Elevated expression of NXPH4 is associated with poor prognosis and immune infiltration in colorectal cancer patients

doi:10.21203/rs.3.rs-3567455/v1

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Elevated expression of NXPH4 is associated with poor prognosis and immune infiltration in colorectal cancer patients

https://doi.org/10.21203/rs.3.rs-3567455/v1

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Objective: NXPH4 is involved in the occurrence and development of various tumors, but its role in colorectal cancer (CRC) remains unknown. The aim of our study was to elucidate the role of NXPH4 in the progression of CRC.

Methods: The expression levels of NXPH4 in pan-cancer and CRC were evaluated using the Tumor Immune Estimate Resource (TIMER), Human Protein Atlas (HPA), and The Cancer Genome Atlas (TCGA). The diagnostic and prognostic value of NXPH4 in CRC was analyzed using the TCGA dataset. A prediction model for overall survival (OS) in CRC patients was constructed and validated based on the protein expression level of NXPH4 and multivariate analysis results. The UALCAN database was used to analyze the promoter methylation level of NXPH4 in CRC. Additionally, the functional roles of NXPH4 were examined through GO analysis, KEGG analysis, gene set enrichment analysis (GSEA), immune infiltration analysis, and immune checkpoint correlation analysis.

Results: NXPH4 was significantly upregulated in CRC compared to adjacent normal colonic tissue. High expression of NXPH4 in colorectal cancer was significantly correlated with T stage, N stage, pathological stage, residual tumor, and lymphatic invasion. Increased expression of NXPH4 was associated with unfavorable overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in CRC. Univariate analysis results indicated that NXPH4 was an independent prognostic factor for OS in CRC. Receiver operating characteristic (ROC) analysis suggested that NXPH4 could serve as a potential diagnostic biomarker for CRC patients. The nomogram constructed based on NXPH4 outperformed individual prognostic factors and could establish long-term survival rates in CRC patients. The promoter methylation level of NXPH4 was significantly elevated in CRC tissues. GO and KEGG analysis results indicated that NXPH4 and its co-expressed genes were involved in cell signaling function and neuroactive ligand-receptor interaction pathways. GSEA revealed that NXPH4 may participate in multiple pathways including tumor formation, cell invasion, inflammatory pathways, head and neck cancer, breast cancer, cytokine interactions, and lipid metabolism. Additionally, NXPH4 was significantly correlated with the infiltration levels of various immune cells including NK CD56bright cells, Cytotoxic cells, aDC, Th1 cells, Macrophages, NK cells, iDC, Tcm, Th17 cells, etc. NXPH4 was closely associated with multiple immune checkpoint markers including TNFRSF9, LAG3, PDCD1, CD276, CD274, IDO1, CTLA4, HHLA2, CD40LG, etc.

Conclusion: Our study revealed a strong correlation between elevated NXPH4 expression and poor prognosis and immune cell infiltration in CRC, suggesting it as a promising independent prognostic biomarker and a potential therapeutic target for CRC.

NXPH4

colorectal cancer

prognosis

biomarker

immune infiltration

Colorectal cancer (CRC) is the second most diagnosed cancer globally, It is the fourth leading cause of cancer-related deaths[1]. with over 1.85 million new cases and 0.85 million deaths reported annually[2]. Approximately 50%-60% of patients present with metastasis at the time of diagnosis, leading to poor prognosis[3]. Various treatment options including primary disease-directed surgery, more aggressive resection of metastatic disease (such as liver and lung metastases), radiation therapy for rectal cancer, as well as neoadjuvant and palliative chemotherapy have provided more choices for patients. However, these new treatment strategies have limited impact on cure rates and long-term survival[4]. Moreover, even after a series of treatments, many patients still experience recurrence, highlighting the need for alternative and effective therapies for CRC[5]. Therefore, it is urgent to explore more detailed molecular mechanisms to identify early screening biomarkers and potential therapeutic targets for CRC.

Neurexophilin-4 (NXPH4) is a synaptic secretory protein belonging to the Neurexophilin (NXPH) family[6]. The NXPH family consists of four members: NXPH1, NXPH2, NXPH3, and NXPH4[7]. NXPH1 has been identified as a potential biomarker for breast cancer invasion and metastasis[8]. The expression of NXPH2 in macrophages is upregulated during immune system dysfunction[9]. Abnormal expression of NXPH1 and NXPH2 in pancreatic ductal adenocarcinoma is associated with tumor location and lymph node metastasis[10]. NXPH3 is highly expressed in the cortex and cerebellar areas and plays a crucial role in sensory motor gating and motor coordination[11]. NXPH4 is a neuro-peptidoglycan widely expressed in human organs including the brain and liver[12]. It is expressed in neurons and is responsible for feeding, emotions, balance, and movement[13]. NXPH4 has been shown to regulate specific cerebellar synapses and motor functions[14]. However, the exact role of NXPH4 in colorectal cancer is unknown.

In this study, we analyzed the expression of NXPH4 in CRC patients and its correlation with clinical pathological features using public databases. We compared the expression levels of NXPH4 mRNA in tumor and corresponding normal tissues. Regression analysis and construction of column graphs were conducted to analyze the relationship between the expression level of NXPH4 in CRC and prognosis. We also compared the methylation level of NXPH4 promoter in CRC and adjacent normal tissues. In addition, we performed gene set enrichment analysis (GSEA) to study the pathways potentially involved in CRC development. Finally, we discussed the relationship between NXPH4 expression, immune cell infiltration, and immune checkpoints in CRC. This study highlights the biomarker potential of NXPH4 in predicting prognosis and its possibility as a therapeutic target in CRC.

TIMER and HPA database analysis

The Tumor Immune Estimate Resource (TIMER) is an online database used for comprehensive analysis of tumor-infiltrating immune cells and different gene expression levels in various cancer types. We used the TIMER database (http://TIMER.cistrome.org) to study the differential expression of NXPH4 in tumors and normal tissues of different cancer types. In addition, protein immunohistochemistry data for human tumor tissue and normal tissue were obtained from the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/).

RNA sequencing data analysis

The Cancer Genome Atlas (TCGA) is a publicly funded project aimed at classifying and discovering major genomic changes that cause cancer, and creating comprehensive cancer genome maps[15]. We obtained standardized RNA-seq data and corresponding clinical pathological information for 647 colon (COAD) and rectal (READ) tumor tissues, as well as 51 normal tissues, from the colorectal cancer (COADREAD) dataset in the TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). The level 3 HTSeq fragments per kilobase per million (FPKM) data format was downloaded. The main clinical pathological characteristics of CRC patients are shown in Table 1. Some patients had incomplete clinical information and were only excluded when comparing specific clinical factors that they were lacking. Gene expression datasets GSE44076 and GSE66229 were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/GEO/) to validate the mRNA expression of NXPH4 in CRC patients[16].

Table 1

The relationship between NXPH4 expression in colorectal cancer patients and clinical pathological parameters.
Characteristic	Low expression of NXPH4	High expression of NXPH4	p
n	322	322
T stage, n (%)			< 0.001
T1	12 (1.9%)	8 (1.2%)
T2	67 (10.5%)	44 (6.9%)
T3	223 (34.8%)	213 (33.2%)
T4	20 (3.1%)	54 (8.4%)
N stage, n (%)			0.004
N0	200 (31.2%)	168 (26.2%)
N1	77 (12%)	76 (11.9%)
N2	44 (6.9%)	75 (11.7%)
M stage, n (%)			0.076
M0	244 (43.3%)	231 (41%)
M1	36 (6.4%)	53 (9.4%)
Pathologic stage, n (%)			0.026
Stage I	67 (10.8%)	44 (7.1%)
Stage II	123 (19.7%)	115 (18.5%)
Stage III	84 (13.5%)	100 (16.1%)
Stage IV	37 (5.9%)	53 (8.5%)
Gender, n (%)			0.155
Female	141 (21.9%)	160 (24.8%)
Male	181 (28.1%)	162 (25.2%)
Age, n (%)			0.811
<=65	136 (21.1%)	140 (21.7%)
> 65	186 (28.9%)	182 (28.3%)
Residual tumor, n (%)			0.005
R0	237 (46.5%)	231 (45.3%)
R1	4 (0.8%)	2 (0.4%)
R2	9 (1.8%)	27 (5.3%)
CEA level, n (%)			0.732
<=5	128 (30.8%)	133 (32%)
> 5	79 (19%)	75 (18.1%)
Perineural invasion, n (%)			0.666
No	98 (41.7%)	77 (32.8%)
Yes	31 (13.2%)	29 (12.3%)
Lymphatic invasion, n (%)			< 0.001
No	198 (34%)	152 (26.1%)
Yes	98 (16.8%)	134 (23%)
Age, median (IQR)	68 (59, 75)	68 (56, 77)	0.911

Values shown in bold are statistically significant (P < 0.05); CRC, colorectal cancer; T: topography distribution; N: lymph node metastasis; M: distant metastasis; CEA: carcinoembryonic antigen.

Diagnosis and prognosis analysis

Based on the TCGA dataset, ROC analysis was used to evaluate the effectiveness of NXPH4 mRNA expression level in distinguishing colorectal adenocarcinoma from normal tissue. Patients were divided into two groups based on high or low expression of NXPH4, and Kaplan-Meier method was used to establish survival analysis. Kaplan-Meier estimation is the simplest method to calculate survival rate over time [17]. Overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) were used to analyze the correlation between NXPH4 expression and prognosis in colorectal cancer patients. The significance threshold was set at Cox P-value < 0.05.

Prediction model development

We performed univariate and multivariate Cox regression analyses to evaluate whether NXPH4 could serve as a protective factor for colorectal cancer. We included clinical parameters such as age, gender, T stage, N stage, M stage, pathological stage, and age. Multivariate Cox regression variables and NXPH4 variable were included, and nomograms and calibration plots were generated using the rms package [version 6.2-0] and survival package [version 3.2–10] to predict 1-year, 3-year, and 5-year overall survival. Nomograms are widely used in the prognosis of cancer[18].The calibration plots were graphically evaluated by mapping the predicted probabilities from the nomograms to observed rates. The diagonal line was used as the best prediction value. The concordance index (C-index) was used to determine discriminative ability.

Promoter methylation analysis

UALCAN(https://ualcan.path.uab.edu/), an easy-to-use interactive web portal, was used to perform in-depth analysis of NXPH4 promoter methylation levels in TCGA gene expression data. which is publicly available [19]. We used the UALCAN tool to detect the promoter methylation level of NXPH4 in CRC and adjacent normal tissues.

GO analysis and KEGG analysis

Based on the TCGA-CADREAD dataset, tumor samples were divided into low-level and high-level NXPH4 groups. The R package DESeq2 [version 1.26.0] was used to obtain differentially expressed genes between the low NXPH4 group and high NXPH4 group. Differentially expressed genes were selected based on |logFC| > 1 and P.adj < 0.05. GO enrichment analysis was performed on differentially expressed genes using three modules: biological processes (BP), molecular functions (MF), and cellular components (CC). In addition, KEGG analysis was performed on differentially expressed genes. The clusterProfiler package [version 3.14.3] was used for enrichment analysis of differentially expressed genes. A p.adjust < 0.05 was considered significant enrichment.

GSEA enrichment analysis

Gene set enrichment analysis (GSEA), widely used to identify significant statistical differences and control large gene sets [20], was used in this study. Data downloaded from the TCGA database were used, and the R package clusterProfiler (version 3.6.0) was used to perform GSEA between high NXPH4 groups. GSEA was run using the MSigDB Collections of c2.all.v7.2.symbols.gmt. A p-value < 0.05 was considered significant.

Immune cell infiltration analysis and immune checkpoint analysis

The correlation between NXPH4 expression and the infiltration levels of immune cells, including NK CD56dim cells, cytotoxic cells, aDC, Th1 cells, NK cells, iDC, Tcm, and Th17 cells, in relation to 24 tumor-infiltrating lymphocytes, was further analyzed. Subsequently, the correlation between NXPH4 and immune checkpoints, including TNFRSF9, LAG3, PDCD1, CD276, CD274, IDO1, CTLA4, HHLA2, and CD40LG, was analyzed.

Statistical analysis

Bioinformatics analysis was performed using R version 3.6.3. Wilcoxon signed-rank test and one-way analysis of variance were used to analyze the differential expression of NXPH4 in normal and tumor tissues. Fisher's exact test, chi-square test, Wilcoxon signed-rank test, and logistic regression were used to evaluate the correlation between NXPH4 expression and clinical pathological features. Receiver operator characteristic (ROC) curve analysis was applied, and the area under the curve (AUC) was used as an indicator of diagnostic accuracy. In addition, Kaplan-Meier method and Cox regression were used to evaluate the role of NXPH4 expression in prognosis. In Cox regression analysis, variables with statistically significant univariate Cox regression were further included significant.

Transcription levels of NXPH4 in colorectal cancer patients

To determine the overall expression level of NXPH4 in different malignant tumors, we first analyzed the expression of NXPH4 in different cancer types using the TIMER database. The results showed differential expression patterns of NXPH4 across various cancers. NXPH4 mRNA expression was significantly upregulated in COAD (colon adenocarcinoma) and READ (rectal adenocarcinoma) compared to normal tissues (Fig. 1A). To further determine the differential expression levels of NXPH4 in colorectal tumors and normal tissues, we collected RNA-seq data and clinical information of 647 colorectal adenocarcinoma tissues and paired non-matched normal tissues from the TCGA-COADREAD dataset. The results showed significant upregulation of NXPH4 in CRC tissues (p < 0.001, Fig. 1B). Moreover, we analyzed the expression levels of NXPH4 in CRC tissues and their matched adjacent normal tissues, and the results showed higher expression of NXPH4 in CRC tissues (p < 0.001, Fig. 1C). To validate these findings, we downloaded the GSE44076 dataset, and the results showed upregulation of NXPH4 in colon adenocarcinoma compared to normal tissues (p < 0.05, Fig. 1D). Additionally, we downloaded the GSE66229 dataset for analysis, and the results showed significant upregulation of NXPH4 in tumor tissues of early-stage CRC patients compared to normal tissues (p < 0.01, Fig. 1E).

The association between high expression of NXPH4 and adverse clinicopathological features in colorectal cancer

Table 1 shows the characteristics of 647 colorectal adenocarcinoma patients with complete clinical and gene expression data collected from the TCGA dataset. Based on the relationship between NXPH4 expression and the mean expression value, CRC patients were divided into a high expression group (n = 322) and a low expression group (n = 322). The association between NXPH4 expression and different clinicopathological features of CRC patients was evaluated, and the results showed a significant correlation between high NXPH4 mRNA expression and higher T stage (p < 0.001), N stage (p = 0.004), pathological stage (p = 0.026), residual tumor (p = 0.005), and lymphatic invasion (p < 0.001). However, there was no statistically significant correlation between NXPH4 mRNA expression and M stage, gender, age, CEA level, or neural invasion (all p > 0.05). Additionally, a similar pattern was observed in Figs. 1F-M, indicating a significant association between high NXPH4 expression and T stage (p < 0.05, Fig. 1F), N stage (p < 0.05, Fig. 1G), pathological stage (p < 0.05, Fig. 1I), residual tumor (p < 0.05, Fig. 1L), and lymphatic invasion (p < 0.001, Fig. 1M). However, there was no significant correlation between NXPH4 mRNA expression and M stage (p > 0.05, Fig. 1H), age (p > 0.05, Fig. 1J), or CEA level (p > 0.05, Fig. 1K). Furthermore, univariate logistic regression analysis was performed (Table 2), which showed a close correlation between NXPH4 mRNA expression and T stage (OR = 1.669, 95% confidence interval (CI): 1.132–2.478, p = 0.010), N stage (OR = 1.486, 95% CI: 1.085–2.038, p = 0.014), pathological stage (OR = 1.511, 95% CI: 1.100-2.079, p = 0.011), residual tumor (OR = 2.289, 95% CI: 1.184–4.655, p = 0.017), and lymphatic invasion (OR = 1.781, 95% CI: 1.275–2.495, p < 0.001).

Table 2

Logistic regression analysis of NXPH4 expression in colorectal cancer and clinical pathological parameters.
Characteristics	Total(N)	Odds Ratio(OR)	P value
T stage (T3&T4 vs. T1&T2)	641	1.669 (1.132–2.478)	0.010
N stage (N1&N2 vs. N0)	640	1.486 (1.085–2.038)	0.014
M stage (M1 vs. M0)	564	1.555 (0.985–2.479)	0.060
Pathologic stage (Stage III&Stage IV vs. Stage I&Stage II)	623	1.511 (1.100-2.079)	0.011
Gender (Male vs. Female)	644	0.789 (0.578–1.075)	0.134
Age (> 65 vs. <=65)	644	0.951 (0.695–1.299)	0.750
CEA level (> 5 vs. <=5)	415	0.914 (0.613–1.361)	0.657
Residual tumor (R1&R2 vs. R0)	510	2.289 (1.184–4.655)	0.017
Perineural invasion (Yes vs. No)	235	1.191 (0.660–2.146)	0.561
Lymphatic invasion (Yes vs. No)	582	1.781 (1.275–2.495)	< 0.001
Colon polyps present (Yes vs. No)	323	0.957 (0.593–1.538)	0.856

Values shown in bold are statistically significant (P < 0.05); CRC, colorectal cancer; T: topography distribution; N: lymph node metastasis; M: distant metastasis; CEA: carcinoembryonic antigen.

NXPH4 expression is a potential diagnostic biomarker for CRC patients.

In the TCGA-CADREAD dataset, the AUC of NXPH4 for distinguishing CRC tissues from normal tissues was 0.927 (95% confidence interval: 0.9-0.954, Fig. 2A), indicating that NXPH4 can serve as a good diagnostic biomarker for CRC patients.

Elevated NXPH4 expression indicates poor prognosis in CRC patients.

Kaplan-Meier survival analysis based on the TCGA-COADEAD dataset was performed to validate the correlation between high NXPH4 expression and prognosis. As shown in Fig. 2B-D, high NXPH4 expression was negatively associated with overall survival (OS) (HR = 1.52, 95% CI = 1.08–2.16, p = 0.018, Fig. 2B). Similarly, upregulation of NXPH4 expression was significantly correlated with shorter progression-free interval (PFI) (HR = 1.50, 95% CI = 1.10–2.04, p = 0.01, Fig. 2C) and disease-specific survival (DSS) (HR = 1.87, 95% CI = 1.18–2.96, p = 0.08, Fig. 2D). Univariate analysis showed that T stage, N stage, M stage, pathological stage, age, and NXPH4 expression level influenced the prognosis of CRC patients (all p < 0.05). In addition, multivariate Cox regression analysis showed that M stage, pathological stage, age, and NXPH4 expression level were independent risk factors for poor overall survival (OS) in CRC patients (Table 3).

Table 3

Univariate and multivariate analysis of clinical pathological factors related to overall survival in CRC patients.
Characteristics	Total(N)	Univariate analysis		Multivariate analysis
Characteristics	Total(N)	Hazard ratio (95% CI)	P value	Hazard ratio (95% CI)	P value
T stage	640
T1&T2	131	Reference
T3&T4	509	2.468 (1.327–4.589)	0.004	2.091 (0.946–4.624)	0.068
N stage	639
N0	367	Reference
N1&N2	272	2.627 (1.831–3.769)	< 0.001	0.425 (0.164–1.105)	0.079
M stage	563
M0	474	Reference
M1	89	3.989 (2.684–5.929)	< 0.001	2.370 (1.467–3.830)	< 0.001
Pathologic stage	622
Stage I&Stage II	348	Reference
Stage III&Stage IV	274	2.988 (2.042–4.372)	< 0.001	5.101 (1.758–14.803)	0.003
Gender	643
Female	301	Reference
Male	342	1.054 (0.744–1.491)	0.769
Age	643
<=65	276	Reference
> 65	367	1.939 (1.320–2.849)	< 0.001	2.797 (1.800-4.347)	< 0.001
NXPH4	643
Low	321	Reference
High	322	1.524 (1.075–2.160)	0.018	1.493 (1.015–2.196)	0.042

Values shown in bold are statistically significant (P < 0.05); CRC, colorectal cancer; OS, overall survival; CI, confidence interval; T: topography distribution; N: lymph node metastasis; M: distant metastasis.

Construction and validation of a nomogram based on NXPH4 expression levels.

To assist clinicians in correctly predicting the prognosis of colorectal cancer patients using a useful quantitative model, we constructed a nomogram. This nomogram was based on NXPH4 expression levels and combined with clinical features (M stage, pathological stage, age, and NXPH4 expression level) that were independently associated with patient survival in the multivariate analysis. The C-index of the nomogram was 0.786. The calibration plot for validating the reliability of this prediction model is shown in Fig. 3B, and the calibration line in the plot is close to the ideal line (also known as the 45-degree line), indicating good consistency between the observed and predicted values. These results suggest that the nomogram is a better model than individual prognostic factors and can establish long-term survival rates (1, 3, and 5 years) for colorectal cancer patients.

Promoter methylation level of NXPH4 in CRC.

Promoter DNA methylation has been shown to affect transcriptional repression and participate in tumor development[21]. By comparing the promoter methylation levels of NXPH4 in colorectal cancer and adjacent normal tissues, the analysis results showed a significant increase in the promoter methylation levels of NXPH4 in colon cancer (Fig. 4A) and rectal cancer (Fig. 4B), with statistically significant differences. These results suggest that upregulation of NXPH4 expression may be due to changes in promoter methylation, which in turn affects the development of colorectal cancer.

Predicted biological functions and pathways of NXPH4 in colorectal cancer.

The potential biological functions were analyzed by selecting co-expressed genes with NXPH4 (|logFC|>1, P.adj < 0.05). Gene ontology (GO) analysis of biological processes (BP) showed significant enrichment in lipid metabolism and protein-lipid complex assembly (Fig. 5A). Analysis of cellular components (CC) showed significant enrichment in lipid metabolism (Fig. 5B). Molecular function (MF) analysis showed significant enrichment in steroid binding, lipase inhibitor activity, haptoglobin binding, intermembrane lipid transfer activity, hormone activity, and DNA-binding transcription activator activity, RNA polymerase II-specific (Fig. 5C). KEGG analysis showed that Neuroactive ligand-receptor interaction was the most significantly enriched pathway (Fig. 5D). Overall, the results suggest that NXPH4 and its co-expressed genes may be involved in cellular signaling functions and the 'Neuroactive ligand-receptor interaction' pathway, thereby regulating the proliferation and invasion.

In addition, we observed the biological pathways that NXPH4 may regulate in the high expression group of NXPH4 based on the standardized enrichment fraction (NES) and FDR (error detection rate) q values. As shown in Table 4, several signal pathways were significantly enriched in the NXPH4 overexpression group, including TUMORIGENESIS (Fig. 6A), CELL MIGRATION (Fig. 6B), INFLAM PATHWAY (Fig. 6C), HEAD AND NECK CANCER (Fig. 6D), INVASIVE BREAST CANCER (Fig. 6E), CYTOKINE CYTOKINE RECEPTOR INTERACTION (Fig. 6F), METABOLIC PATHWAY OF LDL HDL AND TG INCLUDING DISEASES (Fig. 6G), HELLER HDAC TARGETS (Fig. 6H), and VILIMAS NOTCH1 TARGETS (Fig. 6I).

Table 4

Results of Gene Set Enrichment Analysis (GSEA)
Description	setSize	enrichmentScore	NES	pvalue	p.adjust	qvalue	rank
TUMORIGENESIS	23	0.664763507	1.947492735	0.000392764	0.016743068	0.015041376	1961
CELL_MIGRATION	75	0.487544529	1.71510398	0.000991556	0.029497011	0.026499065	2242
INFLAM_PATHWAY	13	0.735215056	1.89105605	0.000806168	0.028250057	0.025378846	1556
HEAD_AND_NECK_CANCER	37	0.569932222	1.821775408	0.001297684	0.03577981	0.032143308	1609
INVASIVE_BREAST_CANCER	124	0.43420764	1.597042651	0.001297937	0.03577981	0.032143308	3151
CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION	104	0.471161679	1.710270294	0.000326884	0.01518819	0.01364453	1936
METABOLIC_PATHWAY_OF_LDL_HDL_AND_TG_INCLUDING_DISEASES	13	0.734936604	1.890339841	0.000845177	0.02829205	0.025416571	513
HELLER_HDAC_TARGETS	156	0.418872121	1.563297394	0.001007445	0.029755608	0.026731379	2441
VILIMAS_NOTCH1_TARGETS	29	0.612661414	1.874769844	0.000877082	0.028556949	0.025654547	2734

Correlation of NXPH4 expression with immune infiltration levels in colorectal cancer.

Tumor-infiltrating lymphocytes can serve as reliable predictive biomarkers for treatment response and metastatic recurrence[22]. Therefore, we further investigated the relationship between NXPH4 expression and immune infiltration in colorectal cancer. Using Spearman correlation analysis and ssGSEA, we found positive correlations between NXPH4 and the levels of immune cell subtypes in CRC, including NK CD56bright cells (r = 0.202, p < 0.001), Cytotoxic cells (r = 0.196, p < 0.001), aDC (r = 0.192, p < 0.001), Th1 cells (r = 0.187, p < 0.001), Macrophages (r = 0.177, p < 0.001), NK cells (r = 0.171, p < 0.001), and iDC (r = 0.156, p < 0.001) infiltration levels (Fig. 7A). In addition, NXPH4 expression was negatively correlated with Tcm (r=-0.193, p < 0.001) and Th17 cells (r=-0.116, p = 0.003) infiltration levels (Fig. 7I-J), suggesting that NXPH4 may regulate the infiltration of lymphocytes in CRC tumors.

Relationship between NXPH4 and immune checkpoints in colorectal cancer.

Immune checkpoint inhibitors have revolutionized the treatment of malignant tumors[23]. We used TCGA data to determine the relationship between NXPH4 expression and the expression of immune checkpoint genes. We further investigated the relationship between NXPH4 expression and ICP to explore the potential of NXPH4 in immune therapy. The results showed a close correlation between NXPH4 expression and most of the 47 ICP genes (Fig. 8A). NXPH4 was highly correlated with TNFRSF9 (r = 0.338, p < 0.001), LAG3 (r = 0.252, p < 0.001), PDCD1 (r = 0.211, p < 0.001), CD276 (r = 0.277, p < 0.001), CD274 (r = 0.133, p < 0.001), IDO1 (r = 0.103, p = 0.009), and CTLA4 (r = 0.126, p = 0.001) (Fig. 8B-H). Moreover, NXPH4 was negatively correlated with HHLA2 (r=-0.091, p = 0.021) and CD40LG (r=-0.118, p = 0.003) (Fig. 8I-J). These findings suggest that NXPH4 regulates tumor immune responses through immune checkpoint activity.

Colorectal cancer (CRC) ranks third in the global incidence of common cancers and fourth in terms of mortality[24]. In recent years, the overall incidence of CRC has decreased in many high-income countries, but the incidence of CRC in individuals under 50 years old has increased significantly[25]. Surgery for early-stage CRC is the mainstay of treatment, but more time is needed to develop new treatment and diagnostic strategies for metastatic tumors[26]. Recent studies have shown that the NXPH family member 4 (NXPH4) plays an important role in the progression of cancer[27]. NXPH4 not only affects the proliferation, migration, and invasion of cancer cells but also influences glycolysis and reactive oxygen species levels[28]. The EZH2/NXPH4/CDKN2A axis has been reported to regulate the proliferation and migration of non-small cell lung cancer cells[29]. In addition, NXPH4 is a biomarker for early diagnosis of hepatocellular carcinoma and a potential prognostic and diagnostic biomarker for breast cancer[30][31]. NXPH4 is also a key gene involved in the occurrence and immune regulation of muscle-invasive bladder cancer[32].

In this study, we first found that the expression of NXPH4 mRNA varied in different types of cancer. We analyzed the expression of NXPH4 in different cancer types using the TIMER database and found that NXPH4 mRNA expression was significantly upregulated in COAD (colon adenocarcinoma) and READ (rectal adenocarcinoma) compared to normal tissues. Furthermore, we investigated the expression of NXPH4 in colorectal cancer based on the TCGA and GEO databases, and the results also showed significant upregulation of NXPH4 mRNA in colorectal cancer tissues compared with normal tissues. The expression level of NXPH4 was significantly associated with T stage, N stage, pathological stage, residual tumor, and lymphatic invasion. In addition, Kaplan-Meier curves and univariate analysis showed that upregulation of NXPH4 was associated with poor overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) in CRC. Importantly, both univariate and multivariate Cox regression analyses indicated that NXPH4 was an independent prognostic factor for CRC patients. ROC analysis showed that NXPH4 expression provided a certain degree of reliability for the diagnosis and prognosis of CRC patients. Furthermore, we developed a nomogram prognostic model based on the expression level of NXPH4 to predict the OS of CRC patients at 1, 3, and 5 years. The promoter methylation level of NXPH4 was significantly increased in colorectal cancer compared to normal tissues. Therefore, NXPH4 may further regulate the progression of CRC by modulating promoter methylation levels.

Next, GO and KEGG analysis were performed on NXPH4 co-expressed genes using TCGA data, and GSEA analysis was conducted on NXPH4. In the GO analysis, biological processes related to cellular signaling functions, including pattern specification process and regionalization, were identified. Cell component (CC) analysis showed blood microparticle. Molecular function (MF) analysis indicated significant enhancement of DNA-binding transcription activator activity, RNA polymerase II-specific, and serine-type peptidase activity. KEGG analysis revealed that neuroactive ligand-receptor interaction was the most significantly enriched pathway. In the GSEA analysis, several pathways corresponding to the high expression phenotype of NXPH4 were significantly enriched, including tumor formation, cell invasion, inflammation pathway, head and neck tumors, invasive breast cancer, cytokine interaction, lipid metabolism-related diseases, increased HDAC target, and increased NOTCH1 target. Cell migration plays an important role in many pathophysiological processes, including cancer invasion [33]. Previous studies have reported the involvement of NXPH4 in the progression of breast cancer [31] and its association with invasive breast cancer pathways.

Furthermore, to describe the immune infiltration level of CRC, we evaluated the relationship between NXPH4 and immune cell populations based on transcriptome data. The results showed that NXPH4 expression was widely associated with immune infiltration, including NK CD56bright cells, cytotoxic cells, aDC, Th1 cells, macrophages, NK cells, iDC, Tcm, and Th17 cells. These findings suggest that the presence of NXPH4 is related to the immune microenvironment of cancer. Studies have shown that the presence of innate immune cells (macrophages, neutrophils, dendritic cells, innate lymphoid cells, myeloid-derived suppressor cells, and natural killer cells) and adaptive immune cells (T cells and B cells) promotes tumor progression [34]. In addition, immune checkpoint inhibition in the form of anticancer immunotherapy is a paradigm shift that has changed the treatment of different types of solid and hematologic cancers [35]. Our current study provides evidence that NXPH4 is involved in regulating immune infiltration in the local microenvironment of CRC. We found that the expression of NXPH4 was positively correlated with immune checkpoint genes TNFRSF9, LAG3, PDCD1, CD276, CD274, IDO1, and CTLA4, and negatively correlated with immune checkpoint gene HHLA2, suggesting its potential as a target for immune therapy in colorectal cancer. Understanding the immune-related mechanisms of tumor cells contributes to a better understanding of tumors and the discovery of new therapeutic approaches. Our study demonstrates the involvement of NXPH4 in the immune environment of CRC. NXPH4 is dysregulated in various tumors and plays different roles in different types of cancer. In this study, an increase in NXPH4 levels was observed in cancer patient samples, and the upregulation of NXPH4 promoted cancer cell proliferation, invasion, and migration.

In conclusion, in this study, NXPH4 was found to be highly expressed in CRC tissues and significantly correlated with poor survival outcomes in CRC. This study suggests that NXPH4 may be considered as a potential biomarker associated with poor prognosis, and further research is needed to determine the mechanisms of interaction between NXPH4 and CRC tumors.

Ethics approval and consent to participate

This study does not involve ethics approval.

Consent for publication

Written informed consent for publication was obtained from all participants.

Data Availability Statement

The data provided in this study can be obtained from the article materials.

Conflicts of Interest

All the authors declare no conflict of interest.

Funding

Not applicable.

Authors’ Contributions

Jinxuan Yu and Qing Cui designed this study.Statistical analysis was conducted by Lingkai Xue, Hanqing Liu.Jinxuan Yu wrote the manuscript.Shuai Lu revised the manuscript. Lingkai Xue and Hanqing Liu are responsible for confirming the authenticity of the data.All authors read and approved the manuscript.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82074061).

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No competing interests reported.

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Elevated expression of NXPH4 is associated with poor prognosis and immune infiltration in colorectal cancer patients

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Abstract

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Introduction

Materials and methods

TIMER and HPA database analysis

RNA sequencing data analysis

Diagnosis and prognosis analysis

Prediction model development

Promoter methylation analysis

GO analysis and KEGG analysis

GSEA enrichment analysis

Immune cell infiltration analysis and immune checkpoint analysis

Statistical analysis

Results

Transcription levels of NXPH4 in colorectal cancer patients

The association between high expression of NXPH4 and adverse clinicopathological features in colorectal cancer

Discussion

Conclusions

Declarations

References

Additional Declarations

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