outcomes in bladder cancer, which is consistent with other cancer types.
The tumor microenvironment (TME) is the supportive nest for tumor cells and can be divided into two parts: tumor cells and the surrounding matrix. The matrix consists of a variety of cells, including immune cells, microvascular cells, lymphoid endothelial cells and fibroblasts, soluble factors, signaling molecules, and extracellular matrix (36). During tumor development, a series of complex changes occurs in the tumor microenvironment, including the immune response and infiltration of various immune cells, especially neutrophils, which affect the initiation and development of tumors (37). Many studies have shown that TINs promote the adhesion of tumor cells to the epithelial monolayer and, thus, accelerate the metastasis of tumor cells (38, 39). In pancreatic ductal adenocarcinoma, TINs promote the EMT process and the invasive growth of tumor cells (40). In epithelial-derived tumors, an increasing number of neutrophils is usually a marker of the systemic inflammatory response that triggers the proliferation and metastasis of tumor cells by inhibiting apoptosis, motivating angiogenesis, and inducing DNA damage (41). Furthermore, neutrophils induce gastric cancer cells to occur EMT, leading to migration and invasion of tumor cells in TME (42). In nasopharyngeal carcinoma, CORO1C promotes cancer cell migration and invasion by induction of EMT (43). We infer that CORO1C may induce EMT in bladder cancer cells by increasing neutrophil activity. Our research first demonstrates that CORO1C positively relates to neutrophil activation, neutrophil degranulation, neutrophil activation involved in the immune response, neutrophil-mediated immunity, and EMT. These results support the view that CORO1C is highly positively correlated with EMT and ultimately leads to poor prognosis in bladder cancer patients.
In the present study, WGCNA is used to construct a co-expression network based on processed TCGA-BLCA data. We also assess clinical features and attempt to find key modules that are most relevant to the advanced stage of bladder cancer. Finally, four modules and their corresponding candidate hub genes are preliminarily identified for further validation. Survival analysis of these candidate hub genes is performed in three data sets (TCGA-BLCA, GSE19915, and an independent cohort of bladder cancer patients from our center). Finally, CORO1C and TMPRSS4 are screened out as hub genes that are significantly correlated with OS. CORO1C and TMPRSS4 might be two meaningful clinical indicators in bladder cancer but appear to have opposite prognostic value.
There are also some limitations in this study. First, the molecular function of CORO1C and TMPRSS4 in EMT need to be verified by biological experiments in vitro, which is in our future research plan. Second, the concrete relationship between CORO1C and neutrophils should be tested by co-culture or by immunohistochemical marker staining. Overall, through the integrated analysis of multiple data sets and the establishment of a co-expression network by WGCNA, CORO1C and TMPRSS4 are found participating in the tumor progression with valuable prognostic information in predicting bladder cancer patients' outcome and might be appealing treatment targets in clinical decision making.
Materials and Methods
Bladder Cancer Data Sources and Processing
The level three RNA-seq data and corresponding clinical information of 412 BC patients were downloaded from the TCGA database (https://cancergenome.nih.gov/). The exclusion criteria were described as follows: (1) neoadjuvant chemotherapy history, (2) unclear histological type of tumor, (3) T0 or T1 stage of tumor, (4) inadequate follow-up information (missing or <1 month), and (5) grade version not belonging to the American Joint Committee on Cancer (AJCC) 6th or 7th edition. Eventually, 274 patients were included in this study to perform WGCNA. The microarray data set GSE19915 consists of 144 BC patients, and related clinical data were downloaded from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) to verify the candidate hub genes found by WGCNA.
Construction of a Weighted Gene Co-expression Network and Identification of Candidate Hub Genes
A matrix includes the expression data of top 5,000 genes ranked by median absolute deviation (MAD), which, retrieved from 274 TCGA-BLCA samples, was used to construct a weighted gene co-expression network by R package “WGCNA” (6). First, to construct a reliable sample tree, the samples with connectivity less than −2.5 were filtered as outliers. Second, multiple soft thresholding powers ranging from 1 to 20 were established to select the appropriate indices (R2 = 0.85) for a scale-free topology network construction with enhanced strong Pearson correlations and weakened weak correlations between paired genes in the adjacency matrix. The adjacency matrix was then converted into a topological overlap matrix (TOM), which could evaluate the network connectivity of a gene, and the corresponding dissimilarity (dissTOM) was generated by subtracting TOM from 1. According to the TOM-based dissimilarity measure, genes were divided into different gene modules through the dynamic tree cut method with a minimum module group size of 30 for the resulting average linkage hierarchical clustering dendrogram, and the modules with low credibility were identified and merged together with a height cutoff of 0.25 in the clustering. The similarity of each module was evaluated by the adjacency of the expression data and the similar modules were clustered together. Later, seven BLCA clinical traits (including patient age, histological subtype of papillary and non-papillary tumors, histological grade, tumor stage, and TNM information) were selected, and the Pearson correlation with co-expression modules was calculated and visualized by a heat map. Finally, the “chooseTopHubInEachModule” function embedded in the “WGCNA” package was used to recognize the candidate hub genes that have the highest connectivity in each module.
Survival Analysis of the Candidate Hub Genes
Prognostic values of the candidate hub genes in TCGA were assessed by Gene expression profiling interactive analysis (GEPIA, http://gepia.cancer-pku.cn) online tools. Survival analysis in the GSE19915 data set was conducted for the candidate hub genes using “survival” R package (https://CRAN.R-project.org/package=survival). Tumor samples within the above data sets were divided based on the candidate hub gene's median expressive value, and the survival analysis was estimated by the Kaplan–Meier (K-M) survival curves of each patient with overall survival (OS) as the end point.
Functional Annotation of the Hub Genes
GSEA was carried out using the Java GSEA v4.0.1 desktop program (http://software.broadinstitute.org/gsea/datasets.jsp). Fifty referenced Hallmark gene sets from MSigDB (http://software.broadinstitute.org/gsea/msigdb/index.jsp) were downloaded to identify significant pathways associated with hub gene expression. Gene ontology (GO) biological process annotation of the hub gene associated modules was implemented by R package “clusterProfiler” (44). In the gene set enrichment analysis, large sets with more than 500 genes and small sets with <15 genes were excluded from the results, and enriched pathways with a cutoff threshold of p < 0.05 and FDR < 0.25 were considered valuable.
Variation Analysis Between the Hub Genes and EMT Gene Sets
Based on the integration of published literature, EPITHELIAL-MESENCHYMAL TRANSITION GENE DATABASE (dbEMT2, http://dbemt.bioinfo-minzhao.org/index.html), EMT-related gene sets, epithelial-related gene sets, and mesenchymal-related gene sets selected from MSigDB (http://software.broadinstitute.org/gsea/msigdb/index.jsp), finally 370 epithelial-related genes and 733 mesenchymal-related genes were collected and further processed into an epithelial-related gene set and a mesenchymal-related gene set, respectively. The enrichment scores of the epithelial-related gene set and mesenchymal-related gene set were calculated by the “GSVA” package (45). Spearman correlation between epithelial or mesenchymal gene set enrichment score and the hub gene Log2 transformed expression data were estimated to evaluate the relationship between the hub genes and the EMT process.
Tumor-Infiltrating Immune Cell Analysis
Immunologic infiltration data were collected from the Tumor Immune Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/) platform to explore the correlation between hub genes and tumor-infiltrating immune components. TIMER is a comprehensive resource for the systematic analysis of immune infiltrates across diverse cancer types. The abundance of six immune cells (B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell) were evaluated by statistical methods mining sequencing data retrieved from TCGA and validated using pathological estimations (46, 47). To calculate the consistency of results retrieved from TIMER, we collected 12 innate immune cells and 12 adaptive immune-related cytotoxic components with the marker genes previously identified by Bindea et al. (48) for further estimating by single sample GSEA (ssGSEA), an extension embedded in the “GSVA” R package. The quantification of RNA-seq abundance was first standardized by calculating the transcripts per million (TPM) to estimate the relative degree of the tumor-infiltrating immune microenvironment among 274 TCGA-BLCA tumor samples; after transforming the ssGSEA scores into a z-score distribution, the normalized enrichment score of each immune component was ranked from high to low by the Pearson correlation with the expression of CORO1C and displayed in a heat map form.
Protein Network Construction and Genetic Alteration of the Hub Genes
We identified 1,273 genes that were highly correlated with the expression of CORO1C and 164 genes that were highly correlated with the expression of TMPRSS4, according to the strict criteria of Pearson r > 0.4 and P < 0.0001. Finally, 163 genes that were regarded as the most closely related genes with CORO1C were obtained from the intersection of 1,273 correlative gene sets and all 394 genes contained in the WGCNA purple module (excluding CORO1C). Another 25 genes most closely related to TMPRSS4 were also determined by the same method as described above. Cytoscape software version 3.5.0 (https://cytoscape.org) was downloaded to visualize the PPI network of the hub genes and their possible co-expressed relationship. The genetic alteration type and mutational frequency map of the hub genes were assessed through the cBioPortal for Cancer Genomics (https://www.cbioportal.org) (49).
Patients and Follow-Up
The study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University with informed consent obtained from all patients. All 124 BC patients have suffered radical resection or TURBT in our hospital between 2010 and 2012. According to the AJCC stage, there were 88 cases of NMIBC and 56 cases of MIBC. The mean follow-up period of patients was 49 months, and no patients were lost to follow-up after surgery. OS was defined as the time between surgical resection and death or the last follow-up.
Immunohistochemistry of Tissue Specimens
After fixation with 10% buffered formalin at room temperature for 24 h, the specimens were embedded in paraffin blocks, and the samples were sliced into 4-μm-thick sections. Then, the sections were deparaffinized using a graded ethanol series. Antigen retrieval was performed by a combination of pressure and heat in citrate buffer (pH 6.0) for 60 s. Subsequently, we incubated the sections with a CORO1C polyclonal antibody (diluted 1:200; PA5-21775; Thermo Fisher Scientific; USA), TMPRSS4 polyclonal antibody (diluted 1:200; ab188816; Abcam; USA), PIK3C2B polyclonal antibody (diluted 1:200; ab231122; Abcam; USA), and ZNF692 polyclonal antibody (diluted 1:200; PA5-63794; Thermo Fisher Scientific; USA) overnight at 4°C. We used the streptavidin-peroxidase method to detect antibodies, and phosphate-buffered saline (PBS) was used as a substitute for the primary antibody as the negative control. Positive controls were used to ensure technique validity. All the sections were performed independently by two experienced pathologists. When evaluating the staining intensity, the staining degrees were classified as 0 (negative or weak), 1 (moderate), or 2 (strong). The percentage of positive cells was determined, and a score of 1 to 3 was defined as follows: 1 (<50%), 2 (50–75%), and 3 (75–100%). The patients were divided into two groups based on the result of multiplying the two scores: high expression (>3) and low expression (≤3).
All statistical analyses were conducted using SPSS 24.0 (Chicago, IL, USA) and R 3.6.0 (https://www.r-project.org/). R and GraphPad Prism 6 (San Diego, CA, USA) were used to draw plots. The Wilcox test was applied to compare the expression of hub gene between different clinical groups. P < 0.05 was considered statistically significant.
Data Availability Statement
Publicly available datasets were analyzed in this study, these can be found in The Cancer Genome Atlas (https://cancergenome.nih.gov/).
The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study.
ZL and TL conceived the study. JY, LY, NW, TH, and ZS participated in statistical analysis. CW performed the experiments. CW and YY wrote the manuscript. LY revised the manuscript. All authors contributed to the article and approved the submitted version.
The study was received funding from National Natural Science Foundation of China (81101938).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2020.00931/full#supplementary-material
Supplementary Figure 1. (A) The expression of CORO1C and TMPRSS4 in normal bladder tissues and bladder cancer tissues in the TCGA and GTEx database. (B) The relationship between two hub genes and tumor stage via GEPIA online tool.
1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. (2018) 68:394–424. doi: 10.3322/caac.21492
2. Botteman MF, Pashos CL, Redaelli A, Laskin B, Hauser R. The health economics of bladder cancer: a comprehensive review of the published literature. Pharmacoeconomics. (2003) 21:1315–30. doi: 10.1007/BF03262330
4. Sylvester RJ, van der Meijden AP, Oosterlinck W, Witjes JA, Bouffioux C, Denis L, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials. Eur Urol. (2006) 49:466–5; discussion 475–467. doi: 10.1016/j.eururo.2005.12.031
5. Davaro F, Schaefer J, May A, Raza J, Siddiqui S, Hamilton Z. Invasive non-urachal adenocarcinoma of the bladder: analysis of the National Cancer Database. World J Urol. (2019) 37:497–505. doi: 10.1007/s00345-018-2411-7
7. Clarke C, Madden SF, Doolan P, Aherne ST, Joyce H, O'Driscoll L, et al. Correlating transcriptional networks to breast cancer survival: a large-scale coexpression analysis. Carcinogenesis. (2013) 34:2300–8. doi: 10.1093/carcin/bgt208
8. Yan X, Guo ZX, Liu XP, Feng YJ, Zhao YJ, Liu TZ, et al. Four novel biomarkers for bladder cancer identified by weighted gene coexpression network analysis. J Cell Physiol. (2019) 234:19073–87. doi: 10.1002/jcp.28546
9. Zhang C, Li Z, Hu J, Qi F, Li X, Luo J. Identification of five long noncoding RNAs signature and risk score for prognosis of bladder urothelial carcinoma. J Cell Biochem. (2019) 121:856–66. doi: 10.1002/jcb.29330
10. Giulietti M, Occhipinti G, Righetti A, Bracci M, Conti A, Ruzzo A, et al. Emerging biomarkers in bladder cancer identified by network analysis of transcriptomic data. Front Oncol. (2018) 8:450. doi: 10.3389/fonc.2018.00450
11. Song R, Song H, Liang Y, Yin D, Zhang H, Zheng T, et al. Reciprocal activation between ATPase inhibitory factor 1 and NF-kappaB drives hepatocellular carcinoma angiogenesis and metastasis. Hepatology. (2014) 60:1659–73. doi: 10.1002/hep.27312
12. Tsai YP, Chen HF, Chen SY, Cheng WC, Wang HW, Shen ZJ, et al. TET1 regulates hypoxia-induced epithelial-mesenchymal transition by acting as a co-activator. Genome Biol. (2014) 15:513. doi: 10.1186/s13059-014-0513-0
13. Ibrahim AA, Schmithals C, Kowarz E, Koberle V, Kakoschky B, Pleli T, et al. Hypoxia causes downregulation of dicer in hepatocellular carcinoma, which is required for upregulation of hypoxia-inducible factor 1alpha and epithelial-mesenchymal transition. Clin Cancer Res. (2017) 23:3896–905. doi: 10.1158/1078-0432.CCR-16-1762
14. Stein JP, Lieskovsky G, Cote R, Groshen S, Feng AC, Boyd S, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. J Clin Oncol. (2001) 19:666–75. doi: 10.1200/JCO.2001.19.3.666
15. Babjuk M, Bohle A, Burger M, Capoun O, Cohen D, Comperat EM, et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2016. Eur Urol. (2017) 71:447–61. doi: 10.1016/j.eururo.2016.05.041
16. Wang L, Saci A, Szabo PM, Chasalow SD, Castillo-Martin M, Domingo-Domenech J, et al. EMT- and stroma-related gene expression and resistance to PD-1 blockade in urothelial cancer. Nat Commun. (2018) 9:3503. doi: 10.1038/s41467-018-05992-x
19. Pastushenko I, Brisebarre A, Sifrim A, Fioramonti M, Revenco T, Boumahdi S, et al. Identification of the tumour transition states occurring during EMT. Nature. (2018) 556:463–8. doi: 10.1038/s41586-018-0040-3
20. van Kampen JGM, van Hooij O, Jansen CF, Smit FP, van Noort PI, Schultz I, et al. miRNA-520f reverses epithelial-to-mesenchymal transition by targeting ADAM9 and TGFBR2. Cancer Res. (2017) 77:2008–17. doi: 10.1158/0008-5472.CAN-16-2609
21. Yu C, Liu Z, Chen Q, Li Y, Jiang L, Zhang Z, et al. Nkx2.8 inhibits epithelial-mesenchymal transition in bladder urothelial carcinoma via transcriptional repression of Twist1. Cancer Res. (2018) 78:1241–52. doi: 10.1158/0008-5472.CAN-17-1545
22. Cheng X, Wang X, Wu Z, Tan S, Zhu T, Ding K. CORO1C expression is associated with poor survival rates in gastric cancer and promotes metastasis in vitro. FEBS Open Bio. (2019) 9:1097–108. doi: 10.1002/2211-5463.12639
23. Larzabal L, Nguewa PA, Pio R, Blanco D, Sanchez B, Rodriguez MJ, et al. Overexpression of TMPRSS4 in non-small cell lung cancer is associated with poor prognosis in patients with squamous histology. Br J Cancer. (2011) 105:1608–14. doi: 10.1038/bjc.2011.432
25. Villalba M, Diaz-Lagares A, Redrado M, de Aberasturi AL, Segura V, Bodegas ME, et al. Epigenetic alterations leading to TMPRSS4 promoter hypomethylation and protein overexpression predict poor prognosis in squamous lung cancer patients. Oncotarget. (2016) 7:22752–69. doi: 10.18632/oncotarget.8045
26. Li XM, Liu WL, Chen X, Wang YW, Shi DB, Zhang H, et al. Overexpression of TMPRSS4 promotes tumor proliferation and aggressiveness in breast cancer. Int J Mol Med. (2017) 39:927–35. doi: 10.3892/ijmm.2017.2893
27. Huang A, Zhou H, Zhao H, Quan Y, Feng B, Zheng M. High expression level of TMPRSS4 predicts adverse outcomes of colorectal cancer patients. Med Oncol. (2013) 30:712. doi: 10.1007/s12032-013-0712-7
28. Guan H, Liang W, Liu J, Wei G, Li H, Xiu L, et al. Transmembrane protease serine 4 promotes thyroid cancer proliferation via CREB phosphorylation. Thyroid. (2015) 25:85–94. doi: 10.1089/thy.2014.0155
29. Jin J, Shen X, Chen L, Bao LW, Zhu LM. TMPRSS4 promotes invasiveness of human gastric cancer cells through activation of NF-kappaB/MMP-9 signaling. Biomed Pharmacother. (2016) 77:30–6. doi: 10.1016/j.biopha.2015.11.002
30. Kim MS, Kuppireddy SV, Sakamuri S, Singal M, Getnet D, Harsha HC, et al. Rapid characterization of candidate biomarkers for pancreatic cancer using cell microarrays (CMAs). J Proteome Res. (2012) 11:5556–63. doi: 10.1021/pr300483r
31. Cheng Y, Wang K, Geng L, Sun J, Xu W, Liu D, et al. Identification of candidate diagnostic and prognostic biomarkers for pancreatic carcinoma. EBioMedicine. (2019) 40:382–93. doi: 10.1016/j.ebiom.2019.01.003
32. Jianwei Z, Qi L, Quanquan X, Tianen W, Qingwei W. TMPRSS4 upregulates TWIST1 expression through STAT3 activation to induce prostate cancer cell migration. Pathol Oncol Res. (2018) 24:251–7. doi: 10.1007/s12253-017-0237-z
33. Wang J, Tsouko E, Jonsson P, Bergh J, Hartman J, Aydogdu E, et al. miR-206 inhibits cell migration through direct targeting of the actin-binding protein coronin 1C in triple-negative breast cancer. Mol Oncol. (2014) 8:1690–702. doi: 10.1016/j.molonc.2014.07.006
34. Lim JP, Shyamasundar S, Gunaratne J, Scully OJ, Matsumoto K, Bay BH. YBX1 gene silencing inhibits migratory and invasive potential via CORO1C in breast cancer in vitro. BMC Cancer. (2017) 17:201. doi: 10.1186/s12885-017-3187-7
35. Mataki H, Enokida H, Chiyomaru T, Mizuno K, Matsushita R, Goto Y, et al. Downregulation of the microRNA-1/133a cluster enhances cancer cell migration and invasion in lung-squamous cell carcinoma via regulation of Coronin1C. J Hum Genet. (2015) 60:53–61. doi: 10.1038/jhg.2014.111
38. Huh SJ, Liang S, Sharma A, Dong C, Robertson GP. Transiently entrapped circulating tumor cells interact with neutrophils to facilitate lung metastasis development. Cancer Res. (2010) 70:6071–82. doi: 10.1158/0008-5472.CAN-09-4442
39. Cools-Lartigue J, Spicer J, McDonald B, Gowing S, Chow S, Giannias B, et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest. (2013) 123:3446–58. doi: 10.1172/JCI67484
40. Gaida MM, Steffen TG, Gunther F, Tschaharganeh DF, Felix K, Bergmann F, et al. Polymorphonuclear neutrophils promote dyshesion of tumor cells and elastase-mediated degradation of E-cadherin in pancreatic tumors. Eur J Immunol. (2012) 42:3369–80. doi: 10.1002/eji.201242628
41. Fang LY, Izumi K, Lai KP, Liang L, Li L, Miyamoto H, et al. Infiltrating macrophages promote prostate tumorigenesis via modulating androgen receptor-mediated CCL4-STAT3 signaling. Cancer Res. (2013) 73:5633–46. doi: 10.1158/0008-5472.CAN-12-3228
42. Li S, Cong X, Gao H, Lan X, Li Z, Wang W, et al. Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells. J Exp Clin Cancer Res. (2019) 38:6. doi: 10.1186/s13046-019-1168-1
43. Fan L, Wei Y, Ding X, Li B. Coronin3 promotes nasopharyngeal carcinoma migration and invasion by induction of epithelial-to-mesenchymal transition. Onco Targets Ther. (2019) 12:9585–98. doi: 10.2147/OTT.S215674
46. Li B, Severson E, Pignon JC, Zhao H, Li T, Novak J, et al. Comprehensive analyses of tumor immunity: implications for cancer immunotherapy. Genome Biol. (2016) 17:174. doi: 10.1186/s13059-016-1028-7
47. Li T, Fan J, Wang B, Traugh N, Chen Q, Liu JS, et al. TIMER: a web server for comprehensive analysis of tumor-infiltrating immune cells. Cancer Res. (2017) 77:e108–10. doi: 10.1158/0008-5472.CAN-17-0307
48. Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf AC, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity. (2013) 39:782–95. doi: 10.1016/j.immuni.2013.10.003
Keywords: bladder cancer (BC), weighted co-expression network construction (WGCNA), epithelial to mesenchymal transition (EMT), CORO1C, TMPRSS4
Citation: Wang C, Yang Y, Yin L, Wei N, Hong T, Sun Z, Yao J, Li Z and Liu T (2020) Novel Potential Biomarkers Associated With Epithelial to Mesenchymal Transition and Bladder Cancer Prognosis Identified by Integrated Bioinformatic Analysis. Front. Oncol. 10:931. doi: 10.3389/fonc.2020.00931
Received: 10 February 2020; Accepted: 12 May 2020;
Published: 30 June 2020.
Edited by:Sheldon L. Holder, Penn State Milton S. Hershey Medical Center, United States
Reviewed by:Roland Arnold, University of Birmingham, United Kingdom
Paul B. Fisher, Virginia Commonwealth University, United States
Copyright © 2020 Wang, Yang, Yin, Wei, Hong, Sun, Yao, Li and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
†These authors have contributed equally to this work and share first authorship


text:
A man who confessed to killing 100 children surrendered at the office of Pakistan’s largest Urdu-language newspaper. Javed Iqbal, 40, had been the target of a massive manhunt since early this month, when he wrote a letter to police that led them to the remains of two bodies as well as the pictures and clothes of many of the 100 boys he said he killed. He walked into the building that houses the Jang newspaper in Lahore and said he wanted to write a confession, editor Shaheen Quereshi said.
Alleged Serial Killer Surrenders at Office
From Times Wire Reports

domain: news


text:
Pretty much the only "ugly" part, if you can even call it that, is the stretch of road through what passes for "downtown" Malibu. Now Malibu itself is beautiful, but from that section of PCH, all you can see is the light retail and residential on the inland side and an unbroken line of houses and fences on the seaward side. The latter houses with their fences block any sight of the beach or the sand, which is exactly how the owners want it to be.
Because the beach is public land in California, the owners of beachfront property have come up with a novel idea to keep people out: simply deny access through the private land between the road and the beach. It's highly effective, but it's not foolproof, especially since many property owners have agreed to provide easements on their land in exchange for making improvements on their property etc. We found the first such easement that had been opened in Malibu, the appropriately named "Zonker Harris Access Way."
For those who don't know and didn't click on the link above, Zonker Harris is the ne'er do well hippie/surfer/champion tanner from the Doonesbury comic strip. Notice how the sign is hidden off the sidewalk? It's only visible from one direction, so if you're northbound from LA, you'd probably never see it or notice the narrow alley that leads to the beach (although some internet guides call it "well marked" or something like that...it isn't that well marked). We missed the Geffen access point that apparently opened last year after Geffen gave up his three year suit against establishing the right of way that he'd originally granted in 1983...
So we went down Zonker Way and were greeted by some nice open beach, which I'd show a picture of except that blogger decided to stop taking my photos here in midpost...:
Anyway, until then I leave you with the words that should ring in everyone's ears concerning Malibu:
"Keep your ugly fuckin' goldbrickin' ass out of my beach community."

domain: geography


text:
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Regent Releasing has acquired worldwide rights to "Freshman Orientation" and plans to release the college comedy in the spring. Regent Worldwide Sales is handling international sales at the American…
Tsui Hark, the Hong Kong-based helmer of "Seven Swords," is to direct "Eye 3," third installment of the Chinese-language horror franchise, which has already given rise to two English-language remakes.
French sales house Celluloid Dreams has picked up world sales rights to "Triangle" a genre pic involving three of Hong Kong's best known directors.
The pic marking the return of "Shall We Dance" helmer Masayuki Suo to the director's chair after 11 years is the newest addition to the AFM lineup for Japanese sales company Pony Canyon.
Myriad Pictures has picked up worldwide rights to comedy "Trailer Park Boys: The Movie," which is based on the long-running Canada Showcase Television skein.
Media 8 Entertainment has inked a deal to acquire international rights to Gener8Xion Entertainment's "One Night With the King," which has hit $10.3 million at the domestic B.O. after three weeks in…
Asian companies are responsible for the significant growth at this year's American Film Market. The number of Asian firms with suites at this year's AFM is 61, up from eight just five years ago.
Indie distrib Magnolia Pictures has picked up all North American rights to Thai martial-arts pic "Dynamite Warrior."
With nuclear weapons tension barely abated, international distributors have been quick to acquire rights to "Crossing The Line," a British-made docu about U.S. Army officer James Dresnok who defected…
TriCoast Studios has partnered with production-distribution exec Ami Artzi to create TriCoast Intl., an international sales and distribution company.
Domestic Film DAILY
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Frozen1Daily: $2.1M Cumulative: $136.3M Disney -79.13%
Out of the Furnace
Out of the Furnace3Daily: $500,820 Cumulative: $5.7M Relativity Media -58.71%


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A family of four are about to die in a car crash when the mother, father, and son become travelers. Charlotte, the daughter, retains her identity when the team's historian's transfer misfires, so MacLaren sends her to stay with her grandparents until the others complete their mission. Trevor, Carly, Marcy, and Philip are abducted and interrogated about the future until Carly kills their guard. David alerts MacLaren when Marcy does not come home, who is able to find and rescue the team with Officer Boyd. MacLaren was forced to cancel the team's mission with the family team, and Carly speculates this was their unidentified captor's goal.
This year is San Antonio’s 300th anniversary, and the city is marking the occasion with events, activities, and new infrastructure. Ahead of the festivities, there’s been a flurry of development: In the past few months, the botanical gardens completed an expansion; the city’s first food hall, the Bottling Department, debuted at the Pearl; and San Antonio’s iconic passenger barges got an upgrade, with colorful electric models replacing the old gas-fueled boats. In January, the city will unveil Confluence Park, an expanse of trails and science-focused education facilities near the convergence of San Pedro Creek and the San Antonio River. A commemorative week is planned for early May, with celebrations at each of San Antonio’s five missions and the dedication of San Pedro Creek Culture Park, a once-unremarkable drainage ditch that’s been transformed into a waterfront promenade with public art and performance spaces. Ruby City, a new David Adjaye–designed art center that will house more than 800 pieces from the Linda Pace Foundation Collection, is expected to be completed at the end of 2018. And boutique stays still in the works — including a Thompson Hotel and the third location of the Saint Hotel — hint that San Antonio’s heyday is just beginning. —Devorah Lev-Tov
Almost all of the Earth's islands are natural and have been formed by tectonic forces or volcanic eruptions. However, artificial (man-made) islands also exist, such as the island in Osaka Bay off the Japanese island of Honshu, on which Kansai International Airport is located. Artificial islands can be built using natural materials (e.g., earth, rock, or sand) or artificial ones (e.g., concrete slabs or recycled waste). Sometimes natural islands are artificially enlarged, such as Vasilyevsky Island in the Russian city of St. Petersburg, which had its western shore extended westward by some 0.5 km in the construction of the Passenger Port of St. Petersburg.
This year, Buenos Aires becomes a hub for art, sports, and politics: the inaugural Art Basel Cities program, the Youth Olympic Games, and the G20 will all take place in the city, beginning with the multi-year Art Basel initiative. Though the Argentinean capital is already home to an eclectic collection of galleries, Art Basel Cities promises to elevate Buenos Aires’ reputation in the global art scene by offering professional support for local artists, as well as lectures and workshops throughout the year to draw art lovers to the city.
Philip is ordered by the Director to attend an "update" with various other Historians, which is where the altered timeline's history is imprinted onto their minds, but it comes with physical and emotional consequences. Trevor tries to help a former football teammate with past trauma. Hall returns from prison and has a new mission assigned to him from the Director. Kat has complications with the pregnancy.
Although the former Soviet republic might seem remote, Uzbekistan once sat at the very center of the world. In the first millennium, no traveler could pass from Asia to Europe without stopping in the Silk Road strongholds of Bukhara and Samarkand, and as a result these cities evolved into rich cultural centers. For intrepid travelers, today’s Uzbekistan is a promised land: a Muslim-majority nation that’s both safe and affordable, with few tourists and an abundance of well-preserved mosques and harems. And since the death of authoritarian president Islam Karimov last year, the new regime has taken steps toward reform that have given both Uzbeks and the international community reason for optimism. Improving relations with Iran could soon bring a rail link to the Persian Gulf, and in 2016, the Afrosiyob high-speed-train line began connecting the country’s major cities. Meanwhile, Uzbekistan should benefit from the so-called Iron Silk Road, or Trans-Asian Railway — a Chinese-funded network of routes knitting together Beijing and Europe — once a segment connecting the country through Kyrgyzstan is completed. Book a customized journey with Exeter International, which specializes in the region. —Heidi Mitchell
Return to the Sea was re-mastered in England for the European version of the album, and was released there by Rough Trade Records on April 3, 2006. In North America, the album was released on the upstart label Equator Records on April 4, 2006. The cover of the album is a painting by Caspar David Friedrich titled The Wreck of the Hope. The album features numerous guest appearances, including members of Arcade Fire and Wolf Parade.
Marcy puts herself in a near-death state to recover her lost memories from when she was reset. The memories she receives include those of the original host Marcy, showing that the host's intellectual disability was caused by Vincent/001 testing Simon's incomplete consciousness transfer technology on her. While she is out of commission, Mac and the rest of the team are ordered to provide Hall with backup in protecting the future 53rd President of the United States, who is currently a prepubescent girl.
Last summer, the museum expanded its artistic footprint by over 130,000 square feet, making it the largest contemporary art space in the country. The new Building 6 houses long-term installations by artists like James Turrell, Louise Bourgeois, and Jenny Holzer. Two new museums are still in the works for North Adams, both spearheaded by former Guggenheim director Thomas Krens. At the Global Contemporary Art Museum, curators will work directly with artists to acquire and commission site-specific pieces, while the Extreme Model Railroad & Contemporary Architecture Museum will showcase works by the likes of Frank Gehry and Zaha Hadid, all built in miniature as part of an elaborate model train system. Design is also top-of-mind at Tourists, a modern take on the roadside motel that’s set to open this summer. The resort — situated on 55 acres at the convergence of the Appalachian and Mohawk Trails — will have 48 rooms, each with floor-to-ceiling windows that put the focus on surrounding vistas. —Fiorella Valdesolo
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In March 2017, Travelers agreed to acquire UK-based Simply Business from Aquiline Capital Partners for approximately $490 million. Simply Business is a leading U.K. distributor of small business insurance policies, offering products online on behalf of a broad panel of carriers. It has more than 425,000 microbusiness customers covering more than 1,000 classes of business, and was named “Best Company to Work For” by The Sunday Times in 2015 and 2016. The transaction adds to Travelers' digital capabilities.
Three high-ranking Travelers are sent back by the Director to put Grace on trial for violating protocols, overwriting a person who was not assigned to be her host, and sabotaging the Grand Plan. Mac and the team must give testimony at the trial that will determine if she should be overwritten or not. Meanwhile, the Faction is trying to replenish its numbers by overwriting people with the stored minds in the Quantum Frame. It transpires the trial was staged by the Director to flush out a Faction member from within the three Travelers judging Grace, which it successfully does, this leading to Mac's team securing the Quantum Frame.


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Ion Optics, Inc. is the emerging technology leader in optical-based MEMS gas detection sensors and wavelength tuned infrared emitters. Ion Optics has developed a patented optical platform technology for controlling (tuning) the infrared emission and absorption wavelengths of a silicon surface using two-dimensional photonic crystals produced through standard, high-volume, semiconductor manufacturing techniques.
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is a provider of next-generation, atomic-level microscopes for the global semiconductor, life sciences and materials sciences industries. ALIS Corporation's disruptive breakthrough in helium ion technology has enabled the company to develop a revolutionary scanning ion microscope and imaging system that allows scientists to see at the atomic level with resolution and material contrast never before seen.


Forged from the same spirit of innovation that led to the diesel engine, today’s diesel offers a whole new level of efficiency and performance. Although the way we use diesel today has…January 23, 2022
Unlike milk, gasoline does not have an expiry date. As a result, predicting how long the gasoline will last might be tricky. Nonetheless, gasoline degrades fast and will inevitably diminish its efficiency.…January 18, 2022
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Finding an Online Casino UK
Online casinos, also known as virtual online casinos, are online versions of real-time traditional casinos. Online casinos allow gamblers around the world to play online casino games and wagers via the web. It really is an extensively popular type of online casino gambling. The growth in online casino gambling could be traced back to the web technology.
Gambling is an extremely common pastime amongst a lot of people. Most people like to enjoy betting by placing bets on horses, football matches, pool matches and lotto games. However, there are some who prefer to play online casinos and play virtual poker, slots, blackjack and other casino games. There are many online casinos which allow gamblers to wager real cash and play these kinds of casino games.
One of the primary differences between an online casino and a land-based casino may be the payment method. Land-based casino platforms allow punters to pay for their winnings using money transfer systems like Debit Cards, Credit Cards, Paypal among others. Online punters do not have the option of using such payment methods. For instance, if a UK player wins a jackpot he would need to profit the prize money in a particular date. Online gamblers therefore, need to cash in their winnings using payment methods that are commonly used in the united kingdom.
Another main distinction between the two is the speed at which a player wins his/her bonus. In the event of online casinos, a player may win his bonus instantly, whereas a land-based casino supplies a certain period of time for a new player to claim his/her bonus amount. This can’t be said about online casinos in which a player can claim his bonus soon after registering with the website.
The reliability of the online casino is another essential aspect that differentiates both gaming platforms. There were instances where players won large sums from online casinos, but later found out that the site they were playing with was fraudulent. This happened to a big player of a multi-valued website. Such incidents create doubt about the reliability of the online casino. On the other hand, if you are dealing with a land-based casino, there is absolutely no way you can lose your cash because there is no way that you can be sure of the dealer.
The third thing that separates online casinos from land-based ones may be the availability of the games that you wish to play. A UK online casino does not have the games that are offered in Las Vegas or Macao, for instance. However, there are several online casinos offering games that are popular in the united kingdom.
The reliability and safety of the online casino also determines your comfort if you are utilizing the site. A UK online casino is highly secured. It is possible for a computer virus to infect an online casino account, and you may lose all of your money. Online casinos use state-of-the-art firewalls to prevent this type of risk, and keep your personal computer safe. There are also some online casinos offering virus protection.
The next time you visit an online casino in the united kingdom, you need to know its reputation. This will make sure that you are playing with an established online casino. It is also important to learn how much it costs to gamble at the online casino. The more popular a niche site is, the more it could cost.
Some online casinos offer free games. They are usually simple casino games such as bingo or poker. Should you have never played within an online casino before, it is best to take a friend along. You may not get the experience that you’ll with other styles of gambling.
Some online casinos will provide you with 007 카지노 로얄 bonuses and referral fees in the event that you refer other players to the website. These can be quite substantial savings. In addition, many online casinos offer discount rates to clients who play their games while on a vacation. The casino may offer you a lower minimum bet, a higher house edge, or even a combination of any of these benefits for playing their games online.
One final consideration when you are choosing an online casino in the united kingdom is whether the casino includes a customer service phone number or address. Many people are uncertain about where you can contact if they have any questions about their online casino. You can usually find information about such a phone number or address on the casino’s website. In fact, if you like, you can call the casino and speak to a live operator to be able to better understand the web casino and the direction they operate it.

domain: entertainment


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Censor is behaving like a homophobic society: Hansal Mehta on Aligarh
By giving his an A certificate to the first trailer of his next film, Aligarh, the Central Board of Film Certification (CBFC) was acting like a cultural police and thwarting its promotions, filmmaker Hansal Mehta has alleged. Starring Manoj Bajpayee and Rajkumar Rao in lead roles, the film which deals with the stigma surrounding homosexuality.bollywood Updated: Jan 31, 2016 16:37 IST
By giving his an A certificate to the first trailer of his next film, Aligarh, the Central Board of Film Certification (CBFC) was acting like a cultural police and thwarting its promotions, filmmaker Hansal Mehta has alleged. Starring Manoj Bajpayee and Rajkumar Rao in lead roles, the film which deals with the stigma surrounding homosexuality.
“This is an attempt to thwart my promotion. They cannot ban the film, because my film does not in any way go against the guidelines of the Cinematograph Act,” he said on the sidelines of the maiden Difficult Dialogues conference in Goa. “This is the cultural police deciding what we should see... I’m furious and very angry, because they’ve violated my expression,” he added.
Aligarh is based on the real life incident of Professor Shrinivas Ramchandra Siras of Aligarh Muslim University, who was terminated from his job because of his sexuality. Rajkummar Rao plays a journalist in the film who is trying to bring his story to the world.
Watch Aligarh trailer
Earlier, speaking at the trailer launch of Aligarh, Hansal Mehta said, “When the censor board saw the film they asked for certain cuts. I am a bit surprised by the cuts they have asked for. The film in many ways is about loneliness, relationships, rejection. It’s about a homophobic society. What has happened is the censor is behaving exactly like the people who suspended professor Siras. They (Censor Board) are behaving like a homophobic society... We are taking the matter to the tribunal. It’s just that you are breaking the back of a film, you are making us run around pillar to post when we should be taking the film to you, showing the film to everyone”
Filmmaker Anurag Kashyap, too, took to Twitter to criticise censor board chief Pahlaj Nihalani for giving the ‘A’ certificate to Aligarh’s trailer. “Aligarh trailer has been given the A certificate because the film is about what all the Pahlaj Nihalani’s of Aligarhdid to one man,” Kashyap posted on twitter.
"Aligarh" trailer has been given the A certificate because the film is about what all the Pahlaj Nihalani's of Aligarh did to one man..— Anurag Kashyap (@anuragkashyap72) January 30, 2016
Mehta expressed his displeasure over the censor board’s decision to give an A certificate to the film’s trailer. “We have only a month to promote the film. It’s not only an important entertainment medium but important film for our nation. They have put a stop to the promotions by giving the promo an A certificate. This promo can now run only with an A-certificate film. I am showing the trailer in protest and absolute anger,” he added.
Mehta, who has directed critically-acclaimed films like Shahid and Citylights in the past, said that there is nothing in the trailer which should attract the A rating and rued that because of the tag, he can only promote his film during screening of adult films and cannot push it during primetime television. He, however, said that the social media is the big hope, which will ensure attempts to suppress the film are not successful.
He said the film is yet to get a nod from the CBFC and the board has asked for some “ridiculous cuts”, which he is contesting. Sounding optimistic about a favourable ruling from the tribunals, Mehta said the film will not miss its February 26 release date.
Follow @htshowbiz for more.
First Published: Jan 31, 2016 16:18 IST

domain: artistic


Unchecked development threatens community
I am responding to your editorial “Realpolitik and the Villas at Canandaigua.”
The claim that “34 condo buildings” actually disturb less ground doesn’t ring true when you consider that each building will contain four 1,250- to 1,850-square-foot townhouses, all with two stall garages. These will have a footprint of approximately 8,000 square feet. Compare 34 of these structures to 44 single-family homes that will average less than a 4,000-square-foot footprint. The issue becomes looking at 34 huge urban-style structures or 44 single-family homes that would be subject to the same conservation subdivision regulations setting aside open space as this development.
You are right that the parcel bordering Routes 5 and 20 doesn’t compare to others closer to the lake. That is why the 2006 amended zoning map allows for one single-family home per acre and the land farther down the lake allows one home per three acres. The town should partner with the city to encourage development that doesn’t just increase sprawl but preserves and enhances the desirability of the entire area.
The requested zoning change deviates too far from existing zoning. This proposal triples the density to 136 townhouses from the 44 single-family homes allowed under existing zoning. This requires real creative zoning changes and exceptions from our existing policy in order to be built.
Wegman Companies wants to change zoning from SCR-1 to R-1-30 to allow incentive zoning. It will then go beyond the R-1-30 rules to increase from 76 houses to 136 and build four- unit townhouse buildings instead of the two-unit structures permitted.
They are offering amenities under incentive zoning that are not permitted in SCR-1 to persuade the Town Board to allow this convoluted manipulation of our zoning laws.
The traffic problems are very real today and are a result of the existing developments. When the Town Board approved these developments, it should have considered not only the increased revenues but also the cost of improvements necessary to support the increased demand for services such as road improvements and pedestrian safety.
The Town Board needs to let all developers know that the town welcomes new development but it must adhere to our comprehensive plan. The reasons that make our town a great place to live are put at severe risk if we allow unchecked development change the face of our beautiful community.
Middle Cheshire Road
The writer is a candidate for Town Board.


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Chala Barrington-CruzLicensed Clinical Social Worker, LCSW, MA-ABA, MS
Raising a child with special needs is undoubtedly taxing mentally and physically.But the emotional and mental fatigue actually gets to most of my counseling clients. I understand when you can't have a fun day at the zoo or the park because you constantly have to scan your kid and the environment. It hurts when someone says something unkind about your child's behavior or appearance . Having to calculate an exit strategy or a plan B everywhere you go, takes a toll on your mental health.
0.0 Miles away

domain: psychology


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The 2nd episode of our Sketch show on HuHa. Featuring Ashens and Amazing Phil.
The Bard warns of the dangers of swearing.
The most childish thing ever.
The 1st episode of Beef House. A fraternity of butcher for hire, falsely accused of selling 3% meat products, fight to clear their names and fight crime.
Evil Guy has the cleaner in. What could go wrong?
Joy Bot is not related to Happy Plane... Well... Maybe a little bit.
Actual, education value with a fat beat and bassline. Word up G-Money.
A song about my sexual prowess and food.
A sketch show we made for the HuHa channel. Not suitable for kids.
Another PSA from the British Advisory Dept. This one covers the topic of sex.
A sloth slothing.
Everyone looks forward to a visit from Happy Plane and Panda Pilot.
A tale of the real happenings at the CERN Science Hole.
From the 2nd episode of BAD Advice comes the full length version of the credits tune.
Another PSA from the B.A.D. This one is about electricity.
It's Summer in the USSR and the Russian Dancing Men are out on...
The newest song from Savlonic from their new album, Red. You...
Cat Face detects some ham in the area.
Once upon a perfect peril...
So here it is! I'm starting Uni this September, and as...
A young boy faces the horrors of crack use through the smooth...


iPhone 15 Pro might sport single, unified volume rocker, mute button
Say farewell to the mute switch
Rumours that Apple will ditch physical buttons on the upcoming iPhone 15 Pro line have swirled for a while, but a new report suggests the company might drop the dual volume buttons and ditch the mute switch.
The detail comes via YouTuber @ZoneOfTech, who posted on Twitter that they are “almost 100% sure” that the iPhone 15 Pro will sport a single unified volume button.
If you look at the regular iPhone 15 CAD, you can see that it clearly has 2 individual button slots with 4 pins in total.
Pretty sure that this means we’ll see a unified volume button on the 15 Pro.
— Daniel (@ZONEofTECH) March 2, 2023
ZoneOfTech made the assertion after comparing previously-leaked CAD renders of the iPhone 15 and iPhone 15 Pro. The 15 Pro shows a single indentation with two pins, whereas the regular iPhone 15 render has two separate slots with four pins total.
Based on the same CAD renders, ZoneOfTech is also “100% sure that the mute switch will also switch to a singular press button” instead of the up/down switch the iPhone uses now.
Admittedly, the CAD renders aren’t much to go on alone. However, 9to5Mac reportedly receive information from a source confirming the single volume button, adding credence to the rumour.
It’ll be interesting to see what else comes out about the iPhone 15 and 15 Pro ahead of the likely September launch. Coupled with rumours about solid-state buttons with haptic feedback for the Pro models, it’s not a stretch to imagine Apple will reimagine other parts of the button too.
Moreover, while some love the mute switch, I could see moving from a switch to a button possibly offering some convenience benefits. For example, it could allow for expanded do not disturb options or automated silencing at specific times since iOS would no longer be restricted by the physical position of the switch.
Source: ZoneOfTech Via: MacRumors, 9to5Mac


C-27J Spartans Grounded.
The Royal Australian Air Force (RAAF) has decided to ground several of its C-27J Spartans after the discovery of structural cracking in these aircraft. This move follows the US Coast Guard’s decision to suspend the use of all 14 of its Spartans, citing the presence of cracks of varying degrees. The US Coast Guard stated that their Spartans would remain grounded until a thorough evaluation could be completed.
The manufacturer, Leonardo, had previously issued an Alert Service Bulletin instructing operators, including the RAAF and US Coast Guard, as well as the military forces of Italy, Mexico, and other nations, to inspect for cracks near the attachment points of the horizontal and vertical stabilizers to the fuselage.
A spokesperson for the Australian Defence said that the Royal Australian Air Force had initiated inspections on its C-27J fleet and identified similar structural issues. Aircraft found to have cracking will not be flown until a comprehensive engineering assessment has been conducted.
Leonardo issued a statement reassuring that the problem is fixable and should have minimal impact on operations. The manufacturer has already identified a repair solution, which can be implemented at customer premises by the operators without requiring significant effort. Leonardo emphasized that the reported issue is not expected to affect planned aircraft operations.
As of now, Australia operates 10 C-27J Spartans through No. 35 Squadron at RAAF Base Amberley. This squadron primarily focuses on peacetime operations such as search and rescue and aeromedical missions. The RAAF initially acquired the C-27J as a replacement for the Caribou, intending to fill the gap between the Chinook and the larger Hercules and C-17 Globemaster aircraft.


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domain: chemistry


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Athena Seafoods sources most of its products in Lofoten. Lofoten is one of the most important fishing areas in Norway. Spectacular scenery and cold clean waters contribute to giving seafood from Lofoten an “exotic touch”.
Eastern European markets are very important for Athena, with Russia being the most important individual market with a substantial potential for further development.
The Far East including Japan, Thailand, Taiwan and Korea, are areas on which Athena is focused. Buyers in this region, perhaps more than anyone, appreciate the high level of service and information, in addition to a stable supply of top quality seafood.
Athena Seafoods’ goal remains to be the quality- and service-minded customer’s preferred supplier of high quality seafood.
Welcome to a cooperation with Athena Seafoods.


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The Fränkische Galerie (Franconian Gallery) is housed in the Rosenberg fortress that dominates the historic district of Kronach. Its collection comprises some 200 masterpieces of painting and sculpture from the Late Gothic and Early Renaissance periods – including paintings by Lucas Cranach the Elder (1472–1553), who was born in Kronach.
As part of a recent upgrade of the exhibition rooms, Katrin Söncksen from lighting design office lichttransfer in Berlin devised an energy-efficient lighting concept that is configured precisely to the requirements of the artwork while creating focal points.
Much like a subtle guidance system, the light directs visitors through the exhibition rooms in the museum. A choreography of high-contrast accent lighting stages the individual objects in each room as required for an optimised visual experience. The lighting designers opted for ERCO’s 12W Optec LED spotlight here, which offers interchangeable Spherolit lenses and diverse light distributions for fine and precise adjustment to the characteristic features of each exhibit: The filigree motifs of the panel paintings and winged altars are rendered realistically and without reflective glare in exceptionally soft light.

domain: design


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Attention is Priceless
In today's world of short attention spans, blaring media, and countless distractions, getting someone's attention is hard work. It may be the hardest work you do, in fact, to cut through the clutter of their lives and responsibilities so they are willingly listening to what you're saying.
Don't squander that rare opportunity, then. Be ready to say what you need to say to move them to a decision.
We can always earn more money; we can never create more time.

domain: literature


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Tuesday afternoon at 3.15-4.15pm
Run by Miss Andrews and Miss Fuentes. Over the period of the winter we will be developing our fundamental basketball skills to enable us to be competitive in the intra schools tournament in February. The children will improve their passing, dribbling, shooting and defensive skills. As well as learning to play as a team.

domain: sports