Metabolic reprogramming in breast cancer is an important driver of tumor progression, yet the key regulatory molecular mechanisms governing this process in estrogen receptor-positive (ER⁺) breast cancer remain unclear. This study aims to investigate the mechanism by which serum and glucocorticoid-regulated kinase 3 (SGK3) maintains glycolysis-associated metabolic phenotypes and promotes proliferation in ER⁺ breast cancer cells by regulating phosphoenolpyruvate carboxykinase 2 (PCK2).

① MCF7 cells were used to construct SGK3 gain-of-function (Tet-On inducible overexpression, +Dox vs -Dox) and loss-of-function (siSGK3 vs siNC) models, followed by proteomics and untargeted metabolomics analyses (n=3). ② Glycolytic metabolic activity was evaluated by measuring phosphoenolpyruvate (PEP), lactate and ATP contents, together with 2-NBDG glucose uptake assay. Mitochondrial membrane potential (ΔΨm) and intracellular reactive oxygen species (ROS) levels were assessed using JC-1 staining and DCFH-DA probe, respectively. ③ TIMER2.0, GEPIA2 and Kaplan-Meier Plotter databases were used to analyze the expression profiles of PCK2 in breast cancer, its correlation with SGK3, and its prognostic value in breast cancer. ④ Western blotting and quantitative realtime PCR were performed to determine PCK2 expression, and a nude mouse xenograft model was constructed to validate the regulatory effect of SGK3 knockdown on PCK2 protein expression. ⑤ Cycloheximide (CHX) chase assay combined with chloroquine (CQ, lysosomal inhibitor) and MG132 (proteasomal inhibitor) treatment were used to analyze PCK2 stability and degradation pathways. ⑥ Co-immunoprecipitation (Co-IP), immunofluorescence (IF) assay and molecular docking were applied to examine the interaction between SGK3 and PCK2. ⑦ Sensitivity assays with 2-deoxy-D-glucose (2-DG, glycolysis inhibitor) and oligomycin (oxidative phosphorylation-inhibitor) were used to evaluate the effect of the SGK3PCK2 axis on metabolic dependency, and colony formation assay was employed to evaluate its effect on cell proliferation.

① Proteomic and metabolomic analyses revealed that SGK3-related differentially expressed proteins were predominantly enriched in the glycolysis/gluconeogenesis and pyruvate metabolism pathways (FDR <0.05). PCK2 was identified as a key candidate regulator, and its catalytic product, pyruvate, was significantly reduced after SGK3 knockdown (P<0.05). ② SGK3 knockdown significantly decreased PEP, lactate and ATP levels (P<0.01), suppressed glucose uptake, accompanied by reduced ΔΨm and increased intracellular ROS levels, whereas SGK3 overexpression produced the opposite effects. ③ TIMER2.0 analysis and Western blotting demonstrated that PCK2 was highly expressed in ER⁺ breast cancer tissues and cell lines (P<0.05). GEPIA2 correlation analysis further confirmed a significant positive correlation between PCK2 and SGK3 expression (r=0.11, P=0.00022). Kaplan-Meier survival analysis showed that high PCK2 expression was significantly associated with poorer overall survival (OS) and relapse-free survival (RFS) (P<0.01). ④ Mechanistically, SGK3 did not affect the mRNA expression of PCK2 (P>0.05) but significantly positively regulated its protein level (P<0.05). In the nude mouse xenograft model with SGK3 knockdown, PCK2 protein levels were also markedly decreased in tumor tissues (P<0.01), further validating the positive regulation of PCK2 by SGK3 in vivo. ⑤ CHX chase assays demonstrated that SGK3 prolonged the half-life of PCK2 protein. Further degradation pathway analysis showed that CQ, but not MG132, restored the reduction of PCK2 induced by SGK3 knockdown, indicating that SGK3 maintains PCK2 protein stability primarily by suppressing lysosome-dependent degradation. ⑥ Co-IP, IF and molecular docking consistently confirmed the interaction between SGK3 and PCK2. ⑦ Metabolic dependency analysis demonstrated that SGK3 overexpression enhanced cellular sensitivity to 2-DG while increased tolerance to oligomycin; these effects were markedly attenuated by PCK2 silence. Functional assays further showed that PCK2 knockdown partially reversed the enhancement of clonogenic formation induced by SGK3 overexpression, supporting the critical role of the SGK3-PCK2 axis in glycolytic metabolic reprogramming and cell proliferation.

SGK3 drives glycolysis-related metabolic phenotpes and promotes cell proliferation in ER⁺ breast cancer cells by interacting with PCK2 and inhibiting its lysosome-dependent degradation, thereby maintaining PCK2 protein stability. The SGK3-PCK2 axis may repesent an important metabolic regulatory node and a potential therapeutic target in ER⁺ breast cancer.

To construct the etoposide (VP-16) combined with cisplatin (DDP) chemotherapy scheme (EP chemotherapy scheme) resistant small cell lung cancer (SCLC) cell line H446/EP and to identify the drug resistance characteristics and explore the mechanism.

NCI-H446 cells were treated with increasing concentrations of VP-16 and DDP to construct an H446/EP cell line. H446/EP and NCI-H446 cells were used as the research objects. The cell viability was detected by MTT assay, and the resistance index (RI) of H446/EP cells was calculated. Cell cloning assay and Incucyte cell proliferation (label-free) assay were used to detect cell proliferation ability. Transcriptome sequencing was performed to analyze the enrichment of differentially expressed genes (DEGs) in the 2 cell lines. Western blotting was applied to detect the protein expression levels of drug resistance, DNA damage repair (DDR), and autophagy markers.

MTT assay showed that the resistance index (RI) of H446/EP cells to VP-16, DDP, and DOX were 6.14, 3.43, and 1.96, respectively. The results of cell cloning assay and Incucyte cell proliferation assay indicated that the proliferation ability was significantly higher in the H446/EP cells than the NCI-H446 cells (P<0.01). Transcriptome sequencing and pathway enrichment analysis displayed that the DEGs between H446/EP and NCI-H446 cells were enriched in tumor chemoresistance, DDR, and autophagy pathways. Western blot results showed the expression levels of MRP1, BCRP, RAD51, γ-H2AX, and LC3-II/LC3-I were significantly increased, and that of p62 was obviously decreased in the H446/EP cells when compared with the NCI-H446 cells (P<0.05).

An EP chemotherapy-resistant H446/EP cell line is successfully constructed. Stronger proliferation ability, increased expression of efflux transporters, and enhanced DDR and autophagy may be the mechanisms of the resistance of SCLC to EP chemotherapy scheme.