High expression of the stemness-associated molecule Nanog in esophageal squamous cell carcinoma tissues promotes tumor invasion and metastasis by activating the TGF‑β signaling pathway

doi:10.12122/j.issn.1673-4254.2024.06.23

Abstract

Abstract:

Objective To investigate the expression of Nanog and its regulatory relationship with MMP-2/MMP-9 proteins in esophageal squamous cell carcinoma (ESCC). Methods We detected Nanog and MMP-2/MMP-9 protein expressions in 127 ESCC tissues and 82 adjacent normal tissues using immunohistochemistry and explored their correlations with the clinicopathological parameters and prognosis of the patients. GEO database was utilized to analyze the pathways enriched with the stemness-related molecules including Nanog, and TIMER online tool was used to analyze the correlations among TβR1, MMP-2, and MMP-9 in esophageal cancer. Results Nanog and MMP-2/MMP-9 proteins were significantly upregulated in ESCC tissues and positively intercorrelated. Their expression levels were closely correlated with infiltration depth and lymph node metastasis of ESCC but not with age, gender, or tumor differentiation. The patients with high expressions of Nanog and MMP-2/MMP-9 had significantly shorter survival time. Bioinformatics analysis showed enrichment of stemness-associated molecules in the TGF-β signaling pathway, and the expressions of MMP-2/MMP-9 and TβR1 were positively correlated. In cultured ESCC cells, Nanog knockdown significantly decreased the expression of TβR1, p-Smad2/3, MMP-2, and MMP-9 and strongly inhibited cell migration. Conclusion The high expressions of Nanog, MMP-2, and MMP-9, which are positively correlated, are closely related with invasion depth, lymph node metastasis, and prognosis of ESCC. Nanog regulates the expressions of MMP-2/MMP-9 proteins through the TGF‑β signaling pathway, and its high expression promotes migration of ESCC cells.

Key words: esophageal squamous cell carcinoma, Nanog, MMP-2/MMP-9, infiltration and metastasis, survival analysis

Chang SUN, Shiyao ZHENG, Mei LI, Ming YANG, Mengyuan QIN, Yuan XU, Weihua LIANG, Jianmin HU, Lianghai WANG, Feng LI, Hong ZHOU, Lan YANG. High expression of the stemness-associated molecule Nanog in esophageal squamous cell carcinoma tissues promotes tumor invasion and metastasis by activating the TGF‑β signaling pathway[J]. Journal of Southern Medical University, 2024, 44(6): 1209-1216.

Figures/Tables 10

Tab.1 Clinical and pathological characteristics of 127 esophageal cancer patients

Clinicopathological parameters	n	Percentage (%)
Age (year)
<60	73	57.48
≥60	54	42.52
Gender
Male	78	61.42
Female	49	38.58
Differentiation
High	40	31.5
Medium	72	56.69
Low	15	11.81
T stage
T1	16	12.6
T2	52	40.94
T3-T4	40	46.46
Lymph node metastasis
+	84	66.14
－	43	33.86

Fig.1 Immunohistochemical staining of Nanog, MMP-2, and MMP-9 proteins in esophageal squamous cell carcinoma tissues and adjacent normal tissues. A: Negative expression of Nanog in adjacent normal tissues. B: Weak positive expression (1+) of Nanog in esophageal squamous cell carcinoma tissues. C: Moderate positive expression (2+) of Nanog in esophageal squamous cell carcinoma tissues. D: Strong positive expression (3+) of Nanog in esophageal squamous cell carcinoma tissues. E: Negative expression of MMP-2 in adjacent normal tissues. F: Weak positive expression (1+) of MMP-2 in esophageal squamous cell carcinoma tissues. G: Moderate positive expression (2+) of MMP-2 in esophageal squamous cell carcinoma tissues. H: Strong positive expression (3+) of MMP-2 in esophageal squamous cell carcinoma tissues. I: Negative expression (-) of MMP-9 in adjacent normal tissues. J: Weak positive expression (1+) of MMP-9 in esophageal squamous cell carcinoma tissues. K: Moderate positive expression (2+) of MMP-9 in esophageal squamous cell carcinoma tissues. L: Strong positive expression (3+) of MMP-9 in esophageal squamous cell carcinoma tissues.

Tab.2 Expression of Nanog, MMP-2 and MMP-9 protein in esophageal squamous cell carcinoma and adjacent tissues

Protein	Tissue samples	n	High expression	Low expression	χ²	P
Nanog	ESCC tissue	122	79	43	70.475	<0.01
Nanog	paraneoplastic tissue	79	4	75
MMP-2	ESCC tissue	99	58	41	35.415	<0.01
MMP-2	paraneoplastic tissue	73	10	63
MMP-9	ESCC tissue	119	60	59	46.605	<0.01
MMP-9	paraneoplastic tissue	81	3	78

Tab.3 Clinical-pathological parameters of patients with esophageal squamous cell carcinoma having high or low Nanog, MMP-2 and MMP-9 protein expressions

Clinicopathological parameters	Nanog			χ²	P	MMP-2			χ²	P	MMP-9			χ²	P
Clinicopathological parameters	n	High expression	Low expression	χ²	P	n	High expression	Low expression	χ²	P	n	High expression	Low expression	χ²	P
Gender
Male	76	50	26	0.095	0.758	57	31	26	0.977	0.41	69	35	34	0.006	1.000
Female	46	29	17			42	27	15			50	25	25
Age (year)
<60	70	45	25	0.016	0.900	60	40	20	4.099	0.060	72	37	35	0.068	0.852
≥60	52	34	18			39	18	21			47	23	24
T stage
T1	15	2	13	23.90	<0.01	8	2	6	6.029	0.049	14	1	13	11.89	0.003
T2	50	31	19			45	31	14			50	28	22
T3-T4	57	46	11			46	25	21			55	31	24
Lymph node metastasis
+	80	55	25	1.626	0.202	66	46	20	10.08	0.020	79	46	33	5.731	0.020
－	42	24	18			33	12	21			40	14	26
Differentiation
High	36	19	17	5.483	0.064	31	18	13	0.268	0.875	13	6	7	2.534	0.282
Medium	71	47	24			59	34	25			67	38	29
Low	15	13	2			9	6	3			39	16	23

Fig.2 Postoperative survival curves of esophageal squamous cell carcinoma patients with low and high Nanog, MMP-2 and MMP-9 protein expressions. A: Survival curves of patients with high and low Nanog expressions (P<0.001). B: Survival curves of patients with high and low MMP-2 protein expression (P=0.004). C: Survival curves of patients with high and low MMP-9 protein expressions (P=0.017).

Tab.4 Correlation between expressions of Nanog and invasion- and migration-related proteins in esophageal cancer

Nanog	MMP-2			r	P	MMP-9			r	P
Nanog	n	High expression	Low expression	r	P	n	High expression	Low expression	r	P
High expression	66	43	23	0.205	0.045	74	46	28	0.307	0.001
Low expression	30	13	17			40	12	28

Fig.3 Bioinformatics analysis. A: KEGG pathway enrichment analysis of differentially expressed genes in the gene chip dataset GSE13834. B: Scatter plot showing the correlation between TβR1 and MMP-2 expression in esophageal cancer. C: Scatter plot showing the correlation between TβR1 and MMP-9 expression in esophageal cancer.

Tab.5 Correlation of Nanog, MMP-2 and MMP-9 protein expressions with expressions of TGF-β signaling pathway proteins in ESCC

Protein			TβR1		r	P		p-Smad2/3		r	P
Protein		n High expression		Low expression	n High expression		Low expression
Nanog	High expression	68	50	18	0.318	0.001	68	50	18	0.301	0.002
Nanog	Low expression	32	13	19			35	15	20
MMP-2	High expression	56	47	9	0.448	<0.001	52	40	12	0.303	0.004
MMP-2	Low expression	47	18	29			55	27	28
MMP-9	High expression	53	39	14	0.202	0.045	54	40	14	0.239	0.015
MMP-9	Low expression	50	26	24			51	27	24

Fig.4 Effect of Nanog knockdown on migration of esophageal squamous cell carcinoma cells. A: qRT-PCR of Nanog mRNA expression levels after Nanog knockdown (P<0.05). B: Western blotting of Nanog protein expression after Nanog knockdown. C: Scratch assay for assessing changes in migration ability of TE-1 cells after Nanog knockdown. *P˂0.05, **P˂0.01 vs NC group.

Fig.5 Expression of TGF-β signaling pathway and invasion- and migration-related molecules after Nanog knockdown. A: qRT-PCR of TβR1, Smad2, MMP-2, and MMP-9 mRNA levels after Nanog knockdown. B: Western blotting of TβR1, p-Smad2/3, MMP-2, and MMP-9 protein levels after Nanog knockdown. *P˂0.05, **P˂0.01, ***P<0.001 vs NC group.

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