An Integrative Pan-Cancer Analysis Revealing ETS1 as an Oncogenic Immune Protein in Tumor Microenvironment

Background Previous research revealed that ETS Proto-Oncogene 1, Transcription Factor (ETS1) might be useful in cancer immunotherapy. However, the processes underlying its therapeutic potential have yet to be thoroughly investigated. The goal of this work was to look into the association between ETS1 expression and immunity and depict its prognostic landscape in pan-cancer. Methods The TCGA provides raw data on 33 different types of cancer. GEO gave GSE67501, GSE78220, and IMvigor210. In addition, we looked at ETS1's genetic changes, expression patterns, and survival studies. The researchers investigated the links between ETS1 and the TME and its linkage to immunological processes/elements and the major histocompatibility complex better to understand the importance of ETS1 in cancer immunotherapy. Meanwhile, three distinct immunotherapeutic cohorts were employed to study the relationship between ETS1 and immunotherapeutic response. Finally, PPI analysis and functional gene enrichment were performed using GSEA.


Introduction
. According to several studies, ETS1 can impede cell differentiation in various settings and boost its cancer-promoting function by keeping cells immature and proliferating. As a result, ETS1 may help convert drug prospects into therapeutic anticancer strategies [ 6] . The link between ETS1 function and carcinogenesis, on the other hand, is yet uncertain, which might be a hot study topic.

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The world is today confronted with a signi cant public health issue: cancer incidence and mortality remain high. Cancer is a problematic sickness because tumors interact with the immune system[ 7-][ a8a].. The tumor microenvironment (TME) comprises many different types of cells, the bulk of which are invading immune cells. Many studies in recent years have found that TME plays an essential role in the occurrence, development, metastasis, and treatment resistance of human cancers[ 9-][ a10a].
Understanding the underlying pathways through which TME interacts with immune cells, on the other hand, remains a challenge. Immunotherapy is gaining popularity in many cancer types as research advances, and various checkpoint blocking drugs are already being used in clinical cancer treatment [ 11] .
As a result, immunophenotypes of tumor-immune interactions and validation of novel immuneassociated tumor therapy targets are critical. However, research on ETS1's role in generalized cancer is sparse.
This work aimed to look at the ETS1 expression landscape in 33 different cancers and the underlying impacts on immunological TME. This researcher concentrated on important immune modulators and dynamic immunological indicators such as tumor mutational burden (TMB) and microsatellite instability (MSI). Furthermore, the link between ETS1 expression and immune checkpoint blockade medication was studied. Considering all of these considerations, ETS1 was shown to be a sign of immunological in ltration and a poor prognosis and potential and promising therapeutic target for cancer.

Genomic Alterations of ETS1 in Cancers
The online cancer genomics database cBioPortal (http://www.cbioportal.org/)[ 13] was used to determine the change in ETS1 status in cancer patients. ETS1's genomic alterations included copy number ampli cation, profound loss, an unknown missense mutation, and mRNA overexpression.

Examining ETS1 Expression in Cancers
The TCGA provides data on ETS1 expression differences between tumor and matched normal tissue.
After extracting the ETS1 data with the limma package, we used log2 (TPM+1) transformed expression data to illustrate the difference analysis ndings in parameter selection.

The relationship between ETS1 and survival and clinical stage
Page 4/24 ETS1's impact on cancer survival was determined using overall survival (OS), disease-speci c survival (DSS), disease-free survival (DFS), and progression-free survival (PFS). Using log-rank and univariate Cox proportional hazards models, P-values and hazard ratios (HR) with 95 % con dence intervals (CI) for Kaplan-Meier curves were obtained. For multivariate Cox regression, clinical variables such as age, gender, grade, and stage were considered. To study the association between ETS1 expression and clinical stage, the stage survival plots module was employed.
2.5 ETS1 Expression and the Role of Immune Cell In ltration and the TME We looked into and calculated the relationship between ETS1 expression and gene markers of tumorin ltrating immune cells (TIICs) in malignant tumors and discovered some immune cell in ltration. After assessing the TME with the ESTIMATE, the stromalscore, immunescore, and ESTIMATE scores were computed. Tumor purity was shown to be negatively related to the previously reported ratings. The limma was then utilized to evaluate the variances in TME in several cancer samples according to the immunological, ESTIMATE, and stroma scores. To measure tumor cell purity, corresponding scatterplots were constructed. The higher the ImmuneScore or StromalScore predicted score, the higher the immune or stromal ratio. It refers to the more signi cant the ratio of the relevant component in TME, the higher the associated score. ESTIMATEScore was the sum of the two, showing the percentage of both components in TME.
TMB is gaining traction as a unique and reliable biomarker for predicting immunotherapy response. TMB is calculated as the total number of mutations per DNA megabase, with recognized changes classi ed as nucleotide insertions, base substitutions, or deletions [ 14] . MSI is a molecular tumor feature characterized by the spontaneous loss or gain of nucleotides from short tandem repeat DNA sequences [ 15] . The fmsb package was used to investigate the relationship between TMB and MSI.

Gene Set Enrichment Analysis
To analyze the biological signaling pathway, gene set enrichment analysis (GSEA) was performed in the high and low-expression groups compared to the median level of ETS1 expression. The top four words from the KEGG and GO analyses were shown. KEGG pathways with signi cant enrichment ndings were found using NES (Net enrichment score), gene ratio, and P-value. Enrichment was evaluated signi cantly for gene sets with |NES|>1, NOM p<0.05, and FDR q<0.05[ 16] .

Network of protein-protein interactions
We constructed an ETS1 protein-protein interaction (PPI) network using the GeneMANIA web tool (http://www.genemania.org) [ 17] . Bioinformatic methodologies distinguish the network integration technique: site prediction, physiological interaction, co-expression, co-localization, gene enrichment analysis, and genetic interaction.

Immunotherapeutic Response Analysis
As previously stated, this study contained and analyzed three primary independent immunotherapeutic cohorts (GSE78220, GSE67501, and IMvigor210). Immunotherapeutic therapies produced four outcomes in general: complete response (CR), partial response (PR), progressing disease (PD), and stable illness (SD). In this study, patients who achieved CR or PR were de ned as responders compared to nonresponders who had symptoms of SD or PD. The Wilcoxon test was then used to compare ETS1 expression levels between responder and non-responder groups.

Results
The study's goal is to look at the link between ETS1 and immunology and determine its predictive value as a possible biomarker in human cancers. We will investigate the genetic anomalies, expression patterns, and survival assessments of ETS1 expression in pan-cancer patients, as well as its relationship with tumor immune in ltration. Finally, we investigated the connection between PPI and gene functional enrichment.

Clinical Landscape of ETS1 Expression
ETS1 was expressed differentially in senior GBM patients, as indicated in Figure.

ETS1's Prognostic Value in Cancer
The forest plots ( Figure.3) revealed a positive relationship between ETS1 expression and OS in KIRP and MESO but a negative relationship in BLCA, KIRC, and THYM. There was a clear positive connection between ETS1 and DFS in KIRP and PAAD. ETS1 expression was a risk factor in KIRP and MESO but a protective factor in BLCA, KIRC, READ, and THYM in DSS. The PFS forest plot also con rmed ETS1 expression's protective impact in CHOL, KIRC, and THCA and its role as a risk factor in KIRP. However, the plot allowed the researchers to identify other cancers where ETS1 expression was deemed a risk factor, such as KIRP and MESO. Although it was not directly related to clinical features, ETS1 expression was strongly associated with survival in various cancers (BLCA, KIRC, and THYM).   The connection between ETS1 expression and immune modulators also was investigated. Figure 6 presents the investigation of 24 various types of immune inhibitors. ETS1 expression was positively related to CSF1R in KICH, KDR in PCPG, and TIGIT in PAAD but negatively connected with PVRL2 in ACC. Correlation investigations of 45 immune stimulators ( Figure.7) found that ETS1 expression was positively associated with IL2RA in CHOL, TMEM173 in ACC, and ICOS in PAAD but negatively associated with TNFRSF25 in READ. Furthermore, as shown in Figure.

Immunotherapeutic markers and response of ETS1
The connection with ETS1 and two novel dynamic markers of immune checkpoint blockage (TMB and MSI) was studied further. Figure. Figure.9 shows no signi cant difference in ETS1 expression between the responder and non-responder groups in the three different cohorts. In the studied cohorts, patients with lower ETS1 expression were shown to be more susceptible to immunotherapy. Figure 9. The correlation between ETS1 and both immunotherapeutic markers and response.

PPI Network of ETS1 in Cancers and GSEA
Following that, we built an ETS1 PPI network to investigate the fundamental mechanisms that ETS1 plays in cancer carcinogenesis ( Figure.10). As seen in the gure, ETS1 made rm physical contact with SP100, required for cancer metastasis. SP100 (SP100 Nuclear Antigen) is a gene that codes for proteins.
Functions as a transcriptional coactivator of ETS1 and ETS2 are involved in various physiological processes such as cell proliferation, differentiation, and apoptosis [ 18] . It may also serve as a corepressor of ETS1, preventing it from binding to DNA under certain situations [ 19] . ETS1 regulation may play a function in angiogenesis by modulating endothelial cell motility and invasion [ 20] . ETS1 was also expected to be related to SP1 and CAMK2G. The functional enrichment of high ETS1 expression versus low ETS1 expression was then determined using GSEA ( Figure.11). According to the KEGG enrichment term, high expression of ETS1 mainly was connected with metabolic-related activities such as cytosolic dna sensing pathway, metabolism of xenobiotics by cytochrome p450, olfactory transduction, retinol metabolism, and steroid hormone biosynthesis. According to the GO enrichment term, high expression of ETS1 is mainly linked with detection of chemical stimulus, detection of stimulus involved in sensory perception, epidermis development, sensory perception of chemical stimulus, and skin development.

Discussion
Contrary to conventional perception, ETS1 is a toxicant-related transcription factor that plays an essential role in the immunological TME and may have immunotherapeutic potential. Thus, more ETS1-related research involving the TME, immune cells, immunological modulators, and the immunotherapeutic response is necessary. This research aimed to understand more about the pathways that may link ETS1 to immune-related factors in pan-cancer. First, the association between ETS1 and clinical variables was investigated, and no signi cant differences in age, gender, or tumor stage were discovered in most cancer types, con rming previous ndings. ETS1 expression, on the other hand, has only a marginal prognostic value in a variety of cancers, including gastric cancer (GC) [ 21] . Similarly, previous research has identi ed ETS1 as a proto-oncogene in various cancers, including hepatocellular carcinoma The link between ETS1 and immune-cell in ltration was examined further to evaluate ETS1's potential use. ETS1 and M2 and M0 macrophages were discovered to have a signi cant connection in THYM. Moreover, some previous study indicates that ETS1 impacts tumor growth and immune responses inside TME-associated macrophages [ 27] . ETS1 may be involved in macrophage polarization and the subsequent activation of an immunosuppressive response [ 28] . KDR would have the most signi cant adverse connection with ETS1 in PCPG. Except for UVM, most immune stimulants and MHC molecules showed a positive relationship with ETS1; this exciting nding may lead to the identi cation of a novel regulatory mechanism in UVM immunotherapy. Enrichment analysis further indicated that high ETS1 expression was mainly associated with metabolic-related activities. Dysregulation of cytokine and adipocytokine expression in adipose tissue characterizes metabolic in ammation [ 29] . ETS1 functions as a transcription factor. Directly regulates the expression of cytokine and chemokine genes in a range of cellular settings [ 30] . This protein may in uence lymphoid cell development, survival, and proliferation and cause in ammatory molecules to clump together, making it easier for macrophages to enter [ 31] . Increased ETS1 expression, according to the current ndings, may impact innate immunity in some cancers by activating metabolic-related mechanisms.
Furthermore, in this study, two immunotherapeutic biomarkers (TMB and MSI) were found to have a signi cant connection with AhR in various cancers. The TMB provides a decent estimate of tumor-neoantigen burden; in general, the more somatic mutations a tumor has, the more likely it is to create neoantigens [ 32] . On the other hand, MSI is described as a robust mutator phenotype caused by poor DNA mismatch repair and is a possible prognostic indicator for immunotherapy.[ 33] ETS1 was adversely associated with TMB and MSI in CHOL, DLBC, HNSC, KIRP, LIHC, PAAD, and THYM; however, it was positively associated with both biomarkers in BLCA, BRCA, CESC, KIRC, LAML, LGG, LUAD, LUSC, PCPG, SARC, TGCT, THCA, and UCEC. This showed that ETS1 might have an indirect effect on the immunotherapeutic response of the previous malignancies. The link between ETS1 and the immunotherapeutic response was investigated, but no signi cant differences were found in any cohorts studied. As a result, our ndings shed insight on ETS1's latent involvement in tumor immunology and its potential application as a cancer biomarker. Meanwhile, only three relevant cohorts were studied in our investigation of immunotherapeutic response, making it di cult to de ne the precise immunotherapeutic response of ETS1. More relevant immunotherapeutic populations should be studied in the future.
This research sheds new information on the role of ETS1 in cancer immunotherapy. It suggests a link between ETS1 and important immunological indicators, which might help us comprehend the possible connections between ETS1 and the immune system. Despite the fact that it is offered for theoretical underpinnings and analysis ideas, our study has limitations. Using the TCGA datasets, we rst generate a validated ETS1 prediction signature. We are frequently unable to obtain adequate external information from several publicly available sources. Second, while the bioinformatics analysis provided us with some valuable insights into the role of ETS1 in cancer, these ndings are preliminary. Biological research, either in vitro or in vivo, is needed to con rm our ndings and improve therapeutic utility. Finally, posttranslational modi cations are essential in altering intracellular signaling and regulatory factor function[ 34-][ a35a]; unfortunately, post-translational modi cation information for LCN2 is not available in these databases. However, to thoroughly understand the facts presented above, we shall do an extra investigation.

Conclusion
In conclusion, our research highlighted the close link and prognostic signi cance of ETS1 expression in many human cancers. ETS1 might be a viable new cancer therapeutic target. Furthermore, our ndings provide insight on ETS1's important role in carcinogenesis and metastasis, as well as a potential mechanism through which ETS1 expression might in uence tumor immunology and metabolic activity. Our ndings are expected to aid in the discovery of the link between ETS1 expression and immunological TME, allowing us to learn more about their potential role in cancer genesis and development and, as a result, offer immuno-based anti-cancer therapy. Patients who granted informed consent to use their data have been uploaded to the public-accessible TCGA databases. At their leisure, users can get and publish relevant articles depending on the needed data. Our study has no ethical problems or con icts of interest because it is based on open-source data.

Ethics approval and consent to participation
This manuscript is not a clinical trial, hence the ethics approval and consent to participation is not applicable.

Consent for publication
All authors have read and approved this manuscript to be considered for publication.

Competing interests
The authors declare no competing nancial interests.