SLC45A4 promotes glycolysis and prevents AMPK/ULK1‐induced autophagy in TP53 mutant pancreatic ductal adenocarcinoma

Abstract Background Somatic mutations of the TP53 gene occur frequently in pancreatic ductal adenocarcinoma (PDA). Solute carrier family 45 member A4 (SLC45A4) is a H+‐dependent sugar cotransporter. The role of SLC45A4 in PDA, especially in TP53 mutant PDA, remains poorly understood. Methods We explored the TCGA datasets to identify oncogenes in TP53 mutant PDA. MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium], colony formation and 5‐ethynyl‐2′‐deoxyuridine (Edu) assays were performed to investigate the function of SLC45A4 in vitro. Glucose consumption, lactate production and ATP production were detected to evaluate glucose utilization. Extracellular acidification rate and oxygen consumption rate assays were used to evaluate glycolysis and oxidative phosphorylation. The subcutaneous xenotransplantation models were conducted to explore the function of SLC45A4 in vivo. RNA‐sequencing and gene set enrichment analysis were employed to explore the biological alteration caused by SLC45A4 knockdown. Western blotting was performed to evaluate the activation of glycolysis, as well as the AMPK pathway and autophagy. Results SLC45A4 was overexpressed in PDA for which the expression was significantly higher in TP53 mutant PDA than that in wild‐type PDA tissues. Moreover, high level of SLC45A4 expression was tightly associated with poor clinical outcomes in PDA patients. Silencing SLC45A4 inhibited proliferation in TP53 mutant PDA cells. Knockdown of SLC45A4 reduced glucose uptake and ATP production, which led to activation of autophagy via AMPK/ULK1 pathway. Deleting SLC45A4 in TP53 mutant HPAF‐II cells inhibited the growth of xenografts in nude mice. Conclusions The present study found that SLC45A4 prevents autophagy via AMPK/ULK1 axis in TP53 mutant PDA, which may be a promising biomarker and therapeutic target in TP53 mutant PDA.


| INTRODUCTION
The pancreatic ductal adenocarcinoma (PDA) is an aggressive disease with high morbidity. TP53 is one of the most frequently mutated genes in PDA. 1,2 The wild-type p53 is a tumor suppressor, which transcriptionally activates target genes to invoke anti-proliferative processes, such as apoptosis, cell cycle arrest and DNA repair. 3 Intriguingly, the mutant p53 obtains a gain-of-function (GOF) activity. [4][5][6] Recent evidence has demonstrated that mutant p53 increases glucose intake and glycolytic activity, which promotes the Warburg effect in cancer cells. [7][8][9][10] Targeting the mutant p53 might be an attractive strategy to reprogram glucose metabolism for cancer therapy. However, the downstream genes of mutant p53 or the genes interplay with mutant p53 in regulating cancer metabolism are largely unknown. The identification of oncogenes that specifically accompany mutant p53 in PDA might provide prognostic information and favor the development of more effective targeted therapies.
Solute carrier family 45 (SLC45) members are H + -dependent sugar cotransporters. 11,12 The four members in this family, A1 to A4, have been confirmed to transport both the monosaccharides and the disaccharide sucrose. SLC45 family members may be involved in glucose metabolism, energy supply, melanin synthesis and osmotic pressure adjustment. [12][13][14] Emerging evidence has indicated that aberrant expression of SLC45 proteins, such as SLC45A2 and SLC45A3, is associated with tumorigenesis and poor prognosis of cancer patients. 15,16 However, the role of this family members in PDA, especially in TP53 mutant PDA, remains poorly understood.
In the present study, we revealed that SLC45A4 was the most highly expressed SLC45 family member in PDA and significantly upregulated in TP53 mutant PDA. Its overexpression was associated with poor prognosis of PDA patients. Intriguingly, knockdown of SLC45A4 inhibited cell growth, uptake of glucose and production of ATP in TP53 mutant PDA cells, but not in TP53 wild-type pancreatic duct cells. Decreased energy production in SLC45A4 knockdown cells induced autophagy through the AMPK/ULK1 axis. Together, our results reveal a novel role of SLC45A4 and provide a promising therapeutic strategy for TP53 mutant PDA.

| Public data collection and bioinformatics analysis
The RNA expression profiles of SLC45 family were obtain from Gene    Real-time PCR was performed using ChamQ SYBR qPCR Master Mix (Q311-02; Vazyme) and analyzed with the Roche LightCycler system (Roche, Basel, Switzerland). Gene expression was standardized to the reference gene β-actin and calculated using the 2 ÀΔΔCT method.

| Clinical specimens and ethical approval
The primer sequences used were: human SLC45A4, 5 0 -AACGTGTCAGAGGAGGCCAA-3 0 (forward) and 5 0 -GTAAACGGGAG TGCGGCTCA-3 0 (reverse); human β-actin, 5 0 -TGGAACGGTGAAG GTGACAG-3 0 (forward) and 5 0 -AACAACGCATCTCATATTTGGAA-3 0 (reverse). (Syngene, Bangalore, India). 2.9 | Glucose utilization assay, lactate production and intracellular ATP production PDA cells were transfected transiently with siRNAs when the cells were 60-80% confluent in a six-well tissue culture plate. For the glucose utilization assay and lactate production, after 12 h, the previous media was replaced with phenol red free RPMI 1640 media supplemented with 1% fetal bovine serum and cultured for 48 h. The glucose concentration was measured with a colorimetric glucose detection kit (EIAGLUC; Thermo Fisher Scientific). The amounts of lactate production were assessed using the lactate assay kit (MAK064; Sigma-Aldrich) in accordance with the manufacturer's instructions. The amounts of ATP production were quantified using an ATP assay kit (S0026; Beyotime, Shanghai, China) in accordance with the manufacturer's instructions. The protein concentration was measured as described above and the normalized ATP level was calculated via the protein concentration.

| Extracellular acidification rate and oxygen consumption rate assays
The oxygen consumption rates (OCR) and the extracellular acidification rates (ECAR) were measured using Glycolysis Stress Test Kit

| Transmission electron microscopy (TEM)
TEM was performed as previously described. 17 Briefly, cells were collected by centrifugation to obtain a pellet with a size in the range 10-30 μl. Then, the cell pellet was fixed with 2.5% glutaraldehyde followed by postfixing in 1% osmium tetroxide, staining in 2% uranyl acetate, dehydrating and infiltrating with resin. The number of autophagic vacuoles was counted for autophagic profiles per cell area on sections with uniform random sampling, and the images were photographed with a JEM-100CX-II transmission electron microscope (Joel, Tokyo, Japan).

| Statistical analysis
All continuous variables were represented as the mean ± SD. Categorical variables were presented as percentages. Non-parametric variables were compared by the chi-squared test. For parametric variables, a two-tailed Student's t test was used to indicate differences between two groups or one-way analysis of variance was used for more than two groups. Kaplan-Meier analysis and a log-rank test were used for survival analysis. p < 0.05 was considered statistically significant.

| Expression of SLC45A4 is associated with TP53 mutation status and poor clinical outcomes of PDA patients
We investigated the gene expression of SLC45 family members in TCGA datasets from the GEPIA web server, 18 which showed that SLC45 family members, especially SLC45A4, were overexpressed in many types of cancer ( Figure 1A). Given that TP53 is one of most frequently mutated genes in PDA, to determine whether the expression of SLC45 family members was associated with TP53 status, we analyzed the TCGA PDA dataset. Among the SLC45 family, the mRNA level of SLC45A4 in TP53 mutant group was significantly higher than that in TP53 wild-type group, whereas the other three members did not show significant results ( Figure 1B).
Then we confirmed that the mRNA expression of SLC45A4 was significantly higher in PDA than that in normal tissues in the TCGA and GTEx dataset ( Figure 1C). The IHC staining data from the HPA database also showed that SLC45A4 protein was highly expressed in PDA tissues ( Figure 1D). Moreover, in our clinical samples, higher expression of SLC45A4 was observed in PDA tissues than in normal tissues ( Figure 1E). Statistical analysis revealed that SLC45A4 expression was associated with the CEA level and lymph node stage (Table 1). Importantly, PDA tissues with higher SLC45A4 expression had significant worse overall survival of patients (hazard ratio = 1.9, P = 0.043) ( Figure 1F).
These data showed that SLC45A4 expression was significantly up-regulated in PDA tissues and its high expression was associated with a poor prognosis of PDA patients.    Figure 4B) and ATP production ( Figure 4C). Furthermore, we measured glycolysis by analysing the ECAR and measured mitochondrial oxidative phosphorylation on the basis of the OCR. We found that SLC45A4 knockdown significantly reduced the glycolytic activity and glycolytic capacity ( Figure 4D). However, there was no significant difference in OCR between SLC45A4 knockdown and control cells, indicating that SLC45A4 knockdown does not affect oxidative phosphorylation ( Figure 4E). HK2 and PKM2 play a critical role in aerobic glycolysis.
Intriguingly, SLC45A4 knockdown also down-regulated the level of HK2 and PKM2 proteins in HPAF-II and MIA PaCa-2 cells ( Figure 4F). These results revealed that inhibition of SLC45A4 expression repressed glucose utilization and ATP production in TP53 mutant PDA cells. Autophagy has been reported to be induced by the energy sensing AMPK/ULK1 pathway. 19,20 As mentioned above, we found that ATP production decreased in SLC45A4 silencing cells ( Figure 4C).

| Down-regulation of SLC45A4 induced autophagy through the AMPK/ULK1 pathway
We proposed that knockdown of SLC45A4 might have activated AMPK/ULK1 signaling. Indeed, phosphorylation of AMPK and ULK1 increased after SLC45A4 knockdown in HPAF-II and MIA PaCa-2 cells ( Figure 5H). Conclusively, these results indicated that silencing SLC45A4 caused energy stress and activated AMPK/ULK1 to induce autophagy in TP53 mutant PDA cells.

| DISCUSSION
In the present study, we found that expression of SLC45A4 was significantly up-regulated and played an oncogenic role in PDA, especially in TP53 mutant PDA cells and tissues. Furthermore, SLC45A4 acted as an essential glucose transporter, and its knockdown induced autophagy in the TP53 mutant PDA cells; thus, it may serve as a novel marker and potential therapeutic target for TP53 mutant PDA.
The role of glucose metabolic reprogramming in cancer has become increasingly recognized. 21,22 Recent studies have showed that some cancer cell lines tend to rely on glycose instead of other nutriments for energy supply. 23 These cells exhibit rapid glucose consumption and lactate secretion. Oncogenic TP53 mutation promotes glucose metabolism in many types of cancer. 7 38 In the present study, GSEA indicated that the autophagy process was significantly affected by SLC45A4 knockdown. Autophagy plays dual roles in tumor promotion and suppression. 39,40 A basal level of autophagy is necessary for cells to maintain cellular homeostasis. However, when it comes to extremely nutrient deprivation, the role of autophagy is controversial. On the one hand, autophagy provides an alternative energy source for cells to rapidly resist nutritional stress. 41,42 On the other hand, the amplitude of autophagy increases above a threshold level, leading to a cell death mechanism. 43,44 The present study revealed that autophagy was induced by SLC45A4 knockdown in TP53 mutant PDA cells. Because inhibition of autophagy with 3-MA intensified the cell growth inhibition caused by SLC45A4 knockdown in TP53 mutant PDA cells, we can infer that the activation of autophagy caused by glucose limitation supported the cell proliferation.
In summary, the present study reveals that SLC45A4 is highly