Introduction
Cancer immunotherapy aims to activate the anti-tumor immune response to kill cancer cells and has greatly changed the paradigm of cancer treatment [
1]. Nowadays, immune checkpoint inhibitors and chimeric antigen receptor T cell therapies have been commonly applied in clinical management [
1]. However, the main obstacle to successful cancer immunotherapy is that cancer cells can develop the capability to evade immune system attacks, and the majority of cancer patients have innated or acquired immunotherapeutic resistance [
2,
3]. Emerging evidence has identified that the infiltration of different immune cell types within the tumor immune microenvironment seems to broadly overlap with developing resistance to immunotherapies [
4]. Furthermore, the mutations generated from genomic instability in cancer may induce different neoantigens and affect the immune cell infiltration in the tumor immune microenvironment, thus leading to cancer immunoediting and immunotherapy resistance [
5]. Therefore, exploring novel biomarkers that are critically involved in regulating cancer immunogenicity and immune microenvironment can pave the way to understand the development of resistance to immunotherapies, and develop novel strategies to overcome such resistance.
Previous research has revealed that the dysregulation of the Wnt signaling pathway is fundamentally involved in tumorigenesis and cancer progression, and ubiquitination is a key regulator of the Wnt signaling pathways in physiological and pathological processes [
6]. As an E3 ubiquitin ligase, RNF43 (E3 ubiquitin-protein ligase RNF43 or RING-type E3 ubiquitin transferase RNF43), which can be stimulated by the activation of the Wnt pathway, can ubiquitinate Wnt receptor complex components frizzled receptors, thereby suppressing the Wnt signaling pathway in turn [
7]. The tumor suppressive roles of RNF43 in different cancer types have been identified in multiple studies. For instance, it has been found that RNF43 can attenuate the stemness of gastric cancer stem-like cells by inhibiting the Wnt/β-catenin pathway and impairing the chemoresistance in vitro [
8]. Moreover, RNF43 is recurrently mutated in several cancer types, and RNF43 mutations can promote the initiation and development of human malignancies, such as gastrointestinal cancers, hepatocellular carcinoma, and pancreatic adenocarcinoma [
9‐
11]. RNF43 loss-of-function mutations are often detected in MSI-type colorectal cancer, and PORCN inhibitor, which can inhibit ligand-mediated activation of the Wnt/β-catenin cascade, can effectively inhibit the progression of RNF43-mutant cell-derived PDX colorectal cancer in vivo [
12]. By constructing a genetically engineered pancreatic ductal adenocarcinoma mouse model via conditional expression of oncogenic Kras and deletion of the catalytic domain of RNF43, loss of RNF43 promotes pancreatic ductal adenocarcinoma tumorigenesis, and contributes to decreased survival time of mice [
13]. Notably, several clinical trials have confirmed the efficacy of RNF43-based immune therapies in colorectal cancer treatment to date [
14,
15]. These findings present that RNF43 may be critically involved in cancer progression and tumor immune microenvironment remodeling. However, there is a lack of comprehensive pan-cancer analysis of the clinical value and genetic mutations of RNF43, and the exact roles of RNF43 in modulating cancer progression and tumor immune microenvironment are still largely unknown.
In the present study, we performed systemic research on the promising roles of RNF43 in predicting the prognosis and immune phenotypes in human cancers. We explored the clinicopathological significance and genetic mutation characteristics of RNF43, and the crosstalk between RNF43 expression and the abundance of immune cells and immune-related molecules in the tumor immune microenvironment. Besides, we further elucidated the predictive value of RNF43 for the immunotherapy efficacy and drug sensitivity in pan-cancer, thus highlighting the promising role of RNF43 as a clinical biomarker and a novel therapeutic target for cancer patients in clinic settings.
Discussion
In recent years, pan-cancer analysis has been of great concern because it can reflect the panorama of human cancers better [
19]. Previous studies have proved that RNF43 expression was commonly downregulated in cancer patients, and RNF43 can serve as a critical tumor suppressor that inhibits various malignant behaviors of cancer cells, such as proliferation, invasion, stemness, epithelial-mesenchymal transition, and drug resistance [
20]. Besides, RNF43 is frequently mutated in colorectal, endometrial cancers, and pancreatic cancer, which can deeply affect the behavior of cancer cells and induce cancerogenesis and progression [
21,
22]. These results indicated that RNF43 plays important roles in tumorigenesis and development, which exhibit promising clinical significance and potential as the novel therapeutic target in the treatment of human cancers. However, a comprehensive investigation of RNF43 is lacking, and the functions of RNF43 in modulating the tumor immune microenvironment and predicting the immunotherapeutic efficacy need to be answered. Hence, our first and comprehensive pan-cancer research focused on the potential roles of RNF43 in predicting the prognosis, tumor immune phenotypes, and the response to immunotherapies and drug sensitivity.
We first investigated the expression-based panoramic picture of RNF43 in different cancer types. We found that the expression of RNF43 was significantly upregulated in several cancer types, such as COAD, LUAD, and STAD, while was significantly downregulated in some cancer types, including GBM, KIRP, and PCPG. These findings are consistent with the existing studies that have reported that RNF43 was upregulated in LUAD [
23] and COAD [
24]. Previous studies have found that the overexpression of RNF43 can directly ubiquitinate E-cadherin, leading to its degradation and thus potentiating metastasis of lung adenocarcinoma through inducible epithelial-to-mesenchymal transition (EMT) [
23]. By single-cell RNA sequencing (scRNA-seq) analysis of primary gastric adenocarcinoma, Zhang et al. have identified 5 cell populations with distinct expression profiles and identified that RNF43 was expressed in a type of well-differentiated gastric adenocarcinoma, GA-FG-CCP which has been reported to be stained positive for PGA3 and MUC6, with the activation of the Wnt/β-catenin signaling pathway in previous studies [
25]. Meanwhile, RNF43 expression was higher in earlier clinical-stage patients than that in an advanced clinical stage in KIRC, KIRP, and UVM. These findings indicated that RNF43 may help the early diagnosis of cancer patients. Furthermore, through Cox regression analysis and curves, we concluded that the high RNF43 expression predicted better survival outcomes in KIRC, UVM, STAD, ESCA, and KIRP, whereas the high expression of RNF43 indicated poor prognosis in DLBC and LIHC. Generally, the expression pattern and prognostic value of RNF43 were distinguished among different cancer types, and RNF43 can function as a critical prognosis-related factor in several cancers.
Several previous studies have identified RNF43 mutations in diverse cancers, such as colorectal cancers [
21], pancreatic ductal adenocarcinoma [
26], gastric cancer [
27], and intrahepatic cholangiocarcinoma [
28]. Previous studies have confirmed the high frequency of RNF43 mutations in colorectal cancer patients, which may play an activating role in the Wnt pathway in colorectal cancer [
21]. The mutations of RNF43 eventually enhance colorectal cancer tumor growth and promote a high recurrence rate in patients [
29]. In pancreatic cancer cell lines with RNF43 loss-of-function mutation, the frizzled level was no longer inhibited. Therefore, the Wnt/β-catenin signaling in acinar cells was increased, which may provide an additional stimulus to facilitate tumorigenesis and development [
22]. The loss-of-function mutations of RNF43 also dampen the activation of DNA damage response, thus preventing apoptosis in gastric cells, and eventually leading to gastric carcinogenesis [
30]. A recent study has presented that RNF43/ZNRF3 depletion mutation can contribute to live cancer tumorigenesis by modulating the differentiation of hepatocytes and the liver lipid metabolic state [
10]. Prostate cancer is the most common non-cutaneous cancer in men worldwide, and multiple studies have detected RNF43 mutations in prostate cancer [
31,
32]. Copy number loss of 17q22 is correlated with lower RNF43 and SRSF1 expression, enzalutamide resistance, and poor prognosis in prostate cancer [
33]. By the investigation of RNF43 mutations on the cBioPortal database, we discovered that RNF43 possessed a high mutation rate of 4%, and truncating mutation was the most alternation type of RNF43 in pan-cancer. We also observed the co-occurrence of several genes, such as TTN, AGAP10P, ALOX12P1, and EIF2S2P4 was more frequent in the RNF43 alteration group. Most importantly, the alteration of RNF43 was significantly associated with PFS and DSS in cancer patients, and among patients who achieved immunotherapies, RNF43-altered patients had a longer OS time. The data unearthed the fact again that specific gene mutations may predict the prognosis and the responses to immunotherapy of cancer patients [
34]. Our findings provide the characteristics of RNF43 mutations in pan-cancer for further understanding the roles of RNF43 mutations in cancer progression and treatment.
Immune cells are critical constituents of the tumor immune microenvironment, and have a remarkable influence on cancer development and survival [
35,
36]. Immunotherapy is a new cancer treatment strategy through harnessing the immune cells to kill the cancer cells, and the plasticity of immune cell infiltration may contribute to the immunosuppressive microenvironment and immunotherapeutic resistance [
37]. Therefore, investigating the correlation between biomolecules and immune cell infiltration can help predict the clinical outcomes and immunotherapy responses [
38]. In this present study, our findings indicated that the expression of RNF43 was related to several immune cells, such as dendritic cells, T cells CD4, T cells CD8, plasma cells, macrophages, and B cells in various cancer types. RNF43 expression was significantly correlated with the immune-associated cell infiltration levels of macrophages in ACC, KICH, LAML, and THYM, as well as the infiltration levels of T cells CD4+ in ACC, KIRP, PCPG, and TGCT. CD4+ T cells and CD8+ cytotoxic T lymphocytes (CTLs) are critical immune cells that play critical roles in cancer development and immunity. Existing studies suggest that T cells CD4+ can target tumor cells in direct or indirect ways, either by eliminating tumor cells through the complex mechanisms, including the regulation of the tumor immune microenvironment, affecting antigen presentation, co-stimulation, and T cell homing [
39,
40]. Our findings showed that RNF43 expression level was positively correlated with the infiltration of T cells CD4+ in KIRP, which is consistent with the mentioned result that a higher expression level of RNF43 is a protective factor in KIRP. In addition, tumor-associated macrophages are also critically involved in mediating tumor cells intrinsic properties and the tumor microenvironment remodeling, and macrophage polarization plays a prognostic role in multiple cancer types [
41]. Therefore, our findings suggest that RNF43 expression may influence the cancer patients’ prognosis through the interaction with the infiltration of T cell CD4+ and T cell CD8+ and macrophage polarization during cancer progression.
Furthermore, we investigated the association between RNF43 expression and the expression levels of immune-related modulators. We detected that RNF43 expression was positively correlated with CXCL14 in LGG, which is mainly involved in regulating immune cell infiltration and serves as a potential mediator of the immune response [
42]. A recent study has indicated that CXCL14 can recruit and activate CD8+ T cells in vitro and in vivo, and favor the anti-tumor CD8+ T-cell response in LGG [
43]. It has been proved that Th1 and Th2 cells play a significant role in predicting the prognosis of cancer patients [
44]. Our study found that RNF43 expression was negatively related to Th1 in UVM and Th2 in COAD, indicating the potential functions of RNF43 in regulating Th1/2 cells in these cancers. Besides, a positive correlation of RNF43 expression with TNFSF13 and ADORA2A was also observed in our investigation. Previous research has clarified that TNFSF13 is closely associated with cancer occurrence and development, and it may be an effective biomarker for predicting the immunotherapy response [
45]. It has also been confirmed that ADORA2A acts as an intrinsic negative regulator in the procedure of NK cell maturation and the anti-tumor immune response [
46]. Above all, RNF43 may interact with various immune-related biomolecules in different cancers, thereby regulating the tumor immune microenvironment and cancer progression.
The results of the GSEA analysis showed that RNF43 was obviously involved in detection of chemical stimulus, detection of stimulus involved in sensory perception, and sensory perception of chemical stimulus in pan-cancer. Besides, olfactory transduction, drug metabolism cytochrome P450, and neuroactive ligand receptor interaction were identified as the most three common signaling pathways in RNF43 biological function in pan-cancer analysis. Previous studies have confirmed that RNF43 is an E3 ubiquitin ligase, and is a critical negative feedback regulators of the Wnt pathway, which plays significant roles in mediating drug resistance [
47,
48]. Notably, cytochrome P450 can influence drug response through metabolism, affecting the therapeutic efficacy and toxicity of drugs [
49]. These results indicated that RNF43 may be critically involved in mediating cancer immunity, drug metabolism and stimulus sensory in diverse cancers.
Recently, immunotherapies have been widely accepted as an effective treatment approach for cancer patients [
50]. However, many cancer patients have no or limited responses to immunotherapies and may gradually develop immunotherapeutic resistance during treatment. It has been observed that higher TMB level can work as an independent predictor of better responses to immunotherapy in multiple cancers [
51,
52]. In addition, MSI can also serve as an effective positive predictor of cancer immunotherapy [
53]. Previous research has indicated that RNF43 mutations are closely correlated with MSI, TMB, and mismatch repair deficiency (dMMR) phenotype in colon cancer patients, indicative of the promising potential of the combination of immune checkpoint inhibitors with Wnt/β-catenin signaling pathways inhibitors as a reasonable therapeutic strategy in cancer treatment [
54]. Our study confirmed that RNF43 expression is significantly correlated with MSI and TMB in several cancer types, such as CESC, LUSC, COAD, and DLBC. These results display that RNF43 may have the potential as a promising biomarker associated with immunotherapy resistance and predict immunotherapeutic responses in cancer patients. To further confirm the effects of RNF43 on cancer immunotherapies, we analyzed the association between RNF43 expression and the immunotherapy efficacy in these datasets that consist of cancer patients who acquired immunotherapies. Our research revealed that cancer patients with high RNF43 expression had better responses to anti-PD-1/PD-L1 treatment. These findings further identify RNF43 as a critical biomarker for predicting the immunotherapeutic efficacy, and dynamic screening RNF43 expression may be an effective approach to predict the responses of cancer patients to immunotherapies.
Targeted therapies have been developed to inhibit specific molecules that are responsible for facilitating tumor growth. Understanding the molecular signatures and genetic mutations involved in cancer initiation and progression is fundamental for developing effective treatments for human malignancies [
55,
56]. For example, Fibroblast Growth Factor Receptor 3 (FGFR3) mutations have been confirmed as potent oncogenic drivers in several cancers, and FGFR3 inhibitors may be a novel therapeutic strategy for patients with FGFR3-altered cancer [
57]. Previous studies have indicated that RNF43 participates in mediating targeted therapy resistance and chemoresistance in several cancers, which may serve as a novel therapeutic target for overcoming drug resistance in cancer management [
22]. In melanoma, RNF43 has been found to inhibit cell invasion and reverse the resistance to BRAF V600E and MEK inhibitors by stimulating the ubiquitination and proteasomal degradation of VANGL2 and inhibiting ROR2 in vitro and in vivo [
58]. It has been found that RNF43 depletion can impair the sensitivity to γ-radiation and chemotherapy by suppressing the activation of DNA damage response via directly targeting phosphorylated H2A histone family member X (γH2AX) in gastric cancer [
30]. In clear cell renal cell carcinoma, RNF43 can inhibit malignant behavior and reverse pazopanib resistance by inhibiting the YAP signaling by decreasing YAP phosphorylation via p-LATS1/2 [
59]. By employing the CellMine
™ database, we found that RNF43 expression was significantly correlated with the sensitivity of several drugs that have been widely applied in cancer clinical treatment or clinical trials, such as cisplatin, erlotinib, cobimetinib, and everolimus. These results further verify that RNF43 exhibits great potential as a predictive biomarker for the response to anti-cancer agents and a promising therapeutic target for overcoming drug resistance. Besides, dynamic monitoring of RNF43 expression may be an effective approach to evaluate the responses of cancer patients to anti-cancer drugs, thus helping develop personalized treatment strategies for individual cancer patients.
However, our study also has several limitations. Firstly, we only validated the expression pattern of RNF43 with our clinical samples, but the clinicopathological significance of RNF43 is assessed based on online databases, which needs to be further verified in our cohorts in the future. In addition, there is a lack of experimental validation for the predicted roles of RNF43 in mediating the tumor immune microenvironment. Besides, large-scale cohorts are urgently warranted to explore the predictive value of RNF43 in pan-cancer outcomes and immunotherapy efficacy, which is time-consuming.