High expression of SLC2A1 inhibits ferroptosis and promotes proliferation and invasion of lung adenocarcinoma cells

doi:10.12122/j.issn.1673-4254.2024.12.17

Abstract

Abstract:

Objective To examine how the glucose transporter SLC2A1 influences the proliferation and migration of lung adenocarcinoma (LUAD) and explore the underlying molecular mechanisms. Methods We examined the differential expression of SLC2A1 between normal and LUAD tissues in the TCGA database and its prognostic implications. Immunohistochemistry was used to detect SLC2A1 protein levels in clinical samples of LUAD and adjacent tissues, and the association of SLC2A1 expression levels with clinicopathological features of the patients was analyzed. In PC9 cells with stable SLC2A1 overexpression or knockdown, the effects of SLC2A1 expression level on cell proliferation and migration were assessed using CCK-8 and Transwell assays, and the changes in expressions of ferroptosis- and autophagy-related proteins were measured; the occurrence of ferroptosis was confirmed using ROS and Fe²⁺ fluorescence staining. Results SLC2A1 expression was significantly higher in LUAD tumor tissues than in normal lung tissues (P<0.05) and was associated with worse pathological parameters and prognosis of the patients (P<0.05). In PC9 cells, SLC2A1 overexpression significantly promoted cell proliferation, invasion and migration, and SLC2A1 knockdown significanty increased cell death and inhibited cell invasion and proliferation. SLC2A1 knockdown caused obvious activation of cell ferroptosis, reduced GPX4 and xCT expressions, and increased intracellular levels of ROS and Fe²⁺. SLC2A1 knockdown also resulted in increased cell autophagy shown by increased LC3B expression, which could be reversed by treatement with 3-MA. Conclusion High SLC2A1 expression is correlated with poor prognosis of patients with LUAD, and inhibiting SLC2A1 can induce ferroptosis and autophagy of LUAD cells, suggesting the potential of SLC2A1 as a target for LUAD diagnosis and treatment.

Key words: lung adenocarcinoma, glucose transporter 1, ferroptosis, autophagy

Hong KUANG, Wenhan CAI, Yiming LIU, Jiaxin WEN, Shuo TIAN, Zhiqiang XUE. High expression of SLC2A1 inhibits ferroptosis and promotes proliferation and invasion of lung adenocarcinoma cells[J]. Journal of Southern Medical University, 2024, 44(12): 2404-2411.

Figures/Tables 9

Fig.1 Expression of SLC2A1 in lung adenocarcinoma (LUAD) and normal lung tissues and cells. A: Expression of SLC2A1 in LUAD and normal lung tissue. B: Expression of SLC2A1 in LUAD patients with different T grades. C: Expression of SLC2A1 in PC9 and BEAS-2B cell lines. *P<0.05, ****P<0.0001.

Tab.1 Relation between SLC2A1 and clinicopathological features of patients with LUAD (n)

Clinicopathological feature	n	SLC2A1 Low	SLC2A1 High	P
Gender				0.0577
Male	238	79	159
Female	278	112	166
Age (year)				<0.0001
<65	365	135	230
≥65	151	67	84
Tumor diameter (cm )				0.0351
<4	280	136	144
≥4	236	62	174
Fuhrman				0.2935
1-2	353	155	198
3-4	163	58	105

Fig.2 Diagnostic and prognostic values of SLC2A1 in LUAD. A: AUC curve feature of SLC2A1 in LUAD. B: Overall survival of LUAD patients with different levels of SLC2A1. C: Progression-free survival of LUAD patients with different levels of SLC2A1.

Tab.2 Univariate analysis of the correlations of SLC2A1 expression and clinicopathologic variables with overall survival of LUAD patients

Variable	n	Mean Survival (months)	P
Gender			0.2986
Male	156	69.2
Female	51	62.3
Age (year)			0.0620
<65	165	70.0
≥65	42	63.6
Tumor diameter ( cm )			0.0053
<4	71	71.2
≥4	136	67.0
Fuhrman			0.0270
1-2	153	71.7
3-4	54	60.1
SLC2A1 expression			0.0079
Low	59	71.7
High	148	69.2
T stage			0.1082
T1	52	71.0
T2	77	70.4
T3	78	65.5

Tab.3 COX multivariate analysis of clinicopathological parameters and overall survival of the patients

Variables	Hazard Ratio (95% CI)	P
Tumor diameter	1.239(1.136-1.352)	0.220
SLC2A1 expression	1.912(1.841-1.988)	0.024
Fuhrman	2.029(1.995-2.063)	0.194

Fig.3 Expression of SLC2A1 protein in LUAD and normal lung tissues. ****P<0.0001.

Fig.4 Knockdown of SLC2A1 inhibits proliferation and migration of LUAD cells. A: Efficiency of SLC2A1 knockdown in PC9 cells. B: qRT-PCR data showing SLC2A1 mRNA expression in PC9 with or without shRNA. C: CCK-8 assay for assessing proliferation of PC9 cells with SLC2A1 downregulation. D: Migration ability of PC9 cells with SLC2A1 downregulation. E: Invasion ability of PC9 cells with SLC2A1 downregulation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.5 Overexpression of SLC2A1 promotes proliferation and migration of LUAD cells.A: Efficiency of SLC2A1 overexpression in PC9 cells. B: qRT-PCR of SLC2A1 mRNA expression in PC9 cells with or without SLC2A1 overexpression. C: CCK-8 for assessing proliferation of PC9 cells overexpressing SLC2A1. D: Migration ability of PC9 cells overexpressing SLC2A1. E: Invasion of PC9 cells overexpressing SLC2A1. Scar bar=100 μm. **P<0.01, ***P<0.001, ****P<0.0001 vs control group.

Fig.6 SLC2A1 knockdown promotes autophagy and iron death in PC9 cells. A: Western blotting of ferroptosis-related proteins in PC9 cells with SLC2A1 knockdown. B: Intracellular Fe2+ level in PC9 cells with SLC2A1 knockdown. C: Mito-Fe2+ level in PC9 cells with SLC2A1 knockdown. D: Intracellular ROS level in PC9 cells with SLC2A1 knockdown. E: Knockdown of SLC2A1 induces autophagy in PC9 cells. F: 3-MA resuced SLC2A1 knockdown-induced autophagy of PC9 cells. G: Knockdown of SLC2A1 induces cell death in PC9 cells. Scar bar=100 μm. **P<0.01, ***P<0.001, ****P<0.0001.

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