The objective of this study was to identify possible biomarkers and to explore the mechanisms of suppression of vemurafenib on melanoma progression.
GSE42872 affymetrix microarray data were downloaded from the Gene Expression Omnibus database for further analysis. Differentially expressed genes (DEGs) between vehicle-treated samples and vemurafenib-treated samples were identified. Gene ontology and pathway enrichment analysis of DEGs were performed, followed by protein-protein interaction (PPI) network construction. Furthermore, the functional modules of the PPI network were screened using BioNet analysis tool. Finally, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed for DEGs in the module.
In total, 794 upregulated transcripts corresponding to 214 genes and 977 downregulated transcripts corresponding to 325 genes were screened. The downregulated DEGs were significantly enriched in pathways such as cell cycle, DNA replication, and p53 signaling pathway. Upregulated DEGs were significantly enriched in phosphatidylinositol signaling system and inositol phosphate metabolism. Significantly enriched functions of downregulated DEGs were mitotic cell cycle, nuclear division, DNA metabolic process, cell cycle, and mitosis. Upregulated DEGs were mainly enriched in single multicellular organism process and multicellular organismal process. Moreover, cell division cycle 6, checkpoint kinase 1 (CHEK1), E2F transcription factor 1 (E2F1), epidermal growth factor receptor (EGFR), and phosphoinositide-3-kinase, regulatory subunit 1-α (PIK3R1) of the module were remarkably enriched in pathways such as cell cycle, apoptosis, focal adhesion, and DNA replication.
Cell division cycle 6, CHEK1, E2F1, EGFR, and PIK3R1 of the module and their relative pathways, cell cycle, and focal adhesion might play important roles of suppression of vemurafenib on melanoma progression.
Tumori 2015; 101(4): 368 - 374
Article Type: ORIGINAL RESEARCH ARTICLE
AuthorsLiangliang Quan, Yang Wang, Jiulong Liang, Jie Shi, Yu Zhang, Kai Tao
- • Accepted on 14/01/2015
- • Available online on 16/05/2015
- • Published in print on 24/07/2015
This article is available as full text PDF.
Melanoma is a deadly skin cancer, which results from the malignant transformation of melanocytes and is characterized by rapid development, high metastasis ability, and limited efficiency of therapeutics (1, 2). The incidence of death from melanoma has increased by 7% from 1990 to 2006 (3). The prognosis is discouraging for patients with metastatic melanoma. About 50% of patients develop metastasis within an average of 36 months, frequently to the liver, and a median survival of only 6 months after metastasis has been reported (4). Additionally, melanoma is resistant to chemotherapy (5). Hence, the need for new and improved therapies is urgent.
Currently, therapeutic drugs targeting for oncogenes and signaling pathways are identified for the treatment of advanced melanoma. Oncogene
Nowadays, gene expression microarray has been widely applied in studying the development and progression of tumors to advance techniques and lower expenses. In the present work, we made use of bioinformatics approaches to analyze the information obtained from GSE42872 in order to investigate the melanoma-related genes and to explore the mechanism of suppression of vemurafenib on melanoma progression. Gene ontology (GO) functions and pathways were identified. Candidate genes and pathways identified by our approach might provide the groundwork for a therapy for melanoma. However, further evaluations of their potential use in the treatment of melanoma are needed.
Methods and materials
Analysis of Affymetrix microarray data
The gene expression profile of
Data preprocessing and identification of differentially expressed genes
The Affy package (15) from Bioconductor and Affy probe annotation files offered by Brain Array Lab were applied to preprocess the gene expression profile data of
Gene ontology enrichment analysis
Based on Gene Ontology Database (
Pathway enrichment analysis
Kyoto Encyclopedia of Genes and Genomes (KEGG) is a bioinformatics database including biochemistry pathways (19). Database for Annotation, Visualization and Integrated Discovery (DAVID) provides analytic tools for extracting biological meaning from a large list of genes (20). The DAVID was used for KEGG pathway enrichment analysis of upregulated and downregulated DEGs, respectively. All pathways were analyzed for significant differences. A p value less than 0.01 was chosen as the cutoff value.
Gene functional annotation
Based on transcription factor (TF) data, we screened and annotated DEGs to determine whether these DEGs had transcriptional regulatory functions. Additionally, tumor suppressor (TS) genes database (21) combined with tumor-associated genes (TAG) database (22) were used to further extract all the known oncogenes and TF genes.
Protein-protein interaction network construction and modules mining
As proteins seldom perform their functions alone, it is important to comprehend the interaction of these proteins by studying larger functional groups of proteins (23). The Search Tool for the Retrieval of Interacting Genes (STRING) database (24) provides both experimental and predicted interaction information. We used the STRING database to annotate functional interactions between DEGs and other genes. Based on this information, a protein-protein interaction (PPI) network was visualized by Cytoscape (25). Node degree ≥20 was selected as the threshold.
The edges and nodes of the PPI network were so complicated that further analysis was needed to expose the enriched functional modules of the PPI network using BioNet analysis tool (26). False discovery rate <0.00005 was regarded as the cutoff criterion. Subsequently, we performed KEGG pathway enrichment analysis of genes in the module.
Identification of DEGs
According to the research criterion FDR <0.01 and |log FC| ≥2, a total of 1771 transcripts were screened, including 794 upregulated transcripts corresponding to 214 genes and 977 downregulated transcripts corresponding to 325 genes. The ratio of the number of upregulated to downregulated genes was 1:1.52 (
The results of the differentially expressed genes in vemurafenib-treated samples
|Transcript counts||Gene counts|
Gene ontology functional enrichment analysis
We used the DAVID to identify GO-enriched functions for significant DEGs. Downregulated DEGs from BRAFV600E A375 melanoma cells treated with vemurafenib were significantly enriched in mitotic cell cycle (p<1.11E-16), nuclear division (p<1.11E-16), DNA metabolic process (p<1.11E-16), cell cycle (p<1.11E-16), and mitosis (p<1.11E-16). These functions were associated with cell cycle regulation (
Top 5 Gene Ontology categories of the differentially expressed genes from BRAFV600E A375 melanoma cells treated with vemurafenib
|Gene ontology ID||Term||Gene counts||p Value|
|Gene counts refer to the number of differentially expressed genes enriched in the gene ontology function.|
|Downregulated genes||GO:0000278||Mitotic cell cycle||82||<1.11E-16|
|GO:0006259||DNA metabolic process||72||<1.11E-16|
|Upregulated genes||GO:0044707||Single multicellular organism process||93||1.92E-05|
|GO:0032501||Multicellular organismal process||95||2.76E-05|
|GO:0055021||Regulation of cardiac muscle tissue growth||5||3.17E-05|
|GO:0044700||Single organism signaling||82||3.90E-05|
Analysis of KEGG pathway
The DAVID was used to determine the profound KEGG pathways to obtain further insights into the function of DEGs. The pathways of the upregulated and downregulated genes were obtained with p value less than 0.01 (
The KEGG pathway of the differentially expressed genes from BRAFV600E A375 melanoma cells treated with vemurafenib
|KEGG pathway||Gene counts||p Value|
|KEGG = Kyoto Encyclopedia of Genes and Genomes.|
|Gene counts refer to the number of differentially expressed genes enriched in the KEGG pathway.|
|Downregulated genes||Cell cycle||18||3.14E-11|
|P53 signaling pathway||7||0.000410847|
|Base excision repair||5||0.000474269|
|Upregulated genes||Ribosome biogenesis in eukaryotes||6||0.005422121|
|Phosphatidylinositol signaling system||5||0.001975|
|Inositol phosphate metabolism||4||0.004102|
|Phosphatidylinositol signaling system||5||0.001975|
The upregulated DEGs were significantly enriched in phosphatidylinositol signaling system (p = 0.001975), inositol phosphate metabolism (p = 0.004102), and phosphatidylinositol signaling system (p = 0.001975).
Gene functional annotation
We mainly observed the expression of TFs and TAGs from A375 melanoma cells treated with vemurafenib. The expression levels of 25 TFs were significantly downregulated in the vemurafenib-treated group. By contrast, only 9 TF levels were upregulated after vemurafenib treatment (
Analysis of transcriptional regulation functions of the differentially expressed genes from BRAFV600E A375 melanoma cells treated with vemurafenib
|TF counts||TF genes|
|TF = transcription factor.|
Moreover, 35 downregulated DEGs were identified to be closely related to the occurrence and development of tumors based on the results of TAG genes, including 10 known oncogenes and 20 known TSs (
Analysis of tumor-associated gene functions of the differentially expressed genes from BRAFV600E A375 melanoma cells treated with vemurafenib
|TAG counts||Oncogene||Tumor suppressor||Other|
|TAG = tumor-associated gene.|
Protein-protein interaction network construction and analysis of KEGG pathway enrichment of the module genes
The STRING tool was used to obtain the PPI relationships of the DEGs. Node degree ≥20 was selected as the threshold. In
Construction of protein-protein interaction network of differentially expressed genes associated with melanoma. Red nodes represent significant upregulated genes and green nodes represent significant downregulated genes.
We then screened the module of the PPI network using BioNet analysis tool (
Protein-protein interaction module of differentially expressed genes (DEGs) related to melanoma. The degrees of color depth of nodes are associated with fold change value of DEGs. Red nodes represent upregulated genes; green nodes represent downregulated genes; white nodes stand for normal genes. Square nodes indicate lower importance of genes in the module; circular nodes indicate significant importance of genes in the module.
We also performed KEGG pathway enrichment analysis of genes in the module. The results are shown in
The KEGG pathway of the differentially expressed genes in the module
|KEGG pathway||Gene counts||p Value||Genes|
|KEGG = Kyoto Encyclopedia of Genes and Genomes.|
|Gene counts refer to the number of differentially expressed genes in the module enriched in the KEGG pathway.|
Melanoma is a deadly skin cancer and its occurrence and mortality have been rising worldwide for the last 30 years (27). In the current study, we investigated gene expression profile
Cancer is characterized by uncontrolled cell proliferation resulting from dysregulation of the cell cycle (28). In the present work, our results argued for an important role of cell cycle out of control in the pathogenesis of malignant melanoma, and several candidate genes were identified to be downregulated after vemurafenib treatment, such as
Checkpoints are present in all stages of the cell cycle and are considered the gatekeepers in order to maintain the integrity of the genome (33, 34). Deregulation of cell cycle checkpoints seems to be a universal phenomenon in human cancers. CHEK1 was found to be a mediator of the checkpoints in G2/M as well as S phase in mammals (35, 36) and to respond to DNA damage by initiating cell cycle arrest. Moreover, CHEK1 was demonstrated to express highly in many types of tumors (37). It is known that the inhibition of CHEK1 enhances the cytotoxicity of DNA-damaging drugs via abrogating of the cell cycle checkpoint (38). Recent study has demonstrated that CHEK1 inhibitors can be used to increase the sensitivity of tumor cells to replication inhibitors
Another major functional pathway, focal adhesion, was identified, involved in
Although we obtained some significant genes and pathways in melanoma treated by vemurafenib, there are some limitations to our study. The study was carried out based on bioinformatics methods and the conclusions have not been proved by experiments. Additionally, the sample size in our study was limited. Hence, more work is warranted to further explore the molecular mechanisms of melanoma and to apply molecular genetic diagnosis to the clinic.
In conclusion, our study sheds new light on the mechanism of suppressing the progression of melanoma after vemurafenib treatment. Cell division cycle 6, CHEK1, E2F1, EGFR, and PIK3R1 of the module and their relative pathways, cell cycle, and focal adhesion might play important roles in vemurafenib inhibition on melanoma progression. Our research may provide a theory model in melanoma treated by vemurafenib. However, the results described should be verified in animal experiments.
- Quan, Liangliang [PubMed] [Google Scholar]
- Wang, Yang [PubMed] [Google Scholar]
- Liang, Jiulong [PubMed] [Google Scholar]
- Shi, Jie [PubMed] [Google Scholar]
- Zhang, Yu [PubMed] [Google Scholar]
- Tao, Kai [PubMed] [Google Scholar] , * Corresponding Author (firstname.lastname@example.org)
Department of Plastic Surgery, General Hospital of Shenyang Military Area Command, PLA, Liaoning - China