Abstract
Background
Leptin is important in physiological and pathological functions in various cancers, however, the significance and mechanisms of leptin in nasopharyngeal carcinoma remain ambiguous.
Methods
Leptin expression was analyzed by QPCR, immunohistochemistry, Western blotting, and TCGA database. The impact of gain- or loss-of-function of leptin were determined by MTT, colony formation, wound healing, and Transwell assays in NPC cells, and by a xenograft tumor model. Leptin-modulated glucose consumption and lactate production were assessed by ELISA. Furthermore, leptin-regulated signaling pathways were examined by QPCR and Western blotting assays. The immunoprecipitation assay was conducted to determine interaction between leptin and EGFR. In addition, miR-874-3p-regulated leptin expression was evaluated using bioinformatics, QPCR, luciferase assay, AGO2-RIP assay, and Western blotting.
Results
In this study, we found that leptin was highly expressed in the sera and tumor tissues of patients with NPC, and elevated leptin expression was associated with advanced clinical features and poor prognosis. Functional assays demonstrated that leptin remarkably promoted NPC cell growth, motility, and glycolysis in vitro and in vivo. Mechanistically, leptin associated with EGFR, resulting in enhanced cell growth through the regulation of cell-cycle related markers, glycolysis-related genes, and EGFR/AKT/c-Myc signaling. Moreover, leptin potentiated the invasive capacity of NPC cells by promoting EMT. We further explored that miR-874-3p influenced leptin-mediated NPC progression. Overexpression of miR-874-3p prevented cell growth, motility, glucose consumption, and lactate production in NPC cells, whereas miR-874-3p inhibition had the opposite effects. AGO-RIP assays confirmed that Argonaute 2 (AGO2), a protein associated with miR-874-3p, regulated leptin expression in NPC cells. The rescue assays indicated that inhibition of leptin suppressed the effects of miR-874-3p inhibitor. In clinical specimens, miR-874-3p was negatively correlated with leptin.
Conclusions
Leptin may serve as a novel prognostic factor and potential therapeutic target for patients with NPC. In addition, a newly discovered regulatory axis of leptin/EGFR/AKT/c-Myc can provide a novel therapeutic strategy for NPC.
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Background
Nasopharyngeal carcinoma (NPC) is a malignant tumor involving the head and neck areas. Unlike other head and neck cancers, NPC is characterized by increased invasiveness and metastasis [1]. Survival outcomes are significantly better in patients with early-stage NPC than in those with late-stage NPC [1, 2]. Although NPC is rare in most parts of the world, it has a high regional incidence in Asia, with that in Southern China and Southeast Asia ranking among the highest in the world [2]. Generally, there are three main types of NPC according to the World Health Organization (WHO), which include keratinizing squamous cell carcinoma, non-keratinizing carcinoma and undifferentiated carcinoma [3]. Epstein-Barr virus (EBV) has been shown to be a risk factor for NPC, especially for non-keratinizing and undifferentiated carcinoma [3,4,5]. The earliest evidence of NPC associated with EBV was identified in 1973 [6]. Although EBV is detected in most patients with NPC, cases of EBV-negative NPC have also been reported [4, 7]. Other lifestyle-associated risk factors and/or host genetic variants may interact with EBV to play roles in the carcinogenesis of NPC [5, 8, 9]. Obesity can occur due to unhealthy lifestyle habits and has been associated with an increased risk for various cancers, including breast, esophageal, pancreatic, and colorectal cancers [10,11,12,13]. Interestingly, several epidemiological studies have shown that patients with NPC have higher BMI, implying that overweight or obese people are tended to have a greater risk of NPC [14, In summary, we conducted a study to understand the effects of leptin on NPC by combining a small-scale clinical analysis, in vitro, in vivo and in silico methods. Our small-scale clinical findings indicated that elevated intra-tumoral leptin was associated with more advanced tumors and poor prognosis of NPC. Our data also suggested that leptin was involved in alteration of diverse molecules related to pathways that regulate EMT and cell proliferation. These results supported the idea that leptin facilitates nasopharyngeal cell survival, proliferation and invasion by influencing the expression levels of EMT-, cell proliferation- and glycolysis-related proteins, which are key to tumor invasiveness and metastasis and the shortened survival of patients with cancer. Similarly, these results may also explain the findings of our clinical research, as well as our mouse model of tumor growth, considering the cancer-promoting effects of leptin in our assays. Furthermore, we utilized in silico miRNA-target prediction to identify miR-874-3p as a novel regulator of leptin and rigorously validated this prediction experimentally. Our data suggests that miR-874-3p targets leptin mRNA to reduce cell survival and proliferation in NPC cells. Overall, this study provides insights into how leptin is involved in the carcinogenesis of NPC with a focus on potential pathological effects of leptin and therapeutic targeting of leptin expression, which may potentially be applied in future clinical settings.Conclusions
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Abbreviations
- NPC:
-
Nasopharyngeal carcinoma
- EBV:
-
Epstein-Barr virus
- AJCC:
-
American Joint Committee on Cancer
- EMT:
-
Epithelial-mesenchymal transition
- QPCR:
-
Quantitative polymerase chain reaction
- TCGA:
-
The Cancer Genome Atlas
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Acknowledgements
We thank Chang Gung Medical Foundation Kaohsiung Chang Gung Memorial Hospital Tissue Bank Core Lab (CLRPG8F1702) for excellent technical support.
Funding
This study was supported by the grants CMRPG8H1011-3 from Chang Gung Memorial Hospital, Kaohsiung, Taiwan, by the grant RD11005 from Chung Shan Medical University, Taichung, Taiwan.
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Contributions
SDL, CCH, and CHC: Experimental design and manuscript draft. SDL, CFH, TJC and SHL: Clinical sample collection, interpretation and analysis. HTT and YLH: Experimental manipulation and analyzed the results related to these assays. CCH: Writing-editing. All authors read and approved the manuscript.
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This study was approved by the Medical Ethics Committee and animal Use and Management Committee of Chang Gung Memorial Hospital. IRB approval (No. 201700700B0) was provided by Chang Gung Memorial Hospital. IACUC approval (No. 2017050402) was provided by the Institutional Animal Care and Use Committee of Kaohsiung Chang Gung Memorial Hospital.
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Written informed consent was obtained from all patients participating in this study.
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Supplementary Information
Additional file 1:
Supplementary Figure S1. Leptin expression profiles are investigated in publicly database and NPC cell lines. (a to d) The mRNA expression levels of leptin in Oncomine HNC databases were determined. (e) Analysis of leptin mRNA and protein levels in NPC cell lines.
Additional file 2:
Supplementary Figure S2. Leptin promotes cell growth in NPC. (a) The mRNA and protein expression levels of leptin were investigated in gain-of-function of leptin in TW02 cells by QPCR and Western blotting. (b) MTS assays was performed to assess the cell proliferation of TW02 cells after transfection of overexpression plasmid. (c) The foci numbers of TW02-leptin transfectants were assessed. The representative images and fold change of foci formation were shown. (d and e) The mRNA and protein expression levels of leptin were investigated in loss-of-function of leptin in TW02 and TW06 cells by QPCR and Western blotting. (f and g). The effect of siLeptin-TW02 on cell proliferation was determined by MTS and colony formation assays. *P<0.05, **P<0.01, ***P<0.001.
Additional file 3:
Supplementary Figure S3. Leptin promotes NPC cells motility by inducing EMT. (a and c) Wound healing assays demonstrated that the overexpression or knockdown of leptin modulated the migratory ability of TW02 cells. (b and d) Transwell assays were performed to examine the change of invasive ability of TW02 cells with leptin overexpression or knockdown. (e) Expressions of EMT markers were detected by Western blotting in TW02 cells with overexpression or knockdown of leptin. *P<0.05, **P<0.01, ***P<0.001.
Additional file 4:
Supplementary Figure S4. Leptin regulates the expressions of cell cycle-related molecules and EGFR/MAPK/c-Myc pathway in TW02 cells. (a) Western blot indicated the expressions of cyclin D1, cyclin E, CDK4, p21 and p27 in leptin-overexpression and leptin-depleted TW02 cells. (b) Western blot analysis was performed to detect the protein levels of p-EGFR, EGFR, p-ERK1/2, ERK1/2, p-AKT, AKT, p-mTOR, mTOR and c-Myc in leptin-overexpression and leptin-depleted TW02 cells.
Additional file 5:
Supplementary Figure S5. miR-874-3p suppresses the malignant properties of TW02 cells. (a and b) MTS assays and colony formation assays were performed to assess the cell proliferation of TW02 cells after transfecting miR-874-3p mimics or miR-874-3p inhibitor and their corresponding negative control. The representative images and fold change of foci formation were shown. (c and d) The migratory and invasive abilities of TW02 cells transfected with miR-874-3p mimics or miR-874-3p inhibitor and their corresponding negative control were assessed by wound healing and Transwell assays. *P<0.05, **P<0.01, ***P<0.001.
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Luo, SD., Tsai, HT., Hwang, CF. et al. Aberrant miR-874-3p/leptin/EGFR/c-Myc signaling contributes to nasopharyngeal carcinoma pathogenesis. J Exp Clin Cancer Res 41, 215 (2022). https://doi.org/10.1186/s13046-022-02415-0
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DOI: https://doi.org/10.1186/s13046-022-02415-0