NSD3 expression pattern across different cancer types
The expression pattern of NSD3 was investigated in different types of cancer using TCGA data. NSD3 expression was compared between cancer specimens and corresponding normal samples. As shown in Fig. 1a, a total of 14 types of cancers showed abnormal NSD3 expression. Among them, NSD3 was highly expressed in cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pheochromocytoma and paraganglioma (PCPG), and stomach adenocarcinoma (STAD). Low NSD3 expression was observed in glioblastoma multiforme (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), prostate adenocarcinoma (PRAD), and thyroid carcinoma (THCA). A few cancer types, such as those with TCGA cancer codes SARC (sarcoma) and CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), had very few normal samples and the diﬀerences were not statistically signiﬁcant; however, the lack of significance was likely due to the small number of normal samples for comparison.
Next, we analyzed NSD3 expression levels in various tumors and ranked them from high expression to low expression (Fig. 1b). All the cancers expressed NSD3 with the highest levels being in acute myeloid leukemia (LAML) and the lowest being in LIHC. We then assessed the 33 cancer types for differential expression of NSD3 based on the cancer stage. Of the 33 types, 10 showed NSD3 expression-stage correlation, including colon adenocarcinoma (COAD), HNSC, KICH, KIRC, LUAD, pancreatic adenocarcinoma (PAAD), (SKCM), STAD, testicular germ cell tumors (TGCT), and THCA (Fig. 2a–2j). Notably, we found the majority of signiﬁcant diﬀerences in NSD3 expression were observed between stage I and other tumor stages. These results suggest that many types of cancers characteristically exhibit NSD3 dysregulation, which is associated with the pathological stage of the cancer.
Amplification of chromosomal region 8p11–12, where NSD3 is located, is a common genetic alteration that has been implicated in the etiology of cancers. Accordingly, we explored the correlation between mRNA expression and DNA-copy-number variation of NSD3 across the 33 tumor types. Interestingly, a positive association was detected for all the cancer types, with the top five being LUSC, breast invasive carcinoma (BRCA), rectum adenocarcinoma (READ), ESCA, and COAD (Fig. 3), indicating the high expression level of NSD3 in cancers is supported by DNA amplification.
Association of NSD3 expression with cancer patient survival
To explore the prognostic value of NSD3 in cancers, we performed a patient survival analysis, including overall survival (OS) and disease-specific survival (DSS) for each of the 33 cancer types. As shown in Fig. 4a, analysis using the Cox proportional hazards model revealed that higher NSD3 expression levels were significantly associated with shorter OS of patients with adrenocortical carcinoma (ACC), KICH, KIRP, PAAD, or uveal melanoma (UVM). Kaplan-Meier survival analysis also demonstrated that high levels of NSD3 was associated with poor OS for patients with ACC (Fig. 4b, P = 0.006), KICH (Fig. 4c, P = 0.005), KIRP (Fig. 4d, P = 0.004), PAAD (Fig. 4e, P < 0.001), and UVM (Fig. 4f, P = 0.043). Furthermore, our results indicated that NSD3 was a high-risk gene for DSS in patients with ACC, PAAD, or UVM, while patients with LUAD and higher NSD3 expression exhibited longer DSS (Fig. 5a). Kaplan-Meier curve analysis validated the predictive value of NSD3 for DSS in patient cohorts of ACC (Fig. 5b, P = 0.006), PAAD (Fig. 5c, P = 0.007), UVM (Fig. 5d, P < 0.001), and LUSC (Fig. 5e, P = 0.043). These results indicated that high NDS3 expression was mainly associated with poor outcomes for patients with cancer.
Correlation of NSD3 with TMB, TMI, and immune checkpoint genes across cancer types
We next analyzed the correlations of NSD3 expression with TMB, MSI, and expression of immune checkpoint genes, all of which have essential connections with ICB sensitivity. Our results indicated that NSD3 expression was positively associated with TMB in six cancer types, ACC, uterine corpus endometrial carcinoma (UCEC), STAD, LUAD, LAML, and HNSC, and negatively associated with TMB in five cancer types, THCA, LIHC, KIRP, KIRC, and BRCA (Fig. 6a). NSD3 expression was related to MSI in the other 10 cancer types (Fig. 6b). As cancer cells can escape immune surveillance by regulating the immune checkpoint gene cytotoxic T-lymphocyte-associated protein 4 (CLAT4), we also calculated the Pearson correlation coefficient between NSD3 expression and immune checkpoint genes across all 33 of the cancers (Fig. 6c). NSD3 was obviously co-expressed with the majority of immune checkpoint genes in most of the cancers, suggesting a vital role of NSD3 in the regulation of immune checkpoints.
Relationship between NSD3 expression and TME
Another essential factor that affects ICB sensitivity is the TME. Accordingly, we used the ESTIMATE algorithm to calculate immune and stromal scores for each tumor type and the relationships between NSD3 expression and these two scores were then assessed. Our results revealed that NSD3 expression negatively correlated with the immune scores of many cancer types, with the top five being GBM, brain lower grade glioma (LGG), SARC, PCPG, and ovarian serous cystadenocarcinoma (OV) (Fig. 7a). NSD3 expression also negatively correlated as with stromal scores in pan-cancer analysis, except for positive correlations with PRAD and KIRC (Fig. 7b). Infiltrating immune cells are an important component of the antitumor immune response. Consequently, we investigated the correlation between NSD3 expression and immune infiltrates in the five cancer types noted above for which NSD3 expression was highly correlated with the immune scores. As shown in Fig. 8, the number of infiltrating immune cells differed significantly between the high and low NSD3 expression populations across these cancer types. NSD3 expression positively correlated with the levels of inﬁltrating regulatory T cells (Tregs), activated mast cells, and M1 macrophages in GBM, LGG, and SARC tumor specimens, but negatively correlated with the levels of infiltrating CD8+ T cells and M2 macrophages. Similarly, NSD3 expression negatively correlated with levels of inﬁltrating activated NK cells, memory B cells, CD8+ T cells, and activated CD4+ memory T cells in PCPG and OV tumor specimens.
Functional annotation of NSD3
To identify the potential biological function of NSD3 in cancers, we performed functional enrichment analyses with NSD3-related genes using gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) algorithms. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms for each cancer type are shown in Fig. 9. NSD3 was associated in diverse cancers with immune-related pathways and glucose metabolism pathways, including antigen processing and presentation, RIG-I-like receptor signaling pathway, TOLL-like receptor signaling pathway, and pentose and glucuronate interconversions (Fig. 9a). NSD3-related genes in each cancer type were mainly enriched in GO terms associated with artery morphogenesis, cell cycle, cell motility, immune response, and inflammatory response (Fig. 9b). GSVA analysis was performed to determine the potential function of NSD3. As shown in Fig. 10, NSD3 expression was positively associated with several immune cell-related and histone methylation-related pathways. In contrast, NSD3 expression negatively correlated with cell metabolism-related, drug transport-related, and drug metabolism-related pathways.