TRPV1 Is a Potential Tumor Suppressor for Its Negative Association with Tumor Proliferation and Positive Association with Antitumor Immune Responses in Pan-Cancer

Background Although numerous studies have shown that the expression and activation of TRPV1 have an important role in cancer development, a comprehensive exploration of associations between TRPV1 expression and tumor proliferation, microenvironment, and clinical outcomes in pan-cancer remains insufficient. Methods From The Cancer Genome Atlas (TCGA) program, we downloaded multiomics data of ten cancer cohorts and investigated the correlations between TRPV1 expression and immune signatures' enrichment, stromal content, genomic features, oncogenic signaling, and clinical features in these cancer cohorts and pan-cancer. Results Elevated expression of TRPV1 correlated with better clinical outcomes in pan-cancer and diverse cancer types. In multiple cancer types, TRPV1 expression correlated negatively with the expression of tumor proliferation marker genes (MKI67 and RACGAP1), proliferation scores, cell cycle scores, stemness scores, epithelial-mesenchymal transition scores, oncogenic pathways' enrichment, tumor immunosuppressive signals, intratumor heterogeneity, homologous recombination deficiency, tumor mutation burden, and stromal content. Moreover, TRPV1 expression was downregulated in late-stage versus early-stage tumors. In breast cancer, bladder cancer, and low-grade glioma, TRPV1 expression was more inferior in invasive than in noninvasive subtypes. Pathway analysis showed that the enrichment of cancer-associated pathways correlated inversely with TRPV1 expression levels. Conclusion TRPV1 upregulation correlates with decreased tumor proliferation, tumor driver gene expression, genomic instability, and tumor immunosuppressive signals in various cancers. Our results provide new understanding of the role of TRPV1 in both cancer biology and clinical practice.


Introduction
Ion channels are important in modulating a variety of biological processes, such as intracellular calcium (Ca 2+ ) functioning in regulating cell motility, cell cycle, and apoptosis [1] and potassium (K + ) channels modifying cell proliferation, cell migration, invasion, and apoptosis [2]. Ion channels are potential pharmacological targets for cancer treatment for their essential roles in tumor development, proliferation, and invasion [3,4]. The nonselective cation channel TRPV1 (transient receptor potential cation channel subfamily V member 1) plays significant roles in cancer onset and advancement [5,6]. TRPV1 was first discovered in 1997 and was defined as a pain and heat receptor [7]. It can be activated by a variety of factors, including capsaicin [7], lipopolysaccharides [8], vanilloids [9], heat [9], protons [9], phosphoinositide 4,5-bisphosphate [10], vitamin D [11], and Toll-like receptor 4 [12]. TRPV1 is also activated in a variety of cancers, such as tongue squamous cell cancer [13], pancreatic cancer [14], breast cancer [15], and prostate cancer [16]. Nevertheless, some studies have revealed a tumor suppressor role of TRPV1 in various cancers. For example, TRPV1 activation can reduce glioma expansion and prolong survival of glioma patients [17]. TRPV1 can inhibit the development of gastric cancer, and its downregulation is associated with poor survival in gastric cancer [18]. TRPV1 can downregulate EGFR levels by inducing EGFR ubiquitination and degradation, thereby inhibiting the EGFR/MAPK signaling in pancreatic cancer cells [19]. In clear cell renal cell carcinoma (ccRCC), TRPV1 expression is associated with immune infiltration and inhibits the progression of ccRCC [20]. The expression and activation of TRPV1 can activate protein tyrosine phosphatase 1B (PTP1B) to inhibit EGFR-associated intestinal tumorigenesis [21]. TRPV1 overexpression can activate p53 and induce apoptosis to inhibit tumor proliferation in melanoma [22].
Although these prior studies have revealed the diverse roles of TRPV1 in cancer, a systematic investigation of its associations with various clinical and molecular features in pan-cancer remains insufficient. To fill this research gap, we explored correlations of TRPV1 expression with immune signatures' enrichment, progression phenotypes, and clinical outcomes in ten cancer types from The Cancer Genome Atlas (TCGA) program. This study is aimed at furnishing novel insights into the role of TRPV1 in both cancer biology and clinical practice.

Calculation of the Enrichment Scores of Immune-Related
Signatures, Phenotypes, and Cancer-Related Pathways. We calculated the enrichment score of an immune-related signature, tumor phenotype, or cancer-related pathway in a tumor sample by the ssGSEA algorithm [24] based on the expression profiles of their marker or pathway gene sets. We presented these gene sets in Supplementary Table S2.

Quantification of Tumor Purity and Stromal
Components. We quantified tumor purity and stromal components for each tumor with the ESTIMATE algorithm [25] with the input of gene expression profiles.

Quantification of Tumor Mutation Burden (TMB), Homologous Recombination Deficiency (HRD), and
Intratumor Heterogeneity (ITH). A tumor's TMB was defined as its total number of somatic mutations. The HRD scores of TCGA cancers were obtained from a previous publication [26]. We used the DEPTH algorithm [27] to evaluate ITH.

Survival Analysis.
We compared the survival rates between different subgroups of cancer patients. A total of four endpoints were compared, including overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), and progression-free interval (PFI). We plotted the Kaplan−Meier survival curves to exhibit the differences in survival rates. The log-rank test was used to assess the significance of survival differences. We utilized the R package "survival" to perform survival analyses.
2.6. Identification of an Interaction Network of TRPV1. The interaction network of TRPV1 was identified by BioGRID [27] with the default parameters in the tool.

Pathway
Analysis. Based on TRPV1 expression levels, we defined the high-TRPV1-expression-level (upper third) and low-TRPV1-expression-level (bottom third) subgroups in pan-cancer. We first identified differentially expressed genes with a threshold of fold change ðFCÞ > 1:5 and the false discovery rate ðFDRÞ < 0:05. We then selected the 500 upregulated genes in high-TRPV1-expression-level tumors and the 500 upregulated genes in low-TRPV1-expression-level tumors with the smallest FDRs. We input both sets of genes into the GSEA web tool [26] to identify the pathways significantly associated with them, respectively, with a threshold of FDR < 0:05.

Statistical Analysis.
We performed two-class comparisons using Student's t-test for normally distributed data. Pearson's or Spearman's correlation test was used to evaluate the correlation between two variables. In analyzing correlations between TRPV1 expression levels and the enrichment scores of tumor immunosuppressive signatures, tumor stemness, epithelial-mesenchymal transition (EMT), and cancerrelated pathways, tumor purity, and stromal scores, we used Spearman's correlation test and reported correlation coefficients (ρ). We used Pearson's correlation test, to analyze correlations between TRPV1 expression levels and the expression levels of a single gene and the ratios of immune signatures, and reported correlation coefficients (r). We employed the Benjamini and Hochberg method [28] to calculate the FDR to correct p values in multiple tests.
We also explored the correlations of TRPV1 expression levels with the enrichment of five cancer-related pathways (p53, mTOR, Wnt, MAPK, and ErbB signaling) in cancer. Our analysis showed that the correlations tended to be negative (p < 0:05) (Figure 1(f)).
In summary, our results suggest that TRPV1 downregulation is associated with unfavorable tumor progression phenotypes in cancer.
Furthermore, we compared TRPV1 expression levels among subtypes of several common cancer types, including BLCA, BRCA, and LGG. In BLCA, TRPV1 expression levels were markedly higher in papillary than in nonpapillary tumors (p < 0:05) (Figure 2(c)). Again, it suggests a positive association between TRPV1 expression and clinical outcomes in BLCA since the papillary subtype has a better prognosis than the nonpapillary subtype (Figure 2(c)). In BRCA, we compared TRPV1 expression levels among breast cancer subtypes defined by the PAM50 assay [35]. Notably, TRPV1    (Figure 2(d)). These results again indicate that TRPV1 is a positive prognostic factor in breast cancer since basal-like and HER2-enriched subtypes have a worse prognosis than luminal A&B subtypes [36]. In LGG, TRPV1 expression was remarkably upregulated in IDH-mutated versus IDH-wild-type tumors (p = 0:0002) (Figure 2(e)). Because the IDH-mutated subtype has a better OS prognosis compared with the IDHwild-type subtype (Figure 2(e)), it suggests a positive relationship between TRPV1 expression and clinical outcomes in LGG.
Taken together, our analysis suggests a significant positive association between TRPV1 expression and clinical outcomes in cancer.

TRPV1 Expression Is Positively Correlated with Tumor
Purity and Negatively Correlated with Stromal Content and Genomic Instability. Our analysis revealed that TRPV1 expression had a marked positive correlation with tumor purity in pan-cancer and seven cancer types (p < 0:01), while it showed a significant negative correlation with stromal content in pan-cancer and nine cancer types (p < 0:05) (Figure 3(a)).

TRPV1 Expression Correlated Inversely with
Immunosuppressive Signatures in Cancer. Our analysis revealed significant negative correlations between TRPV1 expression levels and the enrichment scores of numerous immunosuppressive signatures in pan-cancer and in most cancer types (p < 0:05) (Figure 4(a)). These immunosuppressive signatures included myeloid-derived suppressor cells (MDSCs), T cell exhaustion, PD-L1, anti-inflammatory cytokines, M2 macrophages, TGF-β, and CD4+ regulatory T cells. However, TRPV1 expression levels had a positive correlation with the ratios of immunostimulatory over immunosuppressive signatures (CD8+ T cell/PD-L1) in pan-cancer and in five cancer types (p < 0:05) (Figure 4(b)). These results suggest that TRPV1 may play a role in promoting the antitumor immune response.

Identification of TRPV1-Associated Network and
Pathway. Network analysis by BioGRID [41] uncovered the interaction relationship between TRPV1 and eight proteins/genes ( Figure 5(a)). The eight interactors of TRPV1 included CBL, EGFR, CALM1, HNRNPH1, AKAP5, SYT9, OS9, and SNAPIN. Among those interactors, CBL as a proto-oncogene plays an important role in cancer, whose mutations can enhance the PI3K/AKT signaling [42]. EGFR is a tumor driver factor whose overexpression may promote tumor cell proliferation [43]. TRPV1 promotes the ubiquitination of EGFR by the ubiquitin ligase Cbl, leading to the degradation of EGFR through the lysosomal pathway [44]. It indicates that TRPV1 expression is positively associated with tumor prognosis through multiple mechanisms.  Journal of Oncology GSEA [26] identified 37 and 29 KEGG pathways significantly associated with the top 500 genes upregulated in the high-and low-TRPV1-expression subgroups, respectively. Notably, there were numerous oncogenic pathways upregulated in the low-TRPV1-expression subgroup, including pathways in cancer, small cell lung cancer, Jak-STAT signaling, p53 signaling, and calcium signaling ( Figure 5(b)). It supports the previous results of the negative association between TRPV1 expression and the enrichment of oncogenic pathways.

Discussion
For the first time, we comprehensively analyzed the correlations of TRPV1 expression levels with tumor proliferation, stemness, EMT, genomic instability, ITH, immunity, and various clinical features in pan-cancer and diverse cancer types. We found that TRPV1 expression levels correlated negatively with the expression levels of the tumor proliferation index marker MKI67 and RACGAP1, proliferation score, cell cycle score, tumor stemness, EMT, TMB, HRD, ITH, stromal content, tumor immunosuppressive signatures, and oncogenic pathways' enrichment. As a result, TRPV1 downregulation was associated with unfavorable clinical outcomes in cancer.
Our analysis supports a significant negative correlation between TRPV1 expression and tumor progression in pancancer and multiple individual cancer types. It is in line with previous studies showing that TRPV1 expression correlates negatively with the expression of cancer proliferation and metastasis-related markers (Ki67 and VEGFR) [18] and that the activation of TRPV1 can significantly inhibit cancer cell growth by inducing apoptosis and necrosis [15]. In fact, a previous study has demonstrated that the use of TRPV1 agonists can promote tumor cell proliferation [45], supporting our findings.
The tumor suppressive effect of TRPV1 may be achieved through multiple pathways. First, TRPV1 can regulate the flow of calcium ions, thereby reducing the proliferation of tumor cells. Previous studies have shown that TRPV1 can inhibit the development of cancer by regulating the Ca 2+ / CaMKKβ/AMPK pathway [18]. Second, TRPV1 expression can downregulate the EGFR/MAPK signaling [19], thereby inhibiting the EGFR-induced epithelial cell proliferation [21]. Finally, TRPV1 expression may promote antitumor immunity. It supports a previous study showing that the tumor suppression role of TRPV1 is associated with its positive correlation with antitumor immune infiltration in ccRCC [20]. Our analysis also demonstrates the positive association between TRPV1 expression and antitumor immune responses, as evidenced by TRPV1 expression having a negative association with tumor immunosuppressive signaling and a positive association with the ratio of immunostimulatory to immunosuppressive signatures.
Notably, the p53 pathway is recognized as a tumor suppressor pathway, while our prior study has shown that the tumors highly expressing TP53 have worse prognosis than the tumors lowly expressing TP53 [35]. A potential explanation for this could be that tumor progression stimulates the upregulation of the p53 pathway. Therefore, the negative correlation between the expression levels of TRPV1 and the enrichment scores of the p53 pathway could be attributed to the inhibitory effect of TRPV1 on cancer progression that reduces the stimulatory upregulation of the p53 pathway.
Notably, TRPV1 as a pain and heat receptor is often considered a target for pain relief [46]. However, our study suggests that TRPV1 is likely to act as a tumor suppressor ( Figure 6). Thus, treating pain with a high dose or longterm usage of TRPV1 inhibitors should be cautious for their potential adverse oncogenic effects. This research has several limitations. First, our analyses are merely based on bioinformatics analysis but lack of experimental validation. Second, this research used the mRNA expression data to perform all analyses, which may not fully recapitulate the protein expression profiles of TRPV1 in cancer.

Data Availability
The datasets of RNA-seq, somatic mutations, and clinical information for ten TCGA cancer cohorts can be downloaded from the GDC database (https://portal.gdc.cancer .gov/). The cancer subtype-related data can be downloaded from TCGA with the R function "TCGAquery_subtype" in the R package "TCGAbiolinks".