Comprehensive Analysis of Clinical Prognosis and Molecular Characteristics of LTB4R2 in Clear Cell Renal Cell Carcinoma

Background Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. LTB4 receptor 2 (LTB4R2, also called BLT2), as a chemokine receptor, has been reported to be upregulated in different tumors and associated with tumor cell proliferation and invasion. However, the correlation between BLT2 expression and its prognostic value in ccRCC remain to be explored. Methods We used the TCGA and GEO databases to evaluate the association between BLT2 expression and clinical outcome of ccRCC. The top 300 genes that most positively correlated with BLT2 were selected for functional enrichment analysis. Correlations between BLT2 expression and tumor immune characteristics in ccRCC were evaluated based on TIMER2.0. BLT2 expression was higher in ccRCC than normal tissues. Kaplan-Meier survival analysis indicated that high BLT2 expression was signicantly correlated with poor overall survival (HR = 1.75, p(cid:0)0.001) and disease-speci ﬁ c survival (HR = 1.60, p(cid:0)0.014) for patients with ccRCC. In addition, the ndings revealed that M1 marker genes had no signicant correlations with BLT2 expression in ccRCC, while moderate correlations were observed between BLT2 expression and M2 marker genes. The marker genes of Tregs and T cell exhaustion positively correlated with BLT2 expression in ccRCC (p(cid:0)0.001).


Introduction
Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer, accounting for more than 80% of all diagnoses [1,2] . The primary de ning feature of ccRCC are consistent metabolic abnormalities featuring highly elevated lipid and glycogen deposition. ccRCC is frequently associated with broadly inactivated von Hippel-Lindau tumor suppressor (pVHL) function whose tumour suppressor activity is dependent on its downregulation of hypoxia pathways [3,4] . ccRCC is a heterogeneous and lethal malignancy with a poor prognosis. Surgical resection is currently the primary treatment for localized ccRCC, however, ccRCC is frequently diagnosed at an advanced stage and prone to recur [5] , and develop unpredictable metastases even with proper treatment [6] . The number of targeted pathways in ccRCC therapy was limited, thus no therapeutic options were available for non-responding patients [7] . The prognosis for patients with metastatic ccRCC is poor of which only 8% to 11.7% survive 5 years [8] . Therefore, it is urgent to explore novel candidate genes that serve as prognostic biomarkers or novel potential targets for ccRCC treatment.
There is evidence that unwanted in ammation could affect cancer development and progression. Therefore a growing body of studies deconstruct the roles and outline underlying mechanisms of in ammation in tumors. Gene expression analyses revealed that ccRCC interacted with a particularly in amed tumor microenvironment compared to other tumor types [9] . It was reported that proin ammatory cytokine IL-1β contributed to a pro-tumorigenic microenvironment in ccRCC [10] and chronic in ammation is associated with a poor prognosis and a limited response to sunitinib for ccRCC [11] . Given these signi cant effects of the in ammatory milieu on the prognosis of patients with ccRCC, interrogation of the predictive power and molecular immune characteristics of in ammatory biomarkers in ccRCC prognosis therapy is warranted [12] .
LTB4 is a bioactive lipid cytokine synthesized from arachidonic acid by the sequential action of 5lipoxygenase (5-LO) and leukotriene A4 hydrolase [13] . LTB4 is an important early mediator of neutrophilic in ammation through high-a nity G-protein coupled receptor BLT1, resulting in chemotaxis of in ammatory cells dominated by neutrophils and thus instigating pathological in ammation in a multitude of chronic diseases including cancer [14,15] . The LTB4 receptor 2 (LTB4R2, also called BLT2) shares 45% homology with BLT1 at the amino acid level and BLT2 was less abundant compared to BLT1 [16] . However, BLT2 is more ubiquitously expressed on the surface of various cell types and transduces signals in response to multiple eicosanoids [17] .
BLT2 is upregulated in a variety of human cancers and several in vitro studies explored the roles of BLT2 receptor signaling in different cancer cells [18] . It has been demonstrated that BLT2 is a pro-tumorigenic mediator during cancer progression and BLT2 blockade signi cantly attenuates Ras-induced transformation, suggesting that BLT2 may serve as a therapeutic target for anti-cancer drug development [19] . BLT2 confers invasiveness in aggressive breast cancer cells by regulating IL-8 [19] . Furthermore, it was found that the expression of BLT2 was considerably elevated in the multidrug-resistant MCF-7/DOX cells where BLT2 played a principal role in mediating paclitaxel resistance [20] . In addition, overexpression of BLT2 has been reported in pancreatic cancer cell lines [16] and BLT2 mediate ERK phosphorylation in pancreatic cancer [19] . The activation of BLT2 signaling by LTB4 confers pancreatic cellular proliferation through the MEK/ERK and PI-3 kinase/Akt pathways [21] . Together, these observations implicated BLT2 as a novel target for aggressive cancer. However, the correlation between BLT2 expression and its prognostic value in ccRCC remain to be explored.
In the present study, for the rst time we used the TCGA (The cancer genome atlas) and GEO (Gene expression omnibus) databases to evaluate the association between BLT2 expression and clinical outcome of ccRCC. In addition, the correlation of BLT2 mRNA levels with the abundance of varied immune cells and BLT2-involved molecular mechanisms were investigated. Our ndings revealed the potential role of BLT2 in ccRCC and shed light on the underlying mechanism where BLT2 shaped the tumor immune microenvironment, providing a new off-the-shelf therapeutic strategy for ghting ccRCC.

Methods
Data Source and BLT2 gene expression analysis The TIMER database, a bioinformatics initiative including 10,897 samples across 32 cancer types from The Cancer Genome Atlas (TCGA), provides information of cancer transcriptome data to evaluate the potential value of a candidate gene as therapeutic target [22] . We analyzed BLT2 differential expression between tumor and normal tissue across different types of cancer using TIMER database with web server (https://cistrome. shinyapps.io/timer/). Furthermore, we investigated the differentially expressed BLT2 mRNA levels between tumor and normal tissues across cancer types using Wilcoxon rank sum test in the integrated datasets combined TCGA with GTEx databases. For the above analysis, RNAseq data in terms of Transcripts per Million (TPM) were collected from UCSC XENA (https://xenabrowser.net/datapages/).
The matched and unmatched differential expression of BLT2 between cancerous and normal tissues for ccRCC was analyzed using paired t-test and Wilcoxon rank sum test, respectively. Gene expression data with clinical information for ccRCC were collected from TCGA (https://portal.gdc.cancer.gov/) in terms of FPKM (Fragment Per Kilobase of transcript per Million mapped read). HTSeq-FPKM data were transformed into TPM for further analyses. Moreover, BLT2 overexpression in kidney cancer on the protein level was identi ed using immunohistochemistry (IHC) staining data in The Human Protein Atlas (HPA) (https://www.proteinatlas.org/).

Analysis on the clinical value of BLT2 expression
The correlation between BLT2 expression level and clinicopathologic characteristics were evaluated using a Wilcoxon rank sum test and logistic regression. The distinguishing e cacy of BLT2 expression between normal tissue and ccRCC tumors was estimated using receiver operating characteristic (ROC) curves.
Samples ranking for BLT2 expression above the median were de ned as BLT2-high, below the median as BLT2-low. Kaplan-Meier curves were generated to present the association of BLT2 expression with overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) for patients with ccRCC from TCGA. Log-rank test was used to establish the statistical signi cance of the survival curves. The hazard ratio (HR) with 95% confidence intervals and log-rank p-value were shown in the plots.
Multivariate Cox regression analysis was performed to evaluate the effect of BLT2 expression on OS in the context of other clinical features (T stage, N stage, M stage, pathologic stage, gender and histological grade). The nomogram was constructed using package "rms & survival" to predict overall survival probability for 1-, 3-, and 5-year based on the six independent prognostic factors. All statistical analyses were performed in R package (V3.6.3).
Analysis of DEGs between BLT2-high and BLT2-low Expression ccRCC Groups Differentially expressed genes (DEGs) were detected between BLT2 high and BLT2 low expression ccRCC groups from TCGA datasets using package DESeq2 [23] . All the DEmRNAs, DElncRNAs and DEmiRNAs were identi ed with the adjusted P value <0.05 (Student's t-test) and |log fold change [FC]| >1.0 de ned as the threshold. All the DEGs were presented in volcano plots and the top 15 DEGs were shown in heat maps.

Functional Enrichment Analyses of BLT2-related Genes in ccRCC
The correlations between BLT2 expression and other genes in ccRCC on the mRNA level were investigated using the R stats package. Based on Spearman correlation coe cient, the top 30 genes of the strongest positive or negative correlations with BLT2 in ccRCC are shown in heatmaps. The Venn diagram was utilized to perform an intersection analysis to pick out BLT2-correlated DEGs and top 300 BLT2coexpressed genes. Pearson's correlation analyses based on GEPIA database (http://gepia.cancerpku.cn/) were performed to describe the correlations between BLT2 expression and the common genes from the intersection analysis. Furthermore, the top 300 genes that most positively correlated with BGN were selected to analyze the functional enrichment of BLT2-interacted genes. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using the EnrichGO function and EnrichKEGG function in the R package"clusterPro ler" [24] .
Correlations between BLT2 expression and tumor immune characteristics in ccRCC TIMER2.0 integrates multiple state-of-the-art algorithms for robust estimation of immune infiltration levels for TCGA across diverse cancer types (http://timer.cistrome.org/), including TIMER, xCell, MCPcounter, CIBERSORT, EPIC and quanTIseq [25] . TIMER2.0 provides estimation of the relationship between tumor gene expression and immune infiltrates, and allows users to perform gene-gene co-expression pattern study across TCGA cancer types. In the present study, we investigated the association between BLT2 expression and tumor-immune in ltrations, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and myeloid dendritic cells using the 'Gene Module'. In addition, we explored the coexpression pattern of BLT2 with M1 and M2 marker genes and Treg marker genes in 'Gene Corr Module'.

Results
The Expression Level of BLT2 in ccRCC To further evaluate the expression level of BLT2 in human cancers, we used the TIMER database to analyze BLT2 mRNA expression levels in normal tissue and tumors across all TCGA tumor types. Speci cally, the result indicated that BLT2 mRNA expression was higher in KIRC (kidney renal clear cell carcinoma) than normal tissues ( Figure 1A). Considering the limited samples of tumor-adjacent normal tissues in TCGA, we investigated the differential expression of BLT2 mRNA levels between tumor and normal tissues in the integrated datasets combined TCGA with GTEx databases. The analysis revealed that BLT2 was signi cantly upregulated in KIRC ( Figure 1B). The differential expression of BLT2 in ccRCC tumor samples compared with matched and unmatched tumor-adjacent normal tissues was analyzed. The results of the analyses for ccRCC were consistent with the results described above ( Figure 1C and Figure 1D). In the end, the differential expression of BLT2 on the protein level between kidney cancer tissues and normal kidney tissues was showed ( Figure 1E). Together, the ndings identi ed the overexpression of BLT2 in ccRCC, indicating the potential role of BLT2 during the development and progression of ccRCC.
The clinical value of BLT2 expression in ccRCC Subsequently, we investigated the clinical signi cance of BLT2 in ccRCC. The association of BLT2 with clinicopathological features was shown in Table 1. According to the above results, BLT2 was upregulated in ccRCC. Thus, a ROC curve was constructed to evaluate the distinguishing efficacy of BLT2 between To investigate the prognosis prediction value of BLT2 in patients with ccRCC, we assessed the associations of BLT2 expression levels with the survival for the patients using TCGA database, including OS, DSS and PFI. Notably, Kaplan-Meier survival analysis indicated that high BLT2 expression was signi cantly correlated with poor OS (HR = 1.75, 95% CI =1.30-2.36, p 0.001) and DSS (HR = 1.60, 95% CI = 1.10-2.34, p 0.014) ( Figure 3A and 3B) for patients with ccRCC, not correlated with PFI (p 0.303) ( Figure  3C).
To quantitatively predict the prognosis of patients with ccRCC, a nomogram was constructed based on multivariate Cox analysis of BLT2 expression and independent clinical risk factors. In the nomogram, a straight line was drawn where a point scale was used to assign points for each predictive variable. The sum of the points from each predictor was assigned to the corresponding location on the total point reference line, where a vertical line was drawn down to the clinical outcome axises. In this way, we nd the probability of survival for patients with ccRCC at 1, 3, and 5 years. According to the nomogram, the total points of a ccRCC male patient (5 points) with high BLT2 risk (83.75 points), T4 staging (37.5 points), N1 staging (40 points), and pathologic stage (52.5 points) were up to 218.75. The probabilities of 1-, 3-, 5-year survival based on the nomogram were about 83.5, 60, and 43.5% ( Figure 3D).
Identification of DEGs between BLT2-high and BLT2-low ccRCC samples Samples of ccRCC from TCGA were sorted into BLT2-high and BLT2-low groups by the median value of BLT2 mRNA expression. In the present study, all the DElncRNAs, DEmiRNAs and DEmRNAs were identi ed with the adjusted P value <0.05 (Student's t-test) and | log fold change [FC]| >1.0 as the threshold. A total of 2369 DElncRNAs were identified between the two cohorts, covering 2161 upregulated genes and 208 downregulated genes. In terms of DEmiRNAs, 230 upregulated genes were identi ed without downregulated genes. As for DEmRNAs, a total of 918 DElncRNAs were identified between the two cohorts, covering 423 upregulated genes and 495 downregulated genes. The distribution of DElncRNAs, DEmiRNAs and DEmRNAs were presented in volcano plots ( Figure 4A-4C). The top 15 DEGs (DElncRNAs, DEmiRNAs and DEmRNAs) based on the relative values of expression fold change between the two cohorts were showed in heat maps ( Figure 4D-4F).

Identi cation of BLT2-interacted genes
To understand relevant molecular functions of BLT2 in ccRCC, the correlations between BLT2 and other genes were evaluated using TCGA data. The top 30 genes that negative and positively correlated with BLT2 expression in ccRCC tumor samples were presented in a heat map, respectively ( Figure 5A-5B). Figure 5C showed the intersection of 423 BLT2 expression-sorted DEmRNAs and the top 300 BLT2interacted genes. In the analysis, we screened out the common members such as LTB4R, CCDC17, and NPIPB4. Subsequently, the correlations between BLT2 and the common genes in the intersection were evaluated based on the GEPIA database, and the correlation coe cients were presented as a radar chart ( Figure 5D).

Functional Enrichment Analysis of BLT2-interacted genes
To further explore the potential function and associated pathways of BLT2, the top 300 BLT2-interacted genes were selected for functional enrichment analyses, including GO and KEGG. The results from the GO enrichment analysis showed that BLT2-interactive genes were not signi cantly involved in pathways associated with biological processes (BPs), cellular compositions (CCs), and molecular functions (MFs) ( Figure 6A-6C). The results of KEGG pathway enrichment analysis of BLT2-interacted genes were mainly involved in serotonergic synapse, FcεRI signaling pathway and arachidonic acid metabolism ( Figure 6D).

Correlation of BLT2 expression with immune characteristics in ccRCC
Tumor-infiltrating lymphocytes (TILs) are an independent predictor of cancer survival and responses to immunotherapy [26,27] . Therefore, we evaluated the correlation of BLT2 expression level with the abundance of diverse immune cells in ccRCC based on TIMER2.0, including CD8+ T cells, CD4+T cells, B cells, macrophages, neutrophils, and DCs. BLT2 expression was not significantly correlated with tumor purity in ccRCC. BLT2 expression had significant correlations with the abundance of CD4+ T cells (r = 0.333, P = 2.09e-13) and neutrophils (r = 0.258, P = 1.81e-08), not with CD8+ T cells, B cells, macrophages, and DCs ( Figure 7A).
In addition, we focused on the correlations between BLT2 and immune marker sets of M1 and M2 macrophages in ccRCC based on TIMER2.0. Notably, we found that M1 marker genes (NOS2 and ROS1) had no signi cant correlations with BLT2 expression in ccRCC, while moderate correlations were observed between BLT2 expression and M2 marker genes (CD163 and MRC1) ( Figure 7B-7C). These findings implied that BLT2 may regulate macrophage M2 polarization in ccRCC, thus contributing to an immunosuppressed microenvironment.
Immunotherapy has gained much attraction due to the widespread use of immune checkpoint inhibitors (ICIs) which block tumor-derived immunosuppressed signals, thus amplifying antitumor immunity [28,29] .
The responses to ICIs partly depends on the tumor immune microenvironment such as the abundance of speci c immune cells. Subsequently, we investigated the correlations between the expression of BLT2 and that of over 40 common immune-related genes in ccRCC using TCGA data. Notably, the results showed that in ccRCC BLT2 expression was associated with nearly 33 immune checkpoint markers among these genes, including CD274, PDCD1, CTLA4, LAG3, CD160, IDO2 and so on ( Figure 8A-8B).

Discussion
The main treatment measures for ccRCC are consisted of surgical resection, radiotherapy and chemotherapy, among which ccRCC developed resistance to radiotherapy or conventional chemotherapy [32] . In RCC, surgical resection remains the mainstay of treatment for local tumors. ICIs through the blockade of PD1 and CTLA4 has become a new rst-line standard option for patients with intermediateor poor-risk metastatic RCC [33] . In 2019, FDA has approved the combined approaches of PD1 inhibitors with anti-angiogenic therapy for advanced RCC [34] . Despite recent advances in ICIs, only a minority of patients with RCC will bene t from these therapies, which underscores the need for elucidation of the mechanisms underlying responses to ICIs and for search of reliable biomarkers to predict treatment responses. Genomic and transcriptomic changes in the tumors were closely associated with tumor immune in ltrations, thus providing potential targets for improving ICIs therapy e cacy.
BLT2, as a chemokine receptor, has been reported to be upregulated in different tumors and associated with tumor cell proliferation and invasion. Considering the limited researches had focused on the role of BLT2 in shaping tumor immune microenvironment, in the present study we performed a comprehensive bioinformatics analysis of BLT2 to unearth its biological functions and tumor-immune interactions in ccRCC. For the rst time, we identi ed the upregulation of BLT2 expression in ccRCC tumors compared with adjacent normal tissues on the mRNA and protein levels. In terms of the prognosis analysis, the results showed that high expression of BLT2 was associated with poorer OS and DSS, indicating the potential value of BLT2 as a prognostic biomarker. Simultaneously, high-expressed BLT2 had excellent distinguishing efficacy between ccRCC tissues and normal kidney tissues based on the high value of AUC (0.937). These ndings strongly suggested that BLT2 may act as an oncogene and prognostic biomarker in ccRCC.
To further elucidate the underlying biological function of BLT2 gene in ccRCC, we selected the top 300 genes positively correlated with BLT2 to perform GO and KEGG functional enrichment analysis. The results showed that BLT2-interacted genes were mainly involved in serotonergic synapse, FcεRI signaling pathway and arachidonic acid metabolism. To our knowledge, no study has yet assessed the association of above pathways with cancer progression and responses to immunotherapy. In previous reports, it was reported that the activation of BLT2 signaling by LTB4 conferred pancreatic cellular proliferation through the MEK/ERK and PI-3 kinase/Akt pathways [21] . During the past 10 years, considerable intratumoural heterogeneity has been reported in ccRCC [35] , which was divided into two distinct subtypes ccA and ccB [36] . As previously reported, speci c subtypes of ccRCC are associated with responses to sunitinib and share characteristic molecular features such as the expression of PDL1 [37] . ccA and ccB tumors differed in their gene expression pro les, and genes associated with hypoxia, angiogenesis, organic acid metabolism and fatty acid metabolism were upregulated in ccA tumors [38] . According to the ndings, BLT2 may be a ccA tumor-related gene. The involved functional pathways and underlying molecular mechanisms of BLT2 during ccRCC progression deserve further investigations for providing new approaches for targeted therapy in ccRCC.
The abundance of diverse immune cells and the amount of immunomodulatory molecules in tumor microenvironment in uences cancer progression and therapeutic treatment responses, especially immunotherapy. In cancer, immunogenicity is the ability to induce adaptive immune responses and ccRCC is considered to be an immunogenic tumour based in part on the clinical bene ts for patients from IL-2 and interferon-α (IFNα) treatments [39] . These clinical observations suggested that promoting endogenous anti-tumour immunity could be an effective therapeutic strategy for ccRCC. In the present study, we determined whether BLT2 expression level could in uence the abundance and function of various immune cells in ccRCC, which in turn, favors tumor progression and promotes an immunosuppressive microenvironment. Our results demonstrate that there was a moderate positive relationships between BLT2 expression level and infiltration level of neutrophils, which is in line with its biological function as the receptor of LTB4, a potent neutrophil chemoattractant. Moreover, our results uncover a crucial role of BLT2 in regulating tumor immunity in ccRCC through correlation analyses of BLT2 expression and the marker genes of immune cells (macrophages and T cells). First, M1 marker genes (NOS2 and ROS1) had no signi cant correlations with BLT2 expression in ccRCC, while moderate correlations were observed between BLT2 expression and M2 marker genes (CD163 and MRC1). The nding revealed the potential regulating role of BLT2 in polarization of tumor-associated macrophages (TAM). In addition, our results revealed that BLT2 had the potential to activate Tregs and promote T cell exhaustion in ccRCC. Increased expression of BLT2 positively correlated with the marker gene expression of Treg and T cell exhaustion (FOXP3, CCR8, STAT5B, TGFB1, PD-1, CTLA4, and LAG3). These findings implied that increased expression of BLT2 may contribute to an immunosuppressed microenvironment in ccRCC.
In summary, the ndings of our study uncovered that increased BLT2 expression correlates with poor prognosis. In addition, BLT2 expression potentially contributes to the regulation of TAMs, T cell exhaustion, and Tregs activation in ccRCC. Therefore, BLT2 may serve as a novel prognostic biomarker and shape tumor immune microenvironment in ccRCC. The involved functional pathways and underlying molecular mechanisms of BLT2 during ccRCC progression deserve further investigations for providing new approaches to drive the development of new immunotherapeutic strategies for ccRCC.