Ferroptosis-associated gene CISD2 suppresses colon cancer development by regulating tumor immune microenvironment

Background

In 2020, colon cancer is projected to constitute approximately 6% of all cancer cases, with an expected 2.3 million new occurrences and nearly 1.8 million cancer-related fatalities (Sung et al., 2021). Colon cancer development is a heterogeneous malady stemming from the alteration of multiple pathways, culminating in homeostatic imbalance and subsequent tumor growth (Vogelstein et al., 1988). Due to the lack of suitable early diagnostic tools, roughly 20% of patients are diagnosed with metastatic colon cancer (Ciardiello et al., 2022). Although its pathogenesis remains enigmatic, colon cancer and its complications have deleterious impacts on individuals’ quality of life and life expectancy.

In recent years, substantial advancements have been made in the comprehension of molecular markers and the development of innovative treatment strategies for COAD (Lee & Chan, 2011; Nowak & Hornick, 2016; Punt, Koopman & Vermeulen, 2017; Gonzalez, Washington & Shi, 2017). These breakthroughs have led to a paradigm shift in the management of COAD, with an increasing emphasis on molecular profiling and personalized medicine (Amir-Aslani & Mangematin, 2010). These molecular markers have not only enhanced the prognostic accuracy but have also informed the development of targeted therapies. Some well-established emerging treatment strategies include immune checkpoint inhibitors, such as PD-1/PD-L1 and CTLA-4 blockers, as well as targeted therapies against EGFR, VEGF, and other signaling pathways (Ciardiello et al., 2006; Seidel, Otsuka & Kabashima, 2018; Rotte, 2019; Le et al., 2021). Furthermore, recent research has underscored the significance of ferroptosis and the tumor immune microenvironment in COAD (Xia et al., 2019). A deeper understanding of the interplay between ferroptosis and the immune microenvironment could pave the way for the development of groundbreaking therapeutic approaches, ultimately improving the prognosis for COAD patients.

Ferroptosis, a process necessitating metallic iron, phospholipids containing polyunsaturated fatty acid chains (PUFA-PLs), and reactive oxygen species (ROS), facilitates programmed cell death (Jiang, Stockwell & Conrad, 2021). Intricately connected to internal environmental homeostasis, ferroptosis partakes in diverse tumor progression stages and activates various tumor suppressors, such as p53 and BRCA1-associated protein 1 (BAP1) (Jiang et al., 2015; Zhang et al., 2018; Chen et al., 2021; Zhao, Yang & Yang, 2022). Oncogenes or oncogene signaling-mediated ferroptosis escape incites tumor initiation, progression, metastasis, and treatment resistance (Ubellacker et al., 2020; Yi et al., 2020). Accordingly, ferroptosis-related genes may play a major role in maintaining tumor microenvironment homeostasis. Consequently, ferroptosis-related genes likely play a pivotal role in maintaining tumor microenvironment equilibrium. Prostaglandin E2 (PGE2) impedes CD8+ T cell-mediated immune responses reliant on conventional type 1 dendritic cells (cDC1) and curtails cDC1 infiltration into tumor sites by inhibiting natural killer (NK) cell production of chemokine (C-C motif) ligand 5 (CCL5) and chemokine (C motif) ligand 1 (XCL1) (Zelenay et al., 2015; Böttcher et al., 2018). Ferroptosis has been associated with elevated prostaglandin-endoperoxide synthase 2 (PTGS2) expression and PGE2 release, promoting tumorigenesis when ferroptotic cells reach sufficient numbers (Yang et al., 2014, p. 4).

As malignant cells proliferate, they enlist adjacent nontransformed cells, ultimately forming the tumor microenvironment through the release of various intercellular communication factors, such as cytokines, chemokines, and vesicles (Balkwill, Capasso & Hagemann, 2012). Although metastasis is the primary cause of death in colon cancer patients, no universal mutation is culpable (Jones et al., 2008; Mlecnik et al., 2016). The malignant microenvironment, however, exhibits features like transforming growth factor (TGF)-β, which significantly augments the prevalence of cancer-associated fibroblasts (CAFs)-induced tumor-initiating cells. TGF-β signaling inhibitors can obstruct cross-signaling between cancer cells and the microenvironment, thereby arresting disease progression (Calon et al., 2015). Diminished type 1 T helper cell (TH1) activity or impaired immune cytotoxicity may also portend unfavorable outcomes in colon cancer patients (Galon et al., 2006; Mlecnik et al., 2016). Consequently, the tumor microenvironment, encompassing the immune microenvironment, might contribute more substantially to tumor prognosis than mutations alone.

Cancer immunotherapy, mainly employing immune checkpoint inhibitors and immune cell recombination therapy, has substantially improved patient survival outcomes, emphasizing the crucial significance of immunological and related biomarkers (Sharma & Allison, 2015; Hargadon, Johnson & Williams, 2018; Xu et al., 2021; Sun et al., 2022b; Mei, Li & Kang, 2022; Chen et al., 2022a; Luo et al., 2022). Nevertheless, immunotherapy’s efficacy remains relatively low in cancer patients. The tumor microenvironment comprises cancer cells and infiltrating immune cells, forming a complex and dynamic landscape. Tumor-infiltrating immune cells exhibit numerous connections with ferroptosis, a modality of cancer cell death. This study investigates the role of ferroptosis in maintaining the immune microenvironmental equilibrium in colon cancer. By exploring novel targets, colon cancer can be diagnosed and treated at earlier stages.

Materials and Methods

Results

Ferroptosis and colon cancer associations uncovered through single-cell analysis

The flowchart of this study is shown in Fig. 1. Upon integrating four samples from the GSE110009 dataset through cell clustering (Fig. 2A), cell annotation revealed eight distinct subgroups: mast cells, natural killer cells, T-cells, macrophages, plasma-cells, B-cells, dendritic-cells, and cancer-cells (Figs. 2B and 2C). Among 911 colon cancer marker genes, 19 were found to be associated with ferroptosis (Fig. 2D). Further analysis of the 18 ferroptosis-colon cancer co-associated genes, excluding ATP5MC3, revealed significant correlations (Fig. 2E) and protein-protein interactions (Fig. S1B).

Exploration of ferroptosis-related genes

Following the integration of TCGA and GTEx data and the removal of batch effects (Figs. S1C and S1D), PCA validation confirmed data integration (Figs. S1E and S1F). In COAD pathogenesis, CEBPG, VDAC2, and HNF4A carried high mutational loads and potentially played crucial roles (Fig. 3A). GO enrichment analysis suggested significant associations between ferroptosis-colon cancer co-related gene functions and pathways such as oxidative stress response, toxic substance response, and DNA-binding transcription factor binding (Fig. 3B). Additionally, KEGG enrichment analysis highlighted Glutathione metabolism and Biosynthesis of unsaturated fatty acids pathways (Fig. 3C). Notably, only three genes were statistically significant in the Ferroptosis-Colon Cancer Common Associated Gene Difference Analysis (Fig. 3D). The remaining 15 ferroptosis genes were suggested to play important roles in COAD, and a one-way COX regression analysis showed that only CISD2 had a statistically significant impact on prognosis (Fig. 3E).

Immune microenvironment examination

In colorectal cancer samples, macrophages M0 and M1 displayed greater infiltration compared to normal samples, aligning with general findings (Fig. 4A). Colorectal cancer samples also exhibited increased infiltration of mast cells activated, neutrophils, NK cell resting, T cells CD4 memory activated, and T cells follicular helper. Conversely, CD8 T cells and NK cells activated, which are more effective in killing cancer cells, demonstrated reduced infiltration in colon cancer, potentially contributing to progression and poor prognosis. Colon cancer samples also expressed more immune checkpoints, possibly leading to increased immune escape (Fig. 4B). Cibersort results revealed that CISD2 positively correlated with T cells memory activated (p < 0.001) and negatively correlated with T cells regulatory Tregs and Plasma cells (p < 0.001) (Fig. 4C). CISD2 also showed positive correlations with B cells, neutrophils, macrophages, CD8 T cells, and dendritic cells (p < 0.05) (Fig. 4D), indicating a significant relationship between CISD2 and immune response.

Discrepancies in CISD2 expression

CISD2 expression levels reveal notable distinctions between high and low expression TCGA colon cancer samples, examined through comparative analysis (Fig. 5A). Clinical stratification demonstrates a higher prevalence of CISD2 at advanced T-stages and initial N-stages (p < 0.05) (Figs. 5B–5G). The ROC curve suggests superior diagnostic efficacy of CISD2 over CISD1 (Fig. 5H). Consequently, CISD2 is considered a protective factor for colon cancer, exhibiting a robust immunological connection. CISD2 exhibits a strong association with CISD1 and CISD3, as illustrated by the gene interaction network (Fig. S2A). PPI identifies proteins interacting with CISD2, potentially influencing its expression and function (Fig. S2B). Elevated CISD2 expression correlates with increased protein secretion, MTORC1 signaling activation, and E2F target activation. Thus, high CISD2 expression is intimately linked to cell cycle regulation and growth metabolism. Conversely, low CISD2 expression displays increased KARS signaling DN, hedgehog signaling, myogenesis, and apical surface activation, suggesting interactions with colon cancer-related pathways (Fig. S2C). This insinuates a strong immune response association for CISD2, potentially impeding colon cancer progression (Fig. S2D). In a pan-cancer analysis, CISD2 was highly expressed in most colon tumors (p < 0.001) (Fig. S2E).

CISD2 upregulation impedes proliferation and tumor-promoting activity in colon cancer cells, enhancing 5-FU sensitivity

To explore CISD2’s potential role in colon cancer cell proliferation and carcinogenesis, we established HCT116 cell lines stably expressing CISD2. Western blots detected CISD2 protein expression levels in transfected and control cells. CISD2 overexpression upregulated protein levels, while shCISD2-1 and shCISD2-2 downregulated them (Fig. 6A). OD-CISD2 cells exhibited significantly elevated CISD2 mRNA levels compared to shCISD2 transfected cells, as determined by PCR (p < 0.001, t-test) (Fig. 6B). Assessing CISD2’s impact on colon cancer cell invasiveness, we conducted a wound healing assay. Control cells nearly healed 48 h post-scratch creation, while CISD2 cells did not. Overexpression CISD2 groups displayed fewer healing areas compared to control groups (p = 0.012, t-test) (Fig. 6C). The invasive propensity of colon cancer cells is curtailed by the augmentation of CISD2 expression. The CCK-8 assay demonstrated a notable reduction in the HCT116 cell count overexpressing CISD2 (p = 0.043 vs NC, t-test) (Fig. 6D). An empty vector-transfected control group experienced increased cell growth and proliferation compared to CISD2 overexpressing cells. Notwithstanding the application of FU-5, CISD2 expression persisted in overexpressed cells (p < 0.001, t-test) and remained unaltered by FU-5. Figure 5C depicts the inhibition of scratch healing in CISD2 cells by FU-5 (p < 0.001, t-test). Additionally, Fig. 5C reveals a considerable enhancement of scratch healing following CISD2 overexpression (p = 0.011, t-test). HCT116 cells with CISD2 overexpression were also substantially reduced by FU-5 after the CCK-8 viability assay (p < 0.001, t-test) (Fig. 6D). Consequently, the upregulation of CISD2 may bolster the sensitivity of HCT116 cells to FU-5 treatment.

Oncogenic genetic variations in ferroptosis-associated genes

Ferroptosis genes SCD, CISD1, and CISD2 displayed a greater predisposition to somatic copy number deletions compared to HSPB1, HNF4A, and PRDX6 (Fig. 7A). The correlation between somatic copy number mutations and gene expression in tumors was investigated, as these mutations govern gene expression in neoplasms. The majority of tumors exhibited significant somatic copy number mutations linked to ferroptosis genes (Fig. 7B). These findings imply that copy number aberrations in ferroptosis genes are prevalent in most cancers and can influence gene expression. In most cancers, GPX2, HNF4A, and STMN1 were hypomethylated (Fig. 7C), while the majority of cancer genes exhibited hypermethylation (Fig. 7A). Promoter DNA methylation in most cancers was observed for ferroptosis-cancer co-associated genes based on this outcome. It is plausible that these co-associated genes are less regulated by methylation (Fig. 7D) due to the correlation analysis of methylation levels and gene expression. In contrast, GPX2, CA9, NQO1, and HNF4A displayed the opposite effect. Thus, these findings suggest that methylation may play a more significant role in regulating GPX2, CA9, and HNF4A gene expression than somatic copy number mutations. Mutation analysis revealed elevated mutation rates for IDH1, HNF4A, and ATF4 in LGG, UCEC, and SKCM, as well as higher mutation rates for this ferroptosis-cancer co-associated gene.

Pan-cancer immune microenvironment

In accordance with the correlation analysis of CISD2 and immune cells, CISD2 exhibits a positive association with common lymphoid progenitors, granulocyte-monocyte progenitors, neutrophils, and CD8-positive T cells, providing an extensive insight into its influence on the tumor immune landscape. Nevertheless, a majority of tumors demonstrated negative correlations between CISD2 and endothelial cells, hematopoietic stem cells, and NK T cells. Patients with elevated CISD2 expression exhibited increased infiltration of tumor-eradicating CD8-positive T cells, leading to a more favorable prognosis (Fig. 8A).

Pan-cancer GSEA enrichment investigation

A comprehensive characterization of CISD2 in 20 malignancies revealed significant associations with cancer-related pathways (WNT-beta-catenin signaling, TNFA signaling through NFKB, TGF-beta signature, etc.), and oxidative metabolism-associated pathways (xenobiotic metabolism, reactive oxygen species pathway). However, a substantial correlation with epithelial cells was observed for CISD2 (Fig. 8B). A negative association between CISD2 and epithelial-mesenchymal transition, as well as apical junctions, may impede tumor proliferation and metastasis.

Ferroptosis cancer survival gene analysis

Subsequent survival analysis of the ferroptosis-colon cancer co-associated gene served to determine patient prognosis (Fig. 9A). Heterogeneity may exist among ferroptosis-related genes across tumor types. It was discerned that the prognostic impact of CISD2 varies considerably among distinct tumors based on median expression levels. In patients with LUAD and LGG, the prognosis was markedly superior in the low CISD2 expression cohort, whereas in patients with UCEC and KIRC, a significantly improved prognosis was observed in the high CISD2 expression cohort (Figs. 9B–9E). This study unveiled the overarching prognostic implications of ferroptosis in cancer.

Discussion

Approximately two-thirds of patients with stage III colon cancer, along with certain individuals presenting high-risk stage II disease, undergo adjuvant chemotherapy to mitigate the recurrence risk following colectomy (Kannarkatt et al., 2017; Miller et al., 2022). By examining scRNA-seq samples from the GSE110009 dataset, we identified the crucial ferroptosis gene CISD2, enhancing patient survival outcomes.

CISD2, an enduring gene situated on human chromosome 4q, plays a significant role. Mice with Cisd2 knockout exhibit mitochondrial rupture, dysfunction, and autophagic cell death (Chen et al., 2009). Elevated CISD2 and CISD1 proteins have been observed in human epithelial breast cancer cells; inhibiting CISD2 and CISD1 expression results in increased iron accumulation within mitochondria, autophagy activation, and significant reduction of cell proliferation and tumor growth (Sohn et al., 2013). CISD2 modulates ER Ca²⁺ uptake and Ca²⁺ pump activity to maintain intracellular Ca²⁺ homeostasis through interactions with Serca2b. CISD2 deficiency disrupts calcium homeostasis, endoplasmic reticulum stress, NAFLD, and NASH. CISD2 hemizygous deletions have been identified in numerous human HCCs (Shen et al., 2017). Our analysis of CISD2 gene and copy number variation in colon cancer indicates that CISD2 significantly influences clinical prognosis and is more prone to undergo somatic copy number deletion.

In this investigation, CISD2 was determined to be essential for maintaining immune microenvironment homeostasis in colon cancer. CISD2 infiltration of T cells in colorectal cancer samples exhibited a positive correlation with CISD2 levels. According to the relationship between CISD2 and immune cells, colon cancer patients with high CISD2 expression and CD8+ T cells experienced improved outcomes. Furthermore, CISD2 expression was intimately linked to glioma progression and M2 polarization (Zhang et al., 2022). Consequently, CISD2 is hypothesized to impede tumor growth by promoting immune cell infiltration and preserving endoplasmic reticulum calcium homeostasis.

Moreover, we discerned an inverse correlation between the methylation status of CISD2 and two CpG sites within COAD specimens. It is well-established that aberrant DNA methylation, including hypermethylation of CpG islands in gene promoters, contributes to the silencing of tumor suppressor genes in cancer (Baylin, 2001; Esteller, 2002, 2007). This negative association suggests that heightened methylation of these specific CpG sites may lead to downregulation of CISD2 in COAD samples, potentially impacting the tumor microenvironment and patient prognosis. Nevertheless, further functional investigations are warranted to validate this hypothesis and elucidate the precise mechanisms involved.

Through single-cell and single-gene analyses of colon cancer tumor samples, we identified a correlation between CISD2 and clinical outcomes, with elevated CISD2 expression conferring a protective effect against colon cancer and suppressing the immune system. In this study, we meticulously examined the ramifications of differential CISD2 expression and genetic alterations on the clinical prognosis and associated mechanisms for colon cancer patients at both cellular and genetic levels, which is of paramount importance for investigating the iron regulatory mechanism in colon cancer. However, the specific mechanisms remain elusive, and there is a scarcity of clinical and experimental data. And potential confounding variables, such as patient age, gender, and tumor stage, may have influenced the observed associations between CISD2 expression and clinical outcomes. While we attempted to control for these variables in our analyses, residual confounding may still be present. Additionally, our findings are based on the analysis of existing databases and datasets, which may introduce biases related to data quality, sample selection, and study design. Future studies should incorporate independent cohorts and experimental validation to confirm our observations and further investigate the functional implications of CISD2 methylation and expression in COAD.

Conclusion

By modulating the cell cycle and activating the immune system, CISD2 demonstrates statistical significance for prognosis in colon cancer. Consequently, CISD2 plays a crucial role in controlling the tumor microenvironment by regulating tumor homeostasis. The negative correlation between CISD2 methylation status and 2 CpG sites in COAD samples suggests a potential functional relevance in the downregulation of CISD2, but further studies are necessary to fully understand the underlying mechanisms and confirm these findings.

Supplemental Information