Identi cation of WWTR1 as an immune in ltration- correlated prognostic biomarker in colon cancer

Xinnong Liu (  xinnliu@yzu.edu.cn ) The A liated Hospital of Yangzhou University, Yangzhou University Jianshun Ge The A liated Hospital of Yangzhou University, Yangzhou University Linlin Tian Department of Oncology Dalian Medical University Jie Zhang The A liated Hospital of Yangzhou University, Yangzhou University Rui wang The A liated Hospital of Yangzhou University, Yangzhou University Haining Li The A liated Hospital of Yangzhou University, Yangzhou University Yanbing Ding The A liated Hospital of Yangzhou University, Yangzhou University Guotao Lu The A liated Hospital of Yangzhou University, Yangzhou University Xudong Yin The A liated Hospital of Yangzhou University, Yangzhou University


Introduction
One of the most prevalent malignant tumors in the gastrointestinal tract is colon cancer. Colon cancer has surpassed gastric cancer to become the rst malignant tumor in the gastrointestinal tract, according to the most recent research report1. Although there has been a recent increase in the overall incidence of early-onset colon cancer, the trends in the incidence of early-onset colon cancer vary widely among various populations2. Surgical resection, chemotherapy, and targeted medication therapy are currently the most common treatments for colon cancer. All major treatment modalities have their limitations, and surgical resection is usually the main treatment with adjuvant chemotherapy or targeted therapy3.
Immunotherapy, an emerging treatment strategy for colon cancer, can be used with other treatment modalities, according to reports, to improve colon cancer patients' outcomes4. As a result, the most critical di culty we have today is nding useful indicators and possible immunotherapeutic targets. Many factors, such as metastasis, T stage, and (TGF)-signaling pathways, have been found as crucial in determining cancer prognosis in research5-7. The TGF-β signaling pathway is an important signaling pathway for cancer cell growth and differentiation. Inhibiting tumor function in the early stages, such as cell cycle arrest and apoptosis, and promoting carcinogenesis in the late stages, such as metastasis and chemoresistance, are both possible outcomes of this pathway8-11.
We examined the expression of WWTR1 in colon cancer and its connection with clinical characteristics using the TCGA database in this work. Following that, we veri ed WWTR1's predictive relevance in colon cancer and identi ed it as a gene linked to immune in ltration in colon cancer via TGF signaling.

Page 3/24
Methods Data and information RNA-seq expression data and clinical information of 473 colon cancer tissues and 41 colon normal tissues were acquired from The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/)12. The identi cation of differentially expressed genes with done with |logFC| > 0. 9. Then a total of 54 genes involved in TGF-β signaling were retrieved by the 'HALLMARK_TGF_BETA_SIGNALING' gene set in the Gene set enrichment analysis(GSEA) (http://www.gsea-msigdb.org/gsea/msigdb/cards/HALLMARK_TGF_BETA_SIGNALING.html)13. After survival analysis of the screened TGF-β differentially expressed signaling genes, we obtained 8 genes including WWTR1 through the 'VennDiagram' R software for further study.

Expression levels and prognostic value of WWTR1 in public datasets
The expression pro les and clinicopathological information, involving 473 colon cancer tissues and 41 normal tissues, were obtained from TCGA in order to validate the expression of WWTR1 in colon cancer. We examined the expression of WWTR1 in the 'Expression DIY' module of the GEPIA data base (http://gepia.cancer-pku.cn/index.html )14. Additionally, other expression data, involved the "GSE12945", "GSE40967", "GSE17537" databases, on WWTR1 and clinicopathologic features of colon cancer patients, were screened from the Gene Expression Omnibus(GEO) database(https://www.ncbi.nlm.nih.gov/geo/)15. Moreover, the protein levels of WWTR1 in colon cancer were assessed by The Human Protein Atlas (HPA) (https://www.proteinatlas.org/)16. Then the association between WWTR1 expression and clinical features was assessed using the 'ggpubr' package and Perl language. Next, based on the information on WWTR1 expression and overall survival data of colon cancer patients obtained from the TCGA, GSE12945, GSE40967, GSE17537, Kaplan-Meier (K-M) curves were plotted through Long-term Outcome and Gene Expression Pro ling Database of pancancers (LOGpc)(http://bioinfo.henu.edu.cn/ DatabaseList.jsp). Immunohistochemical gures were obtained from the Human Protein Atlas (HPA) database. Finally, we performed the univariate and multivariate Cox regression analyses in order to further investigate the prognostic value of WWTR1 in colon cancer.

Immune in ltration analysis of WWTR1
The Tumor Immune Estimation Resource (TIMER)(https://cistrome.shinyapps. io/tim er/), a comprehensive website, was used to analyze the correlation between gene expression and the degree of immune cell in ltration with a wide range of cancers in TCGA17. We evaluated WWTR1 expression in colon cancer and its correlation with various conventional immune cells. Then, we carried out a series of correlation analyses to uncover the effect of WWTR1 on markers of immune cells. Gene Expression Pro ling Interactive Analysis (GEPIA) is an online database that contains RNA-sequencing data on 275 colon cancer samples and 349 normal colon samples from the TCGA and Genotype-Tissue Expression (GTEx) projects. Immunocytic differences were further investigated using the CIBERSORT database for in ltration of colon cancer patients, and the results were plotted using the "ggplot2" package in R software for barplot, corHeatmap. Based on the expression level of WWTR1, the 473 samples obtained from the TCGA-COAD cohort were categorized into two groups: high expression and low expression. The CIBERSORT database was used to investigate the in ltration between the two groups, and the results were plotted using the "ggpubr" package in R software.
Identi cation of DCGs of WWTR1 in colon cancer A number of 19828 genes are acquired from LinkedOmics database (http://www. Linked omics.org/), and 12598 genes were screened based on the criteria of |correlation coe cient|>0, FDR<0.05. Then, we used the R language 'limma' program to lter the DCGS with |logFC|>2, and the rst 50 genes positively and negatively associated with WWTR1 were plotted respectively by the 'heatmap' R package.

GO and KEGG enrichment analyses
We used DCGS to perform GO and KEGG by 'clusterpro ler' R software. corrected p-value <0.05 were found to be statistically relevant.
Statistical analysis R software and the Perl language (https://www.perl.org/) were used to perform all statistical analyses. Expression data were normalized by log2 transformation. the survival analysis was completed by logrank test. The correlation of WWTR1 with several biomarkers of immune in ltration was calculated using Spearman's correlation. The relevance of in ltrating immune cells was determined by the following guide for the value of partial cor: 0.00-0.

Results
WWTR1 is an oncogene that has been discovered in many malignancies. Following a comprehensive examination of numerous databases, it was discovered that the expression of WWTR1 is closely linked to the expression of immune in ltration and immune cell markers in colon cancer. As a result, it could be a potential promising biomarker for colon cancer immunotherapy.

Filtration of differentially expressed genes in colon cancer
We obtained differentially expressed genes |LOG>0.9| from the TCGA database (Figure 1(a)). After identi cation transformation, the list of 14198 colon cancer genes was decreased to 3359 genes with o cial gene symbols. 8 intersecting genes were identi ed (intersecting genes between DEGs in the TCGA and TGF-β signaling) (Figure 1(b)).Then we used heatmaps to show these 8 differentially expressed genes ( Figure 1(c)). We discovered that WWTR1 had not been published in bioinformatics in colon cancer, so we decided to investigate it more.

Expression levels of WWTR1 in colon cancer
To verify whether WWTR1 expression affects patients with colon cancer, we analyzed the expression data of TCGA. The boxplot (Figure 2(a)) reveals that the level of WWTR1 expression was lower in colon cancer tissues compared to normal tissues (p<0.05). Moreover, GEPIA is commonly used to examine the expression of WWTR1 in colon cancer versus healthy individuals (Figure 2(b)).
WWTR1 expression in tumor tissues is lower than in normal individuals, according to the ndings. The HPA database was used to assess the expression of WWTR1 protein in colon cancer tissues to better understand how it is expressed. The results showed that, when compared to normal tissues with antibody CAB068248, colon cancer tissues displayed lower amounts of WWTR1 protein (Figure 2

Relationship between WWTR1 and prognosis of colon cancer
We evaluated the association between WWTR1 expression levels and different clinicopathological features of colon cancer to better understand the relationship between WWTR1 and colon cancer prognosis. WWTR1 expression was higher in relatively advanced tumor tissues in the clinical stage ( Figure 3(a)). Similarly, WWTR1 expression was positively correlated with pathological T-stage ( Figure  3(b)) and lymphatic metastasis (Figure 3(c)). Then, on diverse clinical parameters of colon cancer, we ran univariate Cox regression analysis and multifactor Cox regression analysis to con rm the link between WWTR1 and prognosis in colon cancer.  (Figure 3(d)).
WWTR1 expression level was also found to be an independent predictive factor in patients with colon cancer in a multifactorial Cox regression analysis (Figure 3(e)). In conclusion, WWTR1 expression was highly correlated with the prognosis of colon cancer patients with the above clinicopathological features.

Relationship between WWTR1 expression and immune in ltration in colon cancer patients
Immune in ltration has been linked to the development and progression of most tumors, according with literature. In addition, a relationship has been discovered in the case of colon cancer. The association between six immune in ltrates and WWTR1 expression levels was explored to identify a link between immune in ltration and colon cancer.  (Figure 4(a)). We revealed that WWTR1 expression was closely associated with immune in ltration in colon cancer based on these ndings.
In order to obtain a better understanding of the interaction between WWTR1 and immune in ltration, the TIMER database was used to validate the association between WWTR1 and immune marker. We identi ed a positive correlation between WWTR1 and the majority of immune cell markers after adjusting for purity (Table 1) were observed to be tightly linked to WWTR1 expression in colon cancer (Figure 4(b-j)). In addition, the GEPIA database was used to con rm the associations between immune cell markers and WWTR1 expression levels in colon cancer. The results of GEPIA con rmed the accuracy of the previous data ( Table 2).
The CIBERSORT database was then used to investigate the immune in ltration of patients in the TCGA-COAD cohort. Our ndings revealed that the proportion of TICs in patients varies signi cantly, with some having a higher proportion of Macrophages and others having a higher proportion of Neutrophils than others ( Figure 5(a)). We analyzed the relationship between TICs in this dataset and noticed that many  (Figure 5(b)). The speci c linkage needs to be investigated further. Finally, we used the CIBERSORT database to investigate the differences between the WWTR1 high and low expression groups in the tumor microenvironment. There were substantial differences between the two groups in B cells naive, B cells memory, T cells CD4 memory resting, T cells CD4 memory activated, NK cells activated, Monocytes, Macrophages M0, Macrophages M1, Macrophages M2, Dendritic cells activated, and Neutrophils ( Figure  5(c)).
In conclusion, the expression of WWTR1 showed a signi cant correlation with immune in ltration. The intrinsic mechanism needs further study and discussion.

Functional enrichment analysis of WWTR1 DCGs in colon cancer
To explore the mechanisms underlying the role of WWTR1 in colon cancer, 19828 DCGs were identi ed through the LinkedOmics database, which contains 11,579 positively associated genes and 8,249 negatively associated genes (Figure 6(a)). The heat map shows 50 DCGs positively and negatively correlated with WWTR1 ( Figure 6(b-c)). These associated genes were selected for GO and KEGG enrichment analysis and used to explore the important biological pathways and functions between them. Ameboidal-type cell migration, camera-type eye development, eye development, cell-substrate adhesion, visual system development, and sensory system development are among the pathways in which GO enrichment analysis identi es the main involvement of DCGs in biological processes, as shown in the following images. The most enriched categories in the cellular component enrichment analysis were focal adhesion and cell-substrate junction. At the molecular functional level, acting binding was thought to be the most enriched category (Figure 6(d)). DCGs regulates chemical carcinogenesis, reactive oxygen species, nonalcoholic fatty liver disease, diabetic cardiomyopathy, Alzheimer disease, oxidative phosphorylation, thermogenesis, Parkinson disease, Prion disease, Huntington disease, Amyotrophic lateral sclerosis, cardiac muscle contraction, and neurodegeneration pathways, according to KEGG enrichment analysis (Figure 6(e)). The strong association of WWTR1 with non-alcoholic fatty liver disease has been demonstrated in the present study by KEEG enrichment analysis18-20. as novel treatment modalities for colon cancer. Transforming growth factor (TGF)-β signaling pathway is a class of cytokines with multiple biological activities, which are involved in regulating cell proliferation, differentiation, growth, and other life activities26. On the one hand, in pre-malignant cells, TGF-beta signaling mainly acts as a tumor suppressor, and on the other hand, in advanced malignant cells, TGFbeta signaling promotes tumor invasion and metastasis27 , 28. It has been recently documented that TGFβ signaling has a signi cant role in regulating cancer proliferation, migration, and differentiation in a range of cancers, particularly breast, liver, gastric, and bladder cancers29-32. TGF-beta signaling is strongly associated with cancer immunotherapy, and relevant studies have reported that TGF-beta could act as a tumor promoter through several mechanisms, including immunosuppression33, and it also modi es its function via regulation by tumor-in ltrating immune cells (TICs)34. Therefore, de ning the connection between TGF-β signaling and immune in ltration has signi cant and original implications for the immunotherapy of various cancers. WWTR1 is a Hippo pathway transcriptional cofactor that regulates the TGF-β signaling pathway through a mechanism that is independent of Smad3, p38, and MRTF-mediated, but not MRTF-translocated35. Hence, it is essential to investigate the prognostic value of WWTR1 and to assess the e cacy of immunotherapy in colon cancer.

Discussion
In this study, we demonstrated the prognostic value of WWTR1 in colon cancer through data from TCGA, GEO, and TIMER databases. The expression of WWTR1 was much lower in colon cancer tissues compared to normal tissues. In addition, patients with colon cancer with higher WWTR1 expression had lower OS. We found that age, clinical stage, T stage, N stage, M stage, and WWTR1 were all of considerable value in predicting the prognosis of colon cancer in univariate Cox regression analysis. WWTR1 was shown to be an independent prognostic factor in the colon cancer cohort based on multivariate Cox regression analysis.
The colon is an organ containing a wide range of immune cells, and its immune microenvironment is regulated by a number of factors. Crohn's disease (CD), Ulcerative colitis, and various other chronic in ammatory diseases of the colon are considered to be major drivers of colon cancer development36. It has been reported that in ltration of relevant immune cells such as CD8 + T is tightly associated with cancer development37. We found an intimate association of WWTR1 with immune cell in ltration, especially in Dendritic cells, M2, Monocyte, Neutrophils, T cell exhaustion, and TAM (tumor-associated macrophages) cells. Most of the immune cells in the immune microenvironment are used to be activated to ght cancer cells, but not in all of the immune cells38. Immune evasion of tumors is an essential feature of cancer evolution39-41. By inhibiting immunological checkpoints and promoting immune evasion, tumor cells could exploit associated immune cells to boost cancer cell growth and destruction42-44. Consequently, immunotherapy is an essential and well-tolerated treatment option for patients with advanced colon cancer. In our research, we observed that the expression level of WWTR1 in colon cancer was positively correlated with markers of T cell exhaustion, such as PD-1, CTLA4, LAG3, TIM-3. These proteins of markers are essential components of suppressive immunological checkpoints, which help tumor cells evade immune monitoring. This suggests that WWTR1 is critical in the T cell exhaustion process. Furthermore, most immune cell markers were shown to be strongly linked to WWTR1, showing that WWTR1 is involved in the regulation and recruitment of immune in ltrating cells in colon cancer. We used GO and KEGG analysis to learn more about WWTR1's role in colon cancer. Meanwhile, Chemical carcinogenesis reactive oxygen species is identi ed as a key route by KEGG analysis. This is the rst study to use bioinformatics to examine the role of WWTR1 in the immunological microenvironment of colon cancer in relation to immune in ltration function. Despite this, our work has limitations. We evaluated the function of WWTR1 and the predictive level with related colon cancer patients using various databases, but more clinical trials are needed to con rm our ndings.

Conclusion
In conclusion, high levels of WWTR1 expression are associated to a poor prognosis in colon cancer. Further research revealed a relationship between WWTR1 expression and immune in ltration in colon cancer, indicating WWTR1 could be a potential target for colon cancer biotherapy.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. GraphicalAbstract1.jpeg