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.

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“…Li et al found that CALD1 upregulated the expression of PD-L1 through the JAK/STAT signaling pathway and promoted malignant progression of bladder cancer ( 48 ). Several bioinformatics analyses and cellular studies have shown that CALD promoted the proliferation, metastasis, and invasion of CRC cells and is related to a reduction in OS ( 49 ). However, the exact mechanism of CALD involvement in CRC remains to be clarified.…”

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“…Histological and immunofluorescence examination of tumor tissue showed that CaD was associated with vessel architecture in astrocytoma and glioblastoma[ 60 - 63 ]. In stage III/IV mismatch-proficient CRC, CALD1 was upregulated and associated with angiogenesis, as detected by bioinformatics ‘Weighted gene coexpression network analysis’ (WGCNA)[ 41 ].…”

“…A recent study by Zheng et al [ 41 ] utilized a new bioinformatics tool, WGCNA, to clarify the basis of the poor response to immunotherapy in mismatch-proficient, stage III/IV CRC and showed that CALD1 was upregulated and associated with protumorigenic M2 macrophage infiltration. M2 macrophages are believed to be an important contributor to the failure of immunotherapy due to their anti-inflammatory, immunosuppressive, and proangiogenic characteristics[ 103 ].…”

“…M2 macrophages are believed to be an important contributor to the failure of immunotherapy due to their anti-inflammatory, immunosuppressive, and proangiogenic characteristics[ 103 ]. CALD1 was negatively correlated with fractions of plasma cells, CD8 T cells, CD4 memory-activated natural killer cells, and dendritic cells[ 41 ]. High expression of CALD1 was significantly correlated with angiogenesis, TGF-β, and trans-endothelial migration.…”

“…Taken together, these data are consistent with the published literature on the importance of the crosstalk between angiogenesis and TGF-β in macrophage recruitment and M2 polarization[ 104 , 105 ], but the role of CALD1 in this scenario remains to be clarified. Cancer cell proliferation, invasion, and migration abilities were suppressed after reducing CALD1 expression via siRNA silencing in vitro [ 41 ].…”