Weighted Gene Co-expression Network Analysis Identifies CALD1 as a Biomarker Related to M2 Macrophages Infiltration in Stage III and IV Mismatch Repair-Proficient Colorectal Carcinoma

Abstract

Immunotherapy has achieved efficacy for advanced colorectal cancer (CRC) patients with a mismatch-repair-deficient (dMMR) subtype. However, little immunotherapy efficacy was observed in patients with the mismatch repair-proficient (pMMR) subtype, and hence, identifying new immune therapeutic targets is imperative for those patients. In this study, transcriptome data of stage III/IV CRC patients were retrieved from the Gene Expression Omnibus database. The CIBERSORT algorithm was used to quantify immune cellular compositions, and the results revealed that M2 macrophage fractions were higher in pMMR patients as compared with those with the dMMR subtype; moreover, pMMR patients with higher M2 macrophage fractions experienced shorter overall survival (OS). Subsequently, weighted gene co-expression network analysis and protein-protein interaction network analysis identified six hub genes related to M2 macrophage infiltrations in pMMR CRC patients: CALD1, COL6A1, COL1A2, TIMP3, DCN, and SPARC. Univariate and multivariate Cox regression analyses then determined CALD1 as the independent prognostic biomarker for OS. CALD1 was upregulated specifically the in CMS4 CRC subtype, and single-sample Gene Set Enrichment Analysis (ssGSEA) revealed that CALD1 was significantly correlated with angiogenesis and TGF-β signaling gene sets enrichment scores in stage III/IV pMMR CRC samples. The Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm and correlation analysis revealed that CALD1 was significantly associated with multiple immune and stromal components in a tumor microenvironment. In addition, GSEA demonstrated that high expression of CALD1 was significantly correlated with antigen processing and presentation, chemokine signaling, leukocyte transendothelial migration, vascular smooth muscle contraction, cytokine-cytokine receptor interaction, cell adhesion molecules, focal adhesion, MAPK, and TGF-beta signaling pathways. Furthermore, the proliferation, invasion, and migration abilities of cancer cells were suppressed after reducing CALD1 expression in CRC cell lines. Taken together, multiple bioinformatics analyses and cell-level assays demonstrated that CALD1 could serve as a prognostic biomarker and a prospective therapeutic target for stage III/IV pMMR CRCs.

Keywords: M2 macrophages; bioinformatics; colorectal cancer; microsatellite instability; prognosis; tumor microenvironment.

FIGURE 1

The schematic diagram of this study.

FIGURE 2

(A) Violin plot displayed the CIBERSORT TIIC fraction difference between pMMR (85) and dMMR (20) stage III/IV CRC samples; only samples with a CIBERSORT P-value < 0.05 were screened for this analysis. (B) Survival curve of the high- and low-M2 macrophage groups in stage III/IV pMMR CRC patients. (C–F) Co-expression network analysis by WGCNA. (C) Analysis of network topology for optimal soft-threshold power. (D) Dendrogram of genes clustered with dissimilarity based on topological overlap; each color below represented an expression module of highly interconnected groups of genes in the constructed gene co-expression network. Gray indicated that genes were not incorporated into any module. (E) Heatmap of the correlation between each module’s module eigengene and M2 macrophage fractions; each cell contains the Pearson’s correlation coefficient and the corresponding P-value. The red module was the most significant module with the strongest correlation. (F) Scatterplot of gene significance (GS) for M2 macrophage fractions vs. module membership (MM) in the red modules. TIICs, tumor-infiltrating immune cells; pMMR, mismatch-repair-proficient; dMMR, mismatch-repair-deficient; CRC, colorectal cancer; WGCNA, weighted gene co-expression network analysis.

FIGURE 3

(A) Top 10 GO terms, including BP, CC, and MF, respectively, enriched according to the hub genes in the red module; (B) KEGG terms enriched according to the hub genes in the red module; (C) Cytoscape software was used to establish the PPI network of hub genes in the red module based on the STRING website; yellow signifies the hub nodes. GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction.

FIGURE 4

The expressions of CALD1 (A), COL6A1 (B), COL1A2 (C), TIMP3 (D), DCN (E), and SPARC (F) were significantly and positively correlated with M2 macrophage infiltrations.

FIGURE 5

Kaplan–Meier survival curves of six hub genes grouped by their median expression values in the GSE39582 dataset.

FIGURE 6

(A) Spearman correlation matrix of CALD1 expression with the 22 tumor-infiltrating immune cell proportions in 85 stage III/IV pMMR CRC samples of GSE39582. (B) Boxplots of CALD1 expression between dMMR and pMMR stage III/IV CRC samples in both GSE39582 (left) and GSE41258 (right) datasets. (C) Histograms of the distribution characteristics of stage III/IV dMMR and pMMR CRC patients in each CMS subgroup. NA, not assigned. (D) Boxplots exhibiting CALD1 expression was significantly and specifically upregulated in the CMS4 subtype in both GSE39582 (left) and GSE41258 (right) datasets. Significant differences between CMS subgroups were indicated as follows: ns, not significant; ***P < 0.001, ****P < 0.0001 (Kruskal–Wallis test followed by Dunn’s tests). (E,F) Correlation heatmap revealed that CALD1 expression was significantly and positively correlated with angiogenesis and TGF-β signaling ssGSEA enrichment scores, immune and stromal scores, gene markers of TAMs (CCL2 and IL10), and M2 macrophages (CD163, VSIG4, and MS4A4A) but was not clear with gene markers of macrophage M1 (IRF5, NOS2, and PTGS2) in the GSE39582 (E) and GSE41258 (F) datasets. pMMR, mismatch-repair-proficient; dMMR, mismatch-repair-deficient; CRC, colorectal cancer; CMS, consensus molecular subtypes; TAMs, tumor-associated macrophages.

FIGURE 7

Gene Sets Enrichment Analysis (GSEA) revealed that several immune-related (A) and tumor-related (B) pathways were highly enriched in the high CALD1 expression group.

FIGURE 8

CALD1 promotes cell proliferation, migration, and invasion in CRC cells. (A) The protein levels of CALD1 in four kinds of CRC cell lines were measured by Western blot. (B,C) The protein levels of normal, si-NC, and si-CALD1 transfected SW620 (B) and SW480 (C) cells by Western blot; si-CALD1 transfections were performed in duplicate. (D,E) Transwell assays revealed that CALD1 knockdown attenuated SW620 (D) and SW480 (E) cell migration and invasion. (F,G) CCK8 assays were performed to assess cell viability in normal, si-NC, and si-CALD1 transfected SW620 (F) and SW480 (G) cells. *p < 0.05 in comparison with the si-CALD1 group using Kruskal–Wallis one-way analysis of variance (ANOVA). All assays were repeated at least three times.