Transcriptome Profiling Associated with CARD11 Overexpression in Colorectal Cancer Implicates a Potential Role for Tumor Immune Microenvironment and Cancer Pathways Modulation via NF-κB

Abstract

The immune system plays a critical role in inflammation by initiating responses to infections or tissue damage. The nuclear factor-κB (NF-κB) pathway plays a key role in inflammation and innate immunity, as well as other cellular activities. Dysregulation of this well-choreographed pathway has been implicated in various diseases, including cancer. CARD11 is a key molecule in the BCL10-MALT1 complex, which is involved in transducing the signal downstream of the NF-κB pathway. This study aims to elucidate how CARD11 overexpression exacerbates the prognosis of colorectal cancer (CRC). To identify the cellular pathways influenced by CARD11, transcriptomic analysis in both CRC cell lines and patients was carried out on CARD11- overexpressed HCT-116 and HT-29 CRC cell lines alongside empty vector-transfected cell lines. Furthermore, a comparison of transcriptomic data from adenoma and carcinoma CRC patients with low- (CARD11-) and high-(CARD11+) CARD11 expression was carried out. Whole transcriptomics and bioinformatics analysis results indicate that CARD11 appears to play a key role in CRC progression. Absolute GSEA (absGSEA) on HCT-116 transcriptomics data revealed that CARD11 overexpression promotes cell growth and tissue remodeling and enhances immune response. Key genes co-expressed with CARD11, such as EP300, KDM5A, HIF1A, NFKBIZ, and DUSP1, were identified as mediators of these processes. In the HT-29 cell line, CARD11 overexpression activated pathways involved in chemotaxis and extracellular matrix (ECM) organization, marked by IL1RN, MDK, SPP1, and chemokines like CXCL1, CXCL3, and CCL22, which were shown to contribute to the more invasive stage of CRC. In patient samples, adenoma patients exhibited increased expression of genes associated with the tumor immune microenvironment, such as IL6ST, collagen family members, and CRC transition markers, such as GLI3 and PIEZO2, in CARD11+ adenoma patients. Carcinoma patients showed a dramatic increase in the expression of MAPK8IP2 in CARD11+ carcinoma patients alongside other cancer-related genes, including EMB, EPHB6, and CPEB4.

Keywords: CARD11; GSEA; NF-κB pathway; colorectal cancer; tumor immune microenvironment.

Figure 1

Kaplan–Meier overall survival plot for colorectal cancer patients based on CARD11 expression. The analysis ran on 1167 patients; 598 patients had high expression, and 569 patients had low expression of CARD11 (https://kmplot.com (accessed on 17 September 2024)).

Figure 2

Validation of successful overexpression of CARD11 in the CRC (HCT-116 and HT-29) cell lines. (A) CARD11 mRNA expression in empty pcDNA3 vector or pcDNA3-CARD11 transfected HCT-116 and HT-29 cells, as determined by qRT-PCR. Data were normalized to the expression of the housekeeping gene and the 18S rRNA gene, and fold expressions were plotted relative to expression in the empty vector-transfected (control). These data represent the mean ± SD of three independent experiments. *** p < 0.001. (B) Relative CARD11 protein expression was determined with a Western blot. Blots were probed with anti-β-Actin antibody as control, confirming equal loading across the lanes.

Figure 3

CARD11 enhances NF-κB activation in both HCT-116 and HT-29 cell lines. The cells were co-transfected with NF-κB-luc vector (with NF-κB luciferase reporter gene-p65) or CARD11 plasmid alone or together. LPS induction was undertaken for 6 h. NF-κB activation was measured in triplicate experiments and recorded as a fold increase in the vector control.

Figure 4

Volcano plots of differentially expressed genes. Genes that are expressed significantly higher in either empty vector- or CARD11-transfected cell line based on log2-fold change p < 0.05 are highlighted by red dots, p > 0.05 are highlighted by green dots (Log2FC NS), unchanged transcripts are demarcated as grey (NS). The red arrows indicate that the CARD11 expression is significantly upregulated only in the CARD11-transfected cell lines.

Figure 5

Histogram of the selected leading-edge genes based on frequency in CARD11-transfected vs. empty vector-transfected HCT-116 (A), HT-29 (B) cell lines.

Figure 6

(A) Significant enrichment pathways based on frequency in CARD11-transfected vs. empty vector-transfected HCT-116 cell line. Red arrows show the interest-enriched pathways in HCT-116. (B) Leading edge analysis showed that 82 core genes accounted for the significant enrichment in the CARD11-transfected HCT-116 cell line (p < 0.001). The top 15 leading-edge core genes are shown; the frequently found ones are indicated in red.

Figure 7

(A) Significant enrichment pathways based on frequency in a CARD11-transfected vs. empty vector-transfected HT-29 cell line. The red arrows show the significant pathways involved in cancer progression (B) Leading edge analysis showed that 20 core genes accounted for the significant enrichment in the CARD11-transfected HT-29 cell line (p < 0.001). The top 15 leading-edge core genes are shown; the frequently found ones are indicated in red.

Figure 8

Volcano plots of differentially expressed genes. Genes that were expressed significantly higher in either CARD11– and CARD11+ patients based on log2 fold change p < 0.05 are highlighted by red dots, p > 0.05 are highlighted by green dots (Log2FC NS), unchanged transcripts are demarcated as grey (NS).

Figure 9

Histogram of the selected leading-edge genes based on frequency in CARD11− vs. CARD11+ adenoma (A) and carcinoma (B) patient samples.

Figure 10

Significant enrichment pathways based on frequency in CARD11− vs. CARD11+ in adenoma.

Figure 11

(A) Significant enrichment pathways based on frequency in CARD11− vs. CARD11+ in carcinoma. (B) Leading edge analysis showed that there was a significant gene enrichment in the cellular component morphogenesis pathway in CARD11-positive carcinoma patients (p = 0.0019). The top 15 leading-edge core genes are shown; the frequently found ones are indicated in red.

Figure 12

Kaplan–Meier overall survival plot for colorectal cancer patients based on IL6ST (A), GLI3 (B), and MAPK8IP2 (JIP2) (C) expression (https://kmplot.com (accessed on 17 September 2024)).

Figure 13

Comparison of CIBERSORTX immune cell fractions between CARD11-transfected vs. empty vector-transfected for both cell lines, as well as CARD11− versus CARD11+ for both tissue samples.

Figure 14

Flowchart outlining the steps of the bioinformatics approach used to identify differentially expressed genes in CARD11-transfected to empty vector-transfected cell lines and CARD11– to CARD11+ in patient samples. The figure was created using BioRender.com (accessed on 17 September 2024).