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6INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
TARGETING ROLES OF TCF3 AND WNT SIGNALING PATHWAY IN HPV POSITIVE HEAD AND NECK SQUAMOUS CELL CARCINOMA : BIOINFORMATICS ANALYSIS
1Saba Hasan, 2Nishant Kumar Singh, 2Prankur Awasthi, 2Agrika Gupta
1Assistant Professor, Amity University Lucknow Campus 2Ph. D Student, Amity University Lucknow Campus https://doi.org/10.5281/zenodo.13829255
Abstract. A specific category of head and neck cancer (HNC) recognizes the human papillomavirus (HPV) as a significant etiological factor. Transcription factors are significant and appealing biological targets for the development of targeted therapeutic strategies for treating various cancer types in humans. We used various in silico approaches in this paper to identify potential new transcription factors and related pathways that could enhance the therapy for HPV-induced HNSCC. We have performed rigorous literature searches to screen transcription factors related to HNSCC through searches in PUBMED, Google Scholar, etc. After screening transcription factors through literature searches and verifying expressions through UALCAN, we constructed the PPI network using the STRING v12.0 database. We increased the criteria for the maximum number of interactors to 50. Functional enrichment analysis was performed through identified transcription factors (TFs) through the PPI network. Further network was visualized on the cytoscape v3.10.2 for MCODE clustering. Gene set enrichment analysis was done through the Panther v16 database. TCF4, CNOT7, PAX5, SPDEF, and MITF were identified as related HPV-induced HNSCC through a literature search. Expression verification analysis suggested that TCF4, CNOT7, and PAX5 were upregulated and SPDEF and MITF were downregulated. This differential expression is further explored in STRING v12 db. In the PPI network, the number of nodes and number of edges were 56 and 208, respectively. MCODE analysis through cytoscape v3.10.2, given two clusters, in which CNOT6L and TCF3 were the central nodes. To further node degree, gene set enrichment analysis identified TCF3 and Wnt signaling pathways, respectively. Further invitro and in vivo investigation is required to establish TCF3 as a therapeutic target for the therapy of HPV-induced HNSCC.
1. Introduction
This is a group of cancers that can be found in many parts of the upper aerodigestive tract, like the mouth, oropharynx, lips, nasopharynx, hypopharynx, and larynx [24]. This cancer is prevalent globally, with about 330,000 deaths in 650,000 cases per year. The Global Cancer Statistics 2020, prepared by the IARC, indicates that oral cancer constituted around two percent of all cancer cases and 1.8% of all deaths globally due to cancer. South Asian countries have a higher incidence of oral cancer than Western countries have of oropharyngeal and tongue cancers, which are the most common kinds of head and neck cancer (HNC). Over 90% of cancers are found in squamous cells in the mouth, throat, and larynx. The rates of tonsil and oropharyngeal cancer are rising steadily around the world. HNC arises from genetic and environmental influences, especially in individuals with prolonged exposure to risk factors such as tobacco use, excessive alcohol use, and protracted exposure to UV radiation, asbestos, and some viruses. Recognized as a sexually transmitted infection, human papillomavirus (HPV) can spread through cutaneous, oral, and genital contact, and is considered a significant etiological factor in the category of head and
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
neck cancers, particularly oropharyngeal carcinoma [28, 3]. The prevalence of HPV-associated oral malignancies is increasing globally and exhibits unique genetic, pathological, clinical, and epidemiology characteristics [12]. Furthermore, HR- HPV genotypes 16 and 18 generate early proteins E6 and E7, leading to the deregulation of cell cycle control. Researchers have identified HR-HPV16 as a regulator of NF-kB activation and expression in several cancers, including OSCC [8].
TFs are significant molecular targets for the development of targeted therapeutic strategies for the management of many human cancer types [2, 17]. TCF4, CNOT7, PAX5, SPDEF, and MITF were found to link with HPV-induced HNSCC [8, 6, 14, 16, 20, 21]. In this paper, various in silico approaches (protein-protein interaction analysis, functional enrichment analysis, MCODE module analysis, and gene set enrichment analysis) were performed to identify probable new transcription factors and related pathways to improve the therapy of HPV-induced HNSCC.
2. Methods and Materials
2.1. Screening of transcription factors related to HPV induced HNSCC
We have screened transcription factors related to HPV-induced HNSCC through searches in PUBMED, Google Scholar, etc. Key words were "HPV", "HNSCC" and "Transcription Factor" applied [8, 6, 14, 16, 20, 21].
2.2. Protein protein interaction analysis of screened transcription factor
Screened TFs were applied on STRING v12, a tool useful for protein-protein interaction
analysis [27]. The criteria for the interaction score minimally required was 0.400, and the interactors maximum number was increased to 50. It is done to visualize the significant interaction of the proteins.
2.3. Functional enrichment analysis of TFs related to HPV induced HNSCC
ShinyGO v0.80 was applied to find out the major pathways and gene ontology related to
the identified PPI Network genes [10].
2.4. Identification and expression validation of novel TF related to HPV induced HNSCC
PPI Network was transferred to Cytoscape v3.10.2 for further cluster analysis [27]. The MCODE plugin was applied with node score cutoff 0.2, degree cutoff 2, max. depth 100, and K-core 2. GSEA was performed through the Panther v16 tool [19]. Expression validation was done through the UALCAN database [5].
3. Result and Discussion
3.1. Expression verification of screened transcription factors in HNSCC
Transcription factors, including STAT, AP-1, and NF-kB, have been shown to significantly contribute to tumorigenic activities in HNSCC [18, 22]. In HNSCC treatment trials with small-molecule inhibitors, targeting these transcription factors reduces carcinogenic potential and amplifies cytocidal response. [30]. As a result, we conducted rigorous literature searches to screen the transcription factors associated with HPV-induced HNSCC. TCF4, CNOT7, PAX5, SPDEF, and MITF were found to be related to HPV-induced HNSCC [8, 6, 14, 16, 20, 21]. Further, expression validation was performed through the UALCAN database, which contains tumor versus normal expression data from the TCGA database [5]. mRNA expression analysis suggests that TCF4, PAX5, and CNOT7 were upregulated significantly and SPDEF and MITF were downregulated significantly in tumor samples. These results imply that these transcription factors are differentially expressed in HNSCC (Fig. 1).
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Figure 1. Expression Verification of screened transcription factors 3.2. Protein-protein Interaction analysis
Additionally, we pasted these differentially expressed TFs onto STRING v12. In the PPI network, we identified the nodes number: 56, edges number: 208, the node degree in average as 7.43, the clustering coefficient as 0.763, and the expected number of edges as 57 (Figure 2). PPI enrichment p-value was < 1.0e-16. The findings indicate a strong connection between these differentially expressed TFs.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Further functional enrichment analysis was performed through shinyGO v0.80. 56 genes were applied to ShinyGo v0.80. The analysis of gene ontology in biological processes and molecular function links these transcription factors to RNA metabolic processes, transcription-regulated activity, and DNA binding. These genes significantly contribute to RNA degradation and transcriptional mis regulation in cancer, according to further KEGG pathway analysis (Figure 3).
Figure 3. Functional enrichment analysis of TFs identified through PPI network.
3.4. Cluster formation and Gene set enrichment analysis (GSEA)
The PPI network was transferred to Cytoscape v3.10.2 for further analysis. We performed MCODE cluster analysis and identified two significant clusters (Figure 4). Cluster 1 and 2). CNOT6L and TCF3 were the central nodes in cluster 1 and cluster 2, exhibiting strong connections with each other through edges. TCF3 had a very high degree of centrality in the PPI network (Figure 2). TCF 3's node degree was 22 (Table 1). Gene set enrichment analysis of 27 genes from both clusters using the Panther v16 tool revealed 16 pathway hits. The Wnt signaling pathway significantly dominated the chart (Figure 5). TCF3 was present in this WNT signaling pathway. Based on the degree of presence, GSEA and MCODE cluster analysis suggest that TCF3 could be a potential target for further therapy in HPV-induced HNSCC studies. Wnt/p-catenin signaling has been shown to influence the pathobiology of HPV- and HPV+ HNSCC [31]. To get a deeper knowledge of the Wnt/p-catenin signaling pathway in HPV + HNSCC, further study is required [15]. Research on HPV + HNSCC is not as advanced as that on HPV-HNSCC since there are fewer tumor specimens and pertinent clinical data available [4, 1].
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Figure 4. MCODE analysis via Cytoscape v3.10.2
Table 1. Top ten TFs based on degree of centrality representing node degree
Sr. No. node identifier Node degree
1 TCF4 9606.ENSP00000381382 27
2 TCF3 9606.ENSP00000262965 22
3 CNOT7 9606.ENSP00000355279 18
4 CNOT6L 9606.ENSP00000424896 17
5 CNOT8 9606.ENSP00000430493 17
6 CNOT11 9606.ENSP00000289382 16
7 LEF1 9606.ENSP00000265165 16
8 CNOT10 9606.ENSP00000399862 14
9 CNOT6 9606.ENSP00000481893 13
10 CNOT9 9606.ENSP00000486540 13
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Figure 5. Gene set enrichment analysis of MCODE clusters TFs
3.5. Expression validation of identified new transcription factor
Further expression validation of the TCF3 gene was performed on HNSCC samples through the UALCAN database. The mRNA expression of TCF3 was significantly upregulated in HNSCC patient samples, which suggests the importance of TCF3 in the cancer development and progression in HNSCC [Figure 6].
Expression of TCF3 in HNSC based on Sample types
e 00 o
so
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Normal Primary tumor
(n= 44) (n=520)
TCGA samples
Figure 6. Expression validation of TCF3 in HNSCC patient sample 4. Conclusion
In this study, we employed various in silico approaches to explore transcription factors (TFs) involved in HPV-induced head and neck squamous cell carcinoma (HNSCC). We found a number of TFs, such as TCF4, CNOT7, PAX5, SPDEF, and MITF, that are significantly differentially expressed in HPV-induced HNSCC. mRNA expression analysis via the UALCAN database confirmed the upregulation of TCF4, PAX5, and CNOT7, while SPDEF and MITF were significantly downregulated. More research into the PPI network showed that TCF3 is very important and is highly connected, which suggests that it is a key player in the control mechanisms of HPV-induced HNSCC. The Wnt signaling pathway was found to be closely linked to TCF3 through gene set enrichment analysis. This suggests that TCF3 may play a part in the growth and spread of cancer. There is more and more evidence that Wnt/p-catenin signaling affects HPV-positive HNSCC. TCF3 is a new and important TF that needs to be studied further for its therapeutic potential. Overall, this study helps us learn more about how transcription factors affect HPV-related HNSCC and shows that TCF3 could be a good target for future treatments that aim to make this cancer easier to treat.
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS"
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