Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 16:8:232.
doi: 10.3389/fcell.2020.00232. eCollection 2020.

ANXA6 Contributes to Radioresistance by Promoting Autophagy via Inhibiting the PI3K/AKT/mTOR Signaling Pathway in Nasopharyngeal Carcinoma

Qianping Chen  1 , Wang Zheng  1 , Lin Zhu  1 , Dan Yao  1 , Chen Wang  1 , Yimeng Song  1 , Songling Hu  1 , Hongxia Liu  1 , Yang Bai  1 , Yan Pan  1 , Jianghong Zhang  1 , Jian Guan  2 , Chunlin Shao  1
Affiliations

ANXA6 Contributes to Radioresistance by Promoting Autophagy via Inhibiting the PI3K/AKT/mTOR Signaling Pathway in Nasopharyngeal Carcinoma

Qianping Chen et al. Front Cell Dev Biol. .

Abstract

Radiotherapy is a conventional and effective treatment method for nasopharyngeal carcinoma (NPC), although it can fail, mainly because radioresistance results in residual or recurrent tumors. However, the mechanisms and predictive markers of NPC radioresistance are still obscure. In this study, we identified Annexin A6 (ANXA6) as a candidate radioresistance marker by using Tandem Mass Tag quantitative proteomic analysis of NPC cells and gene chip analysis of NPC clinical samples with different radiosensitivities. It was observed that a high expression level of ANXA6 was positively correlated with radioresistance of NPC and that inhibition of ANXA6 by siRNA increased the radiosensitivity. The incidence of autophagy was enhanced in the established radioresistant NPC cells in comparison with their parent cells, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, ANXA6 siRNA (siANXA6) suppressed cellular autophagy by activating the PI3K/AKT/mTOR pathway, ultimately leading to radiosensitization. The combination of siANXA6 and CAL101 (an inhibitor of PI3K, p-AKT, and mTOR, concurrently) significantly reversed the above siANAX6-reduced autophagy. Suppression of PI3K/AKT/mTOR by CAL101 also increased the expression of ANXA6 in a negative feedback process. In conclusion, this study revealed for the first time that ANXA6 could promote autophagy by inhibiting the PI3K/AKT/mTOR pathway and that it thus contributes to radioresistance of NPC. The significance of this is that ANXA6 could be applied as a new predictive biomarker of NPC prognosis after radiotherapy.

Keywords: ANXA6; NPC; PI3K/AKT/mTOR; autophagy; radioresistance.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
High expression of ANXA6 predicts radioresistance of NPC. (A) Survival fractions of HNE2, CNE1, and CNE1R cells after irradiation. (B) Volcano plot of differentially expressed genes between CNE1R and CNE1 cells. (C) Volcano plot of differentially expressed genes in the tumor tissue of NPC radioresistant patients (n = 3) before and after radiotherapy. (D) Heat map of the expression levels of 29 differential genes between the above volcano plots, analyzed by Heml (software for drawing volcano maps). (E) ANXA6 mRNA expression levels in HNE2, CNE1, and CNE1R cells. (F) Western blot assay of ANXA6 protein in HNE2, CNE1, and CNE1R cells. (G) Efficiency of siANXA6 transfection in CNE1 and CNE1R cells. (H) Dose responses of survival factions of CNE1 and CNE1R cells after siANXA6 transfection. * P < 0.05 between indicated groups.
FIGURE 2
FIGURE 2
Autophagy contributes to the radioresistance of NPC cells. (A) Fluorescence images of HNE2, CNE1, and CNE1R cells transfected with mRFP-GFP-LC3 (x 40). (B) Western blot assay of P62 and LC3 proteins in HNE2, CNE1, and CNE1R cells. (C) Efficiency of siLC3 transfection in CNE1R cells. (D) Dose responses of survival factions of CNE1R cells before and after siLC3 transfection. *P < 0.05 between indicated groups.
FIGURE 3
FIGURE 3
Transfection of siANXA6 inhibits autophagy incidence. (A) Fluorescence images of CNE1R cells co-transfected with siANXA6 and mRFP-GFP-LC3 (x40). (B) Western blot assay of ANXA6, P62, and LC3 proteins in CNE1R cells. (C) Western blot assay of ATG12 and Beclin-1 proteins in CNE1R cells. *P < 0.05 between indicated groups.
FIGURE 4
FIGURE 4
ANXA6 contributes to autophagy incidence via inhibition of the PI3K/AKT/mTOR pathway. (A) Western blot assay of PI3K-α, AKT, p-AKT, mTOR, and p-mTOR proteins in HNE2, CNE1, and CNE1R cells. (B) Western blot assay of PI3K-α, AKT, p-AKT, mTOR, and p-mTOR proteins in CNE1R cells with or without siANXA6 transfection. (C) Western blot assay of PI3K-α, AKT, p-AKT, mTOR, p-mTOR, and ANXA6 proteins in siANXA6-transfected CNE1R cells treated with CAL101 or its control (1‰ DMSO). *P < 0.05 between indicated groups.
FIGURE 5
FIGURE 5
A pattern diagram shows that ANXA6 regulates autophagy via inhibiting the PI3K/AKT/mTOR pathway to induce radioresistance of NPC. Irradiation (IR) increases the expression of ANXA6 in NPC cell lines and patient tumor tissues, which, in turn, directly inhibits the expression of PI3K and further reduces the phosphorylation of AKT and mTOR. The reduced p-mTOR increases the expression of Beclin-1 (a key activator of autophagy) and advances the formation of phagophore consisting of ATG16L1 (autophagy related 16-like 1), ATG12, ATG5 (autophagy related protein 5), and LC3 protein. Additionally, P62 binds to autophagosomal membrane protein LC3 and delivers itself to autophagosome, which ultimately leads to a decline of P62. Finally, autophagolysosome formed by the fusion of autophagosome and lysosome can phagocytize injured organelles to maintain/restore metabolic homeostasis, contributing to the radioresistance of NPC. Arrows represent promotion events, and blunt arrows indicate suppression events.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cai C., Min S., Yan B., Liu W., Yang X., Li L., et al. (2019). MiR-27a promotes the autophagy and apoptosis of IL-1beta treated-articular chondrocytes in osteoarthritis through PI3K/AKT/mTOR signaling. Aging 11 6371–6384. 10.18632/aging.102194 - DOI - PMC - PubMed
    1. Chaachouay H., Fehrenbacher B., Toulany M., Schaller M., Multhoff G., Rodemann H. P. (2015). AMPK-independent autophagy promotes radioresistance of human tumor cells under clinical relevant hypoxia in vitro. Radiother. Oncol. 116 409–416. 10.1016/j.radonc.2015.08.012 - DOI - PubMed
    1. Chaachouay H., Ohneseit P., Toulany M., Kehlbach R., Multhoff G., Rodemann H. P. (2011). Autophagy contributes to resistance of tumor cells to ionizing radiation. Radiother. Oncol. 99 287–292. 10.1016/j.radonc.2011.06.002 - DOI - PubMed
    1. Chamcheu J. C., Roy T., Uddin M. B., Banang-Mbeumi S., Chamcheu R. N., Walker A. L., et al. (2019). Role and therapeutic targeting of the pi3k/akt/mtor signaling pathway in skin cancer: a review of current status and future trends on natural and synthetic agents therapy. Cells Basel 8:803. 10.3390/cells8080803 - DOI - PMC - PubMed
    1. Chang L., Graham P. H., Ni J., Hao J., Bucci J., Cozzi P. J., et al. (2015). Targeting PI3K/Akt/mTOR signaling pathway in the treatment of prostate cancer radioresistance. Crit. Rev. Oncol. Hematol. 96 507–517. 10.1016/j.critrevonc.2015.07.005 - DOI - PubMed
开云球赛中国有限公司 | 万搏官方网站 | c7官网登录入口 | 九州平台-九州(中国) | 万搏官方网站 | 千亿球友会·(中国)官方网站 | 米兰体育网址 | 开云手机官方网站 | 江南手机网页版登录入口 |