KCNC3 as a prognostic indicator and a predictive marker for immunotherapy in colorectal cancer

Abstract

Background: Colorectal cancer (CRC) is a heterogeneous disease that is associated with several genetic or somatic mutations. Cancer immunotherapy has become a novel and revolutionary method of treatment for patients with advanced tumors. However, effective biomarkers that can reflect the response of CRC patients to immunotherapy have still not been identified. Our study aimed to explore the expression and functional role of KCNC3 in CRC.

Methods: The correlation between KCNC3 expression levels and CRC progression was explored and validated using data from The Cancer Genome Atlas (TCGA) and patient's samples from the Affiliated Hospital of Nantong University. Univariate and multivariate Cox regression models were developed to determine the predictive value of KCNC3 on the prognosis and immune activation of patients with CRC. We predicted the immunotherapy response in both the high and low KCNC3 expression subgroups. Finally, we confirmed that KCNC3 promotes the proliferation and invasion of colon cancer cells.

Results: In this study, data from TCGA database and clinical patient parameters showed that high KCNC3 expression was associated with tumor immune infiltration and poor prognosis of CRC. KCNC3 expression was positively correlated with the infiltration levels of CD4⁺ cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs), which contributed to the formation of an immunosuppressive tumor microenvironment (TME). The high expression of KCNC3 was accompanied by the upregulation of immune checkpoint molecules, including PDCD1, LAG3, FOXP3, and CTLA4, which stimulated tumor cells to evade immune surveillance. In vitro experiment, KCNC3 knockdown inhibited the growth and metastasis of SW1116 cells.

Conclusions: This study demonstrated that the high expression of KCNC3 contributes to the growth and invasion of CRC and confers with immunosuppressive microenvironment that can promote tumor progression and can be used to predict the poor clinical outcome of CRC patients.

Keywords: KCNC3; colorectal cancer (CRC); prognosis; tumor immune microenvironment.

Figure 1

The expression and prognosis analysis of KCNC3 in CRC. (A) The expression level of KCNC3 in CRC tissues was significantly higher than that in normal tissues. (B) mIHC analysis of the KCNC3 expression between CRC tissues and normal tissues. (C) GEPIA database and clinical data were used to analyze the OS. (D,E) TNM stage and survival functions were analyzed according to the expression level of KCNC3 in CRC, all P<0.001. *, P<0.001. CRC, colorectal cancer; GEPIA, Gene Expression Profiling Interactive Analysis; HR, hazard ratio; mIHC, multiplex immunohistochemistry; OS, overall survival; TNM, tumor-node-metastasis; TPM, transcripts per million.

Figure 2

Network and enrichment analyses. (A,B) GO and KEGG analysis of KCNC3 in CRC. (C) The top 20 interacting genes based on PPI networks were screened out. (D-H) High KCNC3 expression is associated with ECM-receptor interaction, chemokine signaling pathway, cell adhesion molecules, Ras signaling pathway and PD-L1 expression and PD-1 checkpoint pathway in cancer. BP, biological process; CC, cellular component; CRC, colorectal cancer; ECM, extracellular matrix; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; PPI, protein-protein interaction.

Figure 3

ImmuneScore is correlated with the high KCNC3 expression. (A) Analysis of the difference of the immune score between the high and low KCNC3 expression. (B) TME score in high- and low-immune cell score, stroma cell score, and estimate score between the high and low KCNC3 expression. (C) Analysis of the difference of the immune cells between the high and low KCNC3 expression. *, P<0.05; **, P<0.01. (D) mIHC analysis of the CD3 and CD4 expression between CRC tissues and normal tissues. CK, cytokeratin; CRC, colorectal cancer; mIHC, multiplex immunohistochemistry; NK, natural killer; TME, tumor microenvironment.

Figure 4

KCNC3 is associated with immune infiltration and immune activation in CRC. (A) Heatmap showing KCNC3-associated relative abundance of 28 immune cells in CRC. (B) The correlation between the ssGSEA scores of immune cells and the expression of KCNC3 in CRC. (C,D) Correlations between KCNC3 expression and immune cells infiltration levels in TIMER. (E,F) KCNC3 expression was correlated with Treg, macrophage, and MDSC in the TISIDB database. (G) KCNC3 expression was correlated with PDCD1, FOXP3, and CTLA4. ***, P<0.001. aDC, activated dendritic cell; COAD, colon adenocarcinoma; CRC, colorectal cancer; CTLA4, cytotoxic T lymphocyte-associated protein 4; DC, dendritic cell; iDC, immature dendritic cell; MDSC, myeloid-derived suppressor cell; NK, natural killer; pDC, plasmacytoid dendritic cell; READ, rectum adenocarcinoma; ssGSEA, single sample gene set enrichment analysis; Tcm, T central memory cell; TFH, T follicular helper cell; Tgd, T gamma delta cell; Th, T helper; TIMER, Tumor Immune Estimation Resource; TISIDB, tumor-immune system interaction database; TPM, transcripts per million; Treg, regulatory T cell.

Figure 5

The association of KCNC3 with immune molecules was analyzed using mIHC and IHC. (A-D) The correlation between KCNC3 expression levels and CTLA4, LAG3 and CD3⁺ CD4⁺ tumor in CRC patients. (E) Fluorescence-based mIHC was developed to visualize CK, CTLA4, and KCNC3 simultaneously on the same tissue slide. (F) Immunohistochemistry showed LAG3 was high expressed in CRC patients. CK, cytokeratin; CRC, colorectal cancer; CTLA4, cytotoxic T lymphocyte-associated protein 4; IHC, immunohistochemistry; LAG3, lymphocyte activation gene 3; mIHC, multiplex immunohistochemistry.

Figure 6

KCNC3 promotes proliferation and invasion of colon cancer cells. (A) qRT-PCR analysis of KCNC3 mRNA expression in NCM460 and SW1116 cells. (B) Western blot confirmation of KCNC3 in NCM460 and SW1116 cells. (C) In SW1116 cell lines transfected with siRNA, the expression level of KCNC3 was decreased compared to control cells transfected with a negative control plasmid. (D,E) CCK-8 and EdU experiment to verify the effect of KCNC3 knockdown on cell viability and proliferation. (F) The effect of KCNC3 knockdown on cell migration by the wound-healing assay. *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; mRNA, messenger RNA; NC, negative control; OD, optical density; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; si, small interfering RNA.