A comprehensive characterization of the transcriptome in enzalutamide resistance prostate cancer.

BACKGROUND: Prostate cancer (PCa) contributes to more than 1.2 million newly diagnosed cases and more than 350,000 deaths each year, making it the second most common malignancy and the fifth leading cause of cancer-related deaths in men worldwide. Treatment of PCa is further complicated by drug resistance to enzalutamide. The present study comprehensively details the genomic characteristics of enzalutamide-resistant PCa.
METHODS: The determination of enzalutamide-related genes in GSE163240 and GSE136129 was conducted by differential expression analysis, gene set enrichment analysis (GSEA) suggested that these genes were highly correlated with immune-related pathways. Subsequently, network analysis including module analysis and degree analysis and univariable cox analysis were conducted, which led to the identification of both hub genes [contactin 2 (CNTN2) and frizzled class receptor 2 (FZD2)].
RESULTS: GSEA suggested that these genes were highly correlated with immune-related pathways. Subsequently, network analysis, including module analysis and degree analysis, and univariable Cox analysis resulted in the identification of two hub genes, CNTN2 and FZD2, which were further validated using the Gene Expression Omnibus (GEO) and Molecular Signatures Database (MSigDB). GSEA and CIBERSORT indicated that both hub genes were highly correlated with immune-related functions in PCa.
CONCLUSIONS: In conclusion, this study comprehensively described the transcriptome landscape of enzalutamide-resistant PCa and identified two hub genes, CNTN2 and FZD2, that play an important role in enzalutamide-mediated immune infiltration in PCa. 2021 Annals of Translational Medicine. All rights reserved.

Introduction

In 2018, there were 1,276,106 newly diagnosed cases of prostate cancer (PCa) and 358,989 associated deaths, making it the second common malignancy and the fifth leading cause of cancer-related deaths in men worldwide (1). The progression and proliferation of PCa is facilitated by the androgen-signaling axis (2,3) and thus, the main treatment for PCa involves the suppression of androgens (4,5) via anti-androgen deprivation therapy (ADT) (6). However, ADT inevitable leads to the emergence of castration-resistant prostate cancer (CRPC) (7,8).

Enzalutamide, which interferes with the androgen receptor (AR), and abiraterone, which inhibits androgen synthesis, are commonly used for the treatment of PCa. There is increasing evidence to suggest that enzalutamide may have clinical significance in the overall survival (OS) of CRPC patients with metastasis [hazard ratio (HR) =0.63; 95% confidence interval (CI): 0.53 to 0.75; P<0.001] (9) and in metastasis-free survival in patients without metastasis (HR =0.29; 95% CI: 0.24 to 0.35; P<0.001) (10). Indeed, enzalutamide has been approved for both chemotherapy-naïve and chemotherapy-exposed CRPC patients (6,9,11). However, almost all patients of CRPC eventually develops resistance to enzalutamide through complex mechanisms (8,11). Therefore, further understanding the mechanisms by which PCa develops resistance to enzalutamide is urgently required to develop novel treatments for patients with PCa.

Immune “cold tumor” mainly refers to the less infiltration of local immune cells in the tumor and the low expression of PD-L1. If there are ways to improve the tumor immune microenvironment, such as strengthening the role of immune cells, increasing immune cell infiltration, and enhancing immune antigen presentation. Further, it can increase the response rate of PCa to immunotherapy. However, enzalutamide resistance appears to be involved in immune cell infiltration. The current study examined the genomic characteristics of enzalutamide-resistant PCa. Differential gene expression analyses and protein-protein interaction (PPI) network analyses were performed to determine key enzalutamide-related genes. Univariate Cox analysis was used to identify the hub genes, contactin 2 (CNTN2) and frizzled class receptor 2 (FZD2), with prognostic implications. Further validation using the Gene Expression Omnibus (GEO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (12) database suggested that both hub genes were correlated with the AR and enzalutamide. Gene set enrichment analysis (GSEA) demonstrated that both hub genes were positively correlated to immune-related pathways. CIBERSORT (13) was used to confirm the association between the hub genes and immune cell infiltration levels. These hub genes, CNTN2 and FZD2, may be potential biomarkers or immunotherapeutic drug targets for the management of enzalutamide resistance in PCa patients.

We present the following article in accordance with the STREGA reporting checklist (available at https://dx.doi.org/10.21037/atm-21-6191).

Methods

Data source and preprocessing

The RNA-sequencing data of enzalutamide-resistant or sensitive PCa cells was derived from the GSE163240 (14) and GSE136129 (15) datasets in the GEO (16) database. The GSE147976 (17) and GSE150807 (18,19) datasets with gene expression data from enzalutamide-naïve or -exposed cells were also downloaded for validation.

Patient survival-related data were obtained from The Cancer Genome Atlas (TCGA) (20), the International Cancer Genome Consortium (ICGC) (21), cBioPortal (22,23), and the GEO database. Based on the criteria that datasets must have at least 20 samples with complete follow-up data and corresponding gene expression data to implement prognostic analysis, a total of 6 datasets were finally included. The gene expression data including fragments per kilobase of transcript per million (FPKM) values and clinical data from the prostate adenocarcinoma (PRAD) cohort in TCGA (n=493) (20) were downloaded using the R package TCGA biolinks (24,25). Survival data and gene expression data were collated from the PRAD-CA (n=144) and PRAD-FR (n=25) datasets in the ICGC database (21), the MCTP (n=35) (26) and SU2C (n=71) (27) datasets in the cBioPortal database (22,23), and the GSE116918 (n=248) (28) dataset in the GEO.

The raw counts data of PCa and normal control samples were downloaded from TCGA and Genotype-Tissue Expression (GTEx) (29) data cohorts. In addition, RNA expression data related to PCa or control samples were also derived from the GSE80609, GSE56829, and GSE111177 datasets (30). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Enzalutamide-related genes

The DESeq2 software was used to identify the differentially expressed genes between enzalutamide-resistant cells and enzalutamide-sensitive cells with a threshold of |log2FoldChange| >1 and adjusted P value <0.01. Volcano plots generated by ggplot2 (31) were used to depict these differentially expressed genes. A Venn diagram was used to intersect the differentially expressed genes from the GSE163240 and GSE136129 datasets using the ggvenn package (32). To explore the biological process (BP) involved, GSEA was used to implement functional enrichment analysis with reference to Gene Ontology (GO) (33,34), KEGG, and Molecular Signatures Database (MSigDB) (35) using the clusterProfiler (36) package.

PPI network analysis

The STRING database was used to explore the interaction among enzalutamide-related genes. A PPI network was constructed and analyzed using Cytoscape version 3.8.2 (37). Network module analysis and degree analysis were conducted to identify the key nodes using Minimal Common Oncology Data Elements (MCODE) (38) and cytoHubba (39), respectively. The key genes were derived by overlapping nodes in modules from MCODE and nodes with degree >3 from cytoHubba.

Cox analysis and determination of hub genes

Hub genes were defined as genes with prognostic implications in at least two datasets among six cohorts, including TCGA-PRAD, ICGC PRAD-CA and PRAD-FR, cBioPortal MCTP and SU2C, and GSE116918. Univariate Cox analysis was used to determine the prognostic implications with reference to OS, progression-free interval (PFI), or disease-free survival (DFS) (40). OS is the survival time from the date of diagnosis to the date of death by any cause. PFI is defined as the interval from diagnosis to the first emergence of a new tumor event (40). DFS is defined as the interval from a patient’s disease-free status after their first diagnosis and therapy to the first emergence of a new tumor event (40). A P value <0.05 is considered statistically significant.

Exploration of the biological functions of the hub genes

GSEA was applied to examine the BPs in the GO database, and the functional pathways in the KEGG database. The threshold recommended by GSEA (41) includes a minimum gene set size of 15, a maximum gene set size of 500, and an adjusted P value <0.25. The association with hallmark gene sets (35) was also determined. In addition, PCa-special scores [AR scores and neuroendocrine prostate cancer (NEPC) scores] (27,42,43) were analyzed based on the SU2C dataset. The Wilcoxon rank sum and Kruskal-Wallis rank sum tests were used to explore the expression levels of hub genes at various stages of PCa and in normal control samples.

Validation of the association with between the hub genes and enzalutamide

A differential gene expression analysis was conducted in enzalutamide-exposed PCa cells and enzalutamide-naïve PCa cells from the GSE147976 and GSE150807 datasets. Moreover, the enzalutamide-targeted functional pathways were examined using the KEGG database and GSEA was conducted to determine whether the hub genes were enriched in enzalutamide-targeted functional pathways with a threshold of P value <0.05.

Exploration of the association between the PCa hub genes and immune cell infiltration

The immune subtypes related to PCa and the genes that code immunomodulators and chemokines were identified using studies by Thorsson et al. (44) and Charoentong et al. (45), respectively. A total of 4 immune subtypes, including C1 (wound healing), C2 [interferon-gamma (IFNg) dominant], C3 (inflammatory), and C4 (lymphocyte depleted), were identified. The Kruskal-Wallis rank sum test was used to assess the association between immune subtypes and the hub genes. The Spearman correlation coefficient was calculated to explore the association between hub genes and immunomodulators or chemokines. CIBERSORT in the R program was used to enumerate the absolute infiltration levels of 22 types of immune cells in PCa. The differential infiltration immune cells between PCa samples and normal samples were determined by the Wilcoxon rank sum test. Spearman correlation coefficient analysis was performed to investigate the association between immune infiltration levels and hub genes. A P value <0.05 was considered statistically significant.

Statistical analysis

All statistical tests were based on a significant P value <0.05. The Benjamini-Hochberg method was used to adjust the P value. An adjusted P value <0.05 was applied when analyses involved multiple comparisons. The R program (version 4.0.2) (46) was used for all statistical analyses.

A schematic diagram depicting the processes involved in this study is shown in .

Figure 1

The schematic diagram depicting the study process. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction; MCODE, Minimal Common Oncology Data Elements; TCGA, The Cancer Genome Atlas; ICGC, International Cancer Genome Consortium; GEO, Gene Expression Omnibus; GSEA, gene set enrichment analysis; CNTN2, contactin 2; FZD2, frizzled class receptor 2; PCa, prostate cancer.

Results

Identification of the enzalutamide-related genes

Analysis of the GSE163240 dataset comparing enzalutamide-resistant cells and enzalutamide-sensitive cells revealed a total of 686 up-regulated genes and 253 down-regulated genes (). In the GSE136129 dataset, there were 1,246 up-regulated genes and 1,329 down-regulated genes (). A Venn diagram revealed 329 overlapping enzalutamide-related genes identified in both datasets (). A chord diagram constructed using the circlize package (47) showed that the enzalutamide-related genes were significantly negatively correlated with androgen response, L6/JAK/STAT3 signaling, and tumor necrosis factor (TNF)α signaling via nuclear factor (NF) κB (). GO analysis suggested that the differentially expressed enzalutamide-related genes identified in the GSE163240 and GSE136129 datasets were enriched in BPs including cell-cell adhesion via plasma-membrane adhesion molecules, synapse assembly and organization, and granulocyte and neutrophil activation associated with the immune response (). KEGG analysis demonstrated that the differentially expressed enzalutamide-related were mostly associated with pathways including adrenergic signaling in cardiomyocytes, GABAergic synapse and relaxin signaling pathway, cytokine-cytokine receptor interaction, neutrophil extracellular trap formation, and viral protein interaction with cytokines and cytokine receptors ().

Figure 2

Identification of the differentially expressed enzalutamide-related genes. (A) A volcano plot showing the differentially expressed genes between enzalutamide-resistant cells and enzalutamide-sensitive cells. The hub genes, CNTN2 and FZD2, are labeled and circled in yellow. (B) A Venn diagram showing the differentially expressed genes in the GSE163240 and GSE136129 datasets. (C) A chord diagram showing the enrichment results of GSEA with reference to hallmark gene sets in MSigDB. (D) GSEA was applied to characterize the biological functions of the enzalutamide-related genes with reference to BP in GO and functional pathways in KEGG. CNTN2, contactin 2; FZD2, frizzled class receptor 2; GSEA, gene set enrichment analysis; MSigDB, Molecular Signatures Database; BP, biological process; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Development of the PPI network

A total of 329 enzalutamide-related genes were imported into the STRING database. A network including 321 nodes and 355 edges was constructed (), with an enrichment P value of 1.0e-16, suggesting that this network has significantly more interactions than expected. Furthermore, module analysis using MCODE extracted 7 clusters, including 53 genes. The largest module is depicted in . In addition, cytoHubba analysis suggested that there are 70 genes with a degree >3. By overlapping these genes, 37 key genes were identified for further analysis.

Figure 3

The development of the PPI network to analyze enzalutamide-related genes. PPI, protein-protein interaction.

Figure 4

Module analysis of the protein-protein network extracted 7 clusters. The largest modules are depicted.

Determination of hub genes

To obtain genes with prognostic significance to PCa, univariate Cox analysis was applied in the TCGA, PRAD-CA, PRAD-FR, MCTP, SU2C, and GSE116918 datasets.

The TCGA dataset identified 9 genes that were significantly correlated with PFI, including CNTN2 (HR =1.140–5.225; P<0.05), EFNA3 (HR =1.058–1.602; P<0.05), ROR2 (HR =0.686–0.983; P<0.05), FBLN1 (HR =0.665–0.927; P<0.05), NMU (HR =1.408–2.421; P<0.05), MAML2 (HR =0.615–0.950; P<0.05), ROR1 (HR =0.611–0.997; P<0.05), ORM2 (HR =0.842–1.000; P<0.05), and CYP2E1 (HR =1.162–2.567; P<0.05). Analysis of the PRAD-CA dataset revealed that the genes ADRB1 (HR =1.308–11.696; P=0.015) and FZD2 (HR =0.078–0.743; P=0.013) were significantly correlated with OS. The MCTP and SU2C datasets, respectively, demonstrated that FZD2 (HR =1.264–2.603; P=<0.05) and GHRHR (HR =0.335–0.874; P<0.05) were correlated with OS. The GSE116918 dataset further suggested that both CNTN2 (HR =1.053–3.529; P<0.05) and LOX (HR =1.553–5.727; P<0.05) significantly impacted DFS in PCa patients. However, examination of the PRAD-FR dataset revealed that there was no significant correlation among any of the genes (). Therefore, the CNTN2 and FZD2 genes were considered hub genes due to their significant implication in PCa prognosis in at least two datasets.

Figure 5

Determination of the hub genes. Network analysis by MCODE and cytoHubba suggested 37 key nodes. Univariable Cox analysis was used to determine the hub genes with prognostic significance in at least two datasets among the TCGA-PRAD, ICGC PRAD-CA and PRAD-FR, cBioPortal MCTP and SU2C, and GSE116918 cohorts. A P value <0.05 was regarded as the cutoff value. *P<0.05; **P<0.01; ***P<0.001. MCODE, Minimal Common Oncology Data Elements; TCGA, The Cancer Genome Atlas; PRAD, prostate adenocarcinoma; ICGC, International Cancer Genome Consortium; PFI, progression-free interval; DFS, disease-free survival; OS, overall survival.

Biological function of the hub genes

GSEA was used to depict the biological function of the hub genes. As shown in , CNTN2 was strongly correlated with BPs including ATP synthesis coupled proton transport, energy coupled proton transport, down electrochemical gradient, and mitochondrial ATP synthesis coupled proton transport. FZD2 was associated with BPs involving phagocytosis, engulfment, regulation of antigen processing and presentation, and renal filtration. KEGG analysis suggested that both CNTN2 and FZD2 were associated with the intestinal immune network for immunoglobulin (Ig)A production (). Hallmark gene sets revealed that both hub genes were highly associated with immune-related pathways (such as allograft rejection, inflammatory response, IFNg response, IFNα response, IL6/JAK/STAT3 signaling, and complement and coagulation) and proliferation-related pathways (such as mitotic spindle and the p53 pathway) (). Analysis of the SU2C dataset reveal that both hub genes were negatively associated with AR scores [correlation coefficient (R) =−0.172 and P<0.05 for CNTN2; and R=−0.327 and P<0.05 for FZD2; ]. Furthermore, FZD2 was positively correlated to NEPC scores (R=0.176 and P<0.05; ).

Figure 6

Exploration of the biological functions and the relationships with clinical traits of the hub genes. (A) GSEA was applied to analyze the BPs and functional pathways (KEGG) for both hub genes. (B) A heatmap showing the results of GSEA with reference to hallmark gene sets in MSigDB. Grey color indicates insignificance. (C) Spearman correlation analysis was used to explore the association between hub genes and AR score and NEPC score. GSEA, gene set enrichment analysis; BP, biological process; KEGG, Kyoto Encyclopedia of Genes and Genomes; MSigDB, Molecular Signatures Database; AR, androgen receptor; NEPC, neuroendocrine prostate cancer; CNTN2, contactin 2; FZD2, frizzled class receptor 2; BP, biological process.

The importance of the hub genes in PCa

Both CNTN2 and FZD2 hub genes were significantly down-regulated in PCa samples compared to normal healthy tissues obtained from the TCGA and GTEx data cohorts (P<0.05 for CNTN2; P<0.05 for FZD2; ). The GSE80609 dataset also verified the significant differential expression of the hub genes in both low-grade PCa (P=0.00034 and 1.9e-05 for CNTN2 and FZD2, respectively) and advanced PCa (P=8.2e-05 and 0.00033 for CNTN2 and FZD2, respectively) compared to benign prostatic hyperplasia (BPH). In addition, CNTN2 was also significantly differentially expressed in CRPC (P=0.0096; ). However, in the GSE80609 dataset, there was no significant difference in the expression of either hub genes across different stages of PCa (). The MCTP dataset comparing metastatic CRPC (mCRPC) with localized PCa showed statistical difference in the expression of FZD2 (P=0.029; ). Furthermore, the correlation between hub gene expression and castration was examined. The expression of FZD2 was up-regulated after castration (P=8.32e-03; ) and ADT (P=6.91e-03; ). However, in the GSE56829 dataset, there was no significant difference in CNTN2 expression after castration (P=0.894; ), while the GSE111177 dataset indicated that CNTN2 expression was significantly associated with ADT (P=3.59e-04; ).

Figure 7

The importance to hub genes to PCa. The differential expression of hub genes was assessed using (A) the TCGA and GTEx datasets; (B) the GSE80609 dataset; (C) the MCTP dataset (D) the GSE56829 dataset; and (E) the GSE111177 dataset. PCa, prostate cancer; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression; CNTN2, contactin 2; FZD2, frizzled class receptor 2; mCRPC, metastatic castration-resistant prostate cancer; ADT, androgen deprivation therapy.

Validation of the association between the hub genes and enzalutamide

Differential expression analysis in the GSE147976 and GSE150807 datasets showed that FZD2 was significant up-regulated in the enzalutamide-exposed group compared to the enzalutamide-naïve group, while there were no statistical differences in CNTN2 expression (). We got both gene sets targeted by enzalutamide after searching KEGG database (hsa05215: PCa and hsa05200: pathways in cancer) (48). GSEA suggested that both hub genes were enriched in PCa pathways (P<0.05 both for CNTN2 and FZD2) and FZD2 was also related to cancer pathways (P=1.22e-04; ).

Figure 8

Validation of the association between enzalutamide and the two hub genes. (A) Differential expression of CNTN2 and FZD2 in the GSE147976 (left) and GSE150807 (right) datasets. (B) Two gene sets targeted by enzalutamide were searched in KEGG database. GSEA was used to determine the association with the hub genes. P value <0.05 was regarded as the cutoff value. *P<0.05; ***P<0.001. CNTN2, contactin 2; FZD2, frizzled class receptor 2; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis.

Analysis of the immune infiltration in PCa

The Kruskal-Wallis rank sum test indicated that both hub genes were differentially expressed across different immune subtypes (P=1.42e-06 and 6.58e-08 for CNTN2 and FZD2, respectively; ). Interestingly, both hub genes showed positive correlation with most immunomodulators, chemokines, receptors, and major histocompatibility complexes (MHCs) (). CIBERSORT was applied to estimate the infiltration abundance of 22 types of immune cells in PCa and normal control samples (). There was a significant difference in the level of infiltration of regulatory T cells (P=6.71e-05), neutrophils (P=2.56e-11), monocytes (P=1.61e-03), resting mast cells (P=6.04e-03), M0 macrophages (P=7.04e-09), resting dendritic cells (P=4.21e-03), activated dendritic cells (P=0.048), and naïve B cells (P=7.95e-03) between PCa and normal control samples (). Spearman correlation analysis indicated that both hub genes exhibited positive correlation with the infiltration abundance of most of immune cells (resting mast cells, activated dendritic cells, resting dendritic cells, M2 macrophages, M1 macrophages, monocytes, activated natural killer (NK) cells, regulatory T cells, follicular helper T cells, CD4 memory resting T cells, CD8 T cells, plasma cells, and naïve B cells) ().

Figure 9

The association between the hub genes and immune infiltration in PCa. (A) The expression levels of the hub genes was assessed across different immune subtypes, including C1 (wound healing), C2 (IFNg dominant), C3 (inflammatory), and C4 (lymphocyte depleted). (B) The association between hub genes and the expression levels of immunomodulators, chemokines, receptors, and MHC was explored. (C) CIBERSORT was applied to estimate the infiltration abundance of 22 types of immune cells in PCa and normal control samples. (D) The Wilcoxon rank sum test was used to explore the immune infiltration levels of 22 types of immune cells between PCa samples and normal control samples. *P<0.05; **P<0.01; ***P<0.001. PCa, prostate cancer; IFNg, interferon-gamma; MHC, major histocompatibility complex.

Figure 10

Spearman correlation analyses were performed to examine the association between hub genes and the abundance of immune infiltration. CNTN2, contactin 2; FZD2, frizzled class receptor 2; NK, natural killer.

Discussion

PCa is the most common cancer and the second leading cause of cancer-related deaths in men in United States. In 2020, there were an estimated 191,930 new cases and 33,330 related deaths (49). Unfortunately, resistance to enzalutamide presents a significant challenge in the treatment of PCa. The present study explored the underlying molecular characteristics of enzalutamide-resistant PCa using data obtained from the GEO, ICGC, cBioPortal, TCGA, and GTEx databases.

Enzalutamide-related genes were determined by differential expression analysis. As expected, these genes were highly inversely correlated with androgen response in both gene datasets (GSE163240 and GSE136129) (). Previous studies have reported that resistance to enzalutamide is associated with the AR signaling pathway (8,11). In addition, the enrichment of the IL6/JAK/STAT3 signaling pathway in both gene sets indicated that the resistance of enzalutamide may be related to immune-associated functions in PCa. GO and KEGG analysis verified that enzalutamide resistance is related to immune activity (including cell-cell adhesion via plasma-membrane adhesion molecules, granulocyte activation, and neutrophil activation), and this is consistent with previous reports (50,51).

Network analysis and univariate Cox analysis identified two hub genes, CNTN2 and FZD2, both of which were significantly correlated with the AR. The expression of CNTN2 and FZD2 were significantly different at various stages of PCa compared to normal healthy samples. Furthermore, both hub genes were significantly associated with ADT. Interestingly, the expression of FZD2 was significantly up-regulated in enzalutamide-exposed samples, while no statistical difference was found for CNTN2, and this may have been due to the small sample size. These findings suggested that both hub genes, CNTN2 and FZD2, play a vital role in the resistance of PCa cells to enzalutamide.

The GSEA results indicated that both hub genes were highly correlated with immune-associated functional pathways, including allograft rejection, inflammatory response, IFNg response, IFNα response, IL6/JAK/STAT3 signaling, and complement and coagulation. Furthermore, both hub genes showed significantly positive correlations with the expression of almost all immunomodulators, chemokines, receptors, MHCs, and the infiltration of 22 types of immune cells. This suggested that the resistance of enzalutamide may be related to immune infiltration (50,51) and thus, both hub genes may be potential immunotherapeutic targets in PCa.

To date, there have been limit studies relating to CNTN2 and most research have been conducted in gliomas. CNTN2 is highly up-regulated in oligodendrogliomas compared with glioblastomas (52). It has also been reported to coamplify with MDM4, which is involved in the progression of malignant gliomas (53). In addition, CNTN2 has been shown to mediate the RTK/RAS/MAPK pathway in glioma cells (54). Although there is a paucity of data regarding CNTN2 and PCa, studies have suggested that the RAS/MAPK pathway may be involved in the development and progression of PCa and in the resistance to castration (55-57). Indeed, the RAS/MAPK pathway interacting with PI3K-AKT-mTOR has been implicated in many drug-resistance mechanisms including enzalutamide, ipatasertib, and capivasertib (57-59). Therefore, it is possible that CNTN2 may play an important role in mediating enzalutamide resistance in PCa cells.

FZD2, which is a receptor for Wnts, has been implicated in both non-canonical and canonical Wnt signaling pathways in PCa (8,60). FZD2 can regulate the epithelial-mesenchymal transition (EMT) pathway which is characterized by promoting tissue differentiation and shaping (61), and is correlated with the proliferation and metastasis of breast, endometrial, colon, liver, lung, and salivary adenoid cystic carcinomas (62-68). There is strong evidence that the EMT pathway is related to the metastasis and development of NEPC and CRPC (69-71). In addition, the activation of Wnt signaling pathway is one of the mechanisms leading to enzalutamide resistance in PCa cells (8). All these studies suggested that FZD2 may be an effective biomarker for enzalutamide-resistant PCa.

This current study comprehensively explored potential enzalutamide-related biomarkers in PCa. However, there were some limitations to this investigation. Further in vitro and in vivo studies should be conducted to further examine the detailed molecular mechanisms by which CNTN2 and FZD2 mediate enzalutamide-resistance in PCa cells.

In conclusion, this study analyzed the genetic characteristics of enzalutamide-resistant PCa and identified two hub genes, namely, CNTN2 and FZD2, that are related to immune infiltration in PCa. These findings provide crucial information regarding the mechanisms of enzalutamide-resistance in PCa. These hub genes may serve as potential biomarkers or immunotherapeutic drug targets for the management of enzalutamide resistance in PCa.

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