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PSME3 promotes glycolysis and migration of gastric cancer cells via regulating the EGFR/c-myc pathway
1Department of Gastroenterology, Hangzhou Ninth People’s Hospital, 311225 Hangzhou, Zhejiang, China
*Corresponding Author(s):xushasha0820_X@163.com (Shasha Xu)
| History | Submitted: 11 January 2024 | Accepted: 04 February 2024 | Published: 29 February 2024 |
| Copyright: | ©2024 The Author(s). Published by MRE Press. |
Gastric cancer (GC) is a common malignant tumor that is pernicious to the health of patients. Proteasome activator subunit 3 (PSME3) has been shown to exhibit higher expression and aggravate tumorigenesis in cancer progression. A major finding is that PSME3 is also highly expressed in GC tissues, which results in a worse prognosis. Nevertheless, the regulatory functions of PSME3 in GC progression remain unclear. This study aimed to investigate the impacts of PSME3 and related regulatory pathways on GC progression. From the Gene Expression Profiling Interactive Analysis (GEPIA) database, it was noted that PSME3 expression was up-regulated in GC tissues. Our findings suggested that in GC, PSME3 showed higher expression, resulting in a worse prognosis. Functional experiments revealed that PSME3 accelerates cell growth, migration and abnormal glycolysis in GC. PSME3 stimulates the epidermal growth factor receptor (EGFR)/c-myc pathway. In conclusion, GC cells exhibited higher PSME3 expression, which modulated the EGFR/c-myc pathway to facilitate glycolysis and migration. PSME3 might be an effective bio-target for GC treatment.
Cite this article
Liang Han, Qingfeng Yang, Jiacai Lei, Shasha Xu. PSME3 promotes glycolysis and migration of gastric cancer cells via regulating the EGFR/c-myc pathway.Journal of Men's Health,2024,20(2):135-140 DOI:10.22514/jomh.2024.029
1. Introduction
Gastric cancer (GC) is a swart tumor that arises in the gastric mucosa epithelium, whose incidence rate occupies the top spot among diversified cancers in China [1, 2]. Changes in diet structure, increased work pressure, and Helicobacter pylori infection contribute to GC’s inclination towards youth [3, 4]. Surgical resection is the dominant treatment for GC. As GC symptoms are difficult to observe in the early stages, most patients are diagnosed at a later stage, losing the opportunity to undergo resection [5]. Despite chemotherapy being a clinical therapy for GC, GC possesses strong metastatic ability, leading to a poor prognosis [6]. Therefore, understanding the mechanism and process of GC and finding effective molecular targets can be useful in GC treatment and diagnosis.
Proteasome activator subunit 3 (PSME3) is a subunit of 11S proteasome regulators referred to as REGγ and PA28gamma (PA28γ). It modulates the degradation of many key regulatory proteins in cancer [7, 8]. One study revealed that PSME3 increases radiation sensitivity in colon cancer cells, while PSME3 suppression can retard tumorigenesis by reducing the expression of cell cyclin-related proteins (cyclin B1 and cyclin-dependent kinase 1 (CKD1)) [9]. Moreover, PSME3 displays high expression in breast cancer, and PSMES enhances the stem characteristics of tumors, strengthens epithelial-mesenchymal transitions, thereby aggravates the malignant phenotype of breast cancer [10]. Additionally, PSME3 modulates c-myc degradation in pancreatic cancer cells, contributing to abnormal glycolysis and cell proliferation [11]. Importantly, PSME3 is highly expressed in GC, as well as having a poor prognosis among GC patients [12]. However, the detailed regulatory functions of PSME3 and its related molecular mechanism for GC progression remain unclear, calling for further research.
This study examines PSME3’s biological functions in GC progression. Results elucidated that PSME3 exhibited higher expression and facilitated glycolysis and migration of GC cells through modulating the EGFR/c-myc pathway. Our findings may provide a novel target for effective GC therapy.
2. Materials and methods
2.1 Cell lines and cell culture
GC cell lines (AGS, SNU-1 and NCI-N87) and gastric mucosa cell line (GES-1) were obtained from the American Tissue Culture Collection (ATCC, USA). Cell culturing was conducted in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS, 10099-141, Gibco, Grand Island, NY, USA) at 37 °C with 5% CO2 in a moist incubator.
2.2 Cell transfection
PSME3 knockdown plasmids (shPSME3-1# and shPSME3-2#) and an empty plasmid (shNC) were synthesized from GenePharma (Shanghai, China). Cell transfection was performed with Lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA, USA) in line with the manufacturer’s instructions.
2.3 Western blot
The total protein in GC cells was isolated using a radioimmunoprecipitation assay (RIPA) solution (P0013B, Beyotime, Shanghai, China). Protein samples in each lane were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then migrated to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Primary antibodies in membranes were then cultured overnight at 4 °C. After washing, further cultivation of horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:1000 dilution, Abcam) in membranes was performed. The bands were assessed through a chemiluminescence detection kit (89880, Pierce, Rockford, lL, USA).
Primary antibodies: PSME3 (ab157157, 1/1000 dilution, Abcam, Shanghai, China), matrix metalloproteinase 9 (MMP9) (ab76003, 1/1000 dilution), MMP2 (ab92536, 1/1000 dilution), glucose transporter 1 (GLUT1) (ab115730, 1/100,000 dilution), hexokinase 2 (HK2) (ab209847, 1/1000 dilution), Lactate Dehydrogenase A (LDHA) (ab52488, 1/5000 dilution), EGFR (ab40815, 1/500 dilution), EGFR (ab52894, 1/1000 dilution), c-myc (ab32072, 1/1000 dilution) and β-actin (ab6276, 1/5000 dilution).
2.4 CCK-8 assay
The cell counting kit-8 (CCK8) assay was utilized to assess cell viability. AGS cells were seeded into 96-well plates. At 0, 24, 48 and 72 h, 20 μL of CCK8 solution (C0046, Beyotime, Shanghai, China) was added to each well and incubated for another 4 h. An optical density of 450 nm was confirmed.
2.5 EDU assay
The 5-ethynyl-20-deoxyuridine (EDU) assay kit (C10337, Thermo Fisher Scientific, Waltham, MA, USA) was adopted to examine cell proliferation. AGS cells (1 × 105) were seeded into 6-well plates. After 48 h, 100 μL of EDU was added and incubated for 2 h. Cells were mixed with 4% formaldehyde, followed by 0.3% Triton X-100. EDU-positive cells were captured under fluorescence microscopy (IX71, Olympus, Tokyo, Japan).
2.6 Transwell assay
For cell invasion, Transwell chambers (Corning, Tewksbury, MA, USA) coated with Matrigel (Bedford, MA, USA) were used. The upper chamber contained 200 μL of serum-free medium, while the lower chamber contained 15% FBS medium. After 48 h culturing, AGS cells were immobilized in 4% paraformaldehyde (P0899, Sigma-Aldrich, St. Louis, MO, USA) and stained with 0.1% crystal violet (C3886, Sigma-Aldrich, St. Louis, MO, USA). As in the above steps, the migration assay is performed in chambers without Matrigel. The stained, migrated and invaded cells were imaged under a microscope (E200, Nikon, Tokyo, Japan).
2.7 The detection of glucose consumption and lactate production
Measurements of glucose consumption and lactate production in AGS cells were performed using glucose assay kits (KA4086, Abnova, Taiwan, China) and lactate assay kits (ab65330, Abcam, Shanghai, China).
2.8 Statistical analysis
Data were displayed as mean ± standard deviation (SD). The statistical analysis was conducted using GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA). Two groups were compared using Student’s t-test, and multiple groups were compared using one-way analysis of variance (ANOVA). p < 0.05 was deemed statistically significant.
3. Results
3.1 The higher PSME3 expression was discovered in GC
From the GEPIA database, PSME3 expression was up-regulated in GC tissues compared with normal tissues (Fig. 1A). Moreover, GC cell lines (AGS, SNU-1 and NCI-N87) had higher PSME3 protein expression than gastric mucosa cells (GES-1) (Fig. 1B). According to the kmplot database (OS and FP), the prognosis of GC patients with higher PSME3 expression was worse (Fig. 1C). In GC, higher PSME3 expression was observed.
Fig. 1.The higher PSME3 expression was discovered in GC. (A) The expression of PSME3 in normal and GC tissues was obtained from the GEPIA database. (B) By western blot, PSME3 protein expression in gastric mucosa cell line (GES-1) and GC cell lines (AGS, SNU-1 and NCI-N87) was examined. (C) The prognosis of GC patients was verified in the kmplot database (OS and FP). *p < 0.05, **p < 0.01, ***p < 0.001. GC: Gastric cancer; PSME3: Proteasome activator subunit 3; GEPIA: Gene Expression Profiling Interactive Analysis.
3.2 PSME3 accelerated cell growth in GC
Fig. 2A confirms PSME3’s knockdown efficiency. PSME3 inhibition reduced cell viability (Fig. 2B). Furthermore, cell proliferation ability was weakened after PSME3 suppression (Fig. 2C). In GC, PSME3 accelerated cell growth.
Fig. 2.PSME3 accelerated cell growth in GC. (A) PSME3 protein expression was examined after PSME3 knockdown. (B) Cell viability was tested after PSME3 suppression through CCK-8 assay. (C) Cell proliferation was determined after PSME3 inhibition through EDU assay. **p < 0.01, ***p < 0.001. PSME3: Proteasome activator subunit 3; shNC: short hairpin RNA negative control; OD: optical density; EDU: 5-Ethynyl-2′-deoxyuridine; DAPI: 4′,6-diamidino-2-phenylindole.
3.3 PSME3 facilitated cell migration in GC
Silencing PSME3 reduced cell migration and invasion abilities (Fig. 3A,B). In addition, MMP9 and MMP2 protein expressions were down-regulated after PSME3 suppression (Fig. 3C). Overall, PSME3 facilitated cell migration in GC.
Fig. 3.PSME3 facilitated cell migration in GC. (A) Cell migration ability was evaluated after PSME3 silencing through Transwell assay. (B) Cell invasion ability was tested after PSME3 inhibition through Transwell assay. (C) MMP9 and MMP2 protein expressions were examined after PSME3 suppression through western blot. **p < 0.01, ***p < 0.001. PSME3: Proteasome activator subunit 3; shNC: short hairpin RNA negative control; MMP: Matrix Metalloproteinase.
3.4 PSME3 strengthened abnormal glycolysis in GC
PSME3’s regulatory effects on glycolysis were investigated. After PSME3 knockdown, GLUT1, HK2 and LDHA protein expression were all reduced (Fig. 4A). Glucose consumption and lactate production decreased after PSME3 repression (Fig. 4B,C). In brief, PSME3 strengthened abnormal glycolysis in GC.
Fig. 4.PSME3 strengthened abnormal glycolysis in GC. (A) GLUT1, HK2 and LDHA protein expressions were assessed after PSME3 inhibition through western blot. (B) Glucose consumption was detected after PSME3 repression using the commercial kit. (C) Lactate production was evaluated after PSME3 knockdown using the commercial kit. *p < 0.05, **p < 0.01, ***p < 0.001. PSME3: Proteasome activator subunit 3; shNC: short hairpin RNA negative control; GLUT: glucose transporter; HK: hexokinase; LDHA: Lactate Dehydrogenase A.
3.5 PSME3 stimulated the EGFR/c-myc pathway
The protein expressions of p-EGFR/EGFR and c-myc were decreased after restraining PSME3 (Fig. 5), suggesting that PSME3 stimulated the EGFR/c-myc pathway.
Fig. 5.PSME3 stimulated the EGFR/c-myc pathway. Protein expressions of p-EGFR, EGFR and c-myc were measured after inhibiting PSME3 through western blot. **p < 0.01, ***p < 0.001. PSME3: Proteasome activator subunit 3; shNC: short hairpin RNA negative control; EGFR: epidermal growth factor receptor.
4. Discussion
Increasing proteins have been claimed to contribute to GC progression. For example, PRMT1 stimulates the β-catenin pathway in GC to strengthen cell proliferation and metastasis through recruiting MLXIP [13]. Additionally, AFF3 serves as a novel prognostic biomarker that affects immunotherapy in GC [14]. Silencing RACK1 accelerates glutamine addiction and tumor growth by targeting the AKT/mTOR/ASCT2 axis in GC [15]. Besides, NSD1 modulates WNT10B to facilitate cell survival and migration in GC [16]. PSME3 has been shown to display high expression and aggravate tumorigenesis in cancer progression [9, 10, 11]. Importantly, PSME3 is highly expressed in GC tissues, resulting in a poor prognosis [12]. PSME3’s regulatory functions remain dim. In this work, a GEPIA database analysis confirmed upregulation of PSME3 expression in GC tissues. Our findings suggested that PSME3 expression was higher in GC, which led to a worse prognosis as a result.
As a metabolic hallmark of cancer, glycolysis refers to the prioritization of glucose conversion into lactate over oxidative phosphorylation to supply energy requirements [17]. Lactate is the end outcome of glycolysis; the accumulation of it is a feature of glycolysis and drives tumor development [18]. Glycolysis progress is implicated in GC progression regulation. Insulin gene enhancer protein 1 (ISL1) modulates glucose transporter 4 (GLUT4) in GC to enhance glycolysis and tumorigenesis [19]. In addition, protein phosphatase 2a (PP2A) retards MYC signaling to attenuate glycolysis in GC [20]. As a consequence, H19 accelerates aerobic glycolysis and immune escape by targeting the miR-519d-3p/lactate dehydrogenase A (LDHA) axis in GC [21]. The hexokinase domain containing protein-1 (HKDC1) aggravates glycolysis and strengthens chemoresistance in GC [22]. Therefore, it is necessary to explore PSME3’s regulatory functions in glycolysis in further detail. In this study, PSME3 was found to accelerate cell growth, migration and abnormal glycolysis in GC.
A critical pathway in cancer progression is the EGFR/c-myc pathway. Gliomas, for example, display a decrease in miR-524 expression to retard the TGFβ/Hippo/Notch pathway following stimulation of the EGFR/c-myc axis [23]. Furthermore, dihydroconiferyl ferulate reduces breast cancer stemness by regulating nuclear EGFR/c-Myc signaling [24]. Yet, PSME3’s regulatory effects on the EGFR/c-myc pathway in GC progression are still unclear. In this study, PSME3 stimulated the EGFR/c-myc pathway.
5. Conclusions
In conclusion, PMSE3 was demonstrated for the first time to exhibit higher expression, and facilitate glycolysis and migration of GC cells by regulating the EGFR/c-myc pathway. Besides, this study has some limitations, including the need for more experiments on human samples, animal samples and other cytological processes. In the future, the regulatory roles of PSME3 in GC progression will be further investigated.
Availability of data and materials
The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request.
Author contributions
LH and SSX—designed the study and carried them out; prepared the manuscript for publication and reviewed the draft of the manuscript. LH, QFY and JCL—supervised the data collection, analyzed the data, interpreted the data. All authors have read and approved the manuscript.
Ethics approval and consent to participate
Not applicable.
Acknowledgment
Not applicable.
Funding
This work was supported by Hangzhou bio-medicine and health industry development support science and technology project (Grant No. 2023WJC108), (Grant No. 2023WJC219); Hangzhou Medical and Health Technology Project (Grant No. B20200544).
Conflict of interest
The authors declare no conflict of interest.
