LPLUNC1 reduces glycolysis in nasopharyngeal carcinoma cells through the PHB1‐p53/c‐Myc axis

Abstract Cancer cells prefer glycolysis to support their proliferation. Our previous studies have shown that the long palate, lung, and nasal epithelial cell clone 1 (LPLUNC1) can upregulate prohibitin 1 (PHB1) expression to inhibit the proliferation of nasopharyngeal carcinoma (NPC) cells. Given that PHB1 is an important regulator of cell energy metabolism, we explored whether and how LPLUNC1 regulated glucose glycolysis in NPC cells. LPLUNC1 or PHB1 overexpression decreased glycolysis and increased oxidative phosphorylation (OXPHOS)‐related protein expression in NPC cells, promoting phosphorylated PHB1 nuclear translocation through 14‐3‐3σ. LPLUNC1 overexpression also increased p53 but decreased c‐Myc expression in NPC cells, which were crucial for the decrease in glycolysis and increase in OXPHOS‐related protein expression induced by LPLUNC1 overexpression. Finally, we found that treatment with all‐trans retinoic acid (ATRA) reduced the viability and clonogenicity of NPC cells, decreased glycolysis, and increased OXPHOS‐related protein expression by enhancing LPLUNC1 expression in NPC cells. Therefore, the LPLUNC1‐PHB1‐p53/c‐Myc axis decreased glycolysis in NPC cells, and ATRA upregulated LPLUNC1 expression, ATRA maybe a promising drug for the treatment of NPC.


| INTRODUC TI ON
Nasopharyngeal carcinoma (NPC) is a common malignant tumor in Southern China. NPC has high morbidity and mortality in the world and seriously endangers human health and lives. [1][2][3][4] Hence, discovery of new therapeutic targets for the development of therapeutic drugs and understanding the pathogenesis of NPC will be of significance in management of NPC patients.
Tumor cells usually undergo metabolic reprogramming and prefer glycolysis with aberrant activation of the pentose phosphate metabolic pathway (PPP) to support energy, metabolic intermediates, and others to meet their aggressive growth. [5][6][7][8] Previous studies have shown that decreasing glycolysis and increasing oxidative phosphorylation (OXPHOS) can limit the growth of tumor cells. [9][10][11] Thus, improvement of imbalance between glycolysis and OXPHOS may be new therapeutic strategies for the treatment of malignant tumors.
Long palate, lung, and nasal epithelial cell clone 1 (LPLUNC1) is a natural innate immune molecule in nasopharyngeal respiratory epithelium, and has bactericidal and anti-inflammatory effects. 12,13 Our previous study has found that LPLUNC1 expression is significantly downregulated in NPC tissues. LPLUNC1 overexpression can attenuate the IL-6/Stat3 signaling and mitigate the IL-6-stimulated NPC cell proliferation. 14 LPLUNC1 can induce G1 arrest and apoptosis of NPC cells by inhibiting the E2F signaling 15 and can inhibit the invasion and migration of NPC cells and enhance the sensitivity of radiotherapy. 16,17 Moreover, LPLUNC1 can compete with TRIM 21 by binding prohibitin 1 (PHB1) to inhibit PHB1 ubiquitination degradation, upregulate PHB1 protein expression, and block the NF-κB signaling in NPC cells. 18 Accordingly, LPLUNC1 acts as a tumor suppressor of NPC. PHB1 has broad functions, which are crucial for mitochondrial structure and functions, regulating gene transcription and cell energy metabolism, [19][20][21] including glucose metabolism and fat metabolism. 22,23 Our previous studies and those of others have shown that PHB1 expression is downregulated in a variety of tumors, including NPC, which is associated with worse prognosis of tumor patients. 19,24,25 PHB1 overexpression can significantly inhibit the growth of NPC in rodents. [14][15][16][17][18]25 In addition, PHB1 can regulate cell energy metabolism. 26 LPLUNC1 overexpression can downregulate glucose-6-phosphate dehydrogenase (G6PD) in NPC cells. 27 Accordingly, we hypothesize that LPLUNC1 may regulate glycolysis in NPC cells by upregulating PHB1 expression.
P53 and c-Myc are important regulators of cell metabolism, and have opposite effects on glucose metabolism in tumor cells. p53 can inhibit glycolysis to maintain mitochondrial function. 28 In contrast, c-Myc is highly expressed in a variety of tumors and can enhance glycolysis and activation of the PPP in different types of tumor cells. 18,29 Previous studies have shown that PHB1 can downregulate the expression of c-Myc in tumor cells. 30 We hypothesize that LPLUNC1 may modulate p53 and c-Myc expression by enhancing PHB1 to regulate glucose metabolism in NPC cells.
In this study, we investigated whether and how LPLUNC1 or PHB1 overexpression could regulate glycolysis and OXPHOS-related protein expression and alter p53 and c-Myc expression in NPC cells.
Furthermore, we explored whether treatment with all-trans retinoic acid (ATRA) could modulate LPLUNC1 expression to change glucose metabolism in NPC cells. Our findings may provide new insights into the roles of LPLUNC1 in inhibiting NPC growth and aid in designing new therapies for the treatment of NPC.

| Cell culture and transfection
Human NPC HNE1, HNE2, and HONE1 cell lines were maintained and used in our laboratory 14,18 and cultured in RPMI-1640 medium (Gibco) supplemented with 12% fetal bovine serum (FBS; Zeta Life) at 37°C in a humidified incubator of 5% CO 2 .
The nuclear and cytoplasm fractions of individual groups of cells were extracted using PARIS™ Kit Protein and RNA Isolation System (Invitrogen).

| Detection of glucose uptake rate, extracellular lactate secretion rate, and G6P and G6PD activities
The glucose uptake rate, extracellular lactate secretion rate, glucose-6-phosphate (G6P) and G6PD activities in individual cell samples were analyzed using the glucose colorimetric assay kit II (Biovision), lactate colorimetric assay kit II (Biovision), G6PDH activity assay kit with WST-8 (Beyotime), and G6P assay kit with  according to the manufacturer's instructions.

| Seahorse analysis
The glucose metabolism of individual groups of cells in the XF24cell culture plates was characterized by Seahorse analysis, as described in a previous report. 31

| Intracellular ROS levels
After transfection, the three NPC cell lines, HNE1, HNE2, and HONE1, were cultured for 48 hours in six-well plates (1 × 10 6 cells/ well). Their intracellular ROS levels were quantified using Cell ROX Green reagent (Life Technologies). Cells were photographed under a fluorescence microscope (Leica), the average fluorescence signal of individual cells was analyzed with ImageJ software, and histograms were plotted.

| Luciferase
The Luciferase reporter gene plasmid used in the experiments was LPLUNC1-WT-Luc (GENECHEM). LPLUNC1-WT-Luc was transfected into NPC cells, HNE1 and HNE2, as well as 293T cells and treated with 10 μM ATRA drug for 48 hours. The levels of firefly luciferase activities were measured using a luciferase assay system (Promega). 32

| Immunofluorescence (IF)
The different groups of cells were cultured on glass coverslips for the indicated time periods and washed with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes and penetrated with 0.5% Triton X-100 in PBS for 20 minutes, followed by blocking with goat sera from healthy animals. The cells were probed with primary antibodies overnight at 4°C and reacted with the corresponding fluorescently labeled secondary antibodies at room temperature, followed by nuclear staining with DAPI. The cells were examined under a laser confocal microscope Fluorescence intensity of target molecules was observed, and their expression was analyzed and then imaged with a Zeiss LSM900 confocal microscope.

| Immunohistochemistry (IHC) and evaluation
Mouse xenograft NPC tissues were fixed in 10% formalin and embedded in paraffin. The NPC tissue sections (4 μm) were dewaxed, rehydrated, and subjected to antigen retrieval. The sections were incubated with primary antibodies (1:100) at 4°C overnight, and after being washed, the sections were reacted with HRP-conjugated secondary antibodies and visualized with DAB using an IHC kit (ZSGB-Bio). The immune-stained signals were photoimaged under a light microscope. The intensity of immunostaining and the number of positively stained cells were scored in a blinded manner. 33 The intensity of immunostaining was scored as 0, no cell staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The numbers of positively stained cells were scored as 0, no staining; 1, <25% of cells stained; 2, 25%-50% of cells stained; and 3, >50% of cells stained. A final score of one section was the product of the intensity score and positively stained cell number score. A score of <4 was considered low expression, and the rest was assigned as high expression.

| CCK-8 assay and colony formation assay
The proliferation of NPC cells was determined by CCK-8 assay. The cells (5 × 10 3 cells/well) were treated in triplicate with, or without, 1-25 μM ATRA in 96-well plates for 48 hours. Alternatively, the cells were treated with 10 μM ATRA for varying periods of 0-96 hours.
Subsequently, each well of cells was exposed to 5 mg/ml of CCK-8 (Zeta Life) and cultured for another 4 hours. The absorbance of individual wells was measured at 460 nm.
To test the clonogenicity, the cells (300 cells/well) were cultured in triplicate in six-well plates for 14 days. After being washed with PBS, the cells were stained with Giemsa solution (Beyotime).
Individual clones containing more than 50 cells were counted using Image Pro Plus 6.0 in a blinded manner, and the clonogenic rates of individual wells of cells were calculated by the formula (number of clones / number of seeded cells) × 100%.

| Statistical analysis
Data are presented as the mean ± standard deviation (SD), and the difference between groups was analyzed by Student's t test. Oneway ANOVA test was used to analyze the levels of glucose uptake rate, extracellular lactate secretion rate, and G6P and G6PD activities of NPC cells, and the results of clonogenicity after ATRA treatment by using the SPSS version 23.0 (SPSS) software. A P value of <0.05 was considered statistically significant.

| LPLUNC1 and PHB1 inhibit the glycolysis of NPC cells
Studies have shown that LPLUNC1 and PHB1 may act as tumor suppressors of NPC. 14,25 Our previous study has indicated that LPLUNC1 can upregulate PHB1 expression and stabilize PHB1 by F I G U R E 1 LPLUNC1 or PHB1 overexpression inhibits glycolysis in nasopharyngeal carcinoma (NPC) cells. A, B, The levels of glucose uptake, extracellular lactate production, and G6PD activity in wild-type and LPLUNC1-or PHB1-overexpressed HNE1, HNE2, and HONE1 cells. C, Seahorse analysis of ECAR in LPLUNC1-or PHB1 overexpressed HNE2 cells. Data are expressed as the mean ± SD of each group from three separate experiments stated as biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 inhibiting its ubiquitination. 18 Given the key role of PHB1 in cell metabolism, we investigated the regulatory effect of LPLUNC1 on glucose metabolism in NPC cells. We found that LPLUNC1 overexpression significantly reduced the levels of glucose uptake, extracellular lactate production, and G6P and G6PD activities in HNE1, HNE2, and HONE1 cells, except for increased levels of extracellular lactate production in HNE1 cells ( Figure 1A). PHB1 overexpression also had the same effect ( Figure 1B). Both LPLUNC1 and PHB1 overexpression significantly decreased the ECAR in HNE2 cells ( Figure 1C, Figure S1), indicating that both LPLUNC1 and PHB1 inhibited the glycolysis of NPC cells.
Given that inhibition of glycolysis is associated with enhancing OXPHOS in tumor cells, we tested whether LPLUNC1 or PHB1 overexpression could enhance OXPHOS-related protein expression in NPC cells. Western blotting displayed that LPLUNC1 overexpression significantly enhanced the relative levels of PHB1 expression in the tested NPC cells (Figure 2A). Furthermore, both LPLUNC1 and PHB1 overexpression decreased the relative levels of HK2, G6PD, PKM2, and LDHA, but increased OGDH, ACO2, and SDHB expression in NPC cells (Figure 2A

| LPLUNC1 regulates glucose metabolism, dependent on upregulating PHB1 expression in NPC cells
Next, we investigated the importance of PHB1 in the LPLUNC1regulated glucose metabolism in NPC cells. The inhibitory effects of glucose uptake, extracellular lactate production, and G6PD and G6P activities by LPLUNC1 overexpression were significantly mitigated by inducing PHB1 silencing in NPC cells ( Figure 3A, Figure S1B). PHB1 silencing also rescued the levels of ECAR in the LPLUNC1-overexpressed HNE2 cells ( Figure 3B). Moreover, the intracellular ROS levels decreased when silencing PHB1 in LPLUNC1overexpressed NPC cells ( Figure 2C). Further Western blotting revealed that PHB1 silencing partially or completely rescued the decrease in the relative levels of glycolysis-related HK2, G6PD, PKM2, and LDHA expression while abrogated the increase in the relative levels of OGDH, ACO2, and SDHB expression in the LPLUNC1overexpressed NPC cells ( Figure 3D). Similar results were detected in xenograft NPC tumors by IHC ( Figure S2). These results indicated that LPLUNC1 decreased glycolysis and increased OXPHOS-related protein expression in NPC cells, dependent on upregulating PHB1 expression.

| LPLUNC1 enhances the nuclear translocation of PHB1 in NPC cells
To understand how LPLUNC1 increased the levels of PHB1, we performed IF analysis of LPLUNC1-overexpressing HNE2 cells and found that LPLUNC1 overexpression increased PHB1 protein signals predominantly in the nuclei of HNE2 cells, similarly to PHB1 overexpression ( Figure 4A). Similarly, Western blotting revealed that LPLUNC1 overexpression mainly increased the relative levels of PHB1 protein in the nuclei of NPC cells ( Figure 4B). These findings suggest that LPLUNC1 may promote the nuclear translocation of PHB1 protein in NPC cells.
Induction of PHB1 Y259A mutant overexpression abrogated the PHB1 Tyr259 phosphorylation increased by PHB1 overexpression; rescued the levels of HK2, G6PD, PKM2, and LDHA expression; and mitigated the increase in the levels of OGDH, ACO2, and SDHB expression in the PHB1-overexpressed HNE2 cells ( Figure 4G,H).

| LPLUNC1 regulates glucose metabolism via the PHB1-p53/c-Myc signal axis in NPC cells
We tested the hypothesis that LPLUNC1/PHB1 decreased glycolysis and increased OXPHOS-related protein expression by modulating p53 and c-Myc expression in NPC cells. We found that p53 silencing or c-Myc overexpression rescued c-Myc, HK2, G6PD, PKM2, and LDHA expression and reduced OGDH, ACO2, and SDHB expression in LPLUNC1-overexpressed NPC cells ( Figure 5D). As a result, p53 silencing or c-Myc overexpression also partially rescued glucose uptake, extracellular lactate production, and G6PD and G6P activities in LPLUNC1-overexpressed NPC cells ( Figure 6, Figure S3).
Together, our data indicated that LPLUNC1 upregulated PHB1 expression, phosphorylation, and nuclear translocation to enhance p53 expression, but downregulated c-Myc expression, decreasing glycolysis and increasing OXPHOS-related protein expression in NPC cells.

| ATRA enhances LPLUNC1 expression to decrease glycolysis and increase OXPHOS-related protein expression in NPC cells
We screened small-molecule drugs to enhance LPLUNC1 expres-  Figure 7D). Consequently, treatment with ATRA decreased glucose uptake, extracellular lactate production, and G6PD and G6P activities in both wild-type and LPLUNC1-silenced NPC cells ( Figure 7E). Therefore, ATRA enhanced the LPLUNC1/PHB1/p53/c-Myc axis to decrease glycolysis and increase OXPHOS-related protein expression.

| DISCUSS ION
Our previous studies have shown that LPLUNC1 expression is downregulated in NPC and LPLUNC1 overexpression can significantly inhibit the proliferation of NPC cells. [14][15][16][17][18]25 Our recent study reveals that LPLUNC1 can upregulate PHB1 expression and stabilize PHB1 by inhibiting PHB1 ubiquitination mediated by E3 ligase TRIM21. 18 In this study, we further found that LPLUNC1 modulated the glucose metabolism in NPC cells by enhancing the activity of the PHB1/p53/ c-Myc axis. Our findings may shed light on the mechanisms underlying the action of LPLUNC1 in inhibiting the growth of NPC. PHB1 is crucial for mitochondrial structure and functions, regulating gene transcription and cell energy metabolism. 19,40 Previous studies have shown that downregulated PHB1 expression is associated with worse prognosis of several types of cancers, such as liver cancer, ovarian cancer, and lung cancer as well as NPC. 20,25,41,42 PHB1 overexpression can significantly inhibit the growth of tumor F I G U R E 4 LPLUNC1 promotes phosphorylated PHB1 nuclear transport by upregulating 14-3-3σ expression in nasopharyngeal carcinoma (NPC) cells. A, Immunofluorescent analysis of PHB1 expression in the indicated groups of cells. Scale bar: 25 μm. B, Western blotting analysis of the distribution of PHB1 in HNE2 cells. C, The relative levels of PHB1 and 14-3-3σ mRNA transcripts were analyzed by RT-qPCR in the indicated groups of cells. D, Western blotting analysis of the relative levels of PHB1, 14-3-3σ expression, and PHB1 Tyr259 phosphorylation in the indicated groups of NPC cells. E, Immunohistochemistry (IHC) analysis of 14-3-3σ expression in xenograft NPC tumors. Scale bar: 10 μm. F, Western blotting analysis of the relative levels of PHB1 expression and PHB1 Tyr259 phosphorylation in the indicated groups of NPC cells. G, Co-IP analysis of interaction between 14-3-3σ and PHB1 or phosphorylated PHB1 in HNE2 cells. H, Western blotting analysis of the relative levels of glycolysis-related and oxidative phosphorylation (OXPHOS)-related protein expression in the indicated groups of NPC cells. The numerical value is the ratio of the gray value of the target protein to the internal reference protein. Data are representative images or expressed as the mean ± SD of each group from three separate experiments stated as biological replicates. ***p < 0.001 cells, suggesting that PHB1 acts as a tumor suppressor. 18,43,44 Given that LPLUNC1 can significantly enhance PHB1 expression and stabilization, 18,19 we examined whether and how the LPLUNC1/PHB1 could modulate the glucose metabolism in NPC cells. We found that LPLUNC1 overexpression inhibited glycolysis, and the increased expression of OXPHOS-related proteins suggest that LPLUNC1 may enhance OXPHOS in NPC cells. Interestingly, the modulatory effect of LPLUNC1 on glycolysis and OXPHOS was abrogated by PHB1 silencing. Accordingly, LPLUNC1 decreased glycolysis and increased OXPHOS in NPC cells, dependent on upregulating PHB1 expression.
Studies have shown that nuclear PHB1 is crucial for regulating gene transcription. 19,45 Our previous study has indicated that LPLUNC1 can upregulate PHB1 expression and enhance its stabilization by reducing its ubiquitination degradation to attenuate the NF-kB signaling, 18 supporting the notion that PHB1 can affect multiple signal pathways to inhibit tumor cell proliferation. 19,24,29,41 In this study, we found that LPLUNC1 upregulated PHB1 protein expression and promoted phosphorylated PHB1 nuclear translocation by upregulating the expression of 14-3-3σ, an endoplasmic reticulum lipid raft protein. 34,46 Our data indicated that PHB1 Tyr259 phosphorylation was critical for its action in decreasing glycolysis and increasing OXPHOS-related protein expression in NPC cells. Evidently, 14-3-3σ directly interacted with PHB1 and phosphorylated PHB1 and mainly promoted the nuclear translocation of phosphorylated PHB1 at Tyr259 in NPC cells. More interestingly, induction of PHB1 Y259A mutant overexpression abrogated the PHB1-induced decreasing glycolysis and increasing OXPHOS-related protein expression in NPC cells. Previous research showed that PHB1 Tyr114 phosphorylation attenuates the insulin-related signaling 47 and PHB1 exists in lipid rafts of various cell membranes. [48][49][50] We are interested in further investigating how PHB1 is phosphorylated and how phosphorylated PHB1 regulates the glucose metabolic reprogramming in NPC cells. In addition, we found in our last paper that LPLUNC1 delays the progression of the cell cycle from G1 to S phase and inhibits the expression of cyclin D1, CDK4, and phosphorylated Rb. 18 Although there are no relevant studies on NPC in which LPLUNC1 modulates cellular metabolic reprogramming through regulation of the cell cycle, some articles have found that regulation of the cell cycle can cause changes in metabolism, 51 and we also believe that this may be the mechanism by which LPLUNC1 regulates metabolism.
The metabolic effects of LPLUNC1 caused by cell cycle need to be further explored.
It is notable that PHB1 is colocalized with p53 to enhance its transcriptional activity 28  In addition, we also found that treatment with ATRA decreased glycolysis and increased OXPHOS-related protein expression and inhibited the proliferation of NPC cells by upregulating LPLUNC1. These findings suggest that ATRA may be used to restore LPLUNC1 expression for adjuvant treatment of NPC. We also preliminarily showed that ATRA enhanced the activity of the LPLUNC1 promoter by luciferase report assay, and the specific regulatory mechanism deserves further exploration. Our data extended previous findings that ATRA treatment inhibits interstitial fibrosis by upregulating PHB1 expression. 57,58 It is possible that ATRA may first upregulate LPLUNC1 expression to enhance PHB1 expression. Given that ATRA is a relatively safe drug for the treatment of several types of malignant tumors, our novel findings indicate that ATRA may be a promising drug for the treatment of NPC in the clinic.
In conclusion, our data were for the first to indicate that LPLUNC1 modulated the glucose metabolic reprogramming in NPC cells through upregulating PHB1 and promoting its nuclear translocation. In addition, our results demonstrated that ATRA effectively

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

Qianjin Liao
https://orcid.org/0000-0001-9320-3090 F I G U R E 7 All-trans retinoic acid (ATRA) regulates the glucose metabolism of nasopharyngeal carcinoma (NPC) cells by upregulating LPLUNC1 expression. A, Treatment with ATRA significantly reduced the viability of NPC cells in a dose-and time-dependent manner. B, Treatment with 10 μM ATRA inhibited the clonogenicity of the indicated NPC cells. C, Western blotting analysis of LPLUNC1 and PHB1 expression in the NPC cells after ATRA treatment (10 μM for 48 h). D, Western blotting analysis of the relative levels of glycolysis-related and oxidative phosphorylation (OXPHOS)-related protein expression in the indicated groups of NPC cells. E, ATRA treatment decreased glucose uptake, extracellular lactate production, and G6PD and G6P activities in wild-type and LPLUNC1-silenced NPC cells. F, Luciferase reporter assays indicated that ATRA enhanced the LPLUNC1 promoter-controlled luciferase expression in the indicated cells. The numerical value is the ratio of the gray value of the target protein to the internal reference protein. Data are representative images or expressed as the mean ± S.D. of each group from three separate experiments stated as biological replicates. **p < 0.01, ***p < 0.001 versus the control, beside specified