ARNTL inhibits the malignant behaviors of oral cancer by regulating autophagy in an AKT/mTOR pathway-dependent manner

Abstract

Current studies have shown that ARNTL, an important clock gene, plays an anticancer role and is downregulated in certain types of cancer. However, the biological functions and mechanisms of ARNTL in tumors remain largely unknown. This study aimed to elucidate how ARNTL-induced autophagy impacts the biological properties of tongue squamous cell carcinoma (TSCC) cells and the mechanisms by which ARNTL expression activates autophagy. ARNTL was stably overexpressed in TSCC cells to investigate its functions in vitro and in vivo. We found that activation of autophagy induced by ARNTL decreases cell proliferation, enhances cell death, and hinders the migratory ability of TSCC cells. Moreover, ARNTL antagonizes the AKT/mTOR pathway, which potentiates autophagy induction. The manipulation of Akt activation cancels the effects of ARNTL overexpression on the biological behaviors of TSCC cells. Furthermore, after the addition of SC79, the upregulated BAX expression due to ARNTL overexpression and downregulated expressions of BCL-2 and MMP2 were remarkably rescued. In vivo tumorigenicity assays and immunohistochemistry also confirmed that ARNTL overexpression suppresses tumor development. Our study found for the first time that ARNTL inhibits the malignant behaviors of oral cancer cells by regulating autophagy in an AKT/mTOR pathway-dependent manner, which provides a novel theoretical basis for the potential future application of ARNTL to treat patients with oral cancer.

Keywords: ARNTL; autophagy; carcinogenesis; oral cancer; tumor suppressor.

FIGURE 1

Auto and Baf inhibit ARNTL‐induced autophagy. (A) RT‐PCR detection of ARNTL mRNA expression in TSCC cells before and after ARNTL overexpression. (B) Western blot analysis of ARNTL protein expression in TSCC cells before and after ARNTL overexpression. (C) TEM image of intracellular autophagosomes in TSCC cells. The lower picture is the image of the upper picture with the red boxed area under high magnification. The autophagosomes can be distinguished by the double membrane structure and its contents (including mitochondrion, ribosomes, rough endoplasmic reticulum, and the cytoplasm) indicated by red arrows. Scale bars are 5 and 1 μm, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the ARNTL‐OE group. Auto, autophinib; Baf, bafilomycin; NC, negative control; OE, overexpression; TEM, transmission electron microscopy; TSCC, tongue squamous cell carcinoma.

FIGURE 2

Autophagy plays a role in regulating the proliferation and apoptosis of TSCC cells. (A) Cell proliferation measured using flow cytometry. (B) Micrographs of colonies formed by TSCC cells (crystal violet staining). (C) TUNEL assay evaluating TSCC cell apoptosis. Scale bar = 200 um. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the ARNTL‐OE group. Auto, autophinib; Baf, bafilomycin; NC, negative control; OE, overexpression; TSCC, tongue squamous cell carcinoma.

FIGURE 3

Inhibition of autophagy enhances the migration and invasion abilities of TSCC cells. (A) Parallel migration ability of TSCC cells was determined using wound‐healing assay. (B) Vertical migration and invasion abilities of TSCC cells were determined using Transwell assay. Scale bar = 200 um. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the ARNTL‐OE group. Auto, autophinib; Baf, bafilomycin; NC, negative control; OE, overexpression; TSCC, tongue squamous cell carcinoma.

FIGURE 4

ARNTL regulates cell proliferation, apoptosis, migration, and invasion dependent on the AKT/mTOR signaling pathway.(A) The protein expression levels of AKT, p‐AKT, mTOR, p‐mTOR, LC3 II/I, and P62 after adding the AKT activator SC79. (B) The protein expressions of BAX, BCL‐2, and MMP2 after adding the autophagy inhibitors Auto and Baf. (C) The protein expressions of BAX, BCL‐2, and MMP2 after adding the AKT activator SC79. (D) The cell proliferation and apoptotic index measured by flow cytometry after adding the AKT activator SC79. (E) Metastatic cells evaluated using Transwell assay after adding the AKT activator SC79. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the ARNTL‐OE group. Auto, autophinib; Baf, bafilomycin; NC, negative control; OE, overexpression.

FIGURE 5

ARNTL overexpression inhibits TSCC cell tumorigenesis in vivo. (A) Images of subcutaneous xenografts from mice and tumor weights and volumes in the ARNTL‐NC and ARNTL‐OE groups. (B) The expression levels of ARNTL, AKT, p‐AKT, mTOR, p‐mTOR, BAX, BCL‐2, and MMP2 were evaluated using immunohistochemistry H‐score in xenograft tumor tissue sections. Scale bar = 50 um. *P < 0.05, **p < 0.01, ***p < 0.001, compared with the ARNTL‐OE group. NC, negative control; OE, overexpression; TSCC, tongue squamous cell carcinoma.