Gene expression profiling identifies ferroptosis-related genes and pathways in human colon cancers cell lines

Abstract

Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide and the second leading cause of cancer-related deaths. A major challenge in CRC treatment is drug resistance, which limits the efficacy of conventional therapies. Ferroptosis, an iron-dependent form of regulated cell death driven by the accumulation of reactive oxygen species (ROS), has emerged as a promising therapeutic strategy. Erastin (ER), a small-molecule compound, induces ferroptosis through ROS accumulation.

Methods: We performed microarray gene expression analysis on two CRC cell lines, HCT116 and HT-29, to examine the transcriptional response to ER exposure and identify differentially expressed genes and pathways involved in ER-induced ferroptosis.

Results: Our gene expression analysis revealed distinct transcriptional profiles between the two cell lines, and 26 transcripts commonly enriched in response to ER treatment were identified in both HCT116 and HT-29 cells. Notably, several of these genes-including ASNS, PCK2, CHAC1, and DDIT4-were significantly enriched, suggesting a conserved ferroptotic response. The induction of these genes was further confirmed in an additional CRC cell line, DLD-1. Interestingly, SOD1 and NQO1 genes, involved in oxidative stress response, were significantly upregulated by ER in HCT116 cells.

Conclusion: Our findings highlight ASNS, CHAC1, PCK2, DDIT4, and ATF3/4 as potential biomarkers for ferroptosis in CRC. Monitoring the expression of these genes may help identify patients who are responsive to ferroptosis inducers and facilitate the development of personalized treatment strategies.

Keywords: biomarker genes; colon cancer; erastin; ferroptosis; pathway analysis.

FIGURE 1

Cytotoxicity of Erastin in HT-29 and HCT116 cells as determined by CellTiterGlO (Promega) cytotoxicity assay following 72 h ER treatment.

FIGURE 2

Boxplots of (A) PC#1 and (B) PC#2 from the PCA analysis of all HT-29 samples broken down by time (control, treated with ER at 4 h, treated with ER at 24 h).

FIGURE 3

Boxplots of (A) PC#1 and (B) PC#2 from the PCA analysis of all HCT116 samples broken down by time (control, treated with ER at 4 h, treated with ER at 24 h).

FIGURE 4

Venn diagrams of overlay of differentially expressed transcripts (DETs) at 0, 4 and 24 following ER treatment of HT-29 and HCT116 cells.

FIGURE 5

Heat map showing hierarchical clustering DETs of HT-29 [(A) 600 DET’s] and HCT116 [(B) 992 DET’s] following treatment with Erastin at 24 h. Yellow represents upregulated DET’s, and blue indicates downregulated DET’s.

FIGURE 6

A diagrammatic presentation of common pathways modulated in both HT-29 and HCT116 cells by Erastin at 24 h exposure.

FIGURE 7

Landscape Plot depicting the -log10p-values of each hallmark (n = 50) pathway across the four exposure groups. Pathways are ordered in the circular view based on hierarchal clustering of the gene membership (i.e., gene set similarity).

FIGURE 8

RT-PCR results for colon cancer cells following 24 h of Erastin treatment. RNA was isolated, purified, and subjected to RT-PCR as described in the Methods section. Gene expression was normalized to β-actin, and expression levels in each treatment group were compared with those of time-matched controls. Data are presented as fold change relative to β-actin. *, **, and *** indicate p < 0.05, p < 0.005, and p < 0.001, respectively.