Differential expression of monkeypox-related genes in cancers and paracancer and its impact on prognosis
Overall, FOX and EGR1 were significantly low expressed in 14 tumors. In particular, all genes showed significant low expression in BRCA. HMGA2 was significantly highly expressed in LUSC, LUAD, HNSC and THCA (Fig. 1A). Investigation of polygenic expression in different cancers revealed that SMOX, ARC and GFAP were highly expressed in ESCA and LGG (Fig. 1B).
Cnv And Snp Of Monkeypox Related Genes
To detection of frequency and type of variation for each cancer subtype, the monkeypok related genes SNP data were analyzed. This analysis included 1507 tumor samples, and reveal that the SNV frequency of related genes were 100% (1507cases). The SNV frequency of DUOX1, IL4R, ZC3H12C, PTGS2, SPRED1, EGR2, ETV5, PLK3, EGR1, and SMOX were 12, 10, 9, 8, 7,7,7,6,6 and 6%, respectively (Fig. 2A). SNV percentage analysis showed that higher frequency of mutations in multiple monkeypox-related genes in UCEC, SKCM and COAD. DUOX1, EGR2, SPRED1, SMOX, ZC3H12C, IL4R, EGR1, PLK3, USP12 were 41, 34, 32, 30, 29, 26, 25, 25, 25% in UCEC respectively. IL4R, DUOX1, TRIM15, PTGS2, HS3ST1 were 28, 26, 23, 22% in SKCM respectively. DUOX1, EGR2, ZC3H12C, ETV5 were 18, 16, 14, 12% in COAD respectively (Fig. 2B). By evaluating CNV mutation data for monkeypox-related genes in 33 tumors, we found heterozygous amplification or heterozygous deletions were the main mutations. We also observed ARC, RGS16, PTGS16, SMOS and ETVS heterogeneous amplification of the CNV in most tumors. Heterozygous amplification of ARC, RGS16, PTGS16, SMOS was greater than or equal to 50% in UCS and LUSC. In addition, heterozygous deletion of USP12, AREG, ZC3H12C, EGR2, CXCL1, CXCL2, H3S3HT1, SPRY2, SPRY4 and EGR1 was observed more than 50% in TGCA. The amplification and deletion of PRAD, LAML, THCA and THYM tumors were less in 33 tumors (Fig. 2C).
Methylation Of Monkeypox-related Genes And Their Correlation With Expression
To investigate the epigenetic methylation levels of monkeypox-related genes, we performed an analysis of methylation levels. DUSP5, CXCL2, IL4R were hypomethylated in KIRC. And HMGA2, TRIM15 methylation levels were low in UCEC. In addition, DNAJA4, HBEJF, PHLDA1, SPRY2, PTGS2 methylation levels were high in PRAD. PHLDA2, SPRY2, PTGS2 were hypermethylated in BRCA. PHLDA2, SPRY2 and PTGS2 methylation levels were significantly higher in BRCA (Fig. 3A). As a whole, monkeypox-related genes had a negative correlation between methylation level and mRNA expression (Fig. 3B).
Drug Sensitivity Analysis
We performed analysis of drug sensitivity and mRNA expression of monkeypox-related genes. Almost all genes were resistant to FK866, GSK1070916, MP470, CX-5461, 5-Fluorouracil and MPS-1-IN-1, except RGS16 and ARC which were sensitive to these drugs. In addition, CXCL1 and CXCL2 were sensitive to XAV939 (Fig. 4).
Differential Expression Of Monkeypox Score And The Correlation With Staging
FOS and EGR1, HBEGF, EGR2, EEGR1 and HUEGF, EGR2, CACL1 and CXCL2, EREG, AREG and EREG, IE3, LIF and IER3, were significantly and positively correlated overall (Fig. 5A). Monkeypox scores were higher in BLCA, CESC, COAD, ESCA, GBM, KIRC, LGG, OV, PAAD, READ, SKCM, STAD, TGCT, THCA, UCEC, UCS and lower in ACC, BRCA, KICH, LAML, LUAD, LUSC, PRAD (Fig. 5B). The monkeypox score of stage Ⅲ-Ⅳ was significantly higher than stage Ⅰ-Ⅱin TGCT and THCA. Except for TGCT and THCA, there was no significant difference in monkeypox score at different stages in other tumors (Supplementary Fig. 1).
Effect Of Monkeypox Score On Prognosis
By analyzing monkeypox-related genes and OS, we found it is a risk factor in ACC (P = .014), ESCA (P = .003), LGG (P < .001), THCA (P = .027), BLCA (P = .029), GBM (P = .002), LIHC (P = .01), THYM (P = .018), HNSC (P = .001), LUAD (P = .003), CESC (P < .001), LUSC (P < .001), UVM (P = .004), STAD (P = .012), LAML (P = .002) and PAAD (P = .001) (Supplementary Fig. 2). For DSS, the monkeypox-related genes score was considered as a risk factor in ACC (P = .018), ESCA (P < .001), GBM (P = .002), LIHC (P = .02), HNSC (P = .009), LUAD (P = .026), UCEC (P = .01), CESC (P < .001), LUSC (P < .001), CHOL (P = .042) and UVM (P = .005) (Supplementary Fig. 3). By analyzing monkeypox-related genes and PFI, we found it is a risk factor in ACC(P = .005), LGG(P < .001), GBM(P < .001), THYM(P = .049), HNSC(P = .012), LUAD(P = .013), CESC(P = .002), LUSC(P < .001), SARC(P < .012), UVM(P = .039), OV(P = .02), and PAAD(P < .001), TGCT(P < .016) (Supplementary Fig. 4). By OS analysis, we found that monkeypox scores had significantly positive relation with OS in BRCA, COAD, PCPG, PRAD, READ (Fig. 6A). The prognostic value of disease-specific survival(DSS)on monkeypox-related genes was evaluated, and it was found that DSS was significantly associated with poor prognosis in 10 of 33 tumors (Fig. 6B). Besides, monkeypox scores were also better significantly related with better PFI in PCPG, PRAD, READ (Fig. 6C).
The Function Of Monkeypox Score Was Analyzed At A Single-cell Level
By functional analysis at the single cell level, we found that Monkeypox scores were associated with inflammation, quiescence, and hypoxia in most tumors, with a strong correlation in UM, suggesting that Monkeypox methylation scores may be an important factor in tumor metastasis and invasion. We observed that stemness, cell cycle, DNA repair, DNA damage, invasion, proliferation, inflammation, quiescence, EMT, hypoxia, apoptosis, differentiation, angiogenesis, and metastasis in UM promoted Monkeypox function. Stemness, proliferation, inflammation, quiescence, EMT, hypoxia, apoptosis, differentiation, angiogenesis, and metastasis that in RB promote Monkeypox function. Monkeypox function is promoted in most tumors in different cellular functions, however still inhibited in some aspects, such as stemness, cell cycle, DNA repair, DNA damage, invasion, proliferation inhibit Monkeypox function in NSCLC (Fig. 7).
Relationship Between Monkey Score And Tumor Immune Microenvironment
By calculating the abundance of 22 immune cells, we explored the relationship between Monkeypox and tumor immune cells and found that Monkeypox was significantly positively correlated with mast cell resting, macrophage M0, myeloid dendritic cell activated, neutrophil, macrophage M2, T cell CD4 memory resting, and monocyte. In addition, Monkeypox was significantly negatively correlated with activated T cell CD4 naive, B cell memory, and mast cell activated (Fig. 8A). The monkeypox score has significantly positive relationship with monkeypox immune score in GBM, KICH, ACC, PCPG, LGG, LAML, SARC, KIRP, LIHC, LIAD, PRAD, THCA (Fig. 8B). However, monkeypox score was significantly negatively correlated with microenvironment score in STAD, THYM, CESC, HNSC, TGCT and CHOL (Fig. 8C). Meanwhile, monkeypox score was significantly negatively correlated with stroma score in HNSC and THCA (Fig. 8D).
Relationship Between Monkeypox Score And Immune-related Gene
Most immune checkpoints were positively correlated with monkeypox-related genes in most tumors, particularly TNFSF9, TNFSF15, CD276, CD44, CD274, CTLA4, TNFRSF8, NRP1, C10orf54 (Fig. 9A). Monkeypox related genes were positively correlated with most of Immune activating genes especially in IL6, TMEM173, NT5E, PVR and CD276 in most tumors (Fig. 9B). In the immunosuppressive gene correlation analysis PVRL2, IL10RB, TGFBR1, PDCD1LG2, KDR, TGFB1, IL10 and CD274 were found to be positively correlation with monkeypox related genes. But negative correlation with LAG3, CA160, BTLA, BTLA (Fig. 9C). The expression of chemokine was positively correlated with monkeypox related genes in general. And chemokines CXCL3, CXCL2, and CXCL8 were significantly positively correlated with monkeypox related genes in most tumors (Fig. 9D). We observed that chemokine receptors were positively correlated with monkeypox related genes in most tumors overall, and CXCR2, CXCR1, CXCR4, CCR4, CCR1 expression significantly correlated with in most tumor types in testing chemokine receptor expression in tumors (Fig. 9E).
Correlation Between Monkeypox Score And Markers Of Immunotherapy Response
Monkeypox was significantly negatively correlated with TMB in DLBC only, and significantly positively correlated with TMB in most tumors (Fig. 10A). The expression of monkeypox related genes was positively correlated with MSI in PCPG, LAML, KIRC, COAD, THYM, BRCA, KIRP and negatively correlated with GBM, CESC, LIHC, ESCA, TGCT, PRAD, LUSC (Fig. 10B). The monkeypox score has significantly positive relationship with monkeypox infection related genes expressing in CESC, OV.M, KIRC, PAAD, STAD, BRCA, HNSC and LUSC (Fig. 10C). TIDE correlation analysis show that, the expression of monkeypox infection related genes was significantly and positively associate with TIDE score in OV.R, BRCA, HNSC, PAAD, CESC, KIRC, LUSC, OV.M and STAD nine cancers ( Supplementary Fig. 5).
Single-cell Transcriptional Analysis Of Monkeypox In The Kirc Tumor Microenvironment
The results obtained reveal the diversity of KIRC cell types. To investigate the Monkeypox fraction in KIRC tumor microenvironment cells, we used ssGSEA to calculate and compare the differences in Monkeypox fraction in different cell types (Fig. 11A). We observed a significant difference in the expression of marker genes in different cell clusters (Supplementary Fig. 7). We used ScRNA-seq to detect 2 KIRC samples. Then, Seurat was analyzed by containing 13124 high quality single cell transcriptome information after quality control. Cell clustering analysis based on the tSNE algorithm revealed 11 clusters of monocyte 1, monocyte 2, KIRC1, macrophage, KIRC2, KIRC3, CD4 + T cells, mast cells, CD8 + T cells, NK cells and endothelial cells (Fig. 11B). The results obtained reveal the diversity of KIRC cell types. To investigate the Monkeypox fraction in KIRC tumor microenvironment cells, we used ssGSEA to calculate and compare the differences in Monkeypox fraction in different cell types (Fig. 11C). By analyzing the Monkeypox scores of the above 11 cells, we found significant differences between them. The results showed that Monkeypox scores were significantly higher in macrophage within KIRC2 and KIRC3 and significantly lower in CD4 + T cells, and Monkeypox scores were significantly different within different KIRC cell clusters, suggesting that Monkeypox scores reveal potential features of KIRC. Also, this suggests that Monkeypox is significantly different in different cells of the KIRC tumor microenvironment (Fig. 11D).
Construction Of Ppi Network And Hub-gene Definition
PPI network construction showed FOS is the hub gene of monkeypox related gene (Supplementary Fig. 8A). Prognostic analysis revealed monkeypox-related genes as risk factors. Therefore, to verify that similar results existed at the protein level, we performed a protein level analysis. Data from the human protein atlas also showed low expression of the FOS gene in colon adenocarcinoma (COAD), stomach adenocarcinoma (STAD) and Lung squamous cell carcinoma (LUSC), which is consistent with our previous expression analysis (Supplementary Fig. 8B-G), which was consistent with our expression analysis.