Background
Oral squamous cell carcinoma is the most frequent malignant tumor of the head and neck with potential for lymph node metastasis, deep invasion and high recurrence rate, which shows poor prognosis and high mortality [
1,
2]. Despite the constant progression of therapeutic approaches, the 5-year survival rate has stagnated at about 50% [
3]. Hence, illustration of the molecular mechanisms related to OSCC is primarily important to development of original theragnostic plans.
Chronic stress is an ineluctable part of life, and people have always experienced undue stress caused by global issues [
4]. Occupational stress and abnormal adversities are the main sources of chronic stress. Chronic stress underlying negative effects include anxiety, insomnia and depression [
5], and also increasing the risk of mental disease and carcinomatosis [
6]. Chronic stress can alter immune capabilities and regulate the advancement of certain neoplasms by stimulating the hypothalamic–pituitary–adrenal (HPA) axis and freeing neurotransmitter which acts on adrenergic receptors [
7,
8]. Study has revealed that patients with OSCC have an upper incidence of depression and anxiety than normal, and that psychiatric disorders are strongly associated with survival and treatment outcomes of patients with OSCC [
9].
Neurohormonal products originated in chronic stress can affect the behavior of oral cancer cells [
10]. Studies have reported that neurohormone participates in the processes of invasion and advancement of different types of cancer via beta-adrenergic signaling [
11‐
13]. In addition, the increased of norepinephrine (NE) level in microenvironment is predictive for OSCC occurrence [
14]. Whereas, whether chronic stress influences the development of oral squamous cell carcinoma through stress response hormone, the potential mechanism about it remains unclear.
Aldehyde dehydrogenase 3A1 (ALDH3A1), an important member of the ALDHs superfamily, regulates cellular function by affecting the metabolism [
15]. ALDH3A1 is highly expressed in oral epithelium, nasal epithelium and small salivary gland [
16,
17]. Recent research has showed that the expression of ALDH3A1 was upregulated in several cancer types and demonstrated clear association between ALDH3A1 and cancer progression [
18]. However, another study reported that the expression of ALDH3A1 was decreased in OSCC tissue and the low expression of ALDH3A1 is connected with inferior prognosis of patients [
19]. Although ALDH3A1 is known to regulate cell function and cancer prognosis, the effects and underlying mechanisms of chronic stress states remain unclear. In this study, we examined the influence of CRS on OSCC progression, and systematically investigated the role of ALDH3A1 in OSCC cells proliferation and invasion.
Methods
Cell culture
The human OSCC cell lines (HN6 and HSC4) were provided by ATCC (Manassas, USA). All cells were cultured in DMEM of Gibco (Carlsbad, USA) with 10% FBS provided by Invitrogen (Carlsbad, USA).
Tumor xenografts and CRS model
All experiments of animal were approved and supervised by the Ethics Committee of the College of Stomatology, Chongqing Medical University (Approval No. 2021063), and all methods were performed in accordance with the relevant guidelines and regulations. Four-week-old athymic nude mice were acquired by Cavensbiogle (Suzhou, China). All mice were housed under SPF condition. All mice were randomly departed into control (Con) and CRS group, and each group contained six mice. For CRS group, mice were blocked in 50 ml centrifuge tube for 2 h a day for 28 days. After one day of CRS, HN6 cells (2 × 10
6) were injected subcutaneously to the left flanks skin [
20]. The tumor size was measured once a week with vernier calipers, and tumor volumes were computed according to the coming formula: V = 1/2 × a × b
2 (V = volume, a = long diameter, and b = short diameter). After 5 weeks of initial CRS, all mice in the CRS and Con groups were euthanized with carbon dioxide. We collected serum and tumors for further analysis, and recorded the weights of excised tumor tissues. All swatches were analyzed by transcriptomics, metabolomics Enzyme linked immunosorbent assay (ELISA).
Behavioral assessment
Behavioral tests on mice included OFT, TST and FST. Then, video tracking equipment of SMART (Barcelona, Spain) was used for quantitative analysis [
21]. The tracking software system recorded the motion trajectory and counted the travel distance and rest time, and the EthoVision XT 13.0 software was used for analysis. The specified procedures are described in our previous study [
20].
Measurement of NE
The plasma was collected for testing [
20]. Detection of NE level by ELISA kit (JL13969-96 T, Shanghai, China). The measurement was conducted in a single-blinded manner.
Transcriptomics
Total RNA was abstracted from tumor samples with Trizol reagent of Invitrogen (Carlsbad, USA). And RNA integrity was determined by using Agilent 2100 Bioanalyzer of Agilent (CA, USA). Then, RNA samples were sent to platform of Major (Shanghai, China) for NGS analysis. The datasets are available in the cloud.majorbio.com,
https://cloud.majorbio.com/page/v2/project/task.
Tumor tissues were prepared for metabolomics analysis by liquid chromatography–mass spectrometry (LC–MS). The metabolites were extracted and analyzed by Major (Shanghai, China). The specified procedures were afforded in the Supplementary. The datasets generated during the current study are available in the cloud.majorbio.com,
https://cloud.majorbio.com/page/v2/project/task.
The premier metabonomic datum were imported into the XCMS program for analysis, and data missing more than 1/2 of metabolites were excluded. The strategy of PCA analysis in this study was to screen the differential metabolites between groups based on Student’s t-test. Metabolites with a value of
p < 0.05 and VIP (variable importance value, VIP) > 2 among the top 20 expression levels were selected as significant metabolites. R software was used in combination with KEGG and MetaboAnalyst 4.0 (
http://www.MetaboAnalyst.ca/) for metabolic pathway analysis.
RT-PCR
Total RNA was extracted from tumor tissues and treated cells by using RNA extraction kit of Beyotime (Shanghai, China). cDNA was synthesized from total RNA by using Prime Script RT reagent kit of MCE (Shanghai, China). The Real-time PCR was performed by using ABI 7300 system of Biosystems (CA, USA) via SYBR Premium ExTaq kit of MCE (Shanghai, China). Quantity of gene was planned using method 2
−ΔΔCt and normalized to β-actin. Detailed procedures and primer sequences for RT-PCR were listed in supplementary methods and supplementary Table
1.
Western blot
The total protein was extracted from the treated OSCC cells by using RIPA buffer with protease inhibitor (Roche, Basel, Switzerland). The blots were cut prior to hybridization with antibodies during blotting. The detailed protocols, primary antibodies, and full-length original blots are provided in the supplementary.
Cell proliferation
The ability of cell proliferation was explored by using Cell Counting Kit-8 (Tokyo, Japan). Specific procedures were provided in supplementary.
Flow cytometry
The apoptosis and cycle of treated OSCC cells were treated by flow cytometry (BD FACSCanto). Detailed programs were listed in the supplementary.
Cell invasion and migration
The invasive ability of cells was surveyed by transwell assay, and the migration talent of cells was examined by scratch wound healing test. Specified procedures were provided in the supplementary.
Immunofluorescence
Immunofluorescence (IF) was performed on treated cells, as previously reported [
20]. Detailed programs were listed in the supplementary. methods and supplementary Table
1.
Lentiviral transfection
Human ALDH3A1 lentiviral and negative control lentivirus were produced by QingKe (Shanghai, China). ALDH3A1 overexpressed lentiviral was transfected into HN6 and HSC4 cells pursuant to the manufacturer's protocol to construct stable cell lines.
Immunohistochemistry
The transplanted tumor tissues were analyzed by immunohistochemistry (IHC). Detailed programs were supplied in the supplementary.
Adenosine triphosphate (ATP) activity assay
Lv-ALDH3A1 and Lv-Con HN6 cells ATP contents were detected using ATP bioluminescence assay kit of Beyotime (Shanghai, China), pursuant to the manufacturer’s instruction.
Oxygen consumption rate (OCR) Assay
Oxygen consumption rate of Lv-ALDH3A1 and Lv-Con HN6 cells were measured with the Seahorse XFe96 Analyzer (Agilent Technologies, Inc) and Extracellular Oxygen Consumption Assay Kit of Abcam (no. ab197243). The cell inoculation density was 5 × 104 per well. Metabolic inhibitors in the assay included oligomycin, rotenone, and antimycin A. After measurement, data was analyzed with Wave software (Agilent Technologies, Inc).
Statistical analysis
Data were expressed as the mean ± SD. All statistical analyses were performed with GraphPad 8.0 and SPSS25.0. The statistical significance of differences between two groups was analyzed using two-tailed Student’s t-tests. The differences were considered statistically significant at p < 0.05.
Discussion
In this article, we identified the potential pathway and molecular mechanism of CRS regulating the progress of OSCC. Here, we have revealed that regulating ALDH3A1 is the key pathway for CRS to promote OSCC. Additionally, we demonstrated that ALDH3A1 plays a crucial role in reprogramming mitochondrial metabolism and overexpression of ALDH3A1 inhibited the growth and ATP accumulation of OSCC cells.
Since a groundbreaking study focused on the importance of psychology and behavior in the development of the disease [
26], many subsequent studies have focused on the influence of chronic stress on tumor progression [
27,
28]. Previous studies have shown a positive association between chronic stress and carcinoma progression [
13,
29]. In addition, researches have shown that chronic stress can facilitate the increase of catecholamines by activating HPA [
30]. Our study found that CRS promotes OSCC growth, invasion, and metastasis, and significantly increases NE level.
Recently, the effect of nervous system adjustment in the occurrence and progression of cancer has been widely researched and received [
31‐
33]. Sympathetic and parasympathetic excitation stimulates the release of neurotransmitters such as acetylcholine, epinephrine, and substance P. These neurotransmitter stimuli cause downstream signal transduction by activating specific receptors on the cell surface. There was new proof that chronic stress hormone is a risk factor for cancer development and is considered as a marker of malignant progression of tumors [
34,
35]. High levels of stress hormones contributed to carcinogenesis by inducing accumulation of DNA damage, increasing p53 degradation, or other related pathways [
30]. Previous study reported that NE induces the advancement and diversion of gastric cancer via ADRB2 signaling pathway [
7]. Consistent with previous studies [
36,
37], we found that HN6 and HSC4 cultured with NE showed increased proliferation and migration ability compared with the Con group. Distant metastasis is a typical malignant behavior of tumors, and we found that stress response hormone can promote the EMT of OSCC cells. In conclusion, this study revealed that CRS facilitates the growth, motion and invasion of OSCC cells through stress response hormones.
We then further found that CRS and stress response hormone downregulated the expression of ALDH3A1. ALDH3A1 has various biological effects, including maintenance of hematopoietic stem cells, regulation of cell radiation, proliferation and so on [
38,
39]. Recent years, a large number of studies have found that ALDH3A1 can be considered as a marker for predicting tumor prognosis and associated with poor clinical outcomes of various tumors [
40,
41]. In oral mucosa, ALDH3A1 was detected to promote the anti-oxidation and anti-injury ability of epithelial cells, inhibit inflammatory reaction and maintain DNA integrity [
15,
42]. Thus, we explored the effect of ALDH3A1 in OSCC development. In this section, we acquired that overexpression of ALDH3A1 can reverse the effects of stress response hormone on migration and invasion of OSCC cells, this data in accordance with those reported in this research [
19], which demonstrated that overexpression of ALDH3A1 in OSCC cells significantly inhibits cell proliferation and invasion. However, Wu et al. [
18] demonstrated that highly-expressed ALDH3A1 is correlated with gastric cancer malignant progression. Oral squamous cell carcinoma originates from genetic mutation in the upper layer of oral mucosa, and the high expression of ALDH3A1 in normal oral mucosal epithelium may be responsible for these adverse outcomes. Meaningfully, our results suggest that under chronic stress overexpression of ALDH3A1 can restrain the tumorigenic potential of OSCC cells.
The ALDH3A subfamily contains ALDH3A1 and ALDH3A2 enzymes, which are relevant to the oxidation of fats and aromatic aldehydes, as well as the generation of NADPH. Study has shown that ALDH3A1 may be involved in regulating REDOX dependent signal transduction pathways during tumor progression [
43]. Terzuoli et al. reported that ALDH3A1 can affect the stemness and EMT of melanoma and lung tumors by regulating the metabolism of tumor cells [
23]. Mitochondrial metabolism plays a primary role in conditioning whether immune response promotes or suppresses cancer [
44]. Another study found that TLR4 activation reprograms mitochondrial metabolism to meet the promptly increasing energy needs of cells in an inflammatory condition [
45]. Tlr4 induced inflammatory cytokine production and mitochondrial reprogramming require the involvement of STAT3 [
46]. ALDH3A1 inhibits TLR4 activation, leading to the phosphorylation of STAT3, which alters mitochondrial metabolism [
15]. Our study found that CRS induces metabolic changes in OSCC through mitochondrial reprogramming. Furthermore, mitochondrial metabolism was inhibited in OSCC cells by overexpressing ALDH3A1 under chronic stress. These outcomes suggest that regulation of mitochondrial metabolism is one of the underlying mechanisms by which ALDH3A1 inhibits the carcinogenic potential of OSCC cells.
Conclusion
In summary, we demonstrate that inhibition of ALDH3A1 expression by stress response hormones is a key pathway by which CRS enhances the oncogenic potential of OSCC cells. Furthermore, the regulating of mitochondrial metabolism may be involved in this process. Our study affords a new insight into the molecular mechanism by which CRS promotes oral squamous cell carcinoma development. However, the limitation of our study is the establishment of xenograft models in nude mice, so the immune microenvironment is not involved. Therefore, our next study may focus on the role of CRS in OSCC immunomodulation.
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