The metabolic reprogramming of γ-aminobutyrate in oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy. The oncometabolites have been studied in OSCC, but the mechanism of metabolic reprogramming remains unclear. To identify the potential metabolic markers to distinguish malignant oral squamous cell carcinoma (OSCC) tissue from adjacent healthy tissue and study the mechanism of metabolic reprogramming in OSCC. We compared the metabolites between cancerous and paracancerous tissues of OSCC patients by ¹HNMR analysis. We established OSCC derived cell lines and analyzed their difference of RNA expression by RNA sequencing. We investigated the metabolism of γ-aminobutyrate in OSCC derived cells by real time PCR and western blotting. Our data revealed that much more γ-aminobutyrate was produced in cancerous tissues of OSCC patients. The investigation based on OSCC derived cells showed that the increase of γ-aminobutyrate was promoted by the synthesis of glutamate beyond the mitochondria. In OSCC cancerous tissue derived cells, the glutamate was catalyzed to glutamine by glutamine synthetase (GLUL), and then the generated glutamine was metabolized to glutamate by glutaminase (GLS). Finally, the glutamate produced by glutamate-glutamine-glutamate cycle was converted to γ-aminobutyrate by glutamate decarboxylase 2 (GAD2). Our study is not only benefit for understanding the pathological mechanisms of OSCC, but also has application prospects for the diagnosis of OSCC.

Keywords: Metabolic reprogramming; Oral squamous cell carcinoma; γ-aminobutyrate.

Fig. 1

Pathological examination of OSCC patients. The masses of patients with oral squamous cell carcinoma removed surgically were sent for pathological examination. Representative images showed the detection of H&E and immunohistochemistry (Ki67 and CK5). Scale bar, 20 μm

Fig. 2

Analysis of metabolites in cancer and paracancerous tissues of OSCC patients. (A) The metabolites in cancer and paracancerous tissues of 4 OSCC patients (#21-3801, #21-5479, #21-6812 and #21-7123) were analyzed by ¹H-NMR. (B) The signal in the range of 2.9–3.2 ppm was selected to export the original data. The layout was toggled as merged (a) or multiple displayed (b). The arrows show the sites of metabolite with density variation in cancer and paracancerous tissues

Fig. 3

Transcriptional profiling of OSCC patient derived cells. (A) The scheme for establishing OSCC patient derived CA-13 and PA-35 cells (CA, cancer; PA, paracancer). (B) GAB quantities of CA-13 and PA-35 cells were determined by Colorimetric Assay Kit. C and D. The cluster analysis was developed on the differential genes between CA-13 and PA-35 cells. The thresholds used were FDR < 0.05 and the absolute value of log2 (Fold Change) > 1, respectively. The results were showed as heat map (B) or volcano map (C)

Fig. 4

Involvement of GLUL into GAB metabolism

Fig. 5

The diagram of GAB metabolism. Normally, the glutamate is catalyzed by GAD1 to GAB and then metabolized into TCA cycle in mitochondria. In OSCC cells, the glutamate is transpoted out of mitochondria and metabolized to GAB according the alternative pathway