TRAP1 suppresses oral squamous cell carcinoma progression by reducing oxidative phosphorylation metabolism of Cancer-associated fibroblasts

Abstract

Background Glucose metabolism in cancer associated fibroblasts (CAFs) within the tumor microenvironment is a material and energy source for tumorigenesis and tumor development. However, the characteristics and important regulatory mechanisms of glucose metabolism in fibroblasts associated with oral squamous cell carcinoma (OSCC) are still unknown. Methods We successfully isolated, cultured, purified and identified CAFs and normal fibroblasts (NFs). Cell culture, immunohistochemistry (IHC) and CCK8, flow cytometry, Seahorse XF Analyzer, MitoTracker assay, western blotting (WB), transmission electron microscope, Quantitative real-time PCR (qPCR), immunofluorescence (IF), and Label-free quantitative proteomics assay, animal xenograft model studies and statistical analysis were applied in this study. Results We demonstrated that the proliferation activity of CAFs was significantly enhanced as compared to NFs, while the apoptosis rate was significantly decreased. CAFs in OSCC preferentially use oxidative phosphorylation (OXPHOS) rather than glycolysis. Moreover, CAFs showed stronger maximal respiration, a larger substantial mitochondrial spare respiratory capacity (SRC) and higher adenosine triphosphate (ATP) production capacity than NFs. The results of mitotracker green fluorescence staining showed that compared with NFs, CAFs exhibited stronger green fluorescence. The results of WB showed the expression level of Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) obviously increased in CAFs compared to NFs. These results confirmed that CAFs have greater mitochondrial activity and function than NFs. Furthermore, Label-free quantitative proteomics assays showed that both ATP synthase subunit O (ATP5O) and tumor necrosis factor receptor-associated protein 1 (TRAP1) are important differentially expressed proteins in the mitochondria of CAFs/NFs. Overexpression of TRAP1 in CAFs increased basal oxygen consumption rate (OCR), maximal respiration, ATP production and SRC. In vivo, overexpression TRAP1 expression in CAFs suppress tumor growth. Conclusion Taken together, the results indicated that TRAP1 is an important regulatory molecule of CAFs glucose metabolism and promotes OSCC progression by regulating the OXPHOS of CAFs.

Fibroblasts as Turned Agents in Cancer Progression

“…Most investigations with normal fibroblasts are conducted with skin-derived fibroblasts and isolation and characterization are delineated in Dvorankova et al (2019) [ 72 ]. Slightly modified techniques can be applied for the isolation of normal fibroblasts from other normal excised tissue [ 61 , 73 ]. Various biopsy techniques can be used, but the epithelium and adipose tissue must be trimmed away [ 61 , 72 , 73 ].…”

“…Slightly modified techniques can be applied for the isolation of normal fibroblasts from other normal excised tissue [ 61 , 73 ]. Various biopsy techniques can be used, but the epithelium and adipose tissue must be trimmed away [ 61 , 72 , 73 ]. For example, to obtain submucosal gastrointestinal tissue, the gastrointestinal tissue should be dissected away from the muscular layer on the luminal side to generate the lamina propria and the mucosal layer, followed by scrubbing away of the lamina propria to leave submucosal tissue [ 61 ].…”

“…The trypsin solution can be subsequently centrifuged to obtain a tissue pellet. The tissue is then minced into 1–2 cubic mm pieces [ 61 , 72 , 73 ] and cultured at 37 °C with 5% CO 2 in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin, and 250 ng/mL amphotericin B for a short time if heavy yeast contamination is of concern, such as in tissue from oral cavity tumors [ 72 ], and changed every 2–3 days [ 73 ].…”

“…To characterize cells as fibroblasts, they are fixed on coverslips with 4% paraformaldehyde for 5 min and stained with primary antibodies to vimentin, αSMA or phosphate buffer, and confirmed for lack of staining for cytokeratin epithelial marker, leukocyte, and macrophage markers CD45 and CD68, melanocyte markers melan A and HMB-45, and endothelial marker CD34, as controls [ 72 , 73 ]. They are then stained with fluorescence-labeled secondary antibodies and imaged [ 73 ]. Other methods and other control antibodies can be used, as appropriate, for different tissues.…”

Correction to: TRAP1 suppresses oral squamous cell carcinoma progression by reducing oxidative phosphorylation metabolism of Cancer-associated fibroblasts

“…Following publication of the original article [ 1 ], the authors noticed that the funding statement was missing. The funding statement is supplied below:…”