Elesclomol‐Induced Copper Influx Attenuates Lung Adenocarcinoma Progression With Involvement of the ER Stress/PCK2 Axis

ABSTRACT

Copper ionophores such as elesclomol (ES) have been identified as effective agents capable of inducing cuproptosis, a recently characterized form of regulated cell death driven by copper overload. However, the interplay among copper toxicity, endoplasmic reticulum stress, and metabolic rewiring in lung adenocarcinoma (LUAD) remains poorly understood. Here, we demonstrate that ES selectively transports extracellular copper into human LUAD cells, inducing ER stress and upregulating PCK2, a gluconeogenic enzyme with context‐dependent roles in cancer. RNA sequencing and functional assays revealed that PCK2 elevation under copper overload affects glucose metabolism and lipid metabolism to suppress malignant phenotypes. Clinically, high PCK2 expression is correlated with favorable prognosis in LUAD patients, as evidenced by analyses of public datasets. Knocking down PCK2 paradoxically increased proliferation and cell motility, suggesting that PCK2 plays a tumor‐suppressive role under copper stress. KRAS ^G12D transgenic mice‐derived lung cancer organoids were used to validate the in vitro therapeutic effects of ES + Cu by regulating PCK2 expression. In vivo experiments revealed that ES inhibited tumor growth and upregulated PCK2. Taking together, our findings reveal that the ES‐ER stress‐PCK2 axis is a critical mediator of copper‐induced metabolic disruption, providing a rationale for targeting cuproptosis pathways in LUAD therapy.

Schematic diagram of copper‐induced cell death in LUAD. Elesclomol, as a copper ionophore, promotes copper influx into LUAD cells. This copper influx leads to cellular copper overload. Copper overload induces ER stress in LUAD cells. ER stress upregulates PCK2, which in turn inhibits the tricarboxylic acid (TCA) cycle. Collectively, these pathways orchestrate the cellular responses to excessive copper levels in LUAD cells, highlighting the complex interplay between copper homeostasis, metabolic reprogramming, and cell death mechanisms.

1. Introduction

Lung adenocarcinoma (LUAD) is the most common histological subtype of non‐small cell lung cancer and remains a major contributor to cancer‐related mortality worldwide [1, 2, 3, 4]. Despite the emergence of targeted therapies and immune checkpoint inhibitors, the overall survival rate for LUAD patients remains unsatisfactory due to high rates of metastasis, therapeutic resistance, and tumor heterogeneity [2, 4, 5, 6]. These challenges underscore the urgent need for novel therapeutic strategies that exploit intrinsic vulnerabilities in tumor metabolism and stress response pathways.

Recent advances in metal ion biology have revealed copper as a double‐edged regulator of cellular homeostasis [7, 8]. While essential for enzymatic activity and mitochondrial function, excess intracellular copper can trigger a unique form of regulated cell death termed cuproptosis [9, 10, 11, 12, 13, 14]. This process is characterized by copper‐induced proteotoxic stress, mitochondrial dysfunction, and metabolic collapse [15, 16, 17]. Elesclomol (ES), a small‐molecule copper ionophore, has been shown to selectively shuttle extracellular copper into cells, thereby inducing cuproptosis in a variety of cancer models [18, 19, 20]. However, the downstream molecular events linking copper overload to tumor suppression, particularly in LUAD, remain poorly understood.

Endoplasmic reticulum (ER) stress is a critical cellular response to metabolic perturbations and oxidative stress, frequently activated in the tumor microenvironment [21, 22, 23, 24, 25]. The unfolded protein response (UPR), initiated by ER stress sensors such as PERK, IRE1α, and ATF6, orchestrates adaptive or apoptotic outcomes depending on the severity and duration of stress [26, 27, 28, 29]. In cancer, ER stress can promote survival under hypoxic and nutrient‐deprived conditions, but sustained activation may also sensitize cells to therapeutic insults [23, 24, 25, 30]. The intersection between copper‐induced stress and ER signaling remains an underexplored area in LUAD biology.

Phosphoenolpyruvate carboxykinase 2 (PCK2), a mitochondrial isoform of the gluconeogenic enzyme, has emerged as a context‐dependent regulator of cancer metabolism [31, 32]. PCK2 modulates the balance between glycolysis and oxidative phosphorylation, influencing cell proliferation, redox homeostasis, and drug sensitivity [33, 34]. Although downregulated in LUAD tissues, its functional role under copper‐induced stress conditions has not been fully elucidated. Understanding how PCK2 integrates metabolic cues with stress responses may reveal new therapeutic opportunities.

In this study, we investigated the mechanistic link between ES‐induced copper influx, ER stress activation, and PCK2‐mediated metabolic regulation in LUAD. Using transcriptomic profiling, functional assays, and in vivo models, we demonstrate that ES facilitates copper accumulation in LUAD cells, triggering ER stress and upregulating PCK2 expression. PCK2, in turn, modulates glucose and lipid metabolism, contributing to the suppression of malignant phenotypes. Notably, PCK2 knockdown reversed the cytotoxic effects of ES + Cu, highlighting its functional relevance in copper‐induced cell death.

To evaluate translational potential, we employed both xenograft and genetically engineered mouse models. Oral administration of ES in nude mice bearing human LUAD xenografts resulted in tumor growth suppression and PCK2 upregulation, without significant systemic toxicity.

In summary, our study elucidates a novel copper‐dependent regulatory pathway involving ER stress and PCK2 in LUAD. By integrating insights from metal ion biology, stress signaling, and metabolic oncology, we propose a mechanistic framework that may inform future therapeutic strategies. However, further validation in immunocompetent models and clinical settings is essential before considering ES‐based interventions for LUAD patients.

2. Materials and Methods

2.1. Pharmacological Inhibitors and Other Reagents

Elesclomol (#STA‐4783; Selleck Chemicals, Houston, TX, USA), tetrathiomolybdate (#E1166; Selleck Chemicals, Houston, TX, USA), acetylcysteine (N‐acetylcysteine) (#S1623; Selleck Chemicals, Houston, TX, USA), necrostatin‐1 (#S8037; Selleck Chemicals, Houston, TX, USA), pepstatin A (#S7381; Selleck Chemicals, Houston, TX, USA), D‐Boc‐FMK (#HY‐13229; MedChemExpress, NJ, USA), Fer‐1 (ferrostatin‐1) (#S7243; Selleck Chemicals, Houston, TX, USA), Z‐VAD‐FMK (#S7023; Selleck Chemicals, Houston, TX, USA), L‐NAME HCl (#S2877; Selleck Chemicals, Houston, TX, USA), 4‐phenylbutyric acid (#HY‐A0281; MedChemExpress, NJ, USA), thapsigargin (#HY‐13433; MedChemExpress, NJ, USA), CuCl₂ (CAS: 7447‐39‐4; Aladdin, Shanghai, China), CoCl₂ (CAS: 7646‐79‐9; Aladdin, Shanghai, China), FeCl₃ (CAS: 7705‐08‐0; Aladdin, Shanghai, China), NiCl₂ (CAS: 7718‐54‐9; Aladdin, Shanghai, China), and ZnCl₂ (CAS: 7646‐85‐7; Aladdin, Shanghai, China)were used under experimental conditions in vitro and in vivo.

2.2. Cell Lines and Cell Culture

The human immortalized bronchial epithelial cell lines BEAS‐2B (RRID: CVCL_0168), 16HBE (RRID: CVCL_0112), and LUAD cell lines A549 (RRID: CVCL_0023), and H358 (RRID: CVCL_1559) were purchased from Procell Life Science & Technology Co. Ltd. (Wuhan, China). The cells were cultured in RPMI‐1640 medium, Ham's F‐12K medium, high‐glucose DMEM, or MEM (containing NEAA) (Procell Life Science & Technology Co. Ltd., Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Beyotime, Shanghai, China) in a humidified atmosphere with 5.0% CO₂ at 37°C. The genetic characteristics of these cells were also confirmed. All the cell lines were passaged within 6 months.

2.3. ES + Cu Pulse Treatment

The ES + Cu pulse treatment method is consistent with previous research methods [35]. Briefly, ES + Cu (100 nM, 1:1) was used to treat the cells for 2 h, after which the supernatant was aspirated, and the cells were washed twice with PBS. The medium was replaced with fresh medium containing 10% FBS, after which the cells were cultured for subsequent experiments.

2.4. RNA Extraction, cDNA Synthesis, and Quantitative Real‐Time PCR (qRT–PCR) Analysis

The cells or tissues were homogenized in TRIzol Reagent (TaKaRa, Osaka, Japan), and total RNA was extracted according to the standard protocol. The RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was performed using M‐MLV reverse transcriptase (TaKaRa), and qRT–PCR was performed with SYBR Premix ExTaq (TaKaRa) using an ABI Step One Plus Real‐Time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR program was as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The Ct values of the PCK2/HSPA5/CHOP mRNAs were normalized to those of ACTB, which was used as an internal control. The ^△△Ct method was used to determine the relative expression levels of the mRNAs. The sequences of primers used for targeted gene mRNA detection are listed in Table S1. The primers for PCK2/HSPA5/CHOP/ACTB were purchased from Genewiz (Suzhou, China).

2.5. RNA Interference

Two small interfering RNA (siRNA) sequences targeting the coding regions of PCK2 were synthesized by GenePharma (Suzhou, China). Scrambled siRNA served as the negative control (si‐NC). A549 cells were seeded into six‐well cell plates and transiently transfected with 100 pmol of siRNA using the transfection reagent Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). After 72 h of transfection, the cells were harvested for subsequent assays. The si‐NC and target sequences of the siRNAs are listed in Table S2.

2.6. Cu Concentration Determination

A copper assay kit (#MCK4575, MesGen Biotechnology) was used to determine the Cu²⁺ concentration. The cell samples were lysed using an ultrasonic crusher to obtain cell homogenates. After centrifugation at 14 000 rpm for 2 min, the supernatant of the cell homogenate or cell culture supernatant was aspirated for subsequent analysis. The OD was determined by measuring the absorbance at 440 nm.

2.7. Analysis Based on Public Databases

The Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), Kaplan–Meier plotter (https://kmplot.com/analysis/index.php?p=background), and The Cancer Genome Atlas (TCGA) databases (https://portal.gdc.cancer.gov/) were used to obtain publicly available transcriptome data.

2.8. Transcriptomic Profiling and Bioinformatic Analysis

A549 cells were assigned to two distinct conditions: one group received a combined treatment of ES + Cu (100 nM, 1:1), while the other remained untreated as a control. After an 8‐h incubation period, cells were collected in accordance with standardized procedures. At the appropriate confluency, adherent cells were detached either by enzymatic digestion or mechanical scraping. Total RNA was extracted and subjected to high‐throughput sequencing using the Illumina NovaSeq 6000 platform. To identify transcriptional changes, differential gene expression analysis was conducted using established statistical pipelines. Genes exhibiting significant expression alterations were further interrogated for functional relevance. Enrichment analyses based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed to elucidate overrepresented biological processes, molecular functions, and cellular components. For data visualization, z‐score normalized expression values were compiled into a gene‐by‐sample matrix. Heatmaps and statistical plots were generated using the R packages pheatmap and ggpubr (version 4.0.2), facilitating intuitive interpretation of transcriptomic patterns.

2.9. Western Blotting Analysis

The cells were harvested and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with 1% protein protease and phosphatase inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) and incubated for 30 min. The samples were subsequently centrifuged at 12 000 rpm at 4°C for 15 min and subsequently heated for 5 min at 95°C. Western blotting was performed as previously described [36]. The primary antibodies used were PCK2 (#8565S; Cell Signaling Technology, Danvers, MA, USA; RRID:AB_11217628), HSP70 (#66183–1‐Ig; Proteintech Group Inc., Rosemont, IL, USA; RRID: AB_2881578), PARP (#9532; Cell Signaling Technology, Danvers, MA, USA; RRID: AB_659884), CCS (#22802–1‐AP; Proteintech Group Inc., Rosemont, IL, USA; RRID: AB_2879172), and β‐actin (#66009–1‐Ig; Proteintech Group Inc., Rosemont, IL, USA; RRID: AB_2687938).

2.10. Cell Proliferation Analysis and Drug Treatment

A Cell Counting Kit‐8 (CCK‐8) was used for cell proliferation analysis (APExBIO, USA). A549 cells transfected with the target siRNAs or corresponding negative controls were seeded into 96‐well plates. Cell viability was determined according to the manufacturer's instructions. A colony formation assay was performed to assess cell proliferation. Briefly, the cells were diluted with complete culture medium, and 3 × 10³ cells were seeded onto 60‐mm plates. After 7–14 days of incubation, the foci were stained with crystal violet (Beyotime) and counted based on the cell growth rate. The viability of lung cancer organoids was determined using an ATP Assay Kit (#S0026B; Beyotime, Shanghai, China).

2.11. Flow Cytometric Analysis

For the apoptosis assay, both the supernatant and the cells were harvested, washed, and suspended in binding buffer containing Annexin V/FITC or APC and PI (Beyotime, Shanghai, China). A FACSCaliber system (Beckman Coulter, Brea, CA, USA) was used to analyze the stained cells.

2.12. Immunocytochemistry Assays and IHC Score Evaluation

Immunohistochemistry (IHC) was performed as previously described [36]. The primary antibody used was against PCK2 (#8565S; Cell Signaling Technology, Danvers, MA, USA).

The IHC score was calculated on the basis of a combination of the intensity of staining and the percentage of positive cells. Specifically, the staining intensity was graded on a scale from 0 (no staining) to 4 (strong staining), and the percentage of positive cells was estimated in different ranges (0%–20%, 21%–40%, 41%–60%, 61%–80%, and 81%–100%).

2.13. In Vivo Growth Assays

Female BALB/c athymic nude mice were used to establish a xenograft LUAD model. For the in vivo drug treatment assay, A549 cells were suspended in 100 μL of PBS and injected subcutaneously into the armpits of nude mice. After 14 days, the mice were randomly divided into four groups and treated (five mice per group). ES (80 mg/kg) was orally administered to the mice for 2 weeks, and CuCl₂ (20 mg/L) was added into the daily drinking water. Mice in the ES + Cu treatment group and the blank control group were euthanized after 14 consecutive days of administration. The tumor volume (V, mm³) was measured every 2 days using Vernier calipers. At the end of the experiment, the mice were euthanized, and the xenograft tumors were dissected for subsequent experiments. All experimental animal procedures were approved by the Laboratory Animal Center of Soochow University (approval no. 202308A0493).

2.14. Generation and Identification of CC10‐rtTA/TetO‐Cre/LSL‐ KRAS ^G12D Transgenic Mice

CC10‐rtTA/TetO‐Cre/LSL‐KRAS ^G12D conditional mutant mice were generated by crossbreeding CC10‐rtTA/TetO‐Cre and LSL‐KRAS ^G12D mice. Scissors and tweezers were rinsed with 70% ethanol, and the 0.2–1 cm pieces of mouse tails were cut for subsequent identification. Transgenic mice were identified using the Quick Genotyping Assay Kit for Mouse Tail (#D7283M; Beyotime, Shanghai, China). The amplification PCR conditions were as follows: denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 35 s, annealing at 60°C for 45 s, and extension at 72°C for 60 s; and extension at 72°C for 6 min at 72°C. The primer sequences used for targeted region detection are listed in Table S3. Subsequently, the PCR products were subjected to Southern blotting to determine the molecular size (bp) of the target fragments.

2.15. Establishment and Identification of CC10‐rtTA/TetO‐Cre/LSL‐ KRAS ^G12D Transgenic Mice‐Derived LCOs

CC10‐rtTA/TetO‐Cre/LSL‐KRAS ^G12D conditional mutant mice were treated with Dox (1 g/L) for 8 weeks to induce in situ KRAS ^G12D ‐driven LUAD lesions. The LUAD tissues from the mice were dissected and incubated with Tumor Tissue Digestion Solution (#K601003; BioGenous, Jiangsu, China) for 30 min at 100 rpm, 37°C. Tissue suspensions were resuspended in Cancer Organoid Basal Medium (#B213152; BioGenous, Jiangsu, China) to terminate digestion and filtered with 70 μm cell filters (#abs7009; Absin, Shanghai, China) three times. The filtered tissue suspensions were collected and centrifuged at 300 g for 5 min. The precipitated cells were resuspended using Organoid Culture ECM (Reduced Growth Factor) (#M315066; BioGenous, Jiangsu, China) and seeded onto a 24‐well plate to form 3D cultured LCOs. A Lung Adenocarcinoma Organoid Kit (#K2138‐LA; BioGenous, Jiangsu, China) was used for continuous cultivation of LCOs. After the indicated treatments, LCOs were collected, embedded in paraffin, and sliced for subsequent H&E staining and IHC analysis.

2.16. Statistical Analysis

All experiments were independently performed at least in triplicate. All the statistical analyses were performed using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA) and R 4.0.2 software (R Foundation for Statistical Computing). All the data are presented as the means ± SD. Significant differences between two groups were assessed via a non‐paired Student's t test. Significant differences between three or more groups were analyzed using one‐way or two‐way analysis of variance (ANOVA) followed by Bonferroni post hoc correction. All the statistical tests were two‐tailed. p < 0.05 was considered to indicate statistical significance.

3. Results

3.1. ES Induces Copper‐Specific Death in LUAD Cells

To determine the specificity and mechanism of ES‐mediated cytotoxicity in LUAD, we conducted multiple experimental assays to analyze the regulated cell death mediated by ES. CCK‐8 assays revealed that ES combined with copper (Cu) suppressed cell viability of A549 and H358 cells to 20%, whereas combinations with Fe³⁺, Co²⁺, Ni²⁺, or Zn²⁺ had no inhibitory effects (Figure 1A). A 2‐h pulse treatment of A549 and H358 cells with ES + Cu induced persistent suppression of cell viability at 8, 24, and 48 h, suggesting the occurrence of intracellular regulation rather than transient toxicity (Figure 1B). Flow cytometry analysis revealed that the proportion of early‐stage apoptotic A549 and H358 cells did not significantly differ after ES + Cu treatment (Figure 1C,D). Western blot analysis revealed no increase in cleaved‐PARP levels or early apoptosis rates after ES + Cu treatment in A549 and H358 cells, excluding apoptosis as the primary mechanism (Figure 1E). In addition, cell death was irreversible by other regulated cell death inhibitors, including ferroptosis inhibitors (Fer‐1) or ROS scavengers, in A549 and H358 cells but was reversed by the copper chelator TTM, confirming Cu‐dependent cytotoxicity (Figure 1F). These results establish ES + Cu as a Cu‐specific cell death inducer in LUAD cells, which is distinct from classic apoptosis or other regulated cell death pathways.

FIGURE 1.

ES induces copper‐specific death in LUAD cells. (A) CCK‐8 assays revealed that ES (100 nM) inhibited only cell viability (to 20%) when combined with copper ions, whereas it failed to inhibit cell viability when combined with other metal ions (Fe³⁺, Co²⁺, Ni²⁺, and Zn²⁺, 1 μM). (B) CCK‐8 assay results indicating that pulse treatment with ES + Cu (100 nM, 1:1)for 2 h, followed by washing with PBS and replacing with fresh medium, led to persistent inhibition of cell viability at 8, 24, and 48 h. (C, D) Flow cytometry analysis indicated that treatment with ES + Cu (100 nM, 1:1)did not significantly increase the Annexin V⁺/FITC⁺ proportion of early apoptotic cells. (E) Western blot analysis revealed that treatment with ES + Cu (100 nM, 1:1) did not increase the expression level of the apoptosis marker cleaved PARP, suggesting that the cell death mediated by ES + Cu was not via the classic apoptotic pathway. (F) The cell death induced by ES + Cu (100 nM, 1:1) could not be reversed by other cell death inhibitors but could be reversed by the copper chelator TTM, indicating that the cell death mediated by ES + Cu was copper ion dependent and not via other programmed cell death pathways. Before ES + Cu (100 nM, 1:1) treatment, the cells were pretreated overnight with 20 μM necrostatin‐1 (Nec‐1), 10 μM ferrostatin‐1 (Fer‐1), 1 mM N‐acetylcysteine (NAC), 30 μM Z‐VAD‐FMK, 50 μM D‐Boc‐FMK, 20 μM TTM, 300 μM L‐NAME, or 1 μM pepstatin A. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test. ns: p > 0.05, ***p < 0.001.

3.2. ES Facilitates Extracellular Copper Influx

To evaluate ES‐mediated intracellular copper accumulation, we used a copper concentration detection kit to analyze the dynamic changes in the copper concentration. Cu detection assays revealed increased intracellular Cu levels in ES‐treated A549 and H358 cells under CuCl₂ conditions, which could be reversed by the Cu chelator TTM (Figure 2A,B). Consistently, qRT–PCR and Western blot assays revealed the concurrent regulation of the protein CCS, a Cu chaperone whose expression inversely correlates with the intracellular Cu concentration, further confirming the occurrence of Cu influx in A549 and H358 cells (Figure 2C–F). Furthermore, toxicity assessments of the human normal bronchial epithelial cell lines BEAS‐2B and 16HBE demonstrated that ES + Cu treatment exhibited controllable cytotoxicity and was deemed safe for human normal bronchial epithelial cells (Figure 2G,H). CCK‐8 assay and colony formation assay indicated that ES + Cu treatment could significantly inhibit the viability and colony formation ability of A549 and H358 cells, which could be reversed by TTM treatment (Figure 2I–L). In summary, ES acts as a Cu ionophore, driving extracellular Cu into LUAD cells and increasing the intracellular Cu load.

FIGURE 2.

ES facilitates extracellular copper influx in LUAD cells. (A, B) Intracellular Cu levels increased in ES (100 nM)‐treated A549 and H358 cells under CuCl₂ (2 μM), which could be reversed by TTM (10 μM). (C, D) qRT–PCR indicated that ES + Cu treatment led to the downregulation of the copper chaperone CCS, confirming that Cu influx (ES: 100 nM, CuCl₂: 2 μM) could be reversed by TTM (10 μM). (E, F) Western blot analysis revealed that ES + Cu treatment led to the downregulation of the copper chaperone CCS, confirming that Cu influx (ES + Cu: 100 nM or 200 nM, 1:1) could be reversed by TTM (10 μM). (G, H) Cytotoxicity analysis of ES + Cu (100 nM, 1:1) in normal bronchial epithelial cells (BEAS‐2B, 16HBE) indicated the safety of in vivo application. (I, J) CCK‐8 assay indicated that ES + Cu (100 nM, 1:1) treatment could inhibit the cell viability of A549 and H358 cells, and TTM (10 μM) treatment could reverse this inhibitory effect. (K, L) Colony formation assay indicated that ES + Cu (100 nM, 1:1) treatment could inhibit the colony formation viability of A549 and H358 cells, and TTM (10 μM) treatment could reverse this inhibitory effect. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test. ***p < 0.001.

3.3. RNA Sequencing Reveals That ER Stress Is Induced by ES‐Induced Cu Influx

To further identify the transcriptomic alterations underlying ES/Cu cytotoxicity, we performed RNA sequencing in A549 cells to analyze the differentially expressed mRNAs (Figure 3A–C). Gene set enrichment analysis (GSEA) based on RNA‐seq revealed the activation of several important biological pathways, including those related to the cellular response to copper ions, unfolded protein response‐related pathways, and cellular lipid metabolism‐related pathways, in ES + Cu‐treated A549 cells (Figure 3D and Figure S1). These enrichment results indicated that ES + Cu may affect protein homeostasis and lipid metabolic metastasis to inhibit the malignant biological behavior of LUAD cells. By screening for mRNAs that are upregulated upon ES treatment but exhibit concurrent downregulation under two different TTM concentrations, we found that ES + Cu upregulated PCK2 mRNA expression, which was TTM‐reversible (Figure 3E,F). Consistently, qRT–PCR and WB confirmed the Cu‐dependent PCK2 induction at the mRNA and protein levels in A549 and H358 cells (Figure 3G–I). In summary, ES/Cu triggers ER stress via intracellular Cu overload, with PCK2 being a downstream Cu‐responsive gene.

FIGURE 3.

RNA sequencing reveals that ER stress and PCK2 upregulation are induced by ES + Cu. (A) Volcano plot of differentially expressed mRNAs in Ctrl/ES + Cu(100 nM, 1:1)‐treated A549 cells. (B) Volcano plot of differentially expressed mRNAs in ES + Cu(100 nM, 1:1)/ES + Cu + TTM (10 μM)‐treated A549 cells. (C) Volcano plot of differentially expressed mRNAs in ES + Cu(100 nM, 1:1)/ES + Cu + TTM (20 μM)‐treated A549 cells. (D) GO enrichment analysis indicated that treatment of A549 cells with ES + Cu (100 nM, 1:1) activated signaling pathways related to the cellular response to copper ions, unfolded protein response‐related pathways, and cellular lipid metabolism‐related pathways. (E) Differentially expressed mRNAs of A549 cells treated with Ctrl/ES + Cu(100 nM, 1:1)/ES + Cu + TTM (10 μM)/ES + Cu + TTM (20 μM). (F) RNA sequencing revealed that the expression of PCK2 mRNA increased after ES + Cu (100 nM, 1:1) treatment, which was reversed by TTM (10 μM and 20 μM). (G, H) Western blot results revealed that the protein expression of PCK2 increased after ES + Cu (100 nM, 1:1) treatment, which was reversed by TTM (10 μM). (I) qRT–PCR results revealed that the expression of PCK2 mRNA increased after ES + Cu (100 nM, 1:1) treatment, which was reversed by TTM (10 μM and 20 μM). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test. **p < 0.01, ***p < 0.001.

3.4. ER Stress Induction Upregulates PCK2

To validate the role of the ER stress–PCK2 regulatory axis, we used thapsigargin as an ER stress inducer and detected elevated mRNA levels of the ER stress markers HSPA5/CHOP in A549 and H358 cells after thapsigargin treatment (Figure 4A–D). The activation of ER stress led to a significant increase in PCK2 expression, as demonstrated by qRT‐PCR (Figure 4E,F). Consistently, Western blot assays revealed that after the activation of ER stress, the expression level of HSP70 was significantly increased, which was accompanied by the upregulation of PCK2 (Figure 4G,H). Conversely, after treatment with the ER stress inhibitor 4‐PBA, the upregulation of these ER stress markers was reversed (Figure 4I,J). We further analyzed PCK2 mRNA and protein expression and observed upregulation during the induction of ER stress; this upregulation was reversed by 4‐PBA (Figure 4K,L). Taken together, these results indicate that PCK2 expression is correlated with the induction of ER stress.

FIGURE 4.

ER stress induction upregulates PCK2 expression. (A–D) qRT–PCR results showing increased levels of ER stress markers (HSPA5, CHOP) in ES + Cu (100 nM, 1:1) and thapsigargin (0.5 μM)‐treated A549 and H358 cells. (E, F) PCK2 mRNA upregulation upon ER stress induced by ES + Cu (100 nM, 1:1) and thapsigargin (0.5 μM) in A549 and H358 cells. (G, H) Western blot showing elevated HSP70 and PCK2 protein levels after ES + Cu (100 nM, 1:1) and thapsigargin (0.5 μM) treatment in A549 and H358 cells. (I, J) The ER stress inhibitor 4‐PBA (1 mM) reversed the upregulation of HSPA5/CHOP expression mediated by ES + Cu (100 nM, 1:1) and thapsigargin (0.5 μM) in A549 cells. (K, L) 4‐PBA attenuates PCK2 mRNA and protein upregulation mediated by ES + Cu (100 nM, 1:1) and thapsigargin (0.5 μM) treatment in A549 cells. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test. **p < 0.01, ***p < 0.001.

3.5. Impact of PCK2 Knockdown on the Malignant Phenotypes of LUAD Cells

To investigate the role of PCK2 in LUAD progression, we performed siRNA‐mediated PCK2 knockdown in A549 cells. PCR and Western blot analyses indicated a significant reduction in both the mRNA and protein expression levels of PCK2, confirming the efficacy of gene silencing (Figure 5A–E). Functional assays revealed that PCK2 depletion markedly enhanced malignant behaviors: CCK‐8, Transwell, and colony formation assays demonstrated increased cellular proliferation and cell motility in PCK2 knockdown LUAD cells (Figure 5F–J). These findings collectively highlight that PCK2 knockdown disrupts critical oncogenic processes, including proliferation, colony formation, and metastatic potential, suggesting that PCK2 is a promising therapeutic target for mitigating LUAD aggressiveness.

FIGURE 5.

Knocking down PCK2 promotes the malignant phenotype of LUAD cells. (A, B) PCR results showed that treatment with siRNA targeting PCK2 downregulated the mRNA expression of PCK2 in A549 and H358 cells. (C–E) Western blot results showing that treatment with siRNA targeting PCK2 downregulated the protein expression of PCK2 in A549 and H358 cells. (F, G) CCK‐8 assays confirmed that knocking down PCK2 promoted the proliferation of A549 and H358 cells. (H, I) Transwell assays confirmed that knocking down PCK2 increased the migration and invasion of A549 and H358 cells. (J) Colony formation assays confirmed that knocking down PCK2 increased the colony formation of A549 and H358 cells. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test or ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

3.6. Prognostic Relevance of PCK2 in LUAD

To assess the clinical value of PCK2, we conducted multiple bioinformatic analyses via public databases. TCGA data revealed lower PCK2 mRNA in LUAD tissues than in normal tissues (p < 0.05; Figure S2A). Paired analysis of matched normal and LUAD tissues revealed lower PCK2 mRNA expression in LUAD tissues (p < 0.001; Figure S2B). GEO and Kaplan–Meier analyses revealed that high PCK2 expression was associated with increased LUAD patient survival (p = 0.012 and p = 0.023, respectively; Figure S2C,D). STRING database analysis revealed that the PCK2 protein interacts with a group of proteins involved in glucose metabolism and lipid metabolism, suggesting that PCK2 may mediate its regulatory role in lung cancer through these interactions (Figure S2E). In summary, these results indicate that PCK2 serves as a favorable prognostic biomarker in LUAD.

3.7. ES + Cu Induces ER Stress and Affects Viability in LUAD Organoids

To investigate the role of ES + Cu in LUAD, we first established LUAD organoids from inducible CC10‐rtTA/TetO‐Cre/LSL‐KRAS ^G12D mice. As shown in Figure 6A, after an 8‐week induction with doxycycline (Dox, 1 g/L), in situ LUAD was generated. Dissection of LUAD tissues followed by in vitro culture successfully yielded LUAD organoids, and whole‐genome sequencing (WGS) suggested that the organoids and the source tissues were genomically consistent (Figure S3). The morphology of LUAD organoids at passage 13 (P13) was observed under a microscope, presenting typical organoid structures (Figure 6B). We then examined the effect of ES + Cu on LUAD organoid viability. LUAD organoids (P13) were treated with ES + Cu (100 nM, 1:1) for 2 h. Microscopic observation revealed that compared with the control (Ctrl) group, ES + Cu treatment led to obvious structural disruption of LUAD organoids (Figure 6C). Quantification of relative viability showed a significant decrease in the ES + Cu group compared to the Ctrl group. Moreover, the addition of the ER stress inhibitor 4‐PBA partially reversed the reduction in viability caused by ES + Cu, as indicated by the higher viability in the ES + Cu + 4‐PBA group than in the ES + Cu group (Figure 6D, p < 0.001). To explore the molecular mechanism, we detected the expression of ER stress‐related genes. The mRNA expression of CHOP and HSPA5 was significantly upregulated in the ES + Cu treated LUAD organoids compared to the Ctrl group. And 4‐PBA treatment could attenuate this upregulation (Figure 6E,F, p < 0.001). Similarly, the expression of PCK2 was increased in the ES + Cu group and partially restored by 4‐PBA co‐treatment (Figure 6G, p < 0.001). Consistently, Western blot analysis was performed to assess the protein levels of HSP70 and PCK2. As shown in Figure 6H, ES + Cu treatment increased the protein levels of HSP70 and PCK2 in LUAD organoids, further suggesting the induction of ER stress and PCK2 upregulation by ES + Cu. Further, we conducted in vivo experiments using the CC10‐rtTA/TetO‐Cre/LSL‐KRAS ^G12D conditional transgenic mouse LUAD model and compared elesclomol‐treated mice with vehicle controls to analyze the effect of ES on suppressing LUAD progression. The results showed a significant suppression of tumor progression in the ES + Cu (80 mg/kg ES, 20 mg/L CuCl₂ in daily drinking water) treated group, as evidenced by a marked reduction in both the number and size of lung tumor nodules (Figure 6I,J).

FIGURE 6.

ES + Cu induces ER stress and affects viability in LUAD organoids. (A) Schematic of LUAD organoid generation. CC10‐rtTA/TetO‐Cre/LSL‐KRAS ^G12D mice were induced with Dox (1 g/L) for 8 weeks to generate in situ LUAD. LUAD tissues were dissected and cultured to form LUAD organoids. (B) Representative bright‐field images of LUAD organoids at passage 13 (P13). Scale bar = 100 μm. (C) Morphological changes of LUAD organoids (P13) after treatment with ES + Cu (100 nM, 1:1) for 2 h. Ctrl: Control group. Scale bar = 100 μm. (D) Relative viability of LUAD organoids under different treatments: Ctrl, ES + Cu (100 nM, 1:1), ES + Cu (100 nM, 1:1) + 4‐PBA (1 mM). Data are presented as mean ± SD. (E–G) Relative mRNA expression of ER stress‐related genes (CHOP, HSP70) and PCK2 in LUAD organoids after different treatments. Data are presented as mean ± SD. (H) Western blot analysis of HSP70, PCK2 protein levels in LUAD organoids with or without ES + Cu (100 nM, 1:1) treatment. (I, J) HE staining indicated that ES + Cu treatment could suppress the progression of LUAD in vivo. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test. ***p < 0.001.

3.8. ES Exhibits Antitumor Activity and Upregulates PCK2 Expression In Vivo

To further investigate the in vivo antitumor effect of ES and its impact on the expression level of PCK2 in subcutaneous tumors, we conducted in vivo experiments using a xenograft model. A549 cells were injected subcutaneously into one side of each nude mouse. After 1 week of feeding, drug administration commenced. The dosing regimen was 80 mg/kg ES, which was administered for 5 consecutive days followed by a 2‐day break, which lasted for 2 weeks (Figure 7A). Compared with the control, treatment with ES significantly slowed the growth rate of the subcutaneous xenografts (Figure 7B,C). Moreover, the weight of the subcutaneous xenografts was reduced in the ES‐treated group (Figure 7D). Importantly, ES treatment had no significant effect on the body weight of the mice, indicating the safety of its in vivo application (Figure 7E). Additionally, ES upregulated the expression of PCK2 and cuproptosis indicator FDX1 in tumor tissue (Figure 7F–H). In conclusion, these results confirm that ES can exert antitumor effects in vivo, upregulate the expression of PCK2 in tumor tissues, and has potential safety for in vivo applications.

FIGURE 7.

ES exhibits antitumor activity and upregulates PCK2 in vivo. (A) Schematic diagram: A subcutaneous tumorigenesis experiment in nude mice was conducted using A549 cells. A549 cells were injected subcutaneously on one side. After 1 week of feeding, drug administration was started. The specific regimen was 80 mg/kg ES, which was administered continuously for 5 days and then stopped for 2 days, for a total of 2 weeks. (B, C) Compared with the control, treatment with ES slowed the growth rate of the subcutaneously transplanted tumors. (D) Compared with the control treatment, treatment with ES reduced the weight of the subcutaneously transplanted tumors. (E) Compared with the control treatment, treatment with ES had no significant effect on the body weight of the mice, indicating the safety of in vivo application. (F, G) IHC confirming PCK2 upregulation in ES‐treated tumors. (H) IHC staining indicated that ES + Cu treatment could downregulate the expression of FDX1 in vivo, which is a cuproptosis marker. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test or ANOVA. **p < 0.01, ***p < 0.001.

3.9. PCK2 Mediates ES + Cu‐Induced Cell Death Through ER Stress‐Dependent Mechanisms in LUAD Cells

To further investigate the regulatory role of PCK2 in the ES + Cu‐induced cell death, we conducted relevant rescue experiments. Results showed that ES + Cu (100 nM, 1:1) treatment markedly upregulated PCK2 protein expression in A549 and H358 lung adenocarcinoma cells, which was effectively suppressed by siRNA‐mediated knockdown of PCK2 (Figure 8A). Correspondingly, silencing PCK2 significantly rescued ES + Cu‐induced inhibition of cell viability, indicating a functional role of PCK2 in mediating cytotoxicity (Figure 8B). Furthermore, pharmacological inhibition of endoplasmic reticulum (ER) stress using 4‐PBA (1 mM) attenuated ES + Cu‐induced PCK2 upregulation (Figure 8C) and partially restored cell proliferation (Figure 8D), suggesting that ER stress contributes to PCK2‐dependent cell death. IHC analysis of xenograft tissues revealed elevated expression of the ER stress marker HSP70 and PCK2 following ES + Cu treatment, further supporting the involvement of ER stress in the observed phenotype (Figure 8E,F). Collectively, these findings demonstrate that PCK2 mediates ES + Cu‐induced cell death in LUAD cells through ER stress‐dependent mechanisms.

FIGURE 8.

PCK2 mediates ES + Cu‐induced cell death through ER stress‐dependent mechanisms in LUAD cells. (A) Western blot analysis of PCK2 expression in A549 and H358 cells treated with ES + Cu and transfected with either si‐NC or siRNAs targeting PCK2. The upregulation of PCK2 protein levels mediated by ES + Cu (100 nM, 1:1) treatment were markedly reduced upon siRNA‐mediated knockdown. (B) Quantification of relative cell viability (%) in A549 and H358 cells under the same conditions as in (A). Knockdown of PCK2 significantly rescued ES + Cu (100 nM, 1:1)‐induced suppression of cell proliferation. (C) Western blot analysis showing the effect of ER stress inhibition on PCK2 expression. A549 and H358 cells were treated with ES + Cu (100 nM, 1:1) in the presence or absence of 4‐PBA (1 mM). 4‐PBA treatment attenuated ES + Cu‐induced upregulation of PCK2. (D) Cell viability assay under conditions described in (C). Treatment with 4‐PBA partially restored cell proliferation suppressed by ES + Cu (100 nM, 1:1), supporting the involvement of ER stress in PCK2‐mediated cell death. (E, F) Immunohistochemical analysis of lung adenocarcinoma xenograft tissues revealed that treatment with ES + Cu significantly upregulated the expression of the ER stress marker HSP70, accompanied by a concomitant increase in PCK2 expression. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐test. ns: p > 0.05, ***p < 0.001.

4. Discussion

Copper metabolism has gained increasing attention in cancer biology due to its dual role in supporting essential enzymatic functions and inducing cytotoxicity when dysregulated [7, 9, 37, 38]. Elesclomol (ES), a copper ionophore, exploits this vulnerability by facilitating intracellular copper accumulation, leading to cell death in various tumor types [18, 19, 20]. In this study, we demonstrated that ES‐induced copper influx suppresses lung adenocarcinoma (LUAD) progression through activation of endoplasmic reticulum (ER) stress and upregulation of phosphoenolpyruvate carboxykinase 2 (PCK2), revealing a novel mechanistic axis linking metal ion toxicity, proteostasis disruption, and metabolic reprogramming.

Our results confirmed that ES selectively enhances intracellular copper levels in LUAD cells, triggering a form of cell death distinct from apoptosis, ferroptosis, or necroptosis. This specificity was validated by the reversal of cytotoxicity upon treatment with TTM, a copper chelator, and by the lack of rescue with inhibitors targeting other regulated cell death pathways. These findings are consistent with previous studies identifying cuproptosis as a unique modality of cell death driven by copper‐induced mitochondrial stress [8, 35, 39]. However, our data extend this paradigm by implicating ER stress as a critical intermediary in the cytotoxic cascade.

The unfolded protein response (UPR), activated under ER stress conditions, is a key adaptive mechanism that maintains protein folding capacity and cellular homeostasis [26, 27]. In cancer, UPR signaling is frequently upregulated to support tumor survival under hypoxia, nutrient deprivation, and oxidative stress [28, 29]. However, excessive or prolonged ER stress can shift the UPR toward pro‐apoptotic signaling, thereby suppressing tumor growth. In our study, ES + Cu treatment significantly upregulated canonical ER stress markers such as HSPA5 and CHOP, indicating robust activation of the UPR. The use of thapsigargin and 4‐PBA further confirmed that ER stress induction is both necessary and sufficient to modulate downstream gene expression, including PCK2.

PCK2, a mitochondrial isoform of phosphoenolpyruvate carboxykinase, has emerged as a key regulator of metabolic plasticity in cancer cells [31, 32]. It contributes to the replenishment of tricarboxylic acid (TCA) cycle intermediates and supports biosynthetic and antioxidant pathways under stress conditions [31, 32]. Previous studies have shown that PCK2 expression is context‐dependent, with roles in promoting survival under nutrient limitation and in modulating redox balance [40, 41]. Our findings revealed that ES‐induced ER stress leads to significant upregulation of PCK2 at both the mRNA and protein levels. Functional assays demonstrated that PCK2 knockdown enhances LUAD cell proliferation, migration, and colony formation, suggesting that under copper stress, PCK2 acts as a suppressor of malignant phenotypes.

The relationship between copper toxicity and redox homeostasis is particularly relevant in the context of tumor metabolism [38, 42, 43, 44]. Copper ions can catalyze Fenton‐like reactions, generating reactive oxygen species (ROS) and disrupting mitochondrial function [19, 45]. Cancer cells, which often exhibit elevated basal ROS levels due to oncogenic signaling and metabolic reprogramming, rely on antioxidant systems to maintain redox equilibrium [34]. PCK2 has been implicated in supporting NADPH production and glutathione regeneration, thereby contributing to oxidative stress resistance [31, 41].

Importantly, our transcriptomic and bioinformatic analyses revealed that high PCK2 expression is associated with favorable prognosis in LUAD patients. This observation aligns with previous reports indicating that PCK2 expression correlates with reduced tumor aggressiveness and improved survival in certain cancer types. The STRING database analysis further suggested that PCK2 interacts with proteins involved in glucose and lipid metabolism, reinforcing its role as a central node in metabolic regulation. These findings support the notion that PCK2 may serve as both a functional mediator of copper‐induced stress responses and a prognostic biomarker in LUAD.

The translational relevance of our findings was validated using LUAD organoids derived from KRAS ^G12D transgenic mice and xenograft models. ES + Cu treatment significantly reduced organoid viability and tumor growth in vivo, accompanied by increased expression of ER stress markers and PCK2. These results confirm that the ES‐ER stress‐PCK2 axis operates not only in cultured cells but also in physiologically relevant models, underscoring its therapeutic potential.

In summary, this study elucidates a mechanistic framework in which ES‐induced copper influx activates ER stress and upregulates PCK2, leading to suppression of LUAD progression. By integrating metal ion biology, proteostasis regulation, and metabolic control, our findings provide a rationale for targeting the cuproptosis–UPR–metabolism axis in LUAD therapy. Future studies should explore the therapeutic synergy between copper ionophores and agents modulating ER stress or redox pathways, as well as investigate the broader applicability of this strategy across other cancer types. While these findings provide preclinical evidence supporting the ES–ER stress–PCK2 axis as a therapeutic target, we acknowledge that direct clinical translation remains premature. Factors such as pharmacokinetics, copper bioavailability, and tumor‐specific uptake must be rigorously assessed in future studies. Moreover, the use of immunodeficient mouse models limits the evaluation of immune‐mediated effects, which are critical in human cancer therapy.

5. Conclusions

This work bridges metal ion biology, ER stress signaling, and cancer metabolism, offering a conceptual framework for cuproptosis‐driven LUAD therapy. By revealing the ES‐ER stress‐PCK2 axis, we provide mechanistic insights into how copper overload disrupts tumor metabolism while identifying PCK2 as a pivotal regulator of the therapeutic response (Figure 8). These discoveries not only expand the therapeutic arsenal for LUAD but also underscore the importance of context‐dependent metabolic targeting in precision oncology.

Author Contributions

J.Z., and Y.C. performed experiments. J.Z., D.L., Y.Z., J.Z., Z.L. and J.L. participated in the data collection and analysis. J.Z. wrote the manuscript and interpreted data. Z.L. participated in the LUAD tissue collection. J.Z., Y.C. and Y.Z. participated in the animal model. J.H. and Z.L. designed the study and finalized the manuscript. All authors have read and approved the final manuscript.

Ethics Statement

This study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (approval no. 2023‐427). All experimental animal procedures were approved by the Laboratory Animal Center of Soochow University (approval no. 202308A0493).

Consent

Consent for publication was obtained from all the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Functional enrichment analysis on the basis of RNA‐seq data. Gene set enrichment analysis based on RNA‐seq revealed the activation of several important biological pathways in ES + Cu‐treated A549 cells.

Figure S2: Public database analysis suggested that the expression level of the PCK2 gene is lower in LUAD tissues than in normal tissues and that LUAD patients with high PCK2 expression have a better prognosis. (A) TCGA database analysis revealed that, compared with that in normal tissues, the expression level of PCK2 was significantly lower in LUAD tissues. Mann–Whitney U test was used to compare mRNA expression level between groups. (B) Paired expression difference analysis revealed that, compared with that in paired normal tissues, the expression level of PCK2 was significantly lower in LUAD tissues. Mann–Whitney U test was used to compare mRNA expression level between groups. Wilcoxon signed‐rank test was used to compare mRNA expression level between paired‐samples. (C) GEO database analysis indicated that LUAD patients with high PCK2 expression had a better prognosis (p = 0.012). Statistical significance was assessed using the log‐rank test. (D) Kaplan–Meier database analysis indicated that LUAD patients with high PCK2 expression had a better prognosis (p = 0.023, HR = 0.5). Statistical significance was assessed using the log‐rank test. (E) STRING database analysis revealed that the PCK2 protein interacts with a group of proteins involved in glucose metabolism and lipid metabolism.

Figure S3: Genomic variation analyses. (A, B) Venn diagrams illustrating the overlap of single nucleotide polymorphisms (SNPs, A) and insertions/deletions (indels, B) between Kras ^G12D ‐LCOs and in situ LUAD tissues. Numbers represent variant counts in respective regions. (C, D) Circos plots depicting copy number variations (CNVs) in in situ LUAD (C) and Kras ^G12D ‐LCOs (D). Chromosomes (chr) are arranged circularly, with red/blue tracks indicating CNV gain/loss, respectively.

Table S1: Primers used for qRT‐PCR (Quantitative Real‐time PCR).

Table S2: Targeted siRNA (Short interfering RNA) sequence for RNA interference.

Table S3: Primers for targeted region amplification used in transgenic mice identification.

Zhao J., Chen Y., Lu D., et al., “Elesclomol‐Induced Copper Influx Attenuates Lung Adenocarcinoma Progression With Involvement of the ER Stress/PCK2 Axis,” The FASEB Journal 39, no. 22 (2025): e71250, 10.1096/fj.202502105RR.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The raw data of RNA sequencing has been submitted to CNGB Sequence Archive (CNSA) of China National GeneBank DataBase with accession number CNP0007028.