Induction of interleukin-6 by SPZ1-mediated Wnt5a signaling boosts progression of nasopharyngeal carcinoma cells

Abstract

Nasopharyngeal carcinoma (NPC) is a common malignancy in Southeast Asia, and in the Guangxi and Guangdong provinces of China. The spermatogenic transcription factor zip 1 (SPZ1) is a member of bHLH zip family, and promotes tumorigenesis in the liver, colon and breast tissues. However, the role of SPZ1 in the progression of NPC is unclear. In this study, we found that SPZ1 mRNA and protein levels were significantly upregulated in NPC tissues compared to the normal nasopharyngeal tissues. Furthermore, SPZ1 knockdown in NPC cell lines inhibited proliferation, epithelial-mesenchymal transition, migration, and invasion in vitro, and suppressed tumorigenesis in an in vivo model. On the other hand, SPZ1 overexpression facilitated the growth of NPC cells. Mechanistically, SPZ1-driven progression of NPC is dependent on the Wnt5a/interleukin-6 (IL-6) signaling pathway. Consistent with this, IL-6 levels were significantly increased in NPC tissues and correlated positively with SPZ1 expression. Taken together, our findings suggest that SPZ1 mediates NPC progression through Wnt5a/IL-6 signaling, and the SPZ1/Wnt5a/IL-6 axis is a potential therapeutic target for NPC.

Keywords: SPZ1; Wnt5a; interleukin-6; nasopharyngeal carcinoma; tumorigenesis.

Figure 1

SPZ1 expression in NPC tissues. (A and B) Representative images and scores of SPZ1 protein expression in nasopharyngitis (NP) (n=58) and nasopharyngeal carcinoma (NPC) tissues (n=119). Scare bar = 100μm. (C) Relative SPZ1 mRNA levels in 10 paired tumor and normal nasopharyngeal tissues from NPC patients. * p<0.05.

Figure 2

SPZ1 knockdown inhibited NPC cells growth in vitro. (A) SPZ1 protein expression in CNE1, CNE2, HONE1, 5-8F, 6-10B and C666-1 cell lines. (B) SPZ1 protein in CNE1 and HONE1 cells transfected with control siRNA (siCTL) or SPZ1 siRNA (siSPZ1#1 and siSPZ1#2). (C and D) Survival rates of CNE1 and HONE1 cells transfected with siCTL, siSPZ1#1 or siSPZ1#2. (E and F) Representative images and number of colonies formed by NPC cells transfected with siCTL, siSPZ1#1 or siSPZ1#2. (G and H) Representative images and number of Edu-positive proliferative in SPZ1 knockdown NPC cells. The data represent the mean ± SEM of at least three independent experiments. * p<0.05, versus siCTL.

Figure 3

SPZ1 knockdown inhibited NPC cells growth in vivo. (A) Images of tumors and IHC showing SPZ1 expression in the xenografts of CNE1 cells transfected with shScramble or shSPZ1. (B and C) The tumor volume and weight in the xenografts of CNE1 cells. (D) Images of tumors and IHC showing SPZ1 expression in the xenografts of HONE1 cells transfected with shScramble or shSPZ1. (E and F) The tumor volume and weight in the xenografts of HONE1 cells. Scare bar = 100μm. The data represent the mean ± SEM, n=5. * p<0.05, versus shScramble.

Figure 4

SPZ1 knockdown inhibited NPC cells migration and invasion. (A and B) Representative images and summary data of the migration assay in CNE1 and HONE1 cells transfected with siCTL, siSPZ1#1 or siSPZ1#2. (C and D) Representative images and summary data of the invasion assay in CNE1 and HONE1 cells transfected with siCTL, siSPZ1#1 or siSPZ1#2. (E and F) RNA levels of the epithelial-mesenchymal transition (EMT) markers in CNE1 and HONE1 cells transfected with siCTL, siSPZ1#1 or siSPZ1#2. The data represent the mean ± SEM of at least three independent experiments. * p<0.05, versus siCTL.

Figure 5

Overexpression of SPZ1 promoted NPC cells progression. (A) SPZ1 protein in C666-1 and 5-8F cells transfected with SPZ1-overexpression construct and vector. (B and C) Survival rates of C666-1 and 5-8F cells transfected with SPZ1-overexpression construct and vector. (D) Number of colonies formed by SPZ1 overexpression NPC cells. (E and F) RNA levels of SPZ1 and the epithelial-mesenchymal transition (EMT) markers in CNE1 and HONE1 cells C666-1 and 5-8F cells transfected with SPZ1-overexpression construct and vector. The data represent the mean ± SEM of at least three independent experiments. * p<0.05, versus vehicle.

Figure 6

SPZ1 knockdown inhibited Wnt5a expression in NPC cells. (A) Wnt5a protein levels in the supernatants from CNE1 and HONE1 cells transfected with siCTL and siSPZ1. (B) Relative Wnt5a mRNA levels in SPZ1 konckdown NPC cells. (C and D) Survival rates of CNE1 and HONE1 transfected with siSPZ1 and pretreated with Wnt5a (300ng/mL). (E and F) RNA levels of the epithelial-mesenchymal transition (EMT) markers in CNE1 and HONE1 cells transfected with siSPZ1 and pretreated with Wnt5a. The data represent the mean ± SEM of at least three independent experiments. * p<0.05, versus siCTL or siCTL+Control.

Figure 7

Inhibition of SPZ1/Wnt5a suppressed IL-6 expression in NPC cells. (A) IL-6 protein levels in the supernatants from CNE1 and HONE1 cells transfected with siCTL and siSPZ1. (B) Relative IL-6 mRNA levels in SPZ1 konckdown NPC cells. (C) IL-6 protein levels in the supernatants from CNE1 and HONE1 cells transfected with siCTL and siWnt5a. (D) Relative IL-6 mRNA levels in Wnt5a konckdown NPC cells. (E and F) Survival rates and number of colonies formed by NPC cells transfected with siIL-6. (G and H) RNA levels of the epithelial-mesenchymal transition (EMT) markers in NPC cells transfected with siIL-6. The data represent the mean ± SEM of at least three independent experiments. * p<0.05, versus siCTL.

Figure 8

SPZ1-mediated Wnt5a promoted NPC progression through IL-6. (A) Survival rates of CNE1 and HONE1 transfected with siSPZ1 and pretreated with IL-6 (100ng/mL). (B and C) RNA levels of the epithelial-mesenchymal transition (EMT) markers in CNE1 and HONE1 cells transfected with siSPZ1 and pretreated with IL-6 (100ng/mL). (D) Survival rates of CNE1 and HONE1 transfected with siWnt5a and pretreated with IL-6 (100ng/mL). (E and F) RNA levels of the EMT markers in CNE1 and HONE1 cells transfected with siWnt5a and pretreated with IL-6 (100ng/mL). (G and H) Representative images and scores of IL-6 protein expression in nasopharyngitis (NP) (n=58) and nasopharyngeal carcinoma (NPC) tissues (n=119). (F) Pearson correlation of IL-6 and SPZ1 expression levels. Scare bar = 100μm.The data represent the mean ± SEM of at least three independent experiments. * p<0.05, versus siSPZ1+Control, siWnt5a+Control or NP.