CEBPB dampens the cuproptosis sensitivity of colorectal cancer cells by facilitating the PI3K/AKT/mTOR signaling pathway

Abstract

Background:

Cuproptosis is a novel pathway that differs from other forms of cell death and has been confirmed to be applicable for predicting tumor prognosis and clinical treatment response. However, the mechanism underlying the resistance of colorectal cancer (CRC) to cuproptosis at the molecular level has not been elucidated.

Methods:

Using bioinformatics analysis, the expression of CCAAT/enhancer-binding protein beta (CEBPB) in CRC tissues and its enrichment in biological processes were detected. Quantitative reverse transcription polymerase chain reaction and western blotting (WB) were employed to test the expression of CEBPB in CRC cells. WB was utilized to assess the levels of proteins related to cuproptosis and the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. The MTT assay was used to test cell viability. Cell proliferation was assessed by a colony formation assay. Transwell assays were used to measure cell migration and invasion ability. DLAT-aggregate formation was determined by immunofluorescence.

Results:

CEBPB was highly upregulated in CRC cells to enhance cell viability, proliferation, migration, and invasion. CEBPB was strongly implicated in copper ion homeostasis and the mTOR signaling pathway in CRC. In a CRC cuproptosis cell model, rescue experiments revealed that a PI3K/AKT/mTOR pathway inhibitor attenuated the promoting effect of CEBPB overexpression on the PI3K/AKT/mTOR pathway and rescued the sensitivity of CRC to cuproptosis.

Conclusion:

This work demonstrated that CEBPB can activate the PI3K/AKT/mTOR signaling pathway, thereby decreasing the sensitivity of CRC to cuproptosis. These data suggested that targeting CEBPB or the PI3K/AKT/mTOR pathway may enhance the sensitivity of CRC patients to cuproptosis, providing a combined therapeutic strategy for cuproptosis-induced therapy.

INTRODUCTION

Colorectal cancer (CRC) ranks as the third most diagnosed cancer globally and the second most prevalent cause of cancer-related deaths worldwide. CRC was slightly less common than lung cancer in 2020, accounting for 10% of all cancer cases and 9.4% of cancer-related deaths worldwide.[1] Based on projections for population growth, aging, and human development, the number of CRC cases will reach 3.2 million by 2040, with China leading the world (increasing from 555,000 to 912,000).[2] Due to the heterogeneity of CRC,[3] different subtypes of CRC have been identified that can impact patient prognosis and treatment response. The currently defined CRC subtypes still cannot accurately describe the treatment response and patient survival rates for all clinical cases,[2] necessitating further research to elucidate the mechanism of CRC more comprehensively.

Tsvetkov et al.[4] reported the mechanism of copper-induced cell death in 2022 and named it cuproptosis, which is the excessive accumulation of copper ions, leading to abnormal aggregation of sulfur-containing proteins and interfering with mitochondrial respiratory-related Fe-S cluster proteins, causing a protein toxicity stress response and resulting in cell death. They also noted that in addition to copper chelating agents, apoptosis inhibitors, necroptosis inhibitors, reactive oxygen species-induced cell death inhibitors, and ferroptosis inhibitors could not rescue copper-induced cell death, thus proving that cell death induced by copper ion carriers is unrelated to known cell death modes (ferroptosis, pyroptosis, etc.).[4] In tumor progression, cuproptosis may be involved in suppressing cancer cell proliferation and metastasis. For instance, the related stimulation of betel alkaloids may repress cuproptosis in oral squamous cell carcinoma patients who consume betel nuts, thereby increasing the survival of cancer-associated fibroblasts (CAFs).[5] Renal clear cell carcinomas that undergo cuproptosis exhibit reduced angiogenesis and are more sensitive to sunitinib and sorafenib.[6] With the deepening understanding of cuproptosis, cuproptosis inducers that can be applied in targeted therapy have emerged. Elesclomol can deliver Cu to mitochondria, and excessive Cu facilitates the instability of Fe-S cluster proteins and the aggregation of lipids, causing protein toxicity stress and inducing cell death.[4] Elesclomol, a candidate drug, has been shown to increase the therapeutic efficacy of paclitaxel in refractory or recurrent platinum-resistant primary peritoneal cancer, fallopian tube cancer, and ovarian cancer.[7] Unfortunately, cancer cells evolve during tumor development to resist cuproptosis and ensure their survival. Feng et al.[8] put forward that the high expression of SERPINE1 may suppress gastric cancer (GC) cuproptosis and the immune microenvironment through the P53, phosphatidylinositol 3-kinase/protein kinase B (PI3k/Akt), and transforming growth factor β (TGF-β) pathways. Therefore, an in-depth investigation of the mechanisms underlying the resistance of tumors to cuproptosis and the identification of potential targets to enhance tumor sensitivity to cuproptosis will aid in the use of elesclomol for the treatment of patients with chemoresistant cuproptosis.

This study revealed that high expression of CCAAT/enhancer-binding protein beta (CEBPB) facilitates the malignant progression of CRC. Bioinformatics analysis revealed that CEBPB is involved in the modulation of copper ion homeostasis and the mammalian target of rapamycin (mTOR) signaling pathway. We confirmed that CEBPB activated the PI3K/AKT/mTOR signaling pathway and repressed the cuproptosis sensitivity of CRC cells in our CRC cuproptosis cell model. This study partially explained the mechanism of resistance to cuproptosis in CRC and suggested that enhancing tumor cuproptosis sensitivity combined with cuproptosis-induced therapy may help combat adverse outcomes in CRC patients.

MATERIALS AND METHODS

Bioinformatics method

In The Cancer Genome Atlas (TCGA)-CRC cohort, the edge R package was used for differential expression analysis of CEBPB-mRNA in 51 normal samples and 647 tumor samples. Samples in the CRC cohort were clustered into high- and low-expression groups, with the median expression of CEBPB in CRC as the cutoff value. We performed gene set enrichment analysis (GSEA) to investigate whether CEBPB was involved in meaningful biological processes.

Cell transfection and cultivation

Human colonic epithelial cells (NCM460), CRC cells (RKO, DLD-1, Caco-2), and human embryonic kidney cells (293T) were obtained from SUNNCELL. NCM460 and DLD-1 cells were cultured in RPMI-1640 (HyClone, USA). RKO cells were maintained in EMEM (HyClone, USA). Caco-2 and 293T cells were cultivated in DMEM (HyClone, USA). All media were supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA), 100 μg/mL streptomycin (Invitrogen, USA), and 100 U/mL penicillin. The cells were housed in a 5% CO₂ cell culture incubator at 37°C.

We procured the PLVX-puro-CEBPB expression plasmid (oe-CEBPB) and corresponding negative control from RiboBio (China) and transfected the plasmids into the corresponding cells with Lipofectamine 2000 (Thermo Fisher Scientific, USA), followed by 48 h of incubation for later experiments. We plated AML cells in six-well plates, with 20 μl of viral solution and polybrene (5 μg/ml) added 24 h later. Twenty-four hours after infection, fresh complete cell culture medium containing puromycin (2 μg/ml) was used to replace the cell culture medium containing the viral solution. A selective inhibitor of PI3K-dependent AKT phosphorylation and kinase activity, LY294002 (50 ng/ml), was chosen to inhibit the PI3K/AKT/mTOR pathway.[9]

Construction of the cuproptosis model

First, DLD-1 cells were subjected to gradient concentrations of Elesclomol-Cu (Es-Cu) (0, 100, 200, 300, 400, 500 nM) to induce cuproptosis.[10] After 24 h, an MTT assay was designed to measure cell viability, with the optimal Es-Cu concentration identified. Western blotting (WB) was carried out to measure the levels of cuproptosis-related proteins (FDX1, DLTA, and HSP70) in DLD-1 cells at this concentration, and immunofluorescence (IF) was utilized to detect the formation of DLAT aggregates.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cultured cells by utilizing TRIzol regent (Invitrogen, USA). With the use of a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA), RNA purity and concentration were ascertained. Total RNA was subsequently synthesized into cDNA using the PrimeScript™ RT reagent Kit (Takara, Japan). Next, qRT-PCR was performed with AceQ qPCR SYBR Green Master Mix (Vazyme, China) on an Applied Biosystems 7500 rapid real-time fluorescence quantitative PCR system (Thermo Fisher Scientific, USA). GAPDH was used as an endogenous control. By comparison with the 2^-ΔΔCT method, we assessed the relative gene expression. The primer sequences for amplifying CEBPB are displayed in Table 1.

Table 1.

Primers for qRT-PCR

Gene	Sequence
CEBPB	Forward Primer	5’- AAGCTGAGCGACGAGTACAA-3’
	Reverse Primer	5’- GACAGCTGCTCCACCTTCTT-3’
GAPDH	Forward Primer	5’- GGGGAGCCAAAAGGGTCATCATCT-3’
	Reverse Primer	5’- GACGCCTGCTTCACCACCTTCTTG-3’

Western blotting (WB)

Total cellular protein was extracted at 4°C using RIPA lysis buffer (Beyotime, China) supplemented with 1% protease and phosphatase inhibitors (Beyotime, China). The protein concentration was quantified by utilizing a BCA assay kit (Beyotime Biotech, China), and the sample was diluted to the same concentration. Electrophoresis was carried out by utilizing the Omni-Easy™ One-Step PAGE Gel Rapid Preparation Kit (Epizyme, China). Then, the proteins were transferred to PVDF membranes (Millipore, USA). The membrane was blocked with NcmBlot blocking buffer (Ncm Biotech, China) for 10 min. Then, we used the following primary anti-rabbit antibodies: anti-CEBPB (Abcam, UK), anti-FDX1 (Abcam, UK), anti-HSP70 (Abcam, UK), anti-p-PI3K (Abcam, UK), anti-DLAT (ABclonal, China), anti-p-AKT (Abcam, UK), anti-AKT (Abcam, UK), anti-p-mTOR (Abcam, UK), anti-mTOR (Abcam, UK), and anti-GAPDH (Abcam, UK). After the membranes were incubated with the primary antibody at 4°C overnight, they were incubated with the secondary goat anti-rabbit IgG H and L (horseradish peroxidase (HRP)-labeled) secondary antibody at room temperature for 1 h. The bands were visualized and imaged using the ChemiScope 6000 Chemiluminescence Imaging System (Clinx, China) with NcmECL Ultra (NCM Biotech, China).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

Cell viability was assessed by utilizing an MTT assay kit (Solarbio, China). The cells were plated in a 96-well plate (density: 2 × 10³ cells/well). After cell attachment, 10 μl of MTT solution (5 mg/mL) was added to each well on days 0, 1, 2, 3, and 4, and the plates were incubated at 37°C for 4 h. We then removed the supernatant and added 150 μl of DMSO. The absorbance of each well was determined at 570 nm using a microplate reader.

Colony formation assay

At a density of 300 cells/well, we plated the cells in a 12-well plate and cultured them for 2 weeks in a humidified incubator (5% CO₂, 37°C). We rinsed the cells with phosphate-buffered saline (PBS) and fixed them with 4% paraformaldehyde for 10 min. The following 20 min of staining was achieved with 0.5% crystal violet solution. We subsequently counted the cell colonies.

Immunofluorescence (IF)

The cells were first transfected onto six-well plates with coverslips and then incubated for 24 h. We fixed the cells with 4% paraformaldehyde for 10 min, permeabilized them with 0.25% Triton X-100 for 10 min, and blocked them with 3% BSA for 1 h. We incubated the cells with anti-DLAT (1: 50) at 4°C overnight. After three rinses with PBS, the cells were incubated with goat anti-rabbit IgG H and L (Alexa Fluor® 488) (Abcam, UK) at 37°C for 1 h, washed and stained with DAPI solution for 3 min. Finally, the coverslips were sealed with antifluorescence quenching sealing solution (Beyotime, China). The formation of DLAT aggregates was observed and imaged using a Zeiss LSM880 inverted confocal microscope.

For mitochondrial staining, cells were incubated with 200 nM Mito Tracker Red CMXRos (Beyotime, China) for 30 min before fixation with paraformaldehyde.

Transwell

The Transwell upper chamber (Corning, USA) was placed into a 24-well plate. The Matrigel matrix (BD, USA) was diluted with a serum-free medium at a ratio of 1:8. Then, 50 μL of the dilution was added to the Transwell upper chamber (without Matrigel coating during the migration experiment) and incubated for 2 h at 37 °C with 5% CO₂ to set the matrix gel. The cells were placed in the upper chamber, which contained 200 μL of serum-free culture medium (density: 2 × 10⁴ cells per well). We added the culture medium containing 10% FBS as a chemotactic agent to the lower chamber. Migration and invasion assays were performed at 24 h and 48 h, respectively. The cells were fixed in 4% paraformaldehyde at room temperature for 20 min. After they were incubated with 0.1% crystal violet, the cells were stained for 30 min, and the cell numbers were calculated under a microscope (the cell numbers were averaged from three fields of view).

Statistical analysis

Every experiment was independently repeated three times, and the results are presented as the means ± standard deviations. GraphPad 8.0 software was used to analyze notable differences between two independent groups by utilizing Student’s t-test. One-way analysis of variance (ANOVA) was applied to compare differences among more than two groups. P < 0.05 was considered significant.

RESULTS

CEBPB for preventing malignant CRC progression

Based on differential expression analysis and qRT-PCR of the TCGA-CRC cohort, CEBPB was strongly upregulated in CRC tissues and cells (RKO, Caco-2, DLD-1) [Figure 1a and b]. Therefore, we selected DLD-1 and RKO cells with relatively low CEBPB expression to establish an oe-CEBPB (overexpression of CEBPB) cell model with considerably greater CEBPB mRNA expression than the control group [Figure 1c and d]. WB detection revealed consistent mRNA levels of the CEBPB protein in the above cells. Specifically, the level in the oe-CEBPB group was considerably greater than that in the control group [Figure 1e]. Subsequently, we detected the proliferation of DLD-1 and RKO cells and found that the proliferation of both cell lines was strikingly enhanced after treatment with oe-CEPBP [Figure 1f]. Additionally, the colony formation assay revealed that oe-CEBPB promoted colony formation in DLD-1 and RKO cells [Figure 1g]. Finally, the Transwell assay results revealed that oe-CEBPB potentiated the migration and invasion ability of DLD-1 and RKO cells [Figure 1h]. In conclusion, CEBPB promotes the malignant progression of CRC.

Figure 1.

CEBPB facilitates the malignant progression of CRC (a) Expression of CEBPB in CRC tissues; (b) Expression of CEBPB in CRC cells; (c and d) qRT-PCR detection of CEBPB expression; (e) WB detection of CEBPB protein expression; (f) MTT assay; (g) Colony formation assay; (h) Transwell assay; * indicates P < 0.05

Construction of the CRC cell cuproptosis model

GSEA of CEBPB revealed its remarkable concentration in GOBP_COPPER_ION_HOMEOSTASIS [Figure 2a]. To investigate the mechanism of CEBPB in CRC cuproptosis, we established a cuproptosis cell model based on DLD-1 cells. First, the induction effect of gradient concentrations of Es-Cu (0, 100, 200, 300, 400, 500 nM) was determined by cell viability assessment, and the optimal induction concentration (300 nM) was selected [Figure 2b]. Furthermore, WB analysis revealed that the expression of cuproptosis-related proteins (FDX1, DLTA and HSP70) in DLD-1 cells was considerably elevated after treatment with 300 nM Es-Cu [Figure 2c]. In addition, we also observed the formation of DLTA aggregates through IF experiments, revealing that 300 nM Es-Cu treatment substantially triggered DLTA aggregation in DLD-1 cells [Figure 2d]. Overall, 300 nM Es-Cu can induce cuproptosis in DLD-1 cells.

Figure 2.

Construction of the cuproptosis model in CRC cells (a) GSEA results; (b) Selection of the optimal Es-Cu concentration for induction by MTT; (c) WB detection of cuproptosis-related protein expression; (d) IF detection of DLAT aggregate formation; * indicates P < 0.05

CEBPB activates the PI3K/AKT/mTOR signaling pathway to dampen cuproptosis sensitivity in CRC

To determine how CEBPB regulates cuproptosis in CRC, we performed GSEA and revealed that CEPBP was strongly linked to the mTOR signaling pathway [Figure 3a]. The PI3K/AKT/mTOR signaling pathway, in which mTOR is involved, is a highly conserved signal transduction network in eukaryotic cells,[11] and it is tightly linked to the metastasis, occurrence, and progression of CRC.[12] Therefore, we speculated that the PI3K/AKT/mTOR axis in CRC may be connected with cuproptosis regulation. Thus, we treated oe-CEBPB DLD-1 cells with or without a PI3K/AKT/mTOR inhibitor (LY294002) in the presence of 300 nM Es-Cu. WB results demonstrated that oe-CEBPB facilitated the phosphorylation of PI3K, AKT, and mTOR, suggesting that CEBPB promoted the PI3K/AKT/mTOR signaling pathway in DLD-1 cells, which was attenuated by LY294002 treatment [Figure 3b]. In contrast, LY294002 reversed the inhibition of cuproptosis-related protein expression by oe-CEBPB [Figure 3c]. Furthermore, the LY294002 treatment abolished the enhancing effect of oe-CEBPB on the proliferation of DLD-1 cells [Figure 3d]. Moreover, the ability of CEBPB to enhance the proliferation, migration, and invasion of DLD-1 cells was reversed after LY294002 treatment [Figure 3e and f]. Taken together, CEBPB represses the sensitivity of CRC to cuproptosis by triggering the PI3K/AKT/mTOR signaling pathway.

Figure 3.

CEBPB dampens CRC cuproptosis sensitivity by activating the PI3K/Akt/mTOR signaling pathway (a) GSEA results; (b and c) WB detection of PI3K/AKT/mTOR signaling pathway and cuproptosis-related protein expression; (d) MTT assay; (e) Colony formation assay; (f) Transwell assay; * indicates P < 0.05

DISCUSSION

Since Tsvetkov defined cuproptosis, it has been widely studied in cancer, including CRC. Some researchers have divided CRC into different cuproptosis subtypes based on the expression patterns of different cuproptosis-related genes to predict the effects of prognosis and immunotherapy in CRC[13] or have focused on innovating cuproptosis-related lncRNAs that can predict CRC prognosis.[14] In summary, these projects have focused on prognostic models but have failed to elucidate how these predictive factors regulate cuproptosis. The present work is the first to show that CEBPB is highly upregulated in CRC, which facilitates CRC malignant progression. These results are consistent with those of Wang et al.[15] in wild-type IDH1 gliomas. CEBPB is a member of the basic leucine zipper (bZIP) family[16] and is involved in cell proliferation, tumor development, and cell differentiation.[17] CEBPB is considered a promising biomarker for non-small cell lung cancer[18] and a therapeutic target for brain cancer,[19] which fully confirms the carcinogenic role of CEBPB. Nonetheless, we hope to elucidate the underlying mechanism involved. GSEA suggested that CEBPB was strongly involved in copper ion homeostasis and mTOR signaling pathway regulation in CRC, which links CEBPB to the recent hotspot of cuproptosis. Then, a cuproptosis model was built to reveal the relationships among CEBPB, mTOR, and cuproptosis.

As an evolutionarily conserved serine/threonine protein kinase encoded by the mTOR gene in humans, mTOR belongs to the PI3K-related kinase (PIKK) family.[20] When PI3K connects to growth factor receptors, it can alter the protein structure of AKT and activate it, triggering or repressing downstream substrates by phosphorylation to regulate cell proliferation, apoptosis, differentiation, migration, and other phenotypes.[11] mTOR is one of the downstream targets activated by the PI3K/AKT axis.[21] Therefore, the PI3K/AKT/mTOR signaling pathway is often studied together. For example, the PI3K-AKT-mTOR pathway dampens cuproptosis sensitivity in CRC by suppressing fatty acid synthesis mediated by downstream SREBP1/SCD1.[22] Apolipoprotein C-II modulates cuproptosis through the PI3K/AKT/mTOR signaling pathway by binding to CD36, enhancing GC progression and peritoneal metastasis.[9] Consequently, we inferred that CEBPB may modulate mTOR through the PI3K/AKT axis, thereby repressing the sensitivity of CRC to cuproptosis. Using a cuproptosis CRC cell model, we performed rescue experiments and revealed that a PI3K/AKT/mTOR signaling pathway inhibitor enhanced the cuproptosis sensitivity of CEBPB-overexpressing CRC cells and weakened the enhancing effect of CEBPB overexpression on cell migration, proliferation, and invasion. Taken together, these results indicate that CEBPB hindered CRC cuproptosis by activating the PI3K/AKT/mTOR signaling pathway. Targeting CEBPB or the PI3K/AKT/mTOR axis may be an effective approach to increase CRC cuproptosis sensitivity and repress malignant tumor progression.

In conclusion, our study revealed that CEBPB is a key gene that facilitates CRC malignant progression and can trigger the PI3K/AKT/mTOR signaling pathway and decrease the sensitivity of CRC cells to cuproptosis. To our knowledge, cuproptosis is often used to predict clinical outcomes and immune responses in cancer patients. We intend to pioneer the study of the mechanism by which cuproptosis regulates CRC progression, revealing a new approach for adjuvant therapy for CRC patients with poor cuproptosis sensitivity. However, these data only represented the results in DLD-1 cells, necessitating further validation of our conclusions in a broader range of CRC cells and animal models.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.