Inhibition of HIF-1α Affects Autophagy Mediated Glycosylation in Oral Squamous Cell Carcinoma Cells

Purpose. To validate the function of autophagy with the regulation of hypoxia inhibitor-induced glycosylation in oral squamous cell carcinoma cell. Methods. Human Tca8113 cell line was used to detect autophagy and glycosylation related protein expression by western blotting and immunofluorescence with HIF-1α inhibitor. Short interfering RNA (siRNA) transfection blocked human ATG12 and ATG1. Results. HIF-1α inhibitor PX-478 reduced the amount of LC3-II and LC3-I in Tca8113 cells. PX-478 decreased the expression of O-GlcNAc and OGT and increased OGA expression. The tendency of O-GlcNAc showed a similar pattern to OGT. PX-478 gradually decreased OGT expression in Tca8113 cells. Protein level of O-GlcNAc and OGT increased in ATG12 and ATG1 depletion. The expression of OGT decreased at first and then rose slowly with the treatment of Atg12 and Atg1 siRNA and PX-478 fluctuant. Autophagy affected the stability of OGT when HIF-1α signaling was blocked. Conclusions. Autophagy reduced by hypoxic stress inhibited. HIF-1α inhibitor decreased glycosylation. OGT became unstable in the absence of autophagy when HIF-1α signaling was blocked.


Introduction
Autophagy's basic role in the turnover of proteins and organelles, digesting cellular contents to provide cellular energy and building blocks for biosynthesis, possesses multiple physiological and pathophysiological functions in the maintenance of cellular homeostasis. It has early been demonstrated to play a significant role in tumorigenesis, but whether it acts as a promoter or a suppressor during tumorigenesis seems to be context-specific [1].
Glycosylation research is hot now, but the mechanism is not clear yet which may be caused by elevated levels of O-GlcNAcylation by O-linked -N-acetylglucosamine (O-GlcNAc) or O-GlcNAc transferase (OGT). Literatures reported about some cancers indicated that glycosylation played an important role in the development, cell adhesion, and invasion of cancer, such as pancreatic cancer [9], breast tumors [10], and gastric cancer in patients 2 Disease Markers [11]. Absent -dystroglycan ( -DG) expression and likeacetylglucosaminyltransferase (LARGE) deregulation were closely associated with nodal metastasis of tongue cancer [12]. Recent research found that O-GlcNAcylation regulated cancer metabolism signaling via regulation of the HIF-1 pathway. HIF-1 was critical for OGT-mediated regulation of metabolic stress, as overexpression of stable HIF-1 rescues metabolic defects. Human breast cancers with high levels of HIF-1 contain elevated OGT, and lower OGA levels correlate independently with poor patient outcome [13].
To validate the putative participation of autophagy in the regulation of hypoxia inhibitor-induced glycosylation, we examined the effects of hypoxia on autophagic activity of in oral squamous cell carcinoma cells. Then we investigated the HIF-1's function in glycosylation. Finally, we explored potential mechanisms of OGT with both blocked HIF-1 and autophagy.

Short
Interfering RNA (siRNA) Transfection. Doublestranded siRNA targeting human ATG12 and ATG1 (purchased from Invitrogen) were administered simultaneously (30 nM each) to Tca8113 cells in Lipofectamine RNAiMAX reagent. In all experiments, scrambled siRNA served as a control. Tca8113 cells were analyzed 48 h after transfection. Protein knockdown was assessed by western blot analysis.

Statistical Analysis.
Experimental data are reported as mean ± SD of triplicate independent samples. All experiments were performed in triplicate on three independent occasions. Data were analyzed with two-tailed Student'stest. values < 0.05 were considered significant.

HIF-1 Inhibitor PX-478 Reduces Cellular Autophagy in Tca8113
Cells. HIF-1 could regulate autophagy and cell proliferation. MAP-LC3 is a major constituent of the autophagosome. During autophagy, the cytoplasmic form (LC3-I) is processed and recruited to the autophagosomes.
The hallmark of autophagic activation is the formation of cellular autophagosome punctae containing LC3-II, while autophagic activity is measured biochemically as the amount of LC3-II that accumulates in the absence or presence of lysosomal activity [14]. Induction of autophagy induces LC3-I conversion, producing lapidated LC3-II by action of Atg12-Atg5-Atg16L complex [15]. When autophagy was degraded by treatment of HIF-1 inhibitor PX-478 (0, 5, and 25 M), the amount of LC3-II and LC3-I decreased (Figures 1(a) and 1(b)). Autophagosome could be seen decreased with the dose increase of PX-478 (Figure 1(c)). These results suggested that HIF-1 blocking could reduce autophagy. . This result implied that OGT decrease in HIF-1 inhibitor treatment for a time frame might be related to autophagic induction.

Atg12 siRNA and Atg1 siRNA Transfection Increase Glycosylation.
To study whether autophagy affects glycosylation variation, we used Atg12 siRNA and Atg1 siRNA to reduce formation of autophagosome. Conversion of LC3-I to LC3-II decreased in depletion of ATG12 and ATG1 (Figures 3(a)  and 3(b)). Protein level of O-GlcNAc and OGT increased in ATG12 and ATG1 depletion (Figures 3(c) and 3(d)). Our result declared that inhibited autophagosome could induce accumulation of O-GlcNAc and OGT protein in Tca8113 cells. OGT was mainly due to the induction of autophagy at last, partially by the inhibition of HIF-1 at the beginning period. LC3-I and LC3-II were totally inhibited in the 48 hours in immunofluorescence assay (Figure 4(c)). This implied that autophagy still affects the stability of OGT when HIF-1 signaling was blocked.

HIF-1 Inhibitor Reduces Cellular
Autophagy. In this study we were able to link hypoxia and autophagy in Tca8113 tumor cell lines. We found that when HIF-1 was blocked, autophagy reduced with autophagosome and LC3-II/LC3-I decreased. Our result showed that hypoxia positively related to the autophagy in tumor cells. Also, Zhao et al. [16] found that knockdown HIF-1 abrogated hypoxia-induced autophagy activation in osteoclast cells. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient cells [17].
There are many pathways involved in HIF-1 affected autophagy. The resistance against cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway [18]. HIF-1 binds to effectors of chaperone-mediated autophagy (CMA) and is targeted for lysosomal degradation [19].
To study the expression of O-GlcNAc, OGT, and OGA affected by the HIF-1 inhibition in Tca8113 cells, OGT expression was decreased and OGA was increased under PX-478 treatment. These data consisted with the O-GlcNAc modification on proteins expression declined under HIF-1 inhibition, indicating that decreasing of OGT for a time frame with metabolism variation may relate to cell death caused by autophage. Our observations consisted with previous findings that Sp1 was being involved in the basal transcriptional activation of HIF-1 in breast cancer cells [23]. Tumor cells exhibited significant differences from normal cells in their metabolism. The interplay between the cellular metabolic network, oncogenes, tumor suppressors, hypoxia, autophagy, and biosynthetic anabolism is a hot topic [24].

HIF-1 Inhibitor Block Autophagy Mediated Glycosylation
Increasing. In the present study, we investigated glycosylation variation in Atg12 and Atg1 knockdown in Tca8113 cell lines. Our result declared that the inhibition of autophagy induced accumulation of glycosylation. Knockdown Atg normal mRNAs suppress the formation of autophagosome and showed accumulation of O-GlcNAc and OGT. Thus, we considered autophagy to be one of the fundamental degradation ways of OGT. To analyze whether the role of autophagy affecting the glycosylation in tongue squamous cell carcinoma is dependent on the variation of the hypoxia, we designed the OGT protein detected based on the autophagy knockdown while HIF-1 was inhibited. Interestingly, HIF-1 does not seem to play an important role in the autophagy on regulation of glycosylation in the long term although it has the critical effect on the expression of OGT. Our results indicated that OGT became unstable in the absence of autophagy when HIF-1 signaling was blocked. HIF-1 plays a main role at the early stage in the process of this dynamic change.
Hypoxia, autophagy, and glycosylation are all physiological conditions in tissues in vivo. They are all concerned the cells lifetime [25]. The relationship in hypoxia, autophagy, and glycosylation is complicated. Further functional experiments will be needed to support the direct roles of glycosylation mediated autophagy on HIF-1 at molecular and cellular level.

Conflict of Interests
Yi-Ning Li, Ji-An Hu, and Hui-Ming Wang declare that there is no conflict of interests regarding the publication of this paper.