TOM40 regulates the progression of nasopharyngeal carcinoma through ROS-mediated AKT/mTOR and p53 signaling

Abstract

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Southern China, North Africa, and Southeast Asia. The translocase of the outer membrane (TOM) 40 is a transporter of mitochondrial proteins, and is involved in ovarian cancer cell growth. However, its role in the progression of NPC is still unclear. We found that TOM40 levels were upregulated in NPC tissues and multiple NPC cell lines. In addition, high TOM40 expression in the tumor tissues was associated with poor overall survival and disease specific survival. TOM40 knockdown in the NPC cell lines inhibited their proliferation in vitro and in vivo. Furthermore, TOM40 silencing also increased intracellular production of reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP). Mechanistically, the anti-tumor effects of TOM40 silencing were dependent on the inhibition of AKT/mTOR signaling and activation of p53 signaling. To summarize, TOM40 mediates NPC progression through ROS-mediated AKT/mTOR and p53 signaling. Our findings highlight the potential of TOM40 as a therapeutic target for NPC.

Keywords: AKT; Nasopharyngeal carcinoma; ROS; TOM40; p53.

Fig. 1

TOM40 expression in NPC tissues. A The relative TOM40 mRNA expression in the TCGA RNA-seq database. B Overall survival and disease specific survival of patients with high and low TOM40 expression. C Relative TRPV4 mRNA expression in 8 paired tumor and adjacent tissues from NPC patients. D Representative images and scores of TOM40 expression in NP (n = 40) and NPC tissues (n = 117). Scale bar = 50 μm. The data is the mean ± SEM of at least three independent experiments. * p < 0.05, versus NP or adjacent tissue

Fig. 2

TOM40 expression in NPC cell lines. A and B TOM40 mRNA and protein expression in NP69, CNE2, HONE1, 5-8F, 6-10B and C666-1 cell lines. C–F TOM40 protein and mRNA expression in CNE2 and HONE1 cells transfected with siControl, siTOM40#1 or siTOM40#2. The data represent the mean ± SEM of at least three independent experiments. Scale bar = 20 μm * p < 0.05, versus NP69 or siControl

Fig. 3

TOM40 knockdown inhibited proliferation of NPC cells in vitro. A and B Viability of CNE2 and HONE1 cells transfected with siControl, siTOM40#1 or siTOM40#2. C and D Representative images and number of EdU-positive proliferative CNE2 and HONE1 cells transfected with siControl, siTOM40#1 or siTOM40#2. Scale bar = 20 μm. E and F Representative images and number of colonies formed by CNE2 and HONE1 cells transfected with siControl, siTOM40#1 or siTOM40#2. The data represent the mean ± SEM of at least three independent experiments. * p < 0.05, versus siControl

Fig. 4

TOM40 knockdown inhibited proliferation of NPC cells in vivo. A Representative images of tumors and tissues immuno-stained for TMO40 from the xenografts of CNE2 cells transfected with shControl or shTOM40. B Volume of CNE2 xenografts from the indicated groups. C Weight of CNE2 xenografts from the indicated groups. D Representative images of tumors and tissues immuno-stained for TMO40 from the xenografts of HONE1 cells transfected with shControl or shTOM40. E Volume of HONE1 xenografts from the indicated groups. F Weight of HONE1 xenografts from the indicated groups. Scale bar = 50 μm. The data represent the mean ± SEM of at least three independent experiments. * p < 0.05, versus shControl

Fig. 5

TOM40 knockdown induced mitochondrial dysfunction, AKT/mTOR signaling inhibition and p53 signaling activation. A The representative images of CNE2 and HONE1 cells stained with JC-1 showing changes in MMP following TOM40 knockdown. B Representative images of control and TOM40-knockdown CNE2 and HONE1 cells stained with the CMH2DCFDA probe showing ROS levels. C Immunoblot showing levels of p-AKT, AKT, p-mTOR, mTOR, p-p70S6K, pS6, p-4E-BP1, 4E-BP1and ACTB in the indicated groups. D Immunoblot showing levels of p21, p53, cleaved PARP, cleaved Caspase3 and ACTB in the indicated groups. Scale bar = 50 μm

Fig. 6

TOM40 knockdown inhibited cell growth through ROS-mediated AKT/mTOR and p53 signaling. A–C Viability of TOM40 knockdown CNE2 and HONE1 cells treated with NAC, sip53 and Z-VAD-FMK. D–F Caspase 3/7 activities in TOM40 knockdown CNE2 and HONE1 cells treated with NAC, sip53 and Z-VAD-FMK. The data represent the mean ± SEM of at least three independent experiments. $ p < 0.05, versus siTOM40 + Vehicle