Comprehensive Analysis of the Role of Forkhead Box J3 (FOXJ3) in Human Cancers

: Forkhead box J3 ( FOXJ3 ) is a member of the forkhead box ( Fox ) family. Recently, increasing evidence has revealed the rela‐ tionship between Fox family members and cancer. FOXJ3 is involved in various types of cancer, including lung cancer, tongue squamous carcinoma, and prostate cancer; however, a comprehensive pan-cancer analysis of FOXJ3 remains lacking. Here, we explored the function of FOXJ3 across cancers using online websites and databases including TIMER2.0, SangerBox, UALCAN, GEPIA2.0, cBioPortal, Cancer‐ SEA,STRING, BioGRID and Metascape to analyze the role of FOXJ3 in cancers. Abnormal expression of FOXJ3 was found in various tu‐ mors. The genetic alteration percentage in tumors was determined, and elevated FOXJ3 expression was found to be associated with worse overall survival in brain lower grade glioma(LGG), liver hepatocellular carcinoma (LIHC), sarcoma (SARC) and thyroid carcinoma. El‐ evated FOXJ3 expression was related to worse prognosis with disease-free survival in adrenocortical carcinoma, LGG and LIHC. FOXJ3 expression was related to immune infiltration in several cancers. Enrichment analysis showed that histone modification, the TGF-β signal‐ ing pathway, and chromatin organization were the top three enriched ontology clusters of the top 100 similar genes of FOXJ3 . Our pan-cancer analysis provides comprehensive insights into FOXJ3 from the perspective of bioinformatics in different cancers, where it serves as a potential biomarker for prognosis, especially in LGG and LIHC. FOXJ3 is also correlated with immune infiltrates in certain human tu‐ mors.


Introduction
Cancer is a disease with a high incidence rate and mortality, representing one of the main causes of death globally.In terms of appropriate therapy, various novel approaches have been discovered that have obvious ad-vantages compared to traditional therapies.Targeted therapy is the hot spot of current research because of its remarkable clinical success in many cancers, such as pulmonary adenocarcinoma [1] , hepatocellular carcinoma [2] , and anaplastic thyroid carcinoma [3] .Bioinformatics analysis was used to explore new biomarkers that can be used as indicators for cancer diagnosis and prognosis.
The Fox genes encode a family of transcription factors with 100 amino acids, which are characterized by a DNA binding domain comprising approximately 100 amino acids.According to phylogenetic analysis, Fox proteins can be divided into 19 subfamilies [4] .Fox proteins participate in development, differentiation, proliferation, apoptosis, and other multiple biological processes, while increasing research has revealed that they may also participate in the progression of cancer [5] .FOXJ3, located on chromosome 1 [6] .In non-small-cell lung cancer (NSCLC), FOXJ3 is the target of miR-106b-5p, which regulates NSCLC cell proliferation and invasion [7] .miR-517a-3p promotes growth in lung cancer cells by regulating the expression of FOXJ3 by binding to the FOXJ3 promoter [8] .miR-425-5p may contribute to prostate cancer progression by directly targeting FOXJ3 [9] .The expression level of FOXJ3 has been found to be elevated in patients with tongue squamous carcinoma (TSCC), and FOXJ3 could promote migration in SCC15 cells, suggesting that FOXJ3 may be involved in the progression of TSCC [10] .These studies suggest that FOXJ3 may act as a regulator in cancer.In this study, we used several databases to investigate the role of FOXJ3 and its genetic alteration characteristics, as well as its association with prognosis and immune infiltration of multiple cancers.

TIMER Analysis
TIMER2.0 [11] web server (http://timer.cistrome.org/) is a database which provides comprehensive analysis of immune infiltration levels for the Cancer Genome Atlas (TCGA).We used the "cancer exploration" module to investigate the expression of FOXJ3 in various cancers.Cancer-associated fibroblast (CAFs), endothelial cell and eosinophil were chosen for detailed analysis of the association between FOXJ3 expression and immune infiltrates.Estimations were conducted by the EPIC, MCPCOUNTER, XCELL and TIDE algorithms.

UALCAN Analysis
UALCAN (http://ualcan.path.uab.edu.) [13] is a web resource that can analyze cancer.UALCAN provides the protein expression analysis of FOXJ3 through the data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the International Cancer Proteogenome Consortium datasets.The phosphorylation level of FOXJ3 across tumors was also investigated by UALCAN.

Genetic Alteration Analysis
The characteristics of FOXJ3 genetic alterations were searched through cBioPortal database [14] (https:// www.cbioportal.org/) which is an open-access resource provides multidimensional cancer genomics data sets.The alteration frequency was shown in the "Cancer Types Summary" module and the detailed mutation sites were shown in the "Mutations" module.

GEPIA2 Analysis
GEPIA2 [15] was used to obtain overall survival (OS) and disease-free survival (DFS) map data of FOXJ3 across tumors, according to the expression threshold value of the cutoff-high (50%) and cutoff-low (50%).The correlation between FOXJ3 expression and survival probability of patients was explored.Similar genes of FOXJ3 was obtained by GEPIA 2.0.

Single-Cell Analysis
CancerSEA (http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp) [16] was performed to explore the functional states of cancer cells at a single cell resolution.Single cell analysis was performed to investigate the biological functions of FOXJ3 by exploring CancerSEA.

STRING Analysis
STRING [17] (https://string-db.org/.) was used to perform protein-protein interaction (PPI) network analysis on FOXJ3."FOXJ3" was queried in the "protein name", then in the organism module "Homo sapiens" was selected.The parameters as following: meaning of network edges selected "evidence", "experiments" and "database" was chosen in active interaction sources."Low confidence (0.150)" was selected in the minimum required interaction score, and "no more than 50 interactors" in the 1st shell was chosen.

Protein Expression of FOXJ3
Protein expression of FOXJ3 is shown in Fig. 2. Compared with normal tissues, the protein expression of FOXJ3 was increased in breast cancer, ovarian cancer, clear cell renal cell carcinoma, LUAD, and GBM, while it was decreased in UCEC.

Protein Phosphorylation Analysis of FOXJ3
We used UALCAN to explore the phosphorylation levels of FOXJ3 across cancers.The S223 locus of FOXJ3 showed a higher phosphorylation level in breast cancer (p=1.66E− 04), whereas it was decreased in PAAD (p=3.48E− 13) and GBM (p =1.12E − 02).The T216 locus of FOXJ3 showed reduced phosphorylation in clear cell renal cell carcinoma (ccRCC) (p=5.54E− 12) , whereas it was increased in LUAD (p= 1.10E−32) and hepatocellular carcinoma (p=2.56E− 12) (Fig. 3).These results suggest that FOXJ3 protein phosphorylation at S223 and T216 play a vital role in the occurrence and development of cancer.
FOXJ3 gene modifications, including types, sites, and case numbers, are displayed in Fig. 4(b).The main type FOXJ3 alteration was a missense mutation.

Relationship Between Expression of FOXJ3 and Prognosis
GEPIA2 was used to analyze the relationship between FOXJ3 expression and prognosis.The OS significance map obtained by GEPIA2 showed that higher FOXJ3 expression was correlated with worse OS of LGG, LIHC, SARC, and THCA (Fig. 5(a)); while there was a correlation between highly expressed FOXJ3 and poorer DFS of ACC, LGG and LIHC (Fig. 5(b)).Notably, FOXJ3 was a risk factor for both OS and DFS of LIHC and LGG, which was significantly correlated with poor prognosis.

Correlation Between FOXJ3 Expression and Immune Infiltration Across Cancers
In our study, TIMER 2.0 was used to explore the association between immune infiltration and FOXJ3 expression across cancers.According to EPIC, MCPCOUNTER, XCELL and TIDE algorithms, FOXJ3 expression and the estimated infiltration level of cancer associated fibroblast (CAF) was positive in HNSC, HNSC-HPV-and PAAD(Fig.6(a)).FOXJ3 expression and endothelial cell infiltration was positive in COAD, KIRC and THCA through analysis by EPIC, MCPCOUNTER and XCELL algorithms (Fig. 6(b)).FOXJ3 expression and eosinophil was analyzed by CIBERSORT, CIBERSORT-ABS and XCELL algorithms and showed a positive correlation in STAD (Fig. 6(c)).

Biological Functions of FOXJ3
Single-cell analysis was performed to investigate the biological functions of FOXJ3 using CancerSEA.The biological functions associated with FOXJ3 in cancers was revealed by single-cell analysis.The results indicated that FOXJ3 positively regulated the differentiation, angiogenesis, and inflammation of retinoblastoma cells, whereas it negatively regulated the DNA repair, cell cycle, and DNA damage of retinoblastoma cells (Fig. 7).

Related Gene of FOXJ3
The FOXJ3 protein interactions according to Bio-GRID4.4 are shown in Fig. 8(a).The STRING tool was used to obtain the FOXJ3 correlation protein that were

Discussion
Forkhead transcription factors, which have conserved "forkhead" DNA-binding domain, play important roles in the processes of cellular fate determination, proliferation, apoptosis, and differentiation [20] .Members of the forkhead transcription factors could regulate the expression of countless genes by a DNA-binding mechanism or protein-protein interactions.FOXJ3 may play a vital role during the progress of myofiber identity and muscle regeneration in mice by activating Mef2c [21] .FOXA has been shown to activate ABCC2 and ABCC3 transcription in Sf9 cells [22] , while FOX-2 has been shown to interact with ataxin-1 [23] .In cancer, some members of the FOX family may serve as diagnostic and prognostic biomarkers.High expression of FOXD1 is associated with poor OS and DSF in patients with colorectal cancer [24] .The expression of FOXC1 is highly expressed in triple-negative breast cancer, where it may serve as a therapeutic target or biomarker [25] .FOXE1 expression has also been shown to be higher in papillary thyroid cancer tissues than in adjacent non-tumor tissues, and its expression is correlated with clinical prognosis.FOXE1 is a potential therapeutic target and may be a prognosis biomarker for papillary thyroid cancer [26] .FOXM1 has been demonstrated to be upregulated in endometrial cancer, where its high expression is associated with poor prognosis.Silencing the expression of FOXM1 could suppress the growth of AN3CA and ISHIKAWA cells, and FOXM1 may be a prognostic biomarker for endometrial cancer [27] .In recent years, the importance of the FOX family in human cancers has drawn increasing attention.
We analyzed the molecular features of FOXJ3, including gene expression, protein expression, survival prognosis, genetic alteration, and immune infiltration.We explored the expression of FOXJ3 across cancer types by TIMER2.0.The results revealed that FOXJ3 was upregulated in CHOL, ESCA, HNSC, KIRC, LIHC and STAD; whereas it was decreased in BRCA, COAD, GBM, KICH, KIRP, THCA and UCEC.The results showed differences in FOXJ3 expression between tumor and normal tissues based on TCGA.The Clinical Proteomic Tumor Analysis Consortium data from UALCAN provide several types of tumor expression of FOXJ3.FOXJ3 protein expression is upregulated in breast cancer, ovarian cancer, clear cell renal cell carcinoma, LUAD, and GBM but is decreased in UCEC.These findings urged us to further investigate the expression of FOXJ3 across cancers.
The relationship between prognosis and FOXJ3 expression was investigated by GEPIA2, with the results revealing that FOXJ3 expression was significantly correlated with OS and DFS in LGG and LIHC.TIMER2.0TCGA data analysis showed that FOXJ3 expression was upregulated in LIHC, whereas TCGA and GTEx data analyzed by SangerBox showed that FOXJ3 expression was upregulated in both LGG and LIHC.Taken together, these findings suggest that FOXJ3 may serve as a prognostic biomarker for LGG and LIHC.Immunotherapy, as one of the most appealing approaches for treating tumors, has shown great progress recently.The study found that the FOXJ3 level was related to cancer immunity.We found a significant correlation between FOXJ3 expression and CAFs in HNSC, HNSC-HPV, and PAAD.In COAD, KIRC, and THCA, FOXJ3 expression was related to endothelial cell infiltration, while FOXJ3 levels were positive with the eosinophil in STAD.
We also found that histone modification, the TGF-β signaling pathway, and chromatin organization were the top three GO enriched terms in which FOXJ3 and its similar genes may be involved.Histone modifications are included in epigenetic modification, which plays an important role in cancer.Histone modifications are involved in a series of cellular processes and other important biological processes, while altered epigenetic modification is found in cancer.The promoter methylation of BRCA-1 in malignant breast tumors has also been shown to be higher than that in benign breast tumors, and both the methylation of H4K20 and acetylation of H3K18 are lower in malignant breast tumors than in benign breast (GO) enriched terms of FOXJ3 and its 100 most similar genes tumors [28] .In lung cancer cells, the acetylation of both histones H3 and H4 could regulate the expression of MYO18B, and histone deacetylation surrounding the promoter region is related to MYO18B silencing [29] .The detection of epigenetic modifications may be helpful for cancer therapy.The TGF-β signaling pathway is related to various biological processes, and its dysfunction is a hallmark of many human diseases, including cancer.Retinoid X receptor α has been shown to promote the growth of pancreatic cancer cells and suppress their apoptosis by activating the TGF-β/Smad signaling pathway [30] .Moreover, G-protein coupled receptor 34 promotes glioma carcinogenesis by regulating TGF-β/Smad signaling [31] .Chromatin organization is associated with carcinogenesis by affecting gene expression and regional mutation frequencies.The loss of BRG1 has been found to increase the potential of tumorigenicity in NSCLC cells, while the loss of BRG1 is related to variations in chromatin structure [32] .HP1α, a member of the heterochromatin protein 1 (HP1) family, participates in the proliferation of HepG2 cells, while in HP1α -depleted cells, the level of DNA methylation exhibits a global decrease, and perturbed chromatin organization [33] .All of the top three GOenriched terms of FOXJ3 and its similar genes were related to cancer progression, further highlighting its importance in cancer.

Conclusion
Through pan-cancer analysis of FOXJ3, we observed a statistical association between FOXJ3 expression and prognosis, protein phosphorylation, and immune cell infiltration in cancers.These findings will help to further our understanding of FOXJ3 in tumors from various perspectives.

Fig. 1
Fig. 1 The expression of FOXJ3 across cancer and normal tissues (a) The FOXJ3 gene expression in pan cancer from TCGA data; (b) The expression of FOXJ3 gene across cancer based on TCGA and GTEx data, *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2
Fig. 2 Protein expression of FOXJ3 in cancers

Fig. 5
Fig. 5 Correlation between FOXJ3 expression and prognosis of tumors(a) Correlation of FOXJ3 expression and OS in tumors; (b) Correlation of FOXJ3 expression and DFS in tumors

Fig. 6
Fig. 6 Relationship between FOXJ3 expression and infiltration of CAFs, endothelial cells and eosinophil (a) The correlative between the level of FOXJ3 and cancer associated fibroblast was analyzed EPIC, MCPCOUNTER, XCELL and TIDE algorithms; (b) The correlative between the level of FOXJ3 and endothelial cells was analyzed EPIC, MCPCOUNTER and XCELL algorithms; (c)The correlative between the level of FOXJ3 and eosinophil was analyzed by CIBERSORT, CIBERSORT-ABS and XCELL algorithms

Fig. 8
Fig. 8 Related gene of FOXJ3 (a) Protein interactions of FOXJ3 obtained by BioGIRD; (b) The FOXJ3 correlation protein obtained by the STRING tool; (c) Heat map of gene ontology (GO) enriched terms of FOXJ3 and its 100 most similar genes