Phosphoenolpyruvate carboxykinase 2 activation of the AMPK–CEBPB axis to enhance glutamine utilization to promote glycolysis and malignant behavior in adenocarcinomas cells under glucose deprivation

Abstract

Glucose deprivation (Glu‐D) is a critical feature of the tumor microenvironment. Under such conditions, tumor cells seek alternative metabolic resources to maintain rapid growth and proliferation. Glutamine serves as a key alternative resource for cancer cells, yet the metabolic mechanisms involving its transporters in non‐small cell lung cancer remain poorly understood. Lentiviral vectors for overexpression and knockdown of phosphoenolpyruvate carboxykinase 2 (PCK2), solute carrier family 38 member 2 (SLC38A2), and CEBPB were constructed. Transwell, flow cytometry, Western blotting, and dual‐luciferase reporter assays were used to investigate the regulatory relationship between PCK2 and SLC38A2 under Glu‐D, as well as their effects on cellular glutamine metabolism, glycolysis, and malignant cell behaviors. PCK2 and SLC38A2 were highly expressed in human adenocarcinomas tissues. PCK2 upregulated SLC38A2 expression, though this effect was indirect. Under Glu‐D, knockdown of PCK2 or SLC38A2 significantly reduced cellular glutamine utilization, inhibited glycolysis, and suppressed malignant cell behaviors. Treatment with an AMP‐activated protein kinase (AMPK) inhibitor or knockdown of CEBPB produced similar effects. PCK2 activated AMPK, which increased downstream SLC38A2 expression by activating the transcription factor CEBPB. PCK2 upregulates SLC38A2 expression via the AMPK–CEBPB axis, enhancing glutamine utilization to promote glycolysis and malignant behaviors in A549 cells under Glu‐D.

Glucose deprivation is a key characteristic of the tumor microenvironment. In this study, we verified through in vitro experiments that phosphoenolpyruvate carboxykinase 2 (PCK2) upregulates solute carrier family 38 member 2 (SLC38A2) via the AMPK–CEBPB axis, thereby enhancing glutamine utilization in A549 cells, which in turn promotes glycolysis and malignant behaviors of these cells. This uncovers a key metabolic mechanism underlying non‐small cell lung cancer (NSCLC) adaptation to nutritional stress.

1. INTRODUCTION

Non‐small cell lung cancer (NSCLC) is one of the malignant tumors with the highest mortality worldwide. Despite advancements in lung cancer treatment over the past few decades, ¹ its cure rate remains low, underscoring the urgent need for more effective therapies. Lung cancer cells primarily rely on “aerobic glycolysis” or the “Warburg effect” for energy metabolism, preferentially converting glucose to lactate with inefficient ATP production even under sufficient oxygen conditions, rather than generating more ATP through complete glucose oxidation via oxidative phosphorylation. Glucose deprivation (Glu‐D) is reported to be a key feature of the tumor microenvironment, with glucose levels in solid tumor tissues being more than half as low as those in normal tissues. In this context, tumor cells exhibit high metabolic flexibility, adopting alternative metabolic pathways to maintain rapid growth and proliferation. ² , ³

Phosphoenolpyruvate carboxykinase (PEPCK) enhances metabolic flexibility in tumor cells under Glu‐D. ⁴ Its two subtypes, cytosolic PEPCK (PCK1) and mitochondrial PEPCK (PCK2), enable cancer cells to generate glycolytic intermediates for biosynthesis during glucose deficiency, sustaining rapid tumor cell proliferation. PCK2 is the major subtype expressed in NSCLC, with its levels increasing in various NSCLC cell lines under low‐glucose conditions. ⁵ , ⁶ , ⁷ Additionally, studies show that PCK2‐mediated production of phosphoenolpyruvate (PEP) from glutamine‐derived oxaloacetate promotes glucose‐independent proliferation of NSCLC cells. ⁸ The transcriptional regulation of PCK2 is also a critical mechanism by which PGC‐1β and ERRα enhance glutamine metabolism and cancer cell survival. ⁹ Although PCK2 is known as a central molecule linking the tricarboxylic acid cycle, glycolysis, and gluconeogenesis, ⁹ the mechanism of its interaction with glutamine metabolism remains unclear.

Glutamine (Gln) serves as an alternative energy metabolic resource for cancer cells, and its‐mediated metabolic reprogramming is a hallmark of malignant tumors, as cancer cells cannot survive without exogenous glutamine. ¹⁰ Glutamine transporters, key regulators of glutamine metabolism, play important roles in tumor growth and metastasis. Glutamine uptake into cells requires transporters such as SLC1A5/ASCT2 and SLC38A3, most of which have been reported to be associated with lung cancer progression. ¹¹ , ¹² , ¹³ Current anticancer drugs targeting glutamine metabolism mainly focus on enzymes and transporters involved in this pathway, such as CB‐839, 6‐diazo‐5‐oxo‐L‐norleucine, 968, and BPTES. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ However, because tumor metabolism involves not only glutamine pathways but also other molecules and signaling pathways, further exploration of the connections between glutamine transporters and related molecular signals is necessary.

In this study, investigating the relationship and mechanisms between PCK2 and glutamine metabolism in A549 cells revealed that under Glu‐D, PCK2 upregulates the expression of solute carrier family 38 member 2 (SLC38A2) via the AMPK–CEBPB signaling axis. This upregulation enhances glutamine utilization, thereby promoting glycolysis and malignant progression of lung cancer cells. This research highlights the regulatory role of the glutamine transporter SLC38A2 in human adenocarcinomas progression, providing new targets and theoretical foundations for future clinical development of drugs targeting glutamine metabolism and the combined application of related small‐molecule inhibitors to enhance lung cancer treatment efficacy.

2. METHODS

2.1. Reagents

L‐glutamine was purchased from Solarbio, low sugar basal medium, DMEM basal medium, FBS were purchased from Invitrogen. CCK‐8 kit, Annexin V‐FITC apoptosis assay kit, glutamine content, glutamate content, ATP content, pyruvate content, lactate content, and glucose content assay kit were purchased from Beyotime. Glycolysis kit was purchased from Abcam. Dual Luciferase Reporter Gene Assay Kit was purchased from Beyotime. Acadesine (AICAR) and Dorsomorphin (Compound C) were purchased from MCE. Antibodies specific for anti‐PCK2, SLC38A2, p‐AMPK, CEBPB were purchased from Abcam. The specific antibody targeting p‐CEBPB was purchased from Abclonal.

2.2. Bioinformatics analysis

Using data from the GEPIA2 database (http://gepia2.cancer‐pku.cn/#survival) and UALCAN (https://ualcan.path.uab.edu/analysis.html), we analyzed the correlation of six glutamine transporter proteins with survival in patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC).

2.3. Cell culture

A549 (Human NSCLC cell lines, Lineage construction by D. J. Griad by lung cancer tissue transplantation culture derived from a 58‐year‐old white male; RRID: CVCL_0023) was purchased from Wuhan Procell, China, and accessed June 2023. The cell line has recently been supplier verified by STR typing with a 100% match to the ExPASy database, confirming the name of the cell line used in this study as A‐549. Additionally, no cross contamination of human cells was detected in the cell lines identified. In addition, the A549 cell line used in this study ensured no mycoplasma contamination. Control cells were cultured using DMEM complete medium containing 10% fetal bovine serum and 1% penicillin‐streptomycin double antibody. Cells in glucose‐deficient state were maintained in low‐glucose medium. Cells were cultured in a constant temperature incubator at 37°C with 5% CO₂.

2.4. Lentiviral transfected cells

Lentiviral vectors carrying short hairpin RNA (shRNA) sequences targeting PCK2, SLC38A2, and CEBPB and their negative controls (shNC), PCK2, SLC38A2, and CEBPB overexpression (OE) constructs, and the corresponding negative controls (OE‐NC) were purchased from tsingke, Beijing, China. For lentiviral transfection, A549 cells were seeded into 24‐well plates at a density of 5000 cells per well. 24 h later, cells were cultured in lentiviral medium according to the manufacturer's protocol. After 72 h of incubation, the medium was replaced with fresh medium containing 10 μg/mL puromycin to screen for stable cell lines.

2.5. Cell counting kit‐8 (CCK‐8) assay

Cells were inoculated into 96‐well plates, and 5 replicate wells were set in each group. The cells were incubated in a 37°C, 5% CO₂ cell culture incubator for 6 h. The medium of each well was aspirated, and the medium containing different concentrations of drugs was added to each experimental group, and then placed in a 37°C incubator for 24 h. The cells were then incubated for 4 h in a constant temperature incubator. Each well was added with 10 μL CCK8 solution and continued to be incubated in a constant temperature incubator for 4 h. The OD value of the cells in each well was measured at the absorbance of 450 nm on an enzyme meter.

2.6. Cell migration

Cells were seeded into 6‐well plates, and once reaching 70% confluency, a sterile 10 μL pipette tip was used to create vertical scratches through the cell monolayer to form wound gaps. The cells were gently washed three times with sterile PBS to remove detached cells, followed by incubation in serum‐free medium. Images of the wound areas were captured under an inverted microscope at 0 and 24 h post‐scratching. The percentage of wound healing area relative to the initial gap was analyzed using Image J software. The formula for migration rate is: cell migration rate = (initial scratch area − 24 time scratch area)/initial scratch area × 100.

2.7. Cell invasion

Coated Transwell chambers were placed into well plates containing complete medium with 10% FBS. Cell suspensions were adjusted to the desired density using serum‐free medium and added to the upper chambers of the Transwell inserts. At the end of the experiment, cells were fixed with 4% paraformaldehyde for 30 min. After discarding the fixative, chambers were washed once with PBS. Cells were then stained with crystal violet for 10 min, followed by 2–3 PBS washes to remove excess dye, and observed under a microscope.

2.8. Detection of apoptosis by flow cytometry

The cells in each group were stained according to the instructions of the kit and labeled with two fluorescent dyes, PI and FITC, to distinguish normal cells, early apoptotic cells and late apoptotic cells. Subsequently, the labeled cell samples were detected by flow cytometry to accurately measure the apoptosis rate. After obtaining the test data, the professional FlowJo software was used for in‐depth analysis, and the apoptosis data were statistically analyzed.

2.9. Real‐time fluorescence quantitative polymerase chain reaction (RT‐qPCR) assay

Total cellular RNA was extracted using TRIzol reagent. Subsequently, the extracted RNA was reverse‐transcribed into cDNA with the First Strand Synthesis Kit. Then, the cDNA samples were used for qPCR with the SYBR qPCR Master Mix Kit. Primer sequences were as follows: PCK2: Forward primer: 5′‐GTGG GGGA TGAT ATTG CTTG‐3′, Reverse primer: 5′‐TGGT CTCA GCCA CATT GGTA‐3'. SLC38A2: Forward primer: 5′‐AGTT GCCT TTGG TGAT CCAG‐3′, Reverse primer: 5′‐CAGG ACAC GGAA CCTG AAAT‐3'. CEBPB: Forward primer: 5′‐CCCG CCCG TGGT GTTA TTTA‐3′, Reverse primer: 5′‐CACG CGTT CAGC CATG TTTA‐3'. GAPDH: Forward primer: 5′‐CAGC CTCA AGAT CATC AGCA‐3′, Reverse primer: 5′‐ATGA TGTT CTGG AGAG CCCC‐3'. The expression level of the GAPDH gene served as an internal control. The relative expression of the target genes was calculated using the 2‐ΔΔCt method.

2.10. Western blot assay

Lyse cells or tissues (including tumor tissues and paratumor tissues that are more than 3 cm away from the adjacent tumor margin) using RIPA buffer containing a mixture of protease inhibitors and phosphatase inhibitors. After lysis, the protein concentration of the resulting samples was accurately determined using the BCA method. Proteins were separated by electrophoresis on a 4%–20% SDS‐PAGE gel. After electrophoresis, the proteins on the gel were transferred to a PVDF membrane by semi‐dry transfer or wet transfer. After the transfer, closed at room temperature for 1 h. After sealing, the membrane was incubated with the specific primary antibody at 4°C overnight. The next day, the membrane was washed three times with 1 × TBST solution for 10 min each time. After that, the membrane was incubated with the corresponding secondary antibody for 2 h at room temperature. The membrane was developed using ECL detection reagent, and the target protein was detected by chemiluminescence reaction to obtain a clear image of the protein bands. Protein bands were analyzed in grayscale using image J, and β‐actin or GAPDH was used as internal reference for relative quantification.

2.11. Metabolism‐related index content determination

Cells from each group were collected and seeded into 96‐well plates at a density of 2000 cells per well, with an initial culture medium containing 5 mmol/L glucose (lactic acid‐free). After 48 h of incubation, culture supernatants were collected. Glucose and lactate contents were measured using commercial kits: absorbance values were read at the specified wavelengths on a microplate reader, and concentrations were calculated according to the kit protocols.

2.12. Dual luciferase assay and analysis

To construct the luciferase reporter gene vector, the promoter region of SLC38A2 (spanning approximately from −2000 bp upstream to +200 bp downstream relative to the transcription start site, and containing key promoter regulatory elements) was amplified and cloned into the pGL3‐Basic vector. A549 cells were seeded and transfected when the cell confluence reached 50%–70%. The reporter vector, internal reference plasmid, PCK2 or CEBPB overexpression plasmid, and their corresponding negative control vectors were co‐transfected into A549 cells using Lipofectamine 3000. The cells were harvested at 48 h post‐transfection, and the luciferase activity was determined using the dual‐luciferase reporter assay system in strict accordance with the manufacturer's protocol.

2.13. Statistical analysis

All experiments were performed in three independent replications and evaluated using GraphPad Prism 9.0 software to evaluate the processed data. All experiments were replicated independently three times, each replication consisted of three technical replicates, and data were expressed as mean ± SEM. One‐way analysis of variance was used to compare three and more groups of data. p < 0.05 was used as the criterion for statistically significant difference.

3. RESULT

3.1. PCK2 and SLC38A2 are upregulated and positively correlated in LUAD tissues and cell models

To explore the connection between PCK2 and glutamine metabolism, we selected six glutamine‐related genes, including five glutamine transporters (SLC3A2, SLC7A5, SLC1A5, SLC38A2 and SLC38A1) and one key regulator of glutamine metabolism (EIF4B), for survival analysis in adenocarcinomas patients. GEPIA2 analysis (http://gepia2.cancer‐pku.cn/#survival) showed that among these six genes, EIF4B, SLC7A5, and SLC1A5 were not significantly correlated with survival time in adenocarcinomas patients. In contrast, SLC38A2, SLC38A1, and SLC3A2 were significantly associated with LUAD (p < 0.05) but not with LUSC (Figure 1A–C). UALCAN analysis (https://ualcan.path.uab.edu/analysis.html) revealed that only SLC7A5, SLC38A2, and SLC3A2 showed significant correlations with LUAD (p < 0.05) and no significant associations with LUSC (Table 1). Since 85% of NSCLC cases are pathologically classified as LUAD, ²⁰ SLC3A2 and SLC38A2 may play critical roles in NSCLC progression. Although previous studies have reported that SLC3A2 is a key mediator in NSCLC through its molecular mechanisms, ²¹ SLC38A2 remains uninvestigated in the context of NSCLC research. Therefore, we selected SLC38A2 as the target glutamine transporter for subsequent studies.

FIGURE 1.

Correlation of PCK2 and SLC38A2 with NSCLC. (A–C) The relationship between the expression levels of (A) SLC38A2, (B) SLC38A2, and (C) SLC3A2 and the survival of lung cancer patients (LUAD and LUSC) was retrieved and analyzed using the GEPIA2 database. p < 0.05 indicates significant correlation; (D, E) RT‐qPCR for PCK2 overexpression and knockdown efficiency; (F–I) Western blot detection of PCK2 (F, H) overexpression and (G, I) knockdown efficiency; (J, K) Protein expression of SLC38A2 after PCK2 overexpression and knockdown detected by Western blot; (L, M) PCK2 and SLC38A2 mRNA levels detected by RT‐qPCR. (N, O) Western blot detection of protein expression of PCK2 and SLC38A2 in cancer and paracancerous tissues of 10 NSCLC patients; (P, Q) The Western blot bands in Figures N and O were analyzed using Image J software, followed by statistical analysis with GraphPad Prism 9 software. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; N, Normal; NC, Negative control; ns, not significantly different; NSCLC, non‐small cell lung cancer; OE, Overexpression; PCK2, phosphoenolpyruvate carboxykinase 2; RT‐qPCR, Real‐time fluorescence quantitative polymerase chain reaction; SLC38A2, solute carrier family 38 member 2; T, Tumor. *p < 0.05, **p < 0.01, ****p < 0.0001.

TABLE 1.

Correlation of six glutamine‐related genes with LUAD and LUSC, respectively, detected by UALCAN database.

Gene name	LUAD	LUSC
SLC3A2	p = 0.044*	p = 0.56
EIF4B	p = 0.43	p = 0.81
SLC7A5	p = 0.01*	p = 0.26
SLC1A5	p = 0.52	p = 0.81
SLC38A2	p = 0.0053	p = 0.92
SLC38A1	p = 0.19*	p = 0.44

Abbreviations: LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

*p < 0.05 indicates significant correlation.

To determine whether PCK2 affects SLC38A2 expression, A549 cells were subjected to PCK2 overexpression (PCK2‐OE) and knockdown (PCK2‐shRNA) treatments. RT‐qPCR and Western blot analyses confirmed successful PCK2 overexpression and knockdown (Figure 1D–I). The shRNA with the highest knockdown efficiency was selected for detecting SLC38A2 protein expression levels. Western blot results showed that SLC38A2 protein expression was upregulated in A549 cells following PCK2 overexpression; conversely, SLC38A2 expression significantly decreased after PCK2 knockdown (Figure 1J,K). These findings suggest a potential regulatory relationship between PCK2 and SLC38A2 in A549 cells. We next validated mRNA and protein levels of PCK2 and SLC38A2 in clinical NSCLC samples. Results showed that both PCK2 and SLC38A2 expression levels were significantly higher in tumor tissues compared to adjacent non‐tumor tissues from 10 NSCLC patients (Table 2, Figure 1L,Q).

TABLE 2.

Clinicopathological characteristics of 10 patients with non‐small cell lung cancer.

Clinical pathological parameters		Number of patients (n = 10)	Percentage (%)
Age	<60	5	50
	≥60	5	50
Gender	Male	6	60
	Female	4	40
Pathological type	LUAD	9	90
	LUSC	1	10
Occupation	Farmer	5	50
	Freelancer	4	40
	Civil servant	1	10
Lesion location	Left side	7	70
	Right side	3	30
TNM stage	T1b	3	30
	T1c	4	40
	T2a	1	10
	T3	2	20
Clinical stage	Stage IA	7	70
	Stage IB	1	10
	Stage IVA	2	20
Smoking history	Yes	6	60
	No	4	40
Air pollution exposure history	Yes	2	20
	No	8	80
Family history of tumor	Yes	2	20
	No	8	80

3.2. PCK2 promotes glycolysis and malignant behavior in A549 cells by indirectly regulating SLC38A2 expression to increase gln utilization under glucose deficiency conditions

To investigate whether PCK2 affects glutamine utilization in A549 cells via SLC38A2 under Glu‐D, we performed SLC38A2 overexpression and knockdown and measured PCK2/SLC38A2 expression and glutamine/glutamate levels in A549 cells. Results showed successful SLC38A2 overexpression and knockdown (Figure 2A). The results of RT‐qPCR (Figure 2B,C) and Western blot (Figure 2D) showed that supplementation with exogenous glutamine in glucose‐deprived A549 cells significantly upregulated SLC38A2 (Figure 2E) and PCK2 (Figure 2F) expression. SLC38A2 manipulation (knockdown or overexpression) had no effect on PCK2 expression. Knocking down PCK2 significantly reduced SLC38A2 expression, which was reversed by concurrent SLC38A2 overexpression in PCK2‐knockdown cells (Supporting Information Figure S1). Under Glu‐D with exogenous glutamine, knockdown of PCK2 or SLC38A2 led to significantly reduced ATP production (Figure 2G), increased glutamine (Gln) levels (Figure 2H), decreased glutamate (Glu) levels (Figure 2I), decreased lactate/pyruvate levels (end products of glycolysis) (Figure 2J,K), elevated glucose levels (Figure 2L); SLC38A2 overexpression reversed the glutamine/glutamate imbalance caused by PCK2 knockdown and enhanced glycolysis.

FIGURE 2.

PCK2 regulation of solute carrier family 38 member 2 SLC38A2 expression increases Gln utilization under glucose‐deficient conditions. (A), Real‐time fluorescence quantitative polymerase chain reaction for SLC38A2 overexpression and knockdown efficiency. (B, C) Under Glu‐D conditions, the mRNA levels of PCK2 and SLC38A2 were determined by RT‐qPCR; (D–F) Detection of PCK2 and SLC38A2 protein expression by Western blotting under Glu‐D conditions. (G–L) ATP (G), glutamine (H), Glutamate (I), pyruvate (J), lactate (K), and glucose content (L) were detected separately. D, Deprivation; Gln, Glutamine; Glu, Glutamate; Glu‐D, Glucose deprivation; NC, Negative control; ns, not significantly different; OE, Overexpression; PCK2, phosphoenolpyruvate carboxykinase 2; SLC38A2, solute carrier family 38 member 2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Additionally, CCK8, flow cytometry, Transwell, and wound healing assays were used to evaluate the malignant behaviors of A549 cells. Results showed that exogenous glutamine supplementation under Glu‐D enhanced cell invasion (Figure 3A,B), migration (Figure 3C,D), and proliferation while significantly reducing apoptosis (Figure 3E–G); knocking down PCK2 or SLC38A2 under these conditions decreased proliferation, migration, and invasion and increased apoptosis; SLC38A2 overexpression significantly reversed the attenuating effects of PCK2 knockdown on malignant behaviors. Furthermore, a dual‐luciferase reporter assay was conducted to assess PCK2‐mediated regulation of SLC38A2. Results indicated that PCK2 had no obvious transcriptional activity on the SLC38A2 promoter (Figure 3H), suggesting PCK2 may indirectly regulate SLC38A2 expression through other signaling pathways. This result indicates that PCK2 cannot directly regulate SLC38A2, suggesting the existence of intermediate signaling molecules between the two.

FIGURE 3.

Under glucose deprivation conditions the regulation of SLC38A2 expression by PCK2 promotes malignant behaviors of A549 cells. (A, B) Transwell assay for cell invasiveness. (C, D) Detection of cell migration ability by scratch assay. (E, F) Detection of apoptosis by flow cytometry and counting of apoptosis rates in each group. (G) CCK8 assay to detect cell proliferation ability. (H) The dual‐luciferase reporter assay was performed to determine whether PCK2 exerts transcriptional activity on SLC38A2. D, Deprivation; Gln, Glutamine; Glu‐D, Glucose deprivation; NC, Negative control; ns, not significantly different; OE, Overexpression; PCK2, phosphoenolpyruvate carboxykinase 2; SLC38A2, solute carrier family 38 member 2. *p < 0.05, ***p < 0.001, ****p < 0.0001.

3.3. PCK2 regulation of SLC38A2 expression increases gln utilization enhances glycolysis and malignant behavior‐dependent AMP‐activated protein kinase (AMPK) signaling in A549 cells

AMPK is a key sensor and regulator of cellular metabolism. ²² Studies have shown that activation of the PCK2‐AMPK signaling axis can regulate the differentiation of pluripotent stem cells, which has been supported by numerous studies. ²³ However, the association between the PCK2‐AMPK signaling axis and SLC38A2, as well as its role in the occurrence and development of NSCLC, remains unclear. To address this, A549 cells were treated with an AMPK activator (AICAR) and inhibitor (compound C) to assess glycolysis and malignant behaviors under Glu‐D. RT‐qPCR assay results showed that the inhibition of AMPK activity significantly downregulated the mRNA expression of PCK2 and SLC38A2 in A549 cells under normal conditions (Figure 4A,B). Consistent results were obtained via Western blot analysis (Figure 4C–F, Supporting Information Figure S2): knocking down PCK2 led to a notable decrease in both AMPK activity and SLC38A2 expression. Notably, when A549 cells with PCK2 knockdown were treated with an AMPK activator, PCK2 expression did not change significantly compared to the PCK2‐knockdown group, but SLC38A2 expression was significantly upregulated. Inhibiting AMPK activity or PCK2 expression decreased pyruvate/lactate (Figure 4G,H) and ATP levels (Figure 4J) and glutamine utilization (Figure 4K,L), while increasing glucose content (Figure 4I). Activating AMPK in PCK2‐knockdown cells significantly restored glutamine utilization, enhanced glycolysis, and promoted tumor malignant behaviors despite suppressed PCK2 expression.

FIGURE 4.

PCK2 regulates the expression of SLC38A2 and thereby promotes glutamine metabolism in A549 cells via the AMPK signaling pathway. (A, B) The changes in mRNA levels of PCK2 and SLC38A2 in A549 cells under different treatment conditions were determined by RT‐qPCR; (C) Protein expression of PCK2 and SLC38A2 and phosphorylation level of AMPK by western blot; (D–F) Gray‐scale analysis and statistical analysis were performed on the Western blot bands of (D) PCK2, (E) SLC38A2 and (F) p‐AMPK; (G–L), pyruvate (G), lactate (H), glucose (I), ATP (J), glutamate (K) and glutamine (L) content were detected separately. AMPK, AMP‐activated protein kinase; Gln, Glutamine; Glu, Glutamate; ns, not significantly different; PCK2, phosphoenolpyruvate carboxykinase 2; SLC38A2, solute carrier family 38 member 2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In addition, assessment of malignant behaviors in A549 cells revealed that treatment with an AMPK inhibitor or PCK2 knockdown significantly reduced cell proliferation (Figure 5A), invasion, and migration while increasing apoptosis. Conversely, treating PCK2‐knockdown cells with an AMPK activator reversed these malignant phenotypes (Figure 5B–G). These results suggest that PCK2 regulation of SLC38A2 expression promotes glycolysis and malignant behavior‐dependent AMPK signaling pathway in A549 cells. Notably, the metabolic defects induced by PCK2 knockdown were significantly reversed following AMPK activation by AICAR, confirming that the metabolic regulatory function of PCK2 is specifically mediated through the AMPK signaling pathway.

FIGURE 5.

Phosphoenolpyruvate carboxykinase 2 regulates the expression of solute carrier family 38 member 2 and thereby promotes the malignant behaviors of A549 cells via the AMP‐activated protein kinase signaling pathway. (A), CCK8 assay to detect cell proliferation ability; (B, C) Detection of apoptosis by flow cytometry and counting of apoptosis rates in each group; (D, E) Transwell assay for cell invasiveness; (F, G) Detection of cell migration ability by scratch assay. ns, not significantly different; **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.4. PCK2 regulates SLC38A2 expression through AMPK–CEBPB signaling axis under glucose deficiency

CCAAT/enhancer‐binding protein β (CEBPB) is a critical transcription factor belonging to the CEBP gene family. It plays a key role in inflammation, cell proliferation, and differentiation. CEBPB is also a common downstream transcription factor of the AMPK pathway, and AMPK activation enhances its transcriptional activity. ²⁴ Studies have shown that the AMPK signaling pathway can activate CEBPB in human tumor cells via regulating the HuR‐UPA axis. ²⁵ To validate the role of the AMPK–CEBPB signaling axis in regulating SLC38A2 expression in glucose‐deprived NSCLC cells, we overexpressed or knocked down CEBPB and measured SLC38A2 expression changes. RT‐qPCRresults showed that SLC38A2 expression was significantly increased in glucose‐deprived A549 cells overexpressing CEBPB (Figure 6A), whereas the opposite effect was observed after CEBPB knockdown (Figure 6B,C). Results from Western blot analysis demonstrated that the changes in SLC38A2 protein expression levels following CEBPB overexpression or knockdown were consistent with those at the mRNA level (Figure 6D,E). Western blot analysis further demonstrated that in glucose‐deprived A549 cells, sequential PCK2 knockdown followed by CEBPB overexpression resulted in unchanged PCK2 protein levels and AMPK activity, whereas the protein expressions of SLC38A2 and CEBPB as well as CEBPB phosphorylation levels were significantly increased. Treatment with an AMPK activator led to upregulated levels of SLC38A2, CEBPB, and phosphorylated CEBPB (p‐CEBPB), along with enhanced AMPK activity, while PCK2 levels showed no significant alteration. In contrast, compared with CEBPB knockdown alone, concurrent AMPK activation and CEBPB knockdown significantly reduced SLC38A2 and CEBPB expressions as well as CEBPB phosphorylation levels, with no changes observed in PCK2 levels and AMPK activity (Figure 6F–L, Supporting Information Figure S3). Similarly, analogous expression changes in PCK2, SLC38A2, and CEBPB at the mRNA level were also determined via RT‐qPCR (Figure 6M–O). A dual‐luciferase reporter assay confirmed that CEBPB directly activates the promoter region of SLC38A2 (Figure 6P). Collectively, these findings indicate that PCK2 regulates SLC38A2 expression through the AMPK–CEBPB signaling axis.

FIGURE 6.

PCK2 regulates SLC38A2 expression through the AMPK–CEBPB signaling axis. (A, B) Real‐time fluorescence quantitative polymerase chain reaction (RT‐qPCR) for CEBPB overexpression and knockdown efficiency; (C) under glucose deprivation conditions, the mRNA levels of SLC38A2 were determined by RT‐qPCR following CEBPB knockdown or overexpression; (D, E) Western blot for protein expression of SLC38A2; (F) Western blot detection of PCK2, SLC38A2, p‐AMPK level in different treatment groups; (G) the expression and phosphorylation level of CEBPB protein were determined by Western blot analysis; (H–L) gray‐scale analysis of Western blot bands for each protein: (H) PCK2, (I) SLC38A2, (J) p‐AMPK, (K) CEBPB and (L) p‐CEBPB; (M‐O) RT‐qPCR detection of (M) PCK2, (N) SLC38A2 and (O) CEBPB mRNA in different treatment groups; (P) activation of SLC38A2 transcriptional activity by CEBPB detected by dual luciferase assay. D, Deprivation; Glu‐D, Glucose deprivation; NC, Negative control; ns, not significantly different; OE, Overexpression; PCK2, phosphoenolpyruvate carboxykinase 2; SLC38A2, solute carrier family 38 member 2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.5. PCK2 activation of the AMPK–CEBPB signaling axis regulates SLC38A2 expression driving gln utilization under glucose deficiency to promote glycolysis and malignancy in A549 cells

Next, we validated the effect of PCK2 regulating SLC38A2 expression via the AMPK–CEBPB signaling axis on glutamine metabolism and glycolysis in glucose‐deprived NSCLC cells. Results showed that under Glu‐D with exogenous glutamine supplementation, PCK2 knockdown significantly reduced glutamine utilization, decreased ATP production, and suppressed glycolysis; overexpressing CEBPB in these cells abolished these effects. When CEBPB was knocked down and cells were treated with an AMPK activator, glutamine utilization (Figure 7A,B), ATP production (Figure 7C), and glycolytic activity (Figure 7D,E) were significantly lower and glucose levels are significantly higher (Figure 7F) compared to the AMPK activator alone group.

FIGURE 7.

Phosphoenolpyruvate carboxykinase 2 activation of the AMPK–CEBPB signaling axis regulates solute carrier family 38 member 2 expression driving Gln utilization under glucose. (A–F) (A) glutamic acid, (B) glutamine, (C) ATP, (D) pyruvate, (E) lactate, and (F) glucose content were detected separately. D, Deprivation; Glu‐D, Glucose deprivation; ns, not significantly different; OE, Overexpression; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Additionally, under Glu‐D, PCK2 knockdown led to a significant reduction in A549 cell proliferation, migration, and invasion, along with increased apoptosis; these phenotypes were reversed by CEBPB overexpression. Treating CEBPB‐knockdown cells with an AMPK activator resulted in significantly lower proliferation, migration, and invasion, as well as higher apoptosis, compared to the AMPK activator alone group (Figure 8A–G). These results indicate that under Glu‐D conditions, PCK2 can activate the AMPK–CEBPB signaling axis, thereby upregulating SLC38A2 to promote glutamine utilization and metabolic reprogramming in NSCLC cells, and ultimately enhancing malignant cellular behaviors. The schematic diagram of this mechanism is presented in Supporting Information Figure S4.

FIGURE 8.

Activation and regulation of the AMPK–CEBPB signaling axis by phosphoenolpyruvate carboxykinase 2 modulates solute carrier family 38 member 2 expression, thereby promoting glycolysis and malignant behaviors of A549 cells. (A, B) Detection of cell migration ability by scratch assay; (C, D) Transwell assay for cell invasiveness; (E, F) detection of apoptosis by flow cytometry and counting of apoptosis rates in each group; (G) CCK8 assay to detect cell proliferation ability. D, Deprivation, Glu‐D, Glucose deprivation; ns, not significantly different; OE, Overexpression; **p < 0.01, ***p < 0.001, ****p < 0.0001.

4. DISCUSSION

Aerobic glycolysis is a key mechanism for tumor cell survival, providing ample energy and essential metabolic intermediates to meet the needs of rapid proliferation, migration, and invasion. This enhanced pathway is a hallmark of most malignant tumors. In recent years, studies have shown that when the tumor microenvironment deteriorates—such as under Glu‐D—cancer cells can break down glycogen to supply energy and substrates for biosynthesis, playing a critical role in their survival and growth. ²⁶ However, the specific molecular mechanisms underlying this process remain unclear.

Research indicates that Glu‐D in LUAD tumors induces cancer cell dedifferentiation, leading to a more invasive phenotype. Glutamine catabolism is crucial for tumor cell survival and proliferation under Glu‐D. ² In this study, supplementing glucose‐deprived A549 LUAD cells with glutamine significantly enhanced their proliferation, migration, and invasion capabilities. Concurrently, glutamine utilization and glycolytic rates increased, providing more ATP for rapid cell growth—findings consistent with previous reports. ²⁷

Under Glu‐D, cancer cells rely on PCK2 to convert glutamine‐derived PEP into substrates for glucose‐dependent biosynthetic pathways. PCK2 is essential for maintaining tumor cell proliferation in vitro under Glu‐D and tumor growth in vivo. Elevated PCK2 expression has been observed in several human tumor tissues compared to normal tissues, particularly enriched in NSCLC patient tumors. ⁵ Our study mirrored these results: both PCK2 and the glutamine transporter SLC38A2 showed significantly higher mRNA and protein levels in cancer tissues from 10 NSCLC patients compared to adjacent non‐tumor tissues. PCK2 and SLC38A2 expression exhibited a positive correlation, suggesting their roles in LUAD may be interconnected. Previous work has shown that PCK2 loss reduces glutamine availability for glutathione (GSH) synthesis. ²⁸ Our study confirmed that PCK2 knockdown decreased glutamine utilization, reducing energy production to support rapid NSCLC cell growth and attenuating glycolysis and malignant behaviors in A549 cells. Knocking down SLC38A2 yielded identical results, while SLC38A2 overexpression reversed the effects of PCK2 knockdown on glycolysis and cell growth. Notably, dual‐luciferase reporter assays revealed that PCK2 overexpression failed to activate the SLC38A2 promoter, indicating that PCK2 does not directly regulate SLC38A2. Therefore, we conducted a series of functional experiments to identify the intermediate signaling axis connecting PCK2 and SLC38A2. Specifically, PCK2 knockdown led to reduced p‐AMPK levels, downregulation of CEBPB and SLC38A2, and consequent metabolic impairment and suppression of malignant phenotypes. Inhibiting AMPK activity produced effects similar to those of PCK2 knockdown. Conversely, although AMPK activation did not reverse the suppression of PCK2 expression, it successfully rescued the glutamine utilization deficit caused by PCK2 knockdown, enhanced glycolysis, and restored malignant behaviors. These findings demonstrate that AMPK activation is a critical downstream step in the PCK2 pathway.

AMPK, a key energy sensor in eukaryotic cells, maintains intracellular energy homeostasis. Studies have shown that AMPK activation under Glu‐D upregulates phosphoglucomutase 1 to promote glycogenolysis and energy supply for tumor growth. However, AMPK exhibits dual roles in tumor progression—pro‐tumor and anti‐tumor—via different signaling pathways in various cancer cell types and stages. ²⁶ , ²⁹ In our study, AMPK was activated in A549 cells under Glu‐D with glutamine supplementation. Knocking down PCK2 or treating cells with an AMPK inhibitor similarly attenuated glycolysis and malignant behaviors, effects reversed by AMPK activator treatment. These findings indicate that AMPK activation under Glu‐D promotes tumor progression, mediated by PCK2. Additionally, we identified the AMPK–CEBPB axis as a regulator of SLC38A2 in A549 cells: under Glu‐D, PCK2 activates AMPK, which enhances downstream SLC38A2 expression via the transcription factor CEBPB. This upregulation increases glutamine utilization and energy production, boosting glycolysis and strengthening proliferation, migration, and invasion to exacerbate lung cancer malignancy. It should be noted that the use of paratumoral tissues as controls has certain limitations. Paratumoral tissues adjacent to the tumor may undergo certain molecular alterations under the influence of the tumor microenvironment. Therefore, the observed differences between tumor and paratumoral tissues in this study may also reflect the local microenvironmental responses induced by the tumor. However, the conclusion drawn from this study—that PCK2 mediates the progression of NSCLC via the AMPK–CEBPB–SLC38A2 signaling axis under Glu‐D—was derived from rigorous functional experiments conducted in classic lung cancer cell lines. In addition, all paratumoral tissues used in this study have been pathologically confirmed to be free of tumor cell infiltration and precancerous lesions, which provides a reasonable basis for using paratumoral tissues as controls. Future studies can employ techniques such as spatial transcriptomics to further dissect the differential activity of this mechanism in the tumor core region, invasive front, and distal normal tissues.

It is important to note that this study still has several limitations. First, the in vitro experiments were conducted primarily based on the A549 cell line. Although A549 is a widely used classic model in LUAD research, under Glu‐D conditions, the molecular mechanism by which PCK2 regulates SLC38A2 via the AMPK–CEBPB axis still requires further validation in other NSCLC cell lines with different genetic backgrounds to confirm the generalizability of this regulatory pathway. Second, because of resource constraints, this study has not verified the regulatory role of PCK2 in the AMPK–CEBPB axis and its functional relevance to in vivo tumor progression using animal models, and the in vivo mechanistic validation remains to be improved. Although this study has initially confirmed the expression correlation between PCK2 and SLC38A2 in clinical samples, the sample size of the included clinical cohort is relatively limited, and the clinical applicability of the relevant conclusions still needs further verification and confirmation in larger‐scale clinical cohorts. In addition, further analysis of SLC38A2 and glutamine metabolism using techniques such as stable isotope tracing metabolomics to provide direct evidence for the impact of SLC38A2 on metabolic flux would also be highly informative. In subsequent work, we will extend the validation to other NSCLC cell lines (such as H1299, H1975); meanwhile, we will establish nude mouse xenograft models or patient‐derived xenograft models to further clarify the molecular mechanism by which PCK2 regulates SLC38A2 via the AMPK–CEBPB axis to promote LUAD progression from an in vivo perspective, thereby providing a more solid experimental basis for the clinical translation of relevant theories and the development of targeted therapeutic strategies.

5. CONCLUSION

Our study identifies a novel signaling axis by which lung cancer cells enhance glycolysis and malignant behaviors under Glu‐D: PCK2 upregulates SLC38A2 via the AMPK–CEBPB axis to improve glutamine utilization, thereby promoting glycolysis and malignant phenotypes in glucose‐deprived A549 cells. This work is the first to clarify the regulatory mechanism between PCK2 and glutamine transporters in lung cancer cells, providing new target strategies and theoretical foundations for metabolic‐targeted therapies in lung cancer.

AUTHOR CONTRIBUTIONS

Libo Ruan: Study design, data analysis, data interpretation, writing—original draft. Kewang Xu: Data collection, data analysis, writing—original draft, literature analysis. Wenjun Zeng: Study design, data analysis, data interpretation. Ling Xiao: Data collection, data analysis, literature analysis. Minjun Zhao: Study design, funding acquisition, data interpretation, project administration, resources, supervision, writing—review and editing. Haiyan Zhang: Study design, funding acquisition, data interpretation, project administration, resources, supervision, writing—review and editing. All authors have agreed to the final submitted version.

CONFLICT OF INTEREST STATEMENT

The authors declare no potential conflicts of interest.

ETHICS STATEMENT

The study involving human subjects was approved by the Medical Ethics Committee of the First People's Hospital of Yunnan Province (Approval No. KHLL2024‐KY136), and the study was conducted in accordance with local legal and institutional requirements.

CONSENT

Informed consent was obtained from all study participants.

Supporting information

Figure S1

Figure S2

Figure S3

Figure S4

DATA AVAILABILITY STATEMENT

The data involved in this study are labeled in the text, and additional data are available from the corresponding author upon request.