O-GlcNAcylation of TRIM29 and OGT translation forms a feedback loop to promote adaptive response of PDAC cells to glucose deficiency

Abstract

Purpose

Glucose not only provides energy for tumor cells, but also provides various biomolecules that are essential for their survival, proliferation and invasion. Therefore, it is of great clinical significance to understand the mechanism of how tumor cells adapt to metabolic stress and maintain their survival. The aim of this research was to study the critical role of OGT and TRIM29 O-GlcNAc modification driven adaptability of PDAC cells to low glucose stress, which might have important medical implications for PDAC therapy.

Methods

Western blotting, mass spectrometry and WGA-immunoprecipitation were used to examined the levels of OGT and O-GlcNAc glycosylated proteins in BxPC3 and SW1990 cells in normal culture and under glucose deprivation conditions. Crystal violet assay, flow cytometry, RIP, RT-qPCR, protein stability assay, biotin pull down were used to investigate the mechanism of OGT and TRIM29-mediated adaptive response to glucose deficiency in PDAC cells.

Results

The current study found that under the condition of low glucose culture, the levels of OGT and O-GlcNAc glycosylation in PDAC cells were significantly higher than those in normal culture. Moreover, the high expression of OGT has a protective effect on PDAC cells under low glucose stress. This study confirmed that there was no significant change in mRNA level and protein degradation of OGT under low glucose stress, which was mainly reflected in the increase of protein synthesis. In addition, O-GlcNAc modification at T120 site plays a critical role in the metabolic adaptive responses mediated by TRIM29.

Conclusions

Taken together, our study indicated that O-GlcNAcylation of TRIM29 at T120 site and OGT translation forms a loop feedback to facilitate survival of PDAC under glucose deficiency.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-023-00915-5.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) originates from pancreatic ductal epithelial cells, accounting for more than 90% of pancreatic malignant tumors. PDAC is one of the most invasive solid malignant tumors, extensive tumor invasion can occur in the early stage, and the 5-year survival rate below 10% [1]. In recent years, the incidence of PDAC has significantly increased all over the world [2]. At present, the treatment of PDAC is mainly gemcitabine/Abraxane and FOLFIRINOX combined chemotherapy. However, patients usually develop drug resistance within weeks to months after treatment, and the therapeutic effect is not ideal [3–5]. Therefore, the study of the survival mechanism of PDAC cells has important clinical significance for the treatment of pancreatic cancer.

Due to the rapid growth and proliferation of tumor cells, metabolic stress resulting from insufficient angiogenesis and blood supply generally exists in the tumor microenvironment. Tumor cells have a high rate of glucose consumption and glucose metabolism is extremely active, the concentration of glucose in tumor is 3–10 times lower than that in untransformed tissue, so glucose deficiency is the most common metabolic stress of tumor cells [6]. Glucose deficiency can reduce the production of adenosine triphosphate (ATP) and increase the production of reactive oxygen species (ROS), which is not advantageous to cell survival [7]. Therefore, tumor cells need to maintain their survival and proliferation under adverse conditions of metabolic stress [8–10].

In order to resist the adverse environment, solid tumor cells must optimize their nutritional utilization to adapt to the stress environment when resources are scarce. Therefore, metabolic reprogramming is an important sign of tumor cells. The activation of oncogenes and the inactivation of tumor suppressor genes promote the metabolic reprogramming of tumor cells, enhance the nutritional absorption, and support the survival and rapid proliferation of tumor cells by providing energy and a variety of substrates for biosynthesis [11]. Accumulating evidence shows that metabolic reprogramming in tumor microenvironment is very important for the occurrence of PDAC tumors [12].

Hexosamine biosynthesis pathway (HBP) is a branch of glucose and glutamine metabolic pathway, which consumes about 3~5% of total glucose [13, 14]. UDP-GlcNAc is a glycosylated donor modified by O-linked β-N-acetylglucosamine (O-linked β-N-acetylglucosamine) glycosylation [15]. O-GlcNAc glycosylation modification is mainly carried out in the nucleus, cytoplasm and mitochondria, which is a monosaccharide modification and usually is not further extended to form a polysaccharide chain. Similar to the phosphorylation of proteins, O-GlcNAc glycosylation occurs in the serine/threonine of proteins, which is a highly dynamic post-translational modification (PTM). The dynamic balance of O-GlcNAc glycosylation is regulated by two enzymes: O-GlcNAc glycosyltransferase (OGT) and O-GlcNAc glycosidase (OGA) [16]. The level of O-GlcNAc glycosylation modification often fluctuates with the change of intracellular nutrient concentration (such as glucose and glutamine), which plays an important role in maintaining cell nutritional homeostasis [17]. The disruption of the balance of O-GlcNAc glycosylation modification will lead to a variety of diseases, such as diabetes, cardiovascular disease, oncology and neurodegenerative diseases [18–21].

O-GlcNAc glycosylation modification of proteins is closely related to tumorigenesis, development, metastasis, drug resistance, and prognosis [22–24]. Previous studies have shown that both OGA, OGT and total O-GlcNAc protein levels in pancreatic cancer are up-regulated compared with normal pancreatic tissue [18]. Down-regulation of O-GlcNAc glycosylation level in tumor cells had no significant effect on the growth of untransformed pancreatic epithelial cells, but could effectively inhibit the proliferation of PDAC cells and orthotopic transplanted tumor, and promote tumor cell apoptosis [22].

TRIM29 is a member of the TRIM protein family, also known as ataxic telangiectasia D group complementary gene (ataxia telangiectiasia group D complementing gene, ATDC) protein. Although TRIM29 does not contain the common RING-finger domain of TRIM family proteins, it has recently been reported that TRIM29 can also mediate weak E3 ubiquitin ligase activity through the B-box domain [25]. TRIM29 is mainly involved in cell growth and proliferation, and plays a role in immune inflammation regulation, cell signal transduction, protein transport, apoptosis and cell cycle regulation [26–29]. TRIM29 plays a role of tumor suppressor in some types of breast cancer [30] and prostate cancer [31]. However, it plays a tumor-promoting role in hepatocellular carcinoma [32], pancreatic cancer [33] and ovarian cancer [34]. In pancreatic cancer, TRIM29 is closely related to the malignant phenotype of the tumor. TRIM29 knockdown inhibits the growth of pancreatic cancer cells, blocks cell cycle progression, and promotes apoptosis [26]. TRIM29 binds to p53 and antagonizes p53-mediated nuclear function [35]. In addition, TRIM29 is an important regulator of Nrf2 protein stability and antioxidant activity, and can be used as a biomarker for predicting chemotherapy resistance of pancreatic ductal adenocarcinoma [5]. In the process of rapid growth of solid tumors such as PDAC, metabolic stress is often caused by high nutrient consumption and insufficient blood supply, especially glucose deficiency. However, the role of TRIM29 in metabolic stress of tumor cells has not been reported.

In the current study, we found that under the condition of low glucose stress can promote the expression of OGT in PDAC cells. The dynamic balance of O-GlcNAc glycosylation modification levels in PDAC cells is regulated by O-GlcNAc glycosyltransferase OGT. TRIM29 as a cancer factors, expressed in pancreatic ductal adenocarcinoma, and mass spectrometry analysis confirmed that TRIM29 was a substrate of OGT and underwent O-GlcNAc modification during metabolic stress. The current study identified that OGT translation and O-GlcNAc glycosylation of TRIM29 formed a loop feedback to facilitate survival of PDAC under glucose deficiency, which might provide new ideas for the treatment of PDAC.

Results

Low glucose-mediated metabolic stress promotes the expression of OGT in PDAC cells

In order to investigate the effect of O-GlcNAc glycosylation on the survival of PDAC cells in low glucose culture, BxPC3 and SW1990 cells were cultured in the medium with glucose concentration of 25 mM (normal) and 1 mM (low glucose) for 48 h. Compared with normal culture, glucose deficiency promoted the expression of OGT and the level of O-GlcNAc glycosylation of proteins in BxPC3 and SW1990 cells, but there was no significant change in the expression level of OGA (Fig. 1a). In order to explore the potential role of OGT in metabolic stress induced by glucose deficiency, OGT was stably knocked down by lentiviral vector containing specific shRNAs against OGT (shOGT) in BxPC3 and SW1990 cells (Fig. 1b). Cell survival decreased significantly under the condition of low glucose culture, and when OGT was knocked down, the cell survival further decreased (Fig. 1c, d). There was a significant G1/S phase arrest in glucose deficiency, and OGT knockout attenuated this cell cycle arrest (Fig. 1e, f). In addition, AnnexinV and PI double staining followed by cytometry showed that OGT knockdown increased the apoptosis of cells cultured under low glucose (Fig. 1g, h).

Fig. 1.

Low glucose-mediated metabolic stress promotes the expression of OGT in PDAC cells. a O-GlcNAc, OGT and OGA expression were assessed in BxPC3 and SW1990 cells using Western blot under normal culture and glucose deficiency. b OGT was knocked down using CRISPR-Cas 9 system in BxPC3 and SW1990 cells, Western blot was performed using the indicated antibodies. c, d After crystal violet staining, the absorbance of samples at 560 nm was detected by enzyme-labeled instrument, and the survival rates of BxPC3 and SW1990 cells in normal culture and glucose deficiency were analyzed. *, P < 0.05; n.s., not significant. e, f The cell cycles of BxPC3 and SW1990 cells in normal culture and glucose deficiency were analyzed by flow cytometry with PI staining. g, h Apoptosis of BxPC3 and SW1990 cells was detected by flow cytometry with Annexin V and PI double staining in normal culture and glucose deficiency. *, P < 0.05

OGT is upregulated at the translational level under metabolic stress mediated by low glucose

RT-qPCR showed that there was no significant change in the mRNA expression level of OGT under glucose deficiency compared with normal culture (Fig. 2a). Cells were then treated with CHX (10 μg/mL) for the indicated time to suppress synthesis of protein. Compared with normal culture, the stability of OGT protein in low glucose culture was significantly higher than that in normal culture after 8 h of CHX exposure, while CHX had no obvious effect on stability of OGT before (Fig. 2b, c). In order to detect whether the increase in the stability of OGT was caused by the decrease of protein degradation, the cells were treated with protein degradation inhibitors E64D/pepstatinA (10 μg/mL) and MG132 (10 μg/mL) for 24 h to block the lysosomal and proteasomal degradation of OGT respectively (Fig. 2d, e). The results showed that E64D/pepstatin A had no effect on induction of OGT by low glucose (Fig. 2d, e). Although MG132 decreased the extent of OGT induction by low glucose, OGT expression still increased in the presence of MG132 (Fig. 2d, e). Cells were further treated with CHX (10 μg/mL) for 16 h to completely suppress protein translation, then CHX was removed to restart protein synthesis. Compared with normal culture, recovery of OGT protein synthesis was significantly accelerated by low glucose culture (Fig. 2f).

Fig. 2.

OGT is upregulated at the translational level under metabolic stress mediated by low glucose. a The expression levels of OGT mRNA in BxPC3 and SW1990 cells were detected by RT-qPCR under normal culture and glucose deficiency. n.s., not significant. b, c The expression levels of OGT in BxPC3 and SW1990 were detected by Western blot after the cells were treated with actinomycin (CHX) (10 μg/mL) for 0–8 h under normal culture and glucose deficiency conditions. *, P < 0.05; n.s., not significant. d, e The cells were treated with E64D/pepstatin A (10 μg/mL) and MG132 (10 μg/mL) for 24 h, Western blot was used to detect the expression levels of OGT in BxPC3 and SW1990 under normal culture and glucose deficiency conditions. *, P < 0.05; n.s., not significant. f After the cells were treated with CHX for 16 h, the CHX interference was removed, Western blot was used to detect the expression levels of OGT

Recruitment of eIF4G2 and TIA1 to the 5ʹUTR of OGT mRNA is prohibited by low glucose stress

To further explore the potential mechanism underlying OGT translation induced by low glucose stress, luciferase reporter gene plasmids containing 5ʹUTR or 3ʹUTR fragment of OGT mRNA were constructed. Luciferase activity assays demonstrated that luciferase of empty reporter gene vector was significantly decreased when cells were exposure to low glucose culture (Fig. 3a, b), indicating that the total protein synthesis in cells was inhibited when glucose was deficient. Different from suppression of total protein, the luciferase activities of reporter plasmids containing 5ʹUTR or 3ʹUTR fragment of OGT mRNA were significantly increased by low glucose culture (Fig. 3a, b). While activity of the report construct containing 5ʹUTR increased more obviously (Fig. 3a, b), the following investigation was focused on 5ʹUTR. Secondary structure prediction through RNA fold web server demonstrated that 5ʹUTR of OGT mRNA formed a complex structure (Fig. 3c). In addition to its own secondary structure of 5ʹUTR, recruitment of RBPs also affects translational efficiency of mRNAs. 5ʹUTR of OGT mRNA labeled with biotin was then used to carry out RNA pulldown experiment followed by mass spectrometry to identify potential RBPs. Among the ten common RNA binding proteins identified by mass spectrometry, the recruitment of eIF4G2, TIA1 and RBM3 were altered by glucose deficiency. The protein levels of eIF4G2, TIA1 and RBM3 had no obvious changes in glucose deficiency compared with those in normal culture (Fig. 3d). The interaction of eIF4G2 or TIA1 with the 5ʹUTR of OGT mRNA was weakened by glucose deficiency, while RBM3 had no consistent change in BxPC3 and SW1990 cells (Fig. 3e). In accordance with this, RIP confirmed that recruitment of eIF4G2 and TIA1 to the OGT mRNA decreased significantly under low-glucose culture compared with normal culture in both BxPC3 and SW1990 cells (Fig. 3f, g). Recruitment of RBM3 to OGT mRNA increased in BxPC3 cells, while no obvious alteration was observed in SW1990 cells under low glucose exposure (Fig. 3f, g).

Fig. 3.

Recruitment of eIF4G2 and TIA1 to the 5ʹUTR of OGT mRNA is prohibited by low glucose stress. a, b The luciferase reporter plasmid of OGT mRNA 5 ‘UTR and 3ʹ UTR was transfected into BxPC3 and SW1990 cells, and luciferase activity was analyzed. *, P < 0.05. c The secondary structure was predicted by RNAfold web server. d The protein levels of EIF4G2, TIA1 and RBM3 in BxPC3 and SW1990 cells were detected by Western blot. e Biotin pull down assay to detect the interaction of EIF4G2, TIA1, RBM3 and OGT mRNA 5 ‘UTR in normal culture and glucose deficiency. f, g RIP assay was used to detect OGT mRNA recruitment by EIF4G2, TIA1 and RBM3 proteins in BxPC3 and SW1990 cells under normal culture and glucose deficiency. *, P < 0.05; n.s., not significant

Recruitment of eIF4G2 and TIA1 to 67-71nt and 167-171nt of 5ʹUTR respectively, to regulate OGT translation

RNA pulldown followed by spectrometry identified that recruitment of eIF4G2 and TIA1 to the 5ʹUTR of OGT mRNA was significantly reduced under low glucose. RBP site predicted 6 potential binding sites for eIF4G2 or TIA1 on the 5ʹUTR of OGT mRNA, reporter gene plasmids containing deletion of the individual potential binding sites were then constructed. The secondary structure prediction demonstrated that deletion of 67~71nt significantly disrupted the secondary structure of 5ʹUTR of OGT mRNA (Fig. 4a). Deletion of 67~71nt increased the luciferase activity of reporter containing 5ʹUTR of OGT under normal culture condition, while deletion of 167~171nt increased the luciferase activity under low glucose (Fig. 4b, c). According to the bioinformatics prediction results, 67~71nt and 167~171nt are the binding sites of eIF4G2 and TIA1, respectively. These data indicated that the inhibitory effect of eIF4G2 on OGT mRNA 5ʹUTR via 67~71nt binding was relieved when glucose was deficient, while TIA1-mediated inhibition via 167~171nt was released only when dissociation of eIF4G2 occurred. On this basis, we constructed the luciferase reporter plasmid with simultaneous deletion of 67~71nt and 167~171nt of OGT 5ʹUTR. Consistent with the previous results, under normal culture conditions, the luciferase activity of reporter containing 167~171nt deletion did not change significantly, but that of the 67~71nt deletion decreased significantly (Fig. 4d, e). Simultaneous deletion of 67~71nt and 167~171nt significantly increased the luciferase activity, when compared with 67~71nt alone deletion (Fig. 4d, e). However, when glucose is deficient, compared with the 167~171nt deletion alone, the luciferase activity of simultaneous deletion was unaltered (Fig. 4d, e).

Fig. 4.

Recruitment of eIF4G2 and TIA1 to 67-71nt and 167-171nt of 5ʹUTR respectively, to regulate OGT translation. a The secondary structure was predicted by RNAfold web server based on the 5ʹUTR sequences of OGT mRNA. b, c BxPC3 and SW1990 cells were transfected with indicated luciferase reporter construct for 48 h, and luciferase activity was analyzed. *, P < 0.05; n.s., not significant. d, e BxPC3 and SW1990 cells were transfected with indicated luciferase reporter construct for 48 h, and luciferase activity was analyzed. *, P < 0.05; n.s., not significant

TRIM29 improves the adaptive responses of PDAC cells to low glucose stress

Global screening identified potential O-GlcNAc glycosylation of TRIM29 at T120 site (Fig. 5a). Immunoprecipitation using wheat germ agglutinin (WGA) demonstrated that O-GlcNAc glycosylation of TRIM29 increased under low glucose culture (Fig. 5b). Total TRIM29 was unaltered by low glucose (Fig. 5b). In vitro O-GlcNAc modification using recombinant wild type (WT) or T120A mutant TRIM29 demonstrated that WT, but not T120A TRIM29 underwent O-GlcNAc modification by recombinant OGT (Fig. 5c). TRIM29 expression was then downregulated with CRISPR/CAS9 technology (Fig. 5d). TRIM29 interacts with p53, which is a master regulator of metabolic adaptive responses. p53 significantly increased upon low glucose culture, which was unaltered by TRIM29 knockdown in both normal and low glucose culture (Fig. 5d). TRIM29 knockdown had no obvious effect on the survival of cells under normal culture conditions, while under extremely low glucose culture conditions, the survival of TRIM29 knockdown cells decreased obviously (Fig. 5e, f). TRIM29 knockdown attenuated cell cycle arrest caused by glucose deficiency (Fig. 5g, h). In addition, TRIM29 knockdown significantly increased cell apoptosis under low sugar culture conditions (Fig. 5i, j).

Fig. 5.

TRIM29 improves the adaptive responses of PDAC cells to low glucose stress. a Mass spectrometry analysis identified threonine 120 (120 T) as the O-GlcNAc glycosylation site of TRIM29. b The O-GlcNAc glycosylation levels of BxPC3 and SW1990 cells in normal culture and glucose deficiency were determined by immunoprecipitation assay for wheat germ agglutinin (WGA). c Wild type (WT) and T120A mutant TRIM29 were expressed and purified by prokaryotic expression, and O-GlcNAc reaction was performed in vitro. d TRIM29 was knocked down using CRISPR-Cas9 system in BxPC3 and SW1990 cells, Western blot was performed using the indicated antibodies. e, f The cells viability of BXPC3 and SW1990 were determined by crystal violet staining at 560 nm. *, P < 0.05; n.s., not significant. g, h The cell cycles of BxPC3 and SW1990 cells in normal culture and glucose deficiency were analyzed by flow cytometry with PI staining. i, j Apoptosis of BxPC3 and SW1990 cells was detected by flow cytometry with Annexin V and PI double staining in normal culture and glucose deficiency. *, P < 0.05

O-GlcNAc modification at T120 site plays a critical role in the metabolic adaptive responses mediated by TRIM29

To further determine whether the protective effect of TRIM29 under low glucose stress is achieved through O-GlcNAc glycosylation modification, TRIM29 knockdown cells were restored with WT or T120A mutant TRIM29 (Fig. 6a). Both WT and T120A mutant TRIM29 had no obvious effect on p53 expression under both normal and low glucose culture (Fig. 6a). In addition, WT and T120A TRIM29 demonstrated similar extent of interaction with p53 under both normal and low glucose culture (Fig. 6a). WT TRIM29 significantly increased cell viability under low glucose culture, while T120A mutant TRIM29 did not (Fig. 6b, c). Similarly, WT TRIM29 promoted G1/S cell cycle arrest (Fig. 6d, e) and decreased apoptosis (Fig. 6f, g), while T120A mutant TRIM29 had no such effect (Fig. 6d–g).

Fig. 6.

O-GlcNAc modification at T120 site plays a critical role in the metabolic adaptive responses mediated by TRIM29. a Wild-type (WT) and T120A mutant TRIM29 were overexpressed after knockdown of TRIM29 in BxPC3 and SW1990 cells, Western blot was performed using the indicated antibodies. b, c The cells viability of BXPC3 and SW1990 were determined by crystal violet staining at 560 nm. *, P < 0.05; n.s., not significant. d, e The cell cycles of BxPC3 and SW1990 cells in normal culture and glucose deficiency were analyzed by flow cytometry with PI staining. f, g Apoptosis of BxPC3 and SW1990 cells was detected by flow cytometry with Annexin V and PI double staining in normal culture and glucose deficiency. *, P < 0.05

O-GlcNAc modification facilitates interaction of TRIM29 with TIA1 to prohibit its recruitment to the 5ʹUTR of OGT mRNA

TRIM29 demonstrated no obvious effects on p53 induction by low glucose (Figs. 5c and 6a). In addition, WT and T120 mutant TRIM29 interacted with p53 with similar extent (Fig. 6a). Luciferase activity of pGL13 Luc construct containing p53 responsive element significantly increased under low glucose culture, which was unaltered by TRIM29 knockdown (Fig. 7a). In addition, neither WT nor T120A mutant altered the luciferase activity of pGL13 reporter (Fig. 7b). Collectively, these data indicated that TRIM29 might affect the adaptive responses of PDACs to low glucose-mediated metabolic stress in a p53-independet manner. The global TRIM29 interacting partners under normal and low glucose culture conditions were then screened using quantitative mass spectrometry, among altered interacting partners, TIA1 attracted our attention as its implication in translation of OGT. Co-IP confirmed that interaction between TRIM29 and TIA1 was increased under low glucose culture (Fig. 7c). Interaction of TIA1 with WT TRIM29, but not T120A mutant TRIM29 was increased under low glucose culture (Fig. 7d). To investigate whether TRIM29 affect recruitment of TIA1 to OGT mRNA, RIP was performed. Consistent with previous data, recruitment of TIA1 and eIF4G2 to the OGT mRNA decreased upon low glucose culture (Fig. 7e). The decrease in TIA1 recruitment was suppressed by TRIM29 knockdown, while recruitment of eIF4G2 was unaltered (Fig. 7e). In addition, WT but not T120A mutant TRIM29 suppressed recruitment of TIA1 (Fig. 7f). Neither WT nor T120A mutant TRIM29 demonstrated any observed effect on eIF4G2 recruitment (Fig. 7f). Importantly, induction of OGT expression by low glucose was suppressed by TRIM29 knockdown (Fig. 7g). Furthermore, WT TRIM29 facilitated OGT induction under low glucose culture, while T120A mutant TRIM29 had little influence (Fig. 7h).

Fig. 7.

O-GlcNAc modification facilitates interaction of TRIM29 with TIA1 to prohibit its recruitment to the 5ʹUTR of OGT mRNA. a Luciferase reporter genes with pGL13 Luc construct containing p53 response elements were transfected into BxPC3 and SW1990 cells, and the luciferase activity was detected in normal culture and glucose deficiency. b Luciferase reporter genes with pGL13 Luc construct containing p53 response elements were transfected into WT TRIM29 and T120A mutant TRIM29 cells, and the luciferase activity was detected in normal culture and glucose deficiency. c Co-IP was used to detect the interaction of TRIM29 with TIA1 in BxPC3 and SW1990 cells under normal culture and glucose deficiency conditions. d Co-IP was used to detect the interaction between TIA1 and WT TRIM29 and T120A mutant TRIM29. e RIP assay was used to analyze the recruitment of TAI1 and eIF4G2 to OGT mRNA under normal culture and glucose deficiency condition. f RIP assay detected the recruitment of TIA1 and eIF4G2 to OGT mRNA in WT TRIM29 and T120A mutant TRIM29 cells under normal culture and glucose deficiency conditions. g Western blot assay was used to detect OGT expression in CON and TRIM29 KD cells under normal culture and glucose deficiency conditions. h Western blot assay was used to detect the expression of OGT in WT TRIM29 and T120A mutant TRIM29 cells under normal culture and glucose deficiency conditions

Discussion

PDAC is a highly aggressive malignant tumor, which cause local invasion and metastasis in the early stage, and is resistant to most treatments, with poor prognosis and high mortality [36]. During the rapid growth of solid tumors such as PDAC, metabolic stress is often caused by the consumption of nutrients and the shortage of blood supply. The proliferation of tumor cells requires a large amount of glucose for anabolism. The glucose concentration in tumor is usually 3~10 times lower than that in untransformed tissues, so glucose deficiency is one of the main forms of metabolic stress in tumor cells. Glucose not only provides energy for tumor cells, but also provides various biomolecules that are essential for their survival, proliferation and invasion [37]. Therefore, it is of great clinical significance to understand the mechanism of how tumor cells adapt to metabolic stress and maintain their survival [38].

As a branch of glucose and glutamine metabolism, HBP consumes about 3~5% of total glucose and a small part of glutamine to synthesize UDP-GlcNAc. HBP uses fructose-6-phosphate, acetyl coenzyme A, glutamine and UTP as substrates to produce UDP-GlcNAc, thus integrating a variety of metabolic pathways, including glucose, amino acids, fatty acids and nucleotide metabolism. UDP-GlcNAc is the end product of HBP, and it is also the glycosylation donor of protein O-GlcNAc [12, 39]. O-GlcNAc signal is highly sensitive to various forms of cell stress (such as heat shock, hypoxia, nutritional deficiency). Therefore, glycosylation modification of O-GlcNAc is considered as a nutritional and pressure sensor, which is involved in regulating biological activities such as transcription, translation, signal transduction and metabolism [40–43].

Previous studies have confirmed that increasing the concentration of UDP-GlcNAc improves the level of protein O-GlcNAc in vitro in a dose-dependent manner. As the biosynthesis of UDP-GlcNAc by HBP requires the participation of glucose, glutamine, acetyl coenzyme A and UTP, when the availability of these nutrients increases, the glycosylation level of O-GlcNAc in cells increases [40]. However, under the condition of nutrient deficiency, the total glycosylation level of O-GlcNAc in cells increased sharply. As the level of UDP-GlcNAc decreases under nutritional deficiency, this effect cannot be explained by the change of HBP flux. A potential mechanism of this reaction is the up-regulation of OGT expression. Although the level of UDP-GlcNAc decreases, the glycosylation level of O-GlcNAc may still increase [44]. In addition, the accumulation of unfolded protein caused by nutritional deficiency may increase the abundance of OGT substrate, thus increasing the total glycosylation level of O-GlcNAc [40]. In this study, under the condition of low glucose stress, the levels of OGT and O-GlcNAc in PDAC cells were significantly higher than those in normal culture, but the level of OGA did not change significantly. It has been reported that O-GlcNAc glycosylation modification is very important for the growth of tumor cells, but has little effect on untransformed normal cells [22]. Because tumor cells are extremely sensitive to the deletion of OGT, inhibiting OGT in pancreatic cancer cells can reduce tumor formation in vivo and clone formation ability of tumor cells in vitro [45]. Therefore, OGT may become a potential therapeutic target in the future.

Compared with normal culture, the mRNA expression level of OGT demonstrated no obvious change under glucose deficiency, indicating that the expression of OGT induced by glucose deficiency is mainly reflected at protein level. The current study demonstrated that the increase of OGT protein expression induced by low glucose stress is mainly reflected in the increase of protein synthesis, rather than the decrease of protein degradation. Protein synthesis is a highly regulated biological process, which may determine the response of cells to different stress states [46]. The translation process of eukaryotes includes three stages: initiation, extension and termination. Protein synthesis and mRNA translocation are mainly regulated in the initial stage [47–49]. Eukaryotic translation initiation factor (eIF) plays an important regulatory role in the process of translation initiation, and it promotes the occurrence of tumors by inducing abnormal gene expression [50]. The current study found that the RNA binding protein (RBP) eIF4G2 and TIA1 inhibited OGT expression by binding to the 5ʹUTR of OGT mRNA. eIF4G2 (also known as NAT1/p97/DAP5) has close homology with eIF4G1. Unlike eIF4G1, eIF4G2 lacks the binding site of eIF4E. Therefore, eIF4G2 can’t bind to cap binding protein. eIF4G2 is mainly involved in the initiation of cap-independent translation by specifically binding to mRNA 5ʹUTR [51–54]. Studies have confirmed that eIF4G2 can promote the survival of cells during mitosis by promoting cap-independent translation of Bcl-2 and CDK1 [55]. In addition, the abnormal expression of eIF4G2 plays a key role in the progress of many cancers. For example, inhibiting the expression of eIF4G2 can significantly inhibit the progression of acute myeloid leukemia, diffuse large lymphoblastic tumor and osteosarcoma [50]. T-cell intracellular antigen 1 (TIA1), an RNA binding protein, contains three RNA recognition motifs (RRM) that bind to mRNA, and a C- terminal domain rich in glutamine and asparagine [56]. TIA1 is the main regulator of the interaction between nucleus and cytoplasm in eukaryotic cells [56]. It is involved in regulating constitutive splicing and alternative splicing, assisting mRNA transportation and subcellular localization [57], and regulating mRNA stability and translation through direct interaction or competition with other protein and RNA [58, 59]. Under stress conditions, TIA1 protein is also involved in regulating cell proliferation and inflammation [60], tumor growth [61], apoptosis [62] and the recruitment of cytoplasmic mRNA to stress granule (SG), preventing translation [63, 64]. In this study, eIF4G2 and TIA1, as RNA binding proteins, bind to the specific region of 5ʹ UTR of OGT mRNA, and participate in the regulation of OGT translation. This study confirmed that both eIF4G2 and TIA1 inhibited translation of OGT. Under the condition of low glucose stress, the inhibitory effect of eIF4G2 was released, and on this premise, the inhibitory effect of TIA1 on OGT translation was released, which further promoted the translation of OGT. It is worth noting that compared with normal culture, the protein expression level of eIF4G2 and TIA1 has not changed obviously under low glucose stress.

TRIM29, as a member of TRIM family, has increased expression in a variety of tumors [65, 66], and is associated with poor prognosis and overall survival rate decline [67, 68]. Sun et al. have shown that TRIM29 can promote cancer in PDAC [33], and it can promote the metastasis and invasion of PDAC cells by stabilizing β-catenin. In addition, in the early stage of KRAS-induced pancreatic tumor, TRIM29 promoted the invasion of pancreatic tumor tissue and vascular spread by binding and antagonizing p53 [5, 26, 69]. Our previous study also confirmed that down-regulation of TRIM29 expression can promote the expression of CAPN3 and promote the degradation of ISG15, thus inhibiting PDAC stem cell-like phenotype [70]. The current study demonstrated that low glucose stress promoted the expression of OGT protein and the O-GlcNAc glycosylation modification of TRIM29 at Thr120, thereby facilitating the adaptability of PDAC cells to low glucose stress (Fig. 8). Inhibition of TRIM29 expression or its O-GlcNAc modification effectively reduced the survival of PDAC cells under low glucose stress, which further supplements the molecular mechanism of TRIM29 as a cancer-promoting factor and provides a new idea for the treatment of PDAC. In addition, the current study demonstrated that O-GlcNAc modification facilitated interaction of TIRM29 with TIA1. TRIM29 prohibited recruitment of TIA1 to the OGT mRNA, thereby forming a positive feedback loop between OGT translation and TRIM29 O-GlcNAc modification (Fig. 8).

Fig. 8.

OGT/TRIM29/TIA1 pathway facilitates adaptive response of PDAC cells to glucose deficiency

Overall, our findings underscored the critical role of OGT and TRIM29 O-GlcNAc modification driven adaptability of PDAC cells to low glucose stress, which might have important medical implications for PDAC therapy.

Conclusions

Our current study demonstrated that low glucose stress promoted the expression of OGT protein and the O-GlcNAc glycosylation modification of TRIM29 at Thr120, thereby facilitating the adaptability of PDAC cells to low glucose stress. Inhibition of TRIM29 expression or its O-GlcNAc modification effectively reduced the survival of PDAC cells under low glucose stress. Overall, our findings underscored the critical role of OGT and TRIM29 O-GlcNAc modification driven adaptability of PDAC cells to low glucose stress, which might have important medical implications for PDAC therapy.

Materials and methods

Cell culture

The human pancreatic cancer cell lines BxPC3 and SW1990 were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 IU/ml of penicillin and 100 µg/ml of streptomycin. The cells were incubated in a humidified atmosphere at 37 °C with 5% CO₂.

Small hairpin RNA (shRNA)

shRNAs against OGT was purchased from Open Biosystems. Transfection of shRNA oligonucleotide was performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s recommendations.

CRISPR-Cas9 cell line generation

To stably knockout the genes, lentivirus infection experiment was performed. Lentivirus were purchased by Nanjing Jikai Gene Corperation. 2 × 10⁵ cells/well were seeded into six-well plates and cultured for 24 h. For infection, viral solutions according to the multiplicity of infection (MOI) were added into cell culture medium. After incubation for 12 h, the medium was changed by normal medium with 1 μg/ml puromycin for 48 h. Cells were expanded and verified by western blot.

DNA constructs and generation of stable cell lines

Gene encoding TRIM29 was cloned into the into the eukaryotic expression plasmid, pcDNA3, tagged with FLAG to generate WT-TRIM29 construct. TRIM29 (T120A) was constructed in which Thr120 was replaced by Ala (T120A) from WT-TRIM29 by a PCR-based method. BXPC3 and SW1990 cells were transfected with various TRIM29 construct using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction, and stable clonal cell lines were selected with 1 mg/ml G418 and were maintained at 0.2 mg/ml G418. Western blot was used to measure the transfection efficiency.

Co-immunoprecipitation

Cell lysates were pre-cleared with Protein-A/G beads (biomake, B23201) and kept on a rotator for 1 h at 4 °C. Lysates were centrifuged and 2 μg of primary antibody or corresponding IgG was added to the precleared lysatesand kept on a rotator overnight at 4 °C. After incubation, 30 μl of Protein-A/G beads was added and kept on a rotator for 2 h at 4 °C. The immunoprecipitates were washed three times with lysis buffer, then analyzed by Western blot analysis.

Apoptosis assay

To detect apoptotic cell death, cells were washed twice with PBS and then stained using the Annexin V/PI Apoptosis Detection Kit according to the manufacturer’s instructions. After staining with Annexin V and PI for 30 min, samples were analysed using a fluorescence- activated cell scanner flow cytometer.

WGA-immunoprecipitation

Wheat germ agglutinin-coupled agarose beads were purchased from Vector Laboratories (AL-1023). In brief, wheat germ agglutinin-coupled agarose beads were incubated with cell lysates to enrich O-GlcNAc modified proteins, which were subsequently detected by western blot.

Mass spectrometry

The immunoprecipitates were washed three times with lysis buffer. The protein mixture was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then stained with Coomassie brilliant blue, collected the target protein tape, the length and width of the target protein strips should be controlled within 1 × 1 cm. After enzymatic hydrolysis in the gel, the protein mixture in the sample was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the target protein and its interacting proteins could be identified at the same time.

Crystal violet assay for determining viability of cultured cells

The cells were maintained in six-well plates in DMEM medium with normal glucose concentration and glucose deficiency medium culture for 48 h. Discard the medium, add 4% paraformaldehyde to fix for 30 min, and then add crystal violet to stain for 30 min. Add 1 mL of 33% glacial acetic acid to dissolve crystal violet dye solution, and add 100 µL to each well of 96-well plate. The absorbance (wavelength set to 560 nm) was detected using Cytation 5.

Cell cycle assay

The cell cycle assay was performed according to the manufacturer’s instruction (Keygen, KGA512). Briefly, cells suspension was cleaned with PBS and the cells concentration were adjusted to 1 × 10⁶/mL, and fixed with 500 μL cold 70% ethanol 4 °C refrigerator overnight. Add 100 μL RNase A and place in 37 °C for 30 min. Add 400 μL PI, mix well, and react at 4 °C for 30 min in the dark. Flow Cytometry analysis of cell cycle.

Protein stability assay

For the protein stability assay, cells were seeded into 6-well plates. It was cultured in an incubator (5%CO₂, 37 °C saturated humidity) for 24 h. CHX (10 μg/mL) was added into the culture medium of each group, and the treatment time was 0, 2, 4 and 8 h, respectively. Cells were further treated with CHX about 16 h and recovery phase. The cells at each time point were collected successively for Western blot assay.

RNA immunoprecipitation (RIP)

The Magna RIP RBP Immunoprecipitation Kit (EMD Millipore, Germany) was used to perform the RIP procedure according to the manufacturer’s instructions. 2 × 10⁷ cells were collected and lysed with RIPA lysis buffer (Beyotime, China). After centrifugation, the supernatant was immunoprecipitated with specific antibodies and protein A/G magnetic beads. The complexes bound to the magnetic beads were immobilized with a magnet. The unbound components were washed off. Finally, RNA bound to the A/G beads was obtained. The real-time qPCR procedure was performed to detect OGT mRNA in the precipitates.

Luciferase reporter assays

Luciferase activity was measured by Dual Luciferase Reporter Gene Assay Kit (Promega). Experiments were performed in triplicates and repeated for three times independently. The Renilla luciferase activity values that reflect transfection efficiency were utilized to normalize the firefly luciferase activity values.

Western blot analysis

Total cellular proteins were extracted using ice-cold RIPA lysis buffer (50 mM Tris [pH 8.0, 4 °C], 200 mM NaCl, 0.5% NP-40 and complete protease inhibitor). To prepare the cells, the cell lines were washed twice with ice-cold PBS, scraped in ice-cold RIPA lysis buffer. Clarified lysate protein concentrations were determined using Bradford reagent (Bio-Rad) before sample normalization for SDS-PAGE electrophoresis using 4–12% Bis-Tris NuPage gels at 200 V for 60 min. Electrophoresed samples were transferred on nitrocellulose membranes and blocked using 3% BSA dissolved in TBST (50 mM Tris [pH 8.0, 4 °C], 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. Membranes were then incubated with primary antibodies overnight at 4 °C. The next day, membranes were washed three times in TBST before the addition of secondary antibody conjugated with horseradish peroxidase (HRP) or streptavidin-HRP. Membranes were further washed three times with TBST before ECL exposure. The following antibodies were used in this study: TRIM29 (Thermo Fisher), OGT (Cell Signaling Technology), Flag (Cell Signaling Technology),OGA(Thermo Fisher) O-GlcNAc (Thermo Fisher), TIA1(Cell Signaling Technology), p53(Cell Signaling Technology), EIF4G2(Cell Signaling Technology), RBM3(Abcam) and GAPDH (Millipore). Dilutions were made in the proportion recommended by the manufacturer’s instructions.

Biotin pulldown assay

Luciferase vectors containing 5′UTR of OGT mRNA were used as the templates for PCR procedure. All 5′ primers contained the T7 promoter sequence. For the biotin pulldown assay, the amplified DNA was used as the template to transcribe biotinylated RNA using T7 RNA polymerase in the presence of biotin-UTP. The RNA—protein binding reaction was performed using 500 μg of cell lysates and 1 μg of biotin-labeled RNA in a final volume of 20 μL binding buffer A (20 mM HEPES-KOH,2.5 mM MgCl2, 100 mM KCl, 20% glycerol, 0.5 mM dithiothreitol and protease inhibitor; pH 7.5) for 1 h at room temperature. Finally, the complexes were isolated with paramagnetic streptavidin-conjugated DynaBeads (Invitrogen) and further analyzed by Western blot.

Statistical analysis

The statistical significance of the difference was analyzed by ANOVA and post hoc Dunnett’s test. Statistical significance was defined as P < 0.05. All experiments were repeated three times, and data were expressed as the mean ± SD (standard deviation) from three independent experiments.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

JS and HW designed this study; FZ, XC, JW and YY performed the experiments; XC, JS, JW, CL analyzed the data; HW and FZ wrote the manuscript. All authors have contributed to the final version of the manuscript.

Funding

This work was partly supported by National Natural Science Foundation of China (32071258, 82203337); Natural Science Foundation of the Higher Education Institutions of Jiangsu Province, China (22KJB320017); Supporting project of Nanjing University of Chinese Medicine to NSFC (XPT82203337); Doctoral Start-up Foundation of Liaoning Province (2021-BS-093); Doctoral Start-up Foundation of Liaoning Province (2020-BS-101).

Data availability

All data in this study will be available from the corresponding author on reasonable request.

Declarations

Consent for publication

Written informed consent for publication was obtained from all participants.

Conflict of interest

There is no conflict of interests in this work.