Mechanistics of SGK3 promoting proliferation in ER-positive breast cancer cells by stabilizing PCK2 protein to regulate glycolysis

Hu Qinghua; Chen Bo; Huang Guangxu; Chen Xiaojiao; Zhou Weiying

doi:10.16016/j.2097-0927.202606067

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Mechanistics of SGK3 promoting proliferation in ER-positive breast cancer cells by stabilizing PCK2 protein to regulate glycolysis

Monographic Reports | 更新时间：2026-10-01

- Mechanistics of SGK3 promoting proliferation in ER-positive breast cancer cells by stabilizing PCK2 protein to regulate glycolysis
- Journal of Army Medical University Vol. 48, Issue 18, Pages: 2561-2577(2026)
- 作者机构：
  
  1.重庆医科大学药学院，药理学系，重庆
  2.重庆医科大学药学院，药物代谢重点实验室，重庆
  3.重庆医科大学药学院，生物化学与分子药理学重点实验室，重庆
- 作者简介：
  
  Zhou Weiying， E-mail： wyzhou0118@cqmu.edu.cn
- 基金信息：
  
  the National Natural Science Foundation of China(82373901);the Joint Innovation and Development Fund of Natural Science Foundation of Chongqing(CSTB2022NSCQ-LZX0041);the General Project of Natural Science Foundation of Chongqing(CSTB2025NSCQ-GPX1168)
- DOI：10.16016/j.2097-0927.202606067
  CLC： R343;R730.23;R737.9
- Received：16 June 2026，
  
  Revised：2026-07-21，
  
  Accepted：30 July 2026，
  
  Published：30 September 2026
- 稿件说明：
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胡青华，陈波，黄光旭，等.SGK3通过稳定PCK2蛋白调控糖酵解促进ER阳性乳腺癌细胞增殖的机制研究［J］.陆军军医大学学报，2026，48（18）：2561-2577.DOI：10.16016/j.2097-0927.202606067.

Hu Q H，Chen B，Huang G X，et al.Mechanistics of SGK3 promoting proliferation in ER-positive breast cancer cells by stabilizing PCK2 protein to regulate glycolysis［J］.J Army Med Univ，2026，48（18）：2561-2577.DOI：10.16016/j.2097-0927.202606067.
胡青华，陈波，黄光旭，等.SGK3通过稳定PCK2蛋白调控糖酵解促进ER阳性乳腺癌细胞增殖的机制研究［J］.陆军军医大学学报，2026，48（18）：2561-2577.DOI：10.16016/j.2097-0927.202606067. DOI：

Hu Q H，Chen B，Huang G X，et al.Mechanistics of SGK3 promoting proliferation in ER-positive breast cancer cells by stabilizing PCK2 protein to regulate glycolysis［J］.J Army Med Univ，2026，48（18）：2561-2577.DOI：10.16016/j.2097-0927.202606067. DOI：

摘要

目的

乳腺癌代谢重编程是肿瘤进展的重要驱动因素，但其关键调控分子机制在雌激素受体阳性（estrogen receptor-positive，ER

）乳腺癌中仍不清楚。本研究旨在探讨血清和糖皮质激素调节激酶3（serum and glucocorticoid-regulated kinase 3，SGK3）通过调控磷酸烯醇式丙酮酸羧激酶2（phosphoenolpyruvate carboxykinase 2，PCK2）维持糖酵解相关代谢表型及促进ER

乳腺癌细胞增殖的作用机制。

方法

①MCF7细胞分别构建SGK3功能获得模型（Tet-On诱导过表达，+Dox

-Dox）及功

能缺失模型（siSGK3

siNC）。分别进行蛋白质组学与非靶向代谢组学分析（

=3）。② 通过PEP、乳酸及ATP含量测定及2-NBDG葡萄糖摄取实验评估糖酵解代谢活性； JC-1染色检测线粒体膜电位（ΔΨm），DCFH-DA探针检测胞内活性氧（ROS）水平。③利用TIMER2.0、GEPIA2及Kaplan-Meier plotter数据库分析PCK2在乳腺癌中的表达特征、与SGK3的相关性及其预后价值。④Western blot及实时荧光定量PCR检测PCK2表达变化，并在裸鼠移植瘤模型中进一步验证SGK3敲低对PCK2蛋白表达的调控作用。⑤通过放线菌酮（cycloheximide，CHX）追踪实验联合溶酶体抑制剂氯喹（CQ）及蛋白酶体抑制剂MG132处理，分析PCK2蛋白稳定性及降解途径。⑥免疫共沉淀（Co-IP）、免疫荧光（IF）及分子对接分析SGK3与PCK2的相互作用。⑦糖酵解抑制剂2-脱氧葡萄糖（2-deoxy-D-glucose，2-DG）及氧化磷酸化抑制剂寡霉素（Oligomycin）敏感性实验评估SGK3-PCK2轴对细胞代谢依赖模式的影响，并结合克隆形成实验评价其对细胞增殖能力的作用。

结果

①蛋白质组学和代谢组学分析显示，SGK3相关差异分子主要富集于糖酵解/糖异生和丙酮酸代谢通路（FDR

0.05），其中PCK2被鉴定为关键候选调控分子，其催化产物丙酮酸在SGK3敲低后显著下降（

0.05）。②SGK3敲低显著降低PEP、乳酸及ATP水平（

0.01），抑制葡萄糖摄取能力，并伴随线粒体膜电位下降及ROS水平升高；SGK3过表达则呈相反变化。③TIMER2.0及Western blot分析显示，PCK2在ER

乳腺癌组织及细胞系中显著高表达（

0.05）；GEPIA2相关性分析进一步证实PCK2与SGK3表达呈显著正相关（

=0.11，

=0.000 22）。Kaplan-Meier生存分析表明，PCK2高表达与较差的总生存期（OS）及无复发生存期（RFS）显著相关（

0.01）。④机制研究表明，SGK3不影响PCK2 mRNA表达（

0.05），但显著正调控其蛋白水平（

0.05）。在敲低SGK3的裸鼠异种移植瘤模型中，肿瘤组织PCK2蛋白水平同样显著降低（

0.01），进一步在体内验证了SGK3对PCK2蛋白的正向调控作用。⑤CHX追踪实验显示SGK3延长PCK2蛋白半衰期。进一步降解通路分析显示，CQ可逆转SGK3敲低所致PCK2下降，而MG132无明显影响，提示SGK3主要通过抑制溶酶体依赖性降解维持PCK2蛋白稳定性。⑥Co-IP、IF及分子对接结果一致证实SGK3与PCK2存在相互作用。⑦代谢依赖性分析显示，SGK3过表达增强细胞对2-DG的敏感性，并提高对寡霉素的耐受性；PCK2沉默显著削弱上述效应。功能实验显示，PCK2敲低可部分逆转SGK3过表达诱导的克隆形成能力增强，进一步支持SGK3-PCK2轴在糖酵解代谢重编程及细胞增殖调控中的关键作用。

结论

SGK3通过与PCK2相互作用并抑制其溶酶体依赖性降解，从而维持PCK2蛋白稳定性，进而驱动ER

乳腺癌细胞形成糖酵解相关代谢表型并促进细胞增殖。SGK3-PCK2轴可能构成ER

乳腺癌重要的代谢调控节点及潜在治疗靶点。

Abstract

Objective

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 a

ims 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).

Methods

①MCF7 cells were used to construct SGK3 gain-of-function (Tet-On inducible overexpression

+Dox

-Dox) and loss-of-function (siSGK3

siNC) models

followed by proteomics and untargeted metabolomics analyses (

=3). ②Glycolytic 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. ③TIMER2.0

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. ④Western blotting and quantitative real-time 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. ⑤Cycloheximide (CHX) chase assay combined with chloroquine (CQ

lysosomal inhibitor) and MG132 (proteasomal inhibitor) treatment were used to analyze PCK2 stability and degradation pathways. ⑥Co-immunoprecipitation (Co-IP)

immunofluorescence (IF) assay and molecular docking were applied to examine the interaction between SGK3 and PCK2. ⑦Sensitivity assays with 2-deoxy-D-glucose (2-DG

glycolysis inhibitor) and oligomycin (oxidative phosphorylation inhibitor) were used to evaluate the effect of the SGK3-PCK2 axis on metabolic dependency

and colony formation assay was employed to evaluate its effect on cell proliferation.

Results

①Proteomic and metabolomic analyses revealed that SGK3-related differe

ntially 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 (

0.05). ②SGK3 knockdown significantly decreased PEP

lactate and ATP levels (

0.01)

suppressed glucose uptake

accompanied by reduced ΔΨm and increased intracellular ROS levels

whereas SGK3 overexpression produced the opposite effects. ③TIMER2.0 analysis and Western blotting demonstrated that PCK2 was highly expressed in ER

breast cancer tissues and cell lines (

0.05). GEPIA2 correlation analysis further confirmed a significant positive correlation between PCK2 and SGK3 expression (

=0.11

=0.000 22). Kaplan-Meier survival analysis showed that high PCK2 expression was significantly associated with poorer overall survival (OS) and relapse-free survival (RFS) (

0.01). ④Mechanistically

SGK3 did not affect the mRNA expression of PCK2 (

0.05) but significantly positively regulated its protein level (

0.05). In the nude mouse xenograft model with SGK3 knockdown

PCK2 protein levels were also markedly decreased in tumor tissues (

0.01)

further validating the positive regulation of PCK2 by SGK3

in vivo

. ⑤CHX 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. ⑥Co-IP

IF and molecular docking consistently confirmed the interaction between SGK3 and PCK2. ⑦Metabolic 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.

Conclusion

SGK3 drives glycolysis-related metabolic phenotypes 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 represent an important metabolic regulatory node and a potential therapeutic target in ER

breast cancer.

关键词

Keywords

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