DNAJC5B highly expressed in late-stage male ESCC patients
Our research examined the association between DNAJC5B expression levels in ESCC patients and a range of clinical and pathological characteristics. Based on the ascending order of DNAJC5B expression levels, we observed an asymmetry in the distribution of TNM staging status (including T, N, M), tumor grading, gender, smoking, alcohol consumption, and radiation therapy status in the dataset (Fig. 1A, B). We compared baseline characteristics among groups with different levels of DNAJC5B expression. The results indicate that high expression of DNAJC5B was present in samples of esophageal cancer in male patients (Fig. 1D), grades G2-G3 (Fig. 1G), and stages III-IV (Fig. 1H, J).
Additionally, we found that DNAJC5B expression is notably higher in cancer tissues compared to the surrounding non-cancerous tissues. To validate the reliability of this result, we performed immunohistochemical staining experiments on paired clinical samples. The results indicate that DNAJC5B is significantly enriched in the cytoplasm of cancer tissue cells (Figure S2). In summary, these results suggest that DNAJC5B is highly expressed in male patients with advanced ESCC patients.
DNAJC5B in tumor cells regulated immunity via T cell receptors
In an effort to investigate the biological functions linked to DNAJC5B, we selected the most strongly associated genes (Top 500, P<0.05) using Pearson correlation analysis from the database, followed by Gene Ontology (GO) functional enrichment and KEGG pathway analyses of these genes. The results of the analysis suggested that the biological processes closely associated with DNAJC5B predominantly involve adaptive immune responses, immune reactions, cell surface receptor signaling pathways, and T cell activation (Fig. 2A). Additionally, the most significantly related cellular components include the T cell receptor complex, plasma membrane, and class II protein complex (Fig. 2B). In terms of molecular function, the aspects most closely related to DNAJC5B are signal receptor activity, transmembrane signaling receptor activity, and peptide antigen binding function (Fig. 2C). Pathway analysis revealed that these genes are involved in various tumor-infiltrating immune-related pathways, including chemokine signaling pathways, Th1 and Th2 cell differentiation, cell adhesion molecules, and Th17 cell differentiation (Fig. 2D). These results imply that DNAJC5B may regulate immune responses in tumor cells through a T cell receptor-dependent mechanism.
High DNAJC5B expression suppressed T-cell-mediated tumor immunity
In the process of immunogenic cell death in tumor cells, multiple factors contribute to apoptosis or necrosis, encompassing the activation of lymphocytes like NK cells, T cells, B cells, and the release of chemokines and cytokines. Therefore, this study aims to clarify the specific impact of DNAJC5B expression on immune responses and cytokine production. Initially, we conducted gene set variation analysis (GSVA) to acquire enrichment scores of immune processes. Then, we utilized Pearson correlation analysis to determine the correlation coefficients and statistical significance between GSVA scores and gene expression. Our findings reveal that, apart from T-cell-mediated tumor immune responses, the expression of DNAJC5B is positively correlated with most immune functions (Fig. 3). These outcomes imply that high expression of DNAJC5B could inhibit T-cell-mediated tumor immune responses.
High DNAJC5B expression positively correlated with immune checkpoint activation and inflammation dysregulation
Based on the above results, we found that DNAJC5B has a certain association with T-cell immune suppression in tumors. Consequently, we delved deeper into the relationship between DNAJC5B and well-known immune checkpoints, including PD-1, SIGLEC15, TIGIT, HVEM, CD200R1, CTLA-4, HAVCR2, and LAG3. Our study results show that DNAJC5B is significantly positively correlated with the aforementioned immune checkpoints (Fig. 4A). Furthermore, we screened seven inflammation-related gene sets to evaluate the immune status [12-16]. The results indicate that DNAJC5B is positively correlated with these inflammation gene sets and has statistical significance (Fig. 4B). Overall, the high expression of DNAJC5B might be positively associated with the activation of immune checkpoint inhibitors and the dysregulation of inflammation.
DNAJC5B mainly enriched in tumor-infiltrating CD8+ T cells
To further investigate the association between DNAJC5B and T-cell immunity, we downloaded and analyzed the esophageal cancer single-cell dataset GSE196756 from a public database. Utilizing the markers mentioned in the original text for clustering, we successfully extracted the T lymphocyte population. Based on the unique expression characteristics of T lymphocytes, we further subdivided them into five distinct clusters (Fig. 5A). By analyzing the expression of cell markers corresponding to each cluster, we defined clusters 0, 3, and 4 as CD4+ T cells, while clusters 1 and 2 were identified as CD8+ T cells. Interestingly, the enrichment of DNAJC5B was primarily observed in cluster 2, which consists of CD8+ T cells (Fig. 5B). Additionally, cytotoxicity-related genes (GZMA, GZMB, PRF1, and GNLY) were also observed to be concentrated in 0, 2 cell clusters (Fig. 5B and Figure S3).
DNAJC5B affected the biological functions of CD8+ T cells
Additionally, we conducted functional enrichment and pathway analysis on the cells in clusters 1 and 2 to explore the potential impact of DNAJC5B expression on the biological functions of CD8+ T cells. The analysis results showed significant functional differences between these two clusters of cells (Fig. 6). To rule out the impact of differences in cell cycle stages on the outcomes, specific cell cycle markers were used in a supervised analysis to quantify the cell cycle stages of each cell group. The analysis revealed no significant differences among cells in the G1, S, and G2 phases (Figure S4). Overall, these findings imply that DNAJC5B is predominantly enriched in tumor-infiltrating CD8+ T cells, and its expression could significantly influence the biological functions of CD8+ T cells.
DNAJC5B is an independent prognostic factor in ESCC patients
To explore the association between DNAJC5B and patient prognosis, we conducted analyses including Kaplan-Meier survival curves, Receiver Operating Characteristic (ROC) curves, and Cox proportional hazards models. Results indicate that patients with high DNAJC5B expression experienced a significantly shorter overall survival, about half compared to those with low expression (median survival of 681 days compared to 1361 days) (Fig. 7A). The time-dependent ROC analysis demonstrated an area under the curve (AUC) value of 0.76 for 2-year survival and 0.66 for 3-year survival (Fig. 7B). Univariate and multivariate Cox regression analyses indicated that the expression level of DNAJC5B is an independent prognostic factor for ESCC patients in Table 1.
Table 1 Univariate and multivariate regression analysis of OS prognostic indicators
|
Univariate analysis
|
Multivarate analysis
|
Variable
|
HR(95%CI)
|
P-value
|
HR(95%CI)
|
P-value
|
DNAJC5B
|
3.577(1.372-9.331)
|
0.009
|
4.114(1.076-15.728)
|
0.039
|
Age
|
1.029(0.987-1.073)
|
0.173
|
|
|
Gender
|
9.837(1.294-74.759)
|
0.027
|
5.092(0.500-51.883)
|
0.169
|
Stage
|
2.542(1.156-5.589)
|
0.020
|
5.346(1.101-25.966)
|
0.038
|
Smoking
|
2.015(0.686-5.921)
|
0.202
|
|
|
Alcohol
|
2.961(0.690-12.706)
|
0.144
|
|
|
Abbreviations: CI, confidence interval; HR, hazard ratio.