Identification of key genes involved in tumor immune cell infiltration and cetuximab resistance in colorectal cancer

Abstract

Background: The anti-epidermal growth factor receptor (EGFR) antibody introduces adaptable variations to the transcriptome and triggers tumor immune infiltration, resulting in colorectal cancer (CRC) treatment resistance. We intended to identify genes that play essential roles in cetuximab resistance and tumor immune cell infiltration.

Methods: A cetuximab-resistant CACO2 cellular model was established, and its transcriptome variations were detected by microarray. Meanwhile, public data from the Gene Expression Omnibus and The Cancer Genome Atlas (TCGA) database were downloaded. Integrated bioinformatics analysis was applied to detect differentially expressed genes (DEGs) between the cetuximab-resistant and the cetuximab-sensitive groups. Then, we investigated correlations between DEGs and immune cell infiltration. The DEGs from bioinformatics analysis were further validated in vitro and in clinical samples.

Results: We identified 732 upregulated and 1259 downregulated DEGs in the induced cellular model. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, along with Gene Set Enrichment Analysis and Gene Set Variation Analysis, indicated the functions of the DEGs. Together with GSE59857 and GSE5841, 12 common DEGs (SATB-2, AKR1B10, ADH1A, ADH1C, MYB, ATP10B, CDX-2, FAR2, EPHB2, SLC26A3, ORP-1, VAV3) were identified and their predictive values of cetuximab treatment were validated in GSE56386. In online Genomics of Drug Sensitivity in Cancer (GDSC) database, nine of twelve DEGs were recognized in the protein-protein (PPI) network. Based on the transcriptome profiles of CRC samples in TCGA and using Tumor Immune Estimation Resource Version 2.0, we bioinformatically determined that SATB-2, ORP-1, MYB, and CDX-2 expressions were associated with intensive infiltration of B cell, CD4⁺ T cell, CD8⁺ T cell and macrophage, which was then validated the correlation in clinical samples by immunohistochemistry. We found that SATB-2, ORP-1, MYB, and CDX-2 were downregulated in vitro with cetuximab treatment. Clinically, patients with advanced CRC and high ORP-1 expression exhibited a longer progression-free survival time when they were treated with anti-EGFR therapy than those with low ORP-1 expression.

Conclusions: SATB-2, ORP-1, MYB, and CDX-2 were related to cetuximab sensitivity as well as enhanced tumor immune cell infiltration in patients with CRC.

Keywords: Anti‐epidermal growth factor receptor therapy; Colorectal cancer; Drug resistance; Transcriptional alterations; Tumor immune cell infiltration.

Fig. 1

Flow chart of the study

Fig. 2

Identification and enrichment analysis of DEGs related to anti-EGFR antibody resistance in CACO2. a Establishment of a cetuximab-resistant CACO2 cell line (CACO2-CR) from its parental sensitive cell line (CACO2-CS) and validation of the resistance using cell viability tests. b Comparison of total gene expression between duplicate samples from the innate sensitive group (blue) and adaptive resistant (red) group. c Principal component analysis results suggested favorable reproducibility between the sensitive (blue) and resistant (red) groups. d The volcanic map reveals the distributions of downregulated (blue) and upregulated (orange) genes in the resistant group versus the sensitive group. Top 10 of DEGs between two groups were shown. e The heat map indicates the upregulated (red) and downregulated (blue) DEGs between the sensitive (blue) and resistant (red) groups. Each column is a sample and each row is a gene. Top 10 of DEGs between two groups were shown. f GO biological process term enrichment analysis of DEGs (left) and their interrelationships (right). g GO molecular function term enrichment analysis of DEGs (left) and their interrelationships (right). h GO cellular component term enrichment analysis of DEGs (left) and their interrelationships (right). i KEGG pathway enrichment analysis of DEGs (left) and their interrelationships (right). DEGs: Differentially expressed genes. GO: Gene Ontology. KEGG: Kyoto Encyclopedia of Genes and Genomes

Fig. 3

GSEA and GSVA of DEGs. a-h GSEA of downregulated and (i) upregulated DEGs involved in biological states and processes in the resistant group, compared with those in the sensitive group. j Volcanic diagram of the GSVA results for the DEGs, which were enriched in the resistant group or the sensitive group (highlighted orange points and blue points, respectively). DEGs: Differentially expressed genes. GSEA: Gene Set Enrichment Analysis. GSVA: Gene Set Variation Analysis

Fig. 4

Identification of common DEGs and construction of PPI network. a, b Identification of DEGs between the resistant (red) and sensitive (blue) groups from the GSE5851 and GSE59857 datasets, illustrated by a heat map. Each column is a sample and each row is a gene. c The venn diagram shows the intersection of DEGs among GSE5851, GSE59857 and CACO2-CR cellular model. d The Network diagram illustrates the interactions of common DEGs. e The PPI network including 12 DEGs and recognized cetuximab resistance-related genes from the Genomics of Drug Sensitivity in Cancer database. DEGs: Differentially expressed genes. PPI: protein-protein interaction network

Fig. 5

Comparison of the expression levels of core DEGs in GSE56386. a SATB-2, c MYB, d CDX-2, e SLC26A3, and g FAR2 were significantly downregulated and (l)AKR1B10 was apparently upregulated among cetuximab non-responders compared with cetuximab responders. No significant differences were seen in (b)ORP-1, f EPHB2, h VAV3, i ATP10B, j ADH1A, k ADH1C. DEGs: Differentially expressed genes

Fig. 6

Association between the expression level of common DEGs and immune cell infiltration. The relationship between expression levels of (a) SATB-2, (b) ORP-1, (c) MYB, and (d) CDX-2 and tumor purity, infiltrating B cells, CD4⁺ T cells, CD8⁺ T cells, and macrophages was investigated by the online tool Tumor Immune Estimation Resource Version. DEGs: Differentially expressed genes. TIMER: Tumor Immune Estimation Resource Version

Fig. 7

Relationship between core DEGs and tumor-infiltrating immune cell markers in CRC. a The representative immunohistochemical staining images of SATB-2, ORP-1, MYB, CDX-2, CD19, CD4, CD8, and CD68. b Spearman analysis results indicated that core DEGs were positively associated with tumor-infiltrating immune cell markers. DEGs: Differentially expressed genes. CRC: Colorectal cancer

Fig. 8

Exploring the expression of core DEGs in vitro and their prognostic roles in CRC. a SATB-2, ORP-1, MYB, and CDX-2 were downregulated after cetuximab treatment. b Patients with high expression levels of core DEGs in primary tumors exhibited a tendency to experience a longer progression-free survival time. **, p < 0.01, ***, p < 0.001, ns, p > 0.05. DEGs: Differentially expressed genes. CRC: Colorectal cancer