Identification of galactosamine-(N-acetyl)-6-sulfatase (GALNS) as a novel therapeutic target in progression of nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a commonly diagnosed malignancy in southern China and southeast Asia. Previous studies have identified galactosamine-(N-acetyl)-6-sulfatase (GALNS) as a potential biomarker for multiple cancers. However, it is unknown whether GALNS plays a role in NPC development, and the underlying mechanisms remain unclear. In this study, we found that GALNS is overexpressed in NPC cell lines and tissues compared to the normal nasopharyngeal counterparts. Knocking down GALNS expression in the NPC cells significantly decreased their proliferation in vitro, and inhibited xenograft growth in a mouse model. Mechanistically, the anti-proliferative effect of GALNS silencing was the result of autophagy induction via the inhibition of PI3K-AKT-mTOR signaling pathway. Taken together, GALNS drives the progression of NPC via PI3K-AKT-mTOR signaling-mediated autophagy, and is therefore a promising therapeutic target.

Keywords: AKT; GALNS; Nasopharyngeal carcinoma; Progression; mTOR.

Fig. 1

GALNS expression in NPC tissues and cell lines. A Immunoblot showing GALNS expression in NP69 cells and NPC cell lines. B Relative GALNS mRNA expression in NP69 cells and NPC cell lines. C Representative immunofluorescence images showing GALNS expression in NP69 cells and NPC cell lines Scare bar = 20 μm. D, E Representative images and scores of GALNS protein expression in normal nasopharyngeal (n = 27) and NPC tissues (n = 96). Scare bar = 50 μm. The data is the mean ± SEM of at least three independent experiments. *p < 0.05, versus NP69

Fig. 2

GALNS promotes NPC cells growth in vitro. A–C GALNS protein and mRNA expression in CNE2 and HONE1 cells transfected with siCTL, siGALNS#1 or siGALNS#2. D, E Viability of CNE2 and HONE1 cells transfected with siCTL, siGALNS#1 or siGALNS#2. F, G Representative images and number of colonies formed by CNE2 and HONE1 cells transfected with siCTL, siGALNS#1 or siGALNS#2. H, I Representative images and number of Edu-positive proliferative CNE2 and HONE1 cells transfected with siCTL, siGALNS#1 or siGALNS#2. The data represent the mean ± SEM of at least three independent experiments. Scare bar = 20 μm *p < 0.05, versus vehicle or siCTL

Fig. 3

GALNS promotes NPC cells growth in vivo. A Representative images of the xenografts of NPC cells transfected with shCTL or shGALNS. B, C Average tumor volume in the indicated groups. D, E The tumor weight in the indicated groups. F Representative images of tumor tissues showing GALNS expression. Scare bar = 50 μm. The data represent the mean ± SEM of at least three independent experiments. *p < 0.05, versus shCTL

Fig. 4

GALNS knockdown in NPC cells induced autophagy. A Immunoblot showing LC3-II protein expression in GALNS-knockdown CNE2 and HONE1 cells. B, C Immunoblot showing LC3-II expression in GALNS-knockdown CNE2 and HONE1 cells treated with the lysosomal protease inhibitors E64d + pepstatin A (Pep A). D, E Viability of GALNS-knockdown CNE2 and HONE1 cells following CQ treatment. F Representative images and number of colonies formed by the CQ-treated GALNS-knockdown CNE2 and HONE1 cells. The data represent the mean ± SEM of at least three independent experiments. *p < 0.05, versus siGALNS + Vehicle

Fig. 5

The PI3K-AKT-mTOR signaling is required for autophagy induced by GALNS knockdown. A Immunoblot showing expression of the PI3K–AKT–TOR pathway proteins in CNE2 and HONE1 cells transfected with siCTL or siGALNS. B Immunoblot showing LC3-II expression in GALNS-knockdown CNE2 or HONE1 cells treated with MHY1485. C Viability of rapamycin-treated CNE2 and HONE1 cells. D Number of colonies formed by rapamycin-treated CNE2 and HONE1 cells. E Viability of GALNS-knockdown CNE2 or HONE1 cells treated with MHY1485. F Number of colonies formed by GALNS-knockdown CNE2 or HONE1 cells treated with MHY1485. The data represent the mean ± SEM of at least three independent experiments. *p < 0.05, versus vehicle or siGALNS + vehicle