Metabolic changes during malignant transformation in primary cells of oral lichen planus: Succinate accumulation and tumour suppression

Abstract Oral squamous cell carcinoma (OSCC) is usually diagnosed at late stages, which leads to high morbidity. There are evidence that chronic inflammation (eg oral lichen planus [OLP]) was a risk factor of OSCC, but often misdiagnosed or ignored until invasion and metastasis. By applying precision medicine, the molecular microenvironment variations and relevant biomarkers for the malignant transformation from OLP to OSCC can be fully investigated. Several studies pointed out that the metabolic pathway were suppressed in OSCC. However, it remains unclear how the systemic profile of the metabolites change during the malignant transformation. In this study, we examined and compared the mucosa samples from 11 healthy individuals, 10 OLP patients and 21 OSCC patients. Based on the results, succinate, a key metabolite of the tricarboxylic acid cycle pathway, was accumulated in the primary cultured precancerous OLP keratinocytes and OSCC cells. Then, we found that succinate activated the hypoxia‐inducible factor‐1 alpha (HIF‐1α) pathway and induced apoptosis, which could also be up‐regulated by the tumour suppressor lncRNA MEG3. These results suggested the critical roles of succinate and MEG3 in the metabolic changes during malignant transformation from OLP to OSCC, which indicated that succinate, HIF1α and downstream proteins might serve as new biomarkers of precancerous OLP for early diagnosis and therapeutic monitoring. In addition, succinate or its prodrugs might become a potential therapy for the prevention or treatment of OSCC.


| INTRODUC TI ON
Oral squamous cell carcinoma (OSCC) holds 95% of all types of head and neck cancer, and the morbidity increased 50% over the last decade. 1 The primary cause of the high morbidity and low survival rate lies on that OSCC is diagnosed at late stages (stage III or IV) in most cases, 2 which significantly impairs the patients' quality of life. Clinical data demonstrated that oral carcinogenesis is a chronic process, in which multiple stages may take their parts, including inflammation, 3 precancerous lesions, invasion and metastasis. 4 Among the precancerous lesions of OSCC, oral lichen planus (OLP) is one of the frequently happening chronic mucocutaneous inflammatory diseases. Thus, for early diagnosis and precise treatment of OSCC, the mechanism underlying malignant transformation from OLP to OSCC should be fully investigated. 5,6 In recent years, the concept of precision medicine leads can- Plenty of evidence suggest that malignant transformation closely relates to changes in several branches of metabolism (eg TAC). 10 In addition, the accumulation of metabolites such as succinate (SUC), fumaric acid (FUM) and 2-hydroxyglutarate, are associated with the oncogenesis. 11 Although genome and metabolome analyses 9,12,13 shed light on specific metabolic pathways and enzymes changes between OSCC, OLP and healthy control samples, the molecular profile of changes in TAC is still not clear enough for the intervention of OSCC, and the lack of proper biomarkers on this pathway hinders the diagnosis of malignant transformation from OLP to OSCC.
In this research project, we first evaluated the content changes in several important metabolites of TAC during malignant transformation by examining the innovatively primary cultured OLP and OSCC cell lines from patients. Then, we investigated the effects of SUC (a key metabolite of TAC) on the hypoxia-inducible factor-1 alpha (HIF-1α), which could be activated by prolyl hydroxylase (PHD) inhibition. 14 The accumulation of SUC was found to promote apoptosis by activating the HIF-1α pathway and could be induced by lncRNA 01 MEG3. Finally, the changes in succinate-HIF-1α pathway were validated on clinical samples. Our study suggested that the MEG3-SUC-HIF-1α pathway-induced apoptosis might act as a protection against malignant transformation, which made SUC administration has a potential therapy for the prevention or treatment of OSCC. In addition, SUC, HIF-1α and downstream proteins could serve as new biomarkers for the diagnosis of OLP malignant transformation.

| Patients and tissue samples
In total, 30 OLP and 41 OSCC specimens were collected for study which were provided by Huashan Hospital, Fudan University from 2014 to 2017 (Table 1). Prior patient's consents and approval from the Institutional Research Ethics Committee were obtained to use the clinical samples for research purposes.

| Cells and culture
The reticular-type lesion tissue, about 0. 6

| Identification of TAC metabolites by HPLC-ESI-QqQ-MS
A high-performance liquid chromatography (HPLC) system equipped with a diode array detector (DAD) was used to analyse samples. TAC metabolites were achieved on a Phenomenex P/N 00B-4378-B07 Luna NH2 (50 × 2.0 mm) (Agilent, USA). The column was thermostatically controlled at 30°C. The flow rate was set to 1 mL/min and the injection volume was 5 μL. The mobile phase consisted of two solvents: ammonium acetate (A, 100%) and acetonitrile (B, 100%). The solvent gradient in volumetric ratios was set as follows: 70%-75% in 15 minutes; at 45 minutes, the gradient was increased to 100% A; and held at 100% A for an additional 2 minutes. The UV absorbance of the peaks were collected between 200 and 620 nm using a DAD and monitored at wavelengths of 360 nm. A triple quadrupole mass spectrometer (6430 QqQ LC/MS system; Agilent Technologies, USA) equipped with an orthogonal electrosprayionization (ESI) source were used to identify the derivatives. A negative ion mode was selected for data collection 15,16 . Mass spectrometr (MS) parameters were set as follows: the sheath gas was nitrogen and collision gas was helium; drying gas flow rate, 11.0 L/min; drying gas temperature, 300°C; nebulizing gas pressure, 15 psi; the capillary voltage, 4 kV. Peak areas in HPLC chromatograms were converted into mass using the calibration curve of pure standard as the method reported.

| Western blot analysis
Protein was extracted from cells using cell lysis solutions containing protease inhibitors and phosphorylase inhibitors. Equal amounts of protein were fractionated on Tris-glycine SDS-polyacrylamide gels and subjected to electrophoresis and transferred to NC membranes.
After washing in TBS-T, membranes were incubated with fluorescent secondary antibodies. β-Actin was used as the loading control. The signal intensity of primary antibody binding was quantitatively analysed with ImageJ software (WS Rasband, ImageJ, NIH, Bethesda, MD).

| RNA isolation and quantitative reverse transcription-PCR
Total RNA was extracted using Trizol reagent (Invitrogen, NY) according to the manufacturer's instructions from the control group.
The primer sequences are shown in following Table 1

| Cell survival and mitochondrial membrane potential assays
Cell survival assay and mitochondrial membrane potential assays were performed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Wisconsin) and Mitochondrial membrane potential assay kit with JC-1 (Beyotime, China). All the analyses were performed according to the manufacturer's instructions. Luminescence and fluorescence were recorded with a Biotek Synergy2 Luminescent plate reader.

| Statistical analysis
All data were presented as means ± SEM. Data were subjected to one-way ANOVA using the GraphPad Prism software statistical package (GraphPad Software). When a significant group effect was found, post hoc comparisons were performed with the Newman-Keulst test to examine special group differences. Independent group t tests were used for comparing two groups. Significant differences with P < 0.05, P < 0.01, and P < 0.001 are indicated by *, **, ***, respectively. All calculations were performed with the 14.0 spss software package (SPSS Inc).

| TAC was suppressed in the process of carcinogenesis
Tissue samples of normal oral mucosa (NOM), OLP mucosa and OSCC nests were collected from healthy volunteers and patients.
Immunohistochemistry was performed to evaluate the localization and the expression of OGDH, IDH and CS. As shown in Figure

| Succinate was accumulated for OLP and OSCC at both the tissue and cellular level
The suppression of energy metabolism might change the level of its metabolites, which could be essential in the process of carcinogenesis.  (Figure 2A). In contrast, CIT, SUC and MAL were up-regulated in OSCC compared with OLP tissues. But in the cellular level, SUC and FUM were increased in OSCC ( Figure 2B). Taken these together, SUC was accumulated in both OSCC tissues and cells, which suggested that SUC might closely relate with the malignant transformation from OLP to OSCC.

| Succinate-HIF-1α pathway activation could suppress cell proliferation and promote apoptosis of OLP precancerous cells
ATP ELISA was employed to test the cell viability under SUC accumulation. Stimulating OLP cells for 24 hours, 1 mmol/L of SUC reduced the survival rate to below 60%, which continued to drop until about 10% at 5 mmol/L. The proliferation was totally inhibited at 10 mmol/L or higher. This antiproliferation effect became significant by treatment of 10 mmol/L SUC for more than 12 hours ( Figure 3A).
The decrease in mitochondrial membrane potential is a marker of which also exhibited dose and time dependency ( Figure S1). These results indicated that apoptosis was induced by SUC accumulation.

| The accumulation of succinate was pivotal during malignant transformation by activating HIF-1α pathway
It has been reported that SUC acts as an inflammatory signal to induce the activation of HIF-1α, 14 because excess SUC could impair the PHD activity (by product inhibition) leading to HIF-1α stabilization.
To verify the hypothesis that SUC accumulation might activate the HIF-1α pathway during the malignant transformation, we performed Western blot experiments to evaluate the expressions of HIF-1α and its downstream target proteins (ie VEGF, MMP-9, Bax, Bcl-2 and caspase 3) in the OLP keratinocytes incubated with SUC from 5 to 20 mmol/L for different time periods ranging from 0 to 48 hours. As shown in Figure 4A,B, 5 mmol/L of SUC caused the strongest upregulation of the HIF-1α pathway. Its highest expression level was achieved at the 24th hour ( Figure 4C,D).

| LncRNA MEG3/miR-361-5p/SDHD pathway could increase the content of succinate and lead to cell apoptosis
Long noncoding RNA MEG3 has been reported to be a tumour suppressor. 21 It is also involved in regulating glycolysis in the tumours  Figure 5A, 500 ng MEG3 caused apoptosis of more than half of the precancerous cells. Meanwhile, the concentration of SUC was increased to about 800 ng/L, seven times higher than the blank control ( Figure 5B). The survival rate and SUC increase also exhibited dose dependency, which indicated strong association between SUC accumulation and MEG3-induced apoptosis of malignant cells. Succinate dehydrogenase (SDH) is an enzyme complex, which catalyse the accumulation of SUC ( Figure 5C).
Increasing research have confirmed that lncRNAs may be function as a molecular sponge or a ceRNA via competitively binding to miRNA leading to the variation in its targeted mRNA in regulating tumour development and pathogenesis. To explore whether MEG3 had the similar function to regulate certain miRNAs, Starbase was used to predict potential miRNAs that directly interacted with MEG3 and SDH (supplementary table). We found miR-7-5p and miR-361-5p were the most potential targets as both interacted with MEG3 and SDH ( Figure 5D). Furthermore, qRT-PCR showed that miR-361-5p was significantly down-regulated upon MEG3 stimulation, while miR-7-5p remained unchanged ( Figure 5E).
Thus, SUC accumulation and HIF-1α activation-induced apoptosis could be a downstream effect of MEG3.

| Validation of succinate, HIF-1α, VEGF and MMP-9 levels on clinical samples
Additional NOM, OLP mucosa and OSCC nests (10 samples each) were collected to validate the changes in the SUC and HIF-1α pathway. A semi-quantitative analysis of the immunohistochemistry experiment demonstrated that HIF-1α, VEGF and MMP-9 were up-regulated in OLP tissue compared with NOM, and further increased in OSCC tissue ( Figure 6A,B). Succinate ELISA result showed significant accumulation of SUC in OLP tissue (about twice of that in NOM), and also more SUC was observed in OSCC nests ( Figure 6C).

ACK N OWLED G EM ENT
We are grateful to Professor Shimin Zhao for careful reading and valuable comments on this manuscript. We would like to thank all of our laboratory members for their technical support.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interests.