Sphingosine kinase-1 regulates migration and invasion of gastric cancer cells via targeting the nuclear factor-κB signaling pathway

doi:10.12122/j.issn.1673-4254.2024.11.13

Abstract

Abstract:

Objective To investigate the role of sphingosine kinase-1 (SPHK1) in regulating migration and invasion of gastric cancer (GC) cells. Methods TIMER2.0, GEPIA and HPA databases were used to investigate SPHK1 expression in GC, and its association with prognosis of the patients was analyzed using Kaplan-Meier Plotter database. In 40 clinical GC and adjacent tissue samples, SPHK1 and MKI67 expressions were detected with immunohistochemistry, Western blotting, and RT-qPCR. Gene enrichment pathway analysis was conducted to explore the biological functions of SPHK1. In HGC-27 and MGC-803 cells, the effects of lentivirus-mediated SPHK1 knockdown or overexpression on cell migration and invasion and expressions of key proteins in the nuclear factor-κB (NF-κB) signaling were evaluated using cell scratch test, Transwell assays and Western blotting. The changes in tumorigenic capacity of the transfected GC cells were evaluated in nude mice. Results SPHK1 was highly expressed in GC tissues in negative correlation with overall survival, overall survival after progression, and relapse-free survival of the patients (all P<0.001). In clinical GC samples, SPHK1 and MKI67 expressions showed a positive correlation (P=0.00049) and were both significantly up-regulated (P<0.001). Gene enrichment pathway analysis suggested the involvement of SPHK1 in cell adhesion, migration, angiogenesis and the NF-κB pathway (P<0.05). In the cell experiment, SPHK1 knockdown significantly decreased while SPHK1 overexpression enhanced migration and invasion abilities of the GC cells. SPHK1 positively regulated the expressions of phosphorylated P65 (P-P65), VEGFA and IL-17, and blocking the NF-κB pathway by PDTC significantly lowered migration and invasion ability of the cells. In nude mice, the GC cells with SPHK1 knockdown resulted in significantly reduced tumor size and mass, while the SPHK1-overexpressing cells showed enhanced tumorigenicity. Conclusion SPHK1 regulates migration and invasion of GC cells via the NF-κB signaling pathway and may serve as a potential diagnostic marker for GC progression.

Key words: gastric cancer, sphingosine kinase-1, NF-κB, migration, invasion

Qianlong LING, Kai JI, Jinye CHEN, Jiajia GUAN, Ruipeng WANG, Wenjiang MAN, Bing ZHU. Sphingosine kinase-1 regulates migration and invasion of gastric cancer cells via targeting the nuclear factor-κB signaling pathway[J]. Journal of Southern Medical University, 2024, 44(11): 2163-2171.

Figures/Tables 8

Tab.1 Clinicopathological characteristics of GC patients (n=40)

Characteristic	Clinicopathological characteristics
Characteristic	n	Percentage (%)
Gender
Male	28	70
Female	12	30
Age (year)
≥60	24	60
<60	16	40
Tumor size (cm)
≥5.0	22	55
<5.0	18	45
Clinical stage
I+II	14	35
III+IV	26	65
N stage
N0	10	25
N1+N2+N3	30	75

Tab.2 Sequences for RNA interference or gene overexpression

Item	Sequences (5'-3')
shNC	GAGTCTATGACATTGCCTCA
shSPHK1#1	CTTCGTGTCAGATGTTGGATAT
shSPHK1#2	GCTTTGCCCTCACCCTTACAT
shSPHK1#3	GCTTTGCCCTCACCCTTACAT
oeNC	CGCATCTAGCCTGTCAGTCC
oeSPHK1#1	CTCGTGTCAGATATTGGTTAT
oeSPHK1#2	ATTGTGTGAGACATCCGTAAG
oeSPHK1#3	TTGAGTCCTGCTTCTTCATTG

Fig.1 Expression of SPHK1 in GC tissues. A: Expression of SPHK1 in pan-cancer based on data retrieved from TIMER2.0 database. B: Expression of SPHK1 and MKI67 in GC tissues based on data retrieved from GEPIA database. C: GEPIA database analysis of the correlation between SPHK1 and MKI67. D, E: Immunohistochemical staining for SPHK1 and MKI67 in GC tissues from HPA database. *P<0.05, **P<0.01, ***P<0.001 vs Tumor group.

Fig.2 Association of SPHK1 expression with overall survival (OS; A), post-progression survival (PPS; B) and recurrence-free survival (RFS; C) of GC patients predicted using Kaplan-Meier Plotter database.

Fig.3 SPHK1 expression is upregulated in GC tissue. A: Immunohistochemical staining for detecting SPHK1 and MKI67 in clinical tissue samples (Original magnification:×200). B: Immunohistochemical score of SPHK1 and MKI67. ***P<0.001. C: Correlation between SPHK1 and MKI67 expressions. D: Western blotting for detecting the expression of SPHK1 protein. E: qRT-PCR for detecting the level of SPHK1 mRNA. *P<0.05, **P<0.01, ***P<0.001 vs GES-1 group.

Fig.4 Effect of SPHK1 knockdown or overexpression on GC cell migration and invasion. A: Western blotting for assessing efficiency of lentivirus-mediated SPHK1 knockdown and overexpression. B: Western blotting for detecting MKI67 protein levels. C, E: Cell scratch assay for assessing migration ability of the cells (×40). D, F: Transwell assays for assessing invasion ability of the cells (×200). G: Size of the subcutaneous tumors in nude mice. F: Weight of the subcutaneous tumors. *P<0.05, **P<0.01, ***P<0.001 vs NC group.

Fig.5 SPHK1 targets the NF-κB signaling pathway. A: Results of GO enrichment analysis. B: Results of KEGG pathway enrichment analysis. C, E: Expression levels of NF-κB signaling pathway proteins after SPHK1 knockdown. D, F: Expression levels of the proteins after SPHK1 overexpression. *P<0.05, **P<0.01, ***P<0.001 vs NC group.

Fig.6 Effect of blocking the NF-κB signaling pathway on migration and invasion ability of GC cells with SPHK1 knockdown or overexpression. A, B: Transwell assay for assessing migration and invasion of GC cells treated with 100 nmol/L PDTC (a NF-κB pathway inhibitor) or DMSO for 24 h (×200). C-F: Western blotting for detecting the levels of P65, p-P65, VEGFA and IL-17 proteins.*P<0.05, **P<0.01, ***P<0.001.

References 39

1	冯金华, 刘亚江, 颜慧玲, 等. 四君子汤对胃癌肿瘤干细胞标志物活性及TFAP4蛋白的作用机制[J].中国老年学杂志,2024,44(7):1739-44.
2	Wang X, Wu WKK, Gao J, et al. Autophagy inhibition enhances PD-L1 expression in gastric cancer[J]. J Exp Clin Cancer Res, 2019, 38(1): 140.
3	Yang WJ, Zhao HP, Yu Y, et al. Updates on global epidemiology, risk and prognostic factors of gastric cancer[J]. World J Gastroenterol, 2023, 29(16): 2452-68.
4	Yeong J, Teo CB, Tay RYK, et al. Reply to: letter to editor on the article "Choice of PD-L1 immunohistochemistry assay influences clinical eligibility for gastric cancer immunotherapy"[J]. Gastric Cancer, 2022, 25(6): 1133-5.
5	Tang L, Guo CM, Li X, et al. TAF15 promotes cell proliferation, migration and invasion of gastric cancer via activation of the RAF1/MEK/ERK signalling pathway[J]. Sci Rep, 2023, 13(1): 5846.
6	Qin ZJ, Tong H, Li TH, et al. SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis[J]. Int J Mol Med, 2021, 48(5): 204.
7	Bernacchioni C, Squecco R, Gamberi T, et al. S1P signalling axis is necessary for adiponectin-directed regulation of electrophysio-logical properties and oxidative metabolism in C2C12 myotubes[J]. Cells, 2022, 11(4): 713.
8	张文路, 徐春燕. SphK1/S1P在肿瘤恶性生物学行为中的研究进展[J]. 实用医学杂志, 2023, 39(19): 2551-5, 2560. DOI: 10.3969/j.issn.1006-5725.2023.19.024
9	Long JT, Yao ZJ, Sui Y, et al. SphK1 promotes cancer progression through activating JAK/STAT pathway and up-regulating S1PR1 expression in colon cancer cells[J]. Anticancer Agents Med Chem, 2022, 22(2): 254-60.
10	Wu JN, Lin L, Luo SB, et al. SphK1-driven autophagy potentiates focal adhesion paxillin-mediated metastasis in colorectal cancer[J]. Cancer Med, 2021, 10(17): 6010-21.
11	Liu SQ, Xu CY, Wu WH, et al. Sphingosine kinase1 promotes the metastasis of colorectal cancer by inducing the epithelial-mesenchymal transition mediated by the FAK/AKT/MMPs axis[J]. Int J Oncol, 2019, 54(1): 41-52.
12	Yu MS, Zhang KN, Wang S, et al. Increased SPHK1 and HAS2 expressions correlate to poor prognosis in pancreatic cancer[J]. Biomed Res Int, 2021, 2021: 8861766.
13	Ma Y, Xing X, Kong R, et al. SphK1 promotes development of non-small cell lung cancer through activation of STAT3[J]. Int J Mol Med, 2021, 47(1): 374-86.
14	卢芳, 蒋鑫, 徐晓敏, 等.分子对接与体内验证联合探讨穿山龙复方对痛风模型大鼠TLR4/MyD88/NF-kB信号通路的影响[J].中药药理与临床, 2023, 39(08): 25-31.
15	Wang MD, Li HT, Peng LX, et al. TSPAN1 inhibits metastasis of nasopharyngeal carcinoma via suppressing NF-kB signaling[J]. Cancer Gene Ther, 2024, 31(3): 454-63.
16	Yang WH, Liu L, Li CX, et al. TRIM52 plays an oncogenic role in ovarian cancer associated with NF-kB pathway[J]. Cell Death Dis, 2018, 9(9): 908.
17	张浩, 张震, 王秋生, 等. FJX1在胃癌组织中高表达并与不良预后相关[J]. 南方医科大学学报, 2023, 43(6): 975-84.
18	Sukocheva OA, Furuya H, Ng ML, et al. Sphingosine kinase and sphingosine-1-phosphate receptor signaling pathway in inflammatory gastrointestinal disease and cancers: a novel therapeutic target[J]. Pharmacol Ther, 2020, 207: 107464.
19	刘加蒙, 李明. S1PR1在胃癌组织中的表达及其临床意义[J]. 现代肿瘤医学, 2024, 32(1): 93-5.
20	Xiong HP, Wang JC, Guan HY, et al. SphK1 confers resistance to apoptosis in gastric cancer cells by downregulating Bim via stim-ulating Akt/FoxO3a signaling[J]. Oncol Rep, 2014, 32(4): 1369-73.
21	Jia ZW, Tang XF, Zhang XC, et al. MiR-153-3p attenuates the development of gastric cancer by suppressing SphK2[J]. Biochem Genet, 2022, 60(5): 1748-61.
22	Huo FC, Zhu ZM, Zhu WT, et al. METTL3-mediated m⁶A methylation of SPHK2 promotes gastric cancer progression by targeting KLF2[J]. Oncogene, 2021, 40(16): 2968-81.
23	Uxa S, Castillo-Binder P, Kohler R, et al. Ki-67 gene expression[J]. Cell Death Differ, 2021, 28(12): 3357-70.
24	Leibold J, Tsanov KM, Amor C, et al. Somatic mouse models of gastric cancer reveal genotype-specific features of metastatic disease[J]. Nat Cancer, 2024, 5(2): 315-29.
25	Ruan Q, Wang CJ, Wu YT, et al. Exosome microRNA-22 inhibiting proliferation, migration and invasion through regulating Twist1/CADM1 axis in osteosarcoma[J]. Sci Rep, 2024, 14(1): 761.
26	Bhadwal P, Randhawa V, Vaiphei K, et al. Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance[J]. Sci Rep, 2022, 12(1): 18239.
27	Jin L, Zhu J, Yao LY, et al. Targeting SphK1/2 by SKI-178 inhibits prostate cancer cell growth[J]. Cell Death Dis, 2023, 14(8): 537.
28	Chen D, Wu JN, Qiu XZ, et al. SPHK1 potentiates colorectal cancer progression and metastasis via regulating autophagy mediated by TRAF6-induced ULK1 ubiquitination[J]. Cancer Gene Ther, 2024, 31(3): 410-9.
29	Long XL, Hu YK, Duan SY, et al. MRGBP promotes colorectal cancer metastasis via DKK1/Wnt/β-catenin and NF-kB/p65 pathways mediated EMT[J]. Exp Cell Res, 2022, 421(1): 113375.
30	Mirzaei S, Saghari S, Bassiri F, et al. NF-κB as a regulator of cancer metastasis and therapy response: a focus on epithelial-mesenchymal transition[J]. J Cell Physiol, 2022, 237(7): 2770-95.
31	冯晓创. POTEE通过调控SPHK1转录激活p65磷酸化促进结直肠癌进展的机制研究[D]. 广州: 南方医科大学, 2020.
32	Hou CX, Mao GY, Sun QW, et al. Metabolomic analysis reveals that SPHK1 promotes oral squamous cell carcinoma progression through NF-κB activation[J]. Ann Surg Oncol, 2022, 29(12): 7386-99.
33	Zhang JX, Chen ZH, Chen DL, et al. Retraction Note: LINC01410-miR-532-NCF2-NF-kB feedback loop promotes gastric cancer angiogenesis and metastasis[J]. Oncogene, 2023, 42(14): 1158.
34	Li YF, Qiao YQ, Wang HH, et al. Intraperitoneal injection of PDTC on the NF-kB signaling pathway and osteogenesis indexes of young adult rats with anterior palatal suture expansion model[J]. PLoS One, 2021, 16(7): e0243108.
35	Mei L, Zheng YM, Song TY, et al. Rieske iron-sulfur protein induces FKBP12.6/RyR2 complex remodeling and subsequent pulmonary hypertension through NF‑κB/cyclin D1 pathway[J]. Nat Commun, 2020, 11(1): 3527.
36	Yang KX, Li JT, Zhu JH, et al. HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis[J]. Cell Death Discov, 2024, 10(1): 33.
37	Zeng K, Xie WW, Wang CY, et al. USP22 upregulates ZEB1-mediated VEGFA transcription in hepatocellular carcinoma[J]. Cell Death Dis, 2023, 14(3): 194.
38	李燕, 王琦, 郑路, 等.白细胞介素-17基因多态性与山西东南地区汉族人群2型糖尿病的相关性[J].实用医学杂志, 2024, 40(05): 695-701.
39	Korbecki J, Simińska D, Gąssowska-Dobrowolska M, et al. Chronic and cycling hypoxia: drivers of cancer chronic inflammation through HIF-1 and NF‑κB activation: a review of the molecular mechanisms[J]. Int J Mol Sci, 2021, 22(19): 10701.

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