Autocrine INSL 5 promotes tumor progression and glycolysis via activation of STAT 5 signaling
2020; Springer Nature; Volume: 12; Issue: 9 Linguagem: Inglês
10.15252/emmm.202012050
ISSN1757-4684
AutoresShibing Li, Yanyan Liu, Yuan Li, Mingfang Ji, Ao Zhang, Huiyu Li, Lin‐Quan Tang, Shuogui Fang, Hua Zhang, Shan Xing, Manzhi Li, Qian Zhong, Shaojun Lin, W Liu, Peng Huang, Yi‐Xin Zeng, Yuming Zheng, Zhi‐Qiang Ling, Jianhua Sui, Mu‐Sheng Zeng,
Tópico(s)RNA modifications and cancer
ResumoArticle12 July 2020Open Access Source DataTransparent process Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling Shi-Bing Li Shi-Bing Li State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Yan-Yan Liu Yan-Yan Liu Department of Nephrology, Division of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Li Yuan Li Yuan State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Ming-Fang Ji Ming-Fang Ji Cancer Research Institute of Zhongshan City, Zhongshan, China Search for more papers by this author Ao Zhang Ao Zhang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Hui-Yu Li Hui-Yu Li National Institute of Biological Sciences, Beijing, China Search for more papers by this author Lin-Quan Tang Lin-Quan Tang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Shuo-Gui Fang Shuo-Gui Fang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Hua Zhang Hua Zhang School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Shan Xing Shan Xing State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Man-Zhi Li Man-Zhi Li State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Qian Zhong Qian Zhong orcid.org/0000-0003-1071-6996 State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Shao-Jun Lin Shao-Jun Lin Department of Radiation Oncology, Fujian Provincial Cancer Hospital, Fuzhou, China Search for more papers by this author Wan-Li Liu Wan-Li Liu State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Peng Huang Peng Huang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Yi-Xin Zeng Yi-Xin Zeng State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Yu-Ming Zheng Yu-Ming Zheng Department of Clinical Laboratory, Wuzhou Red Cross Hospital, Wuzhou, China Search for more papers by this author Zhi-Qiang Ling Zhi-Qiang Ling Zhejiang Cancer Hospital, Hangzhou, China Search for more papers by this author Jian-Hua Sui Jian-Hua Sui National Institute of Biological Sciences, Beijing, China Search for more papers by this author Mu-Sheng Zeng Corresponding Author Mu-Sheng Zeng [email protected] orcid.org/0000-0003-3509-5591 State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Shi-Bing Li Shi-Bing Li State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Yan-Yan Liu Yan-Yan Liu Department of Nephrology, Division of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Li Yuan Li Yuan State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Ming-Fang Ji Ming-Fang Ji Cancer Research Institute of Zhongshan City, Zhongshan, China Search for more papers by this author Ao Zhang Ao Zhang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Hui-Yu Li Hui-Yu Li National Institute of Biological Sciences, Beijing, China Search for more papers by this author Lin-Quan Tang Lin-Quan Tang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Shuo-Gui Fang Shuo-Gui Fang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Hua Zhang Hua Zhang School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Shan Xing Shan Xing State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Man-Zhi Li Man-Zhi Li State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Qian Zhong Qian Zhong orcid.org/0000-0003-1071-6996 State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Shao-Jun Lin Shao-Jun Lin Department of Radiation Oncology, Fujian Provincial Cancer Hospital, Fuzhou, China Search for more papers by this author Wan-Li Liu Wan-Li Liu State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Peng Huang Peng Huang State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Yi-Xin Zeng Yi-Xin Zeng State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Yu-Ming Zheng Yu-Ming Zheng Department of Clinical Laboratory, Wuzhou Red Cross Hospital, Wuzhou, China Search for more papers by this author Zhi-Qiang Ling Zhi-Qiang Ling Zhejiang Cancer Hospital, Hangzhou, China Search for more papers by this author Jian-Hua Sui Jian-Hua Sui National Institute of Biological Sciences, Beijing, China Search for more papers by this author Mu-Sheng Zeng Corresponding Author Mu-Sheng Zeng [email protected] orcid.org/0000-0003-3509-5591 State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Search for more papers by this author Author Information Shi-Bing Li1,‡, Yan-Yan Liu2,‡, Li Yuan1,‡, Ming-Fang Ji3,‡, Ao Zhang1, Hui-Yu Li4, Lin-Quan Tang1,5, Shuo-Gui Fang1,6, Hua Zhang7, Shan Xing1, Man-Zhi Li1, Qian Zhong1, Shao-Jun Lin8, Wan-Li Liu1, Peng Huang1, Yi-Xin Zeng1, Yu-Ming Zheng9,‡, Zhi-Qiang Ling10,‡, Jian-Hua Sui4,‡ and Mu-Sheng Zeng *,1,‡ 1State Key Laboratory of Oncology in South China, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China 2Department of Nephrology, Division of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 3Cancer Research Institute of Zhongshan City, Zhongshan, China 4National Institute of Biological Sciences, Beijing, China 5Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, China 6Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou, China 7School of Medicine, Sun Yat-sen University, Guangzhou, China 8Department of Radiation Oncology, Fujian Provincial Cancer Hospital, Fuzhou, China 9Department of Clinical Laboratory, Wuzhou Red Cross Hospital, Wuzhou, China 10Zhejiang Cancer Hospital, Hangzhou, China ‡These authors contributed equally to this work ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +86 208734 3191; E-mail: [email protected] or [email protected] EMBO Mol Med (2020)12:e12050https://doi.org/10.15252/emmm.202012050 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Metabolic reprogramming plays important roles in development and progression of nasopharyngeal carcinoma (NPC), but the underlying mechanism has not been completely defined. In this work, we found INSL5 was elevated in NPC tumor tissue and the plasma of NPC patients. Plasma INSL5 could serve as a novel diagnostic marker for NPC, especially for serum VCA-IgA-negative patients. Moreover, higher plasma INSL5 level was associated with poor disease outcome. Functionally, INSL5 overexpression increased, whereas knockdown of its receptor GPCR142 or inhibition of INSL5 reduced cell proliferation, colony formation, and cell invasion in vitro and tumorigenicity in vivo. Mechanistically, INSL5 enhanced phosphorylation and nuclear translocation of STAT5 and promoted glycolytic gene expression, leading to induced glycolysis in cancer cells. Pharmaceutical inhibition of glycolysis by 2-DG or blockade of INSL5 by a neutralizing antibody reversed INSL5-induced proliferation and invasion, indicating that INSL5 can be a potential therapeutic target in NPC. In conclusion, INSL5 enhances NPC progression by regulating cancer cell metabolic reprogramming and is a potential diagnostic and prognostic marker as well as a therapeutic target for NPC. Synopsis This study reveals that INSL5 promotes tumor progression by regulating cancer cell metabolic reprogramming. INSL5 is a potential diagnostic and prognostic marker as well as a therapeutic target for nasopharyngeal carcinoma (NPC). INSL5 expression levels are elevated in NPC tumor tissue and in the plasma of NPC patients. INSL5 plasma levels are associated with NPC patient outcome. INSL5 promotes tumor progression through an autocrine mechanism involving binding to its receptor GPCR142. INSL5 activates JAK1-STAT5 signal pathway and promotes glycolytic gene expression, which in turn induces metabolic reprogramming in cancer cells. INSL5-GPCR142 axis can be a potential therapeutic target for NPC. The paper explained Problem Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor with a unique geographic distribution, as it is mainly found in Southern China and South-East Asia. Metabolic reprogramming plays important roles in development and progression of NPC, but the potential key molecular targets involved in modulating NPC metabolism remain to be identified and the underlying mechanism has not been completely defined. Additionally, the specific functions of INSL5 in tumors remain to be elucidated. Results Our study characterized INSL5 as a valuable biomarker for NPC diagnosis and prognosis. EBV infection induces increased expression of INSL5 in NPC. INSL5 can physically bind to the receptor GPCR142 to activate JAK1 and ERK1/2 to enhance STAT5 phosphorylation and transcriptional activity in NPC cells. INSL5 exerts its oncogenic function by reprogramming glycolysis, which is promoted by activation of the STAT5 signaling pathway. The INSL5-GPCR142 axis can be a potential therapeutic target for NPC treatment. The anti-INSL5 neutralizing antibody, anti-GPCR142 neutralizing antibody, and glycolysis inhibitor could be attractive therapeutic approaches for INSL5-overexpressing NPC. Impact These results shed light on the mechanism of INSL5 in mediating glucose metabolism reprogramming and tumor promotion in NPC and highlight a novel biomarker for NPC diagnosis, prognosis, and targeted therapy. Introduction Cancer cells require abundant metabolic intermediates and energy to fuel cell growth and division (Cairns et al, 2011). To meet these elevated requirements, cancer cells often take up large amounts of glucose and generally limit their energy metabolism to glycolysis for ATP generation despite the presence of oxygen, which is termed aerobic glycolysis (also called the Warburg effect; Hsu & Sabatini, 2008; Vander Heiden et al, 2009; Schulze & Harris, 2012). This metabolic shift toward aerobic glycolysis enables cancer cells to convert glucose more efficiently into macromolecules, which are needed for rapid cell growth. On the one hand, cancer cells enhance glycolytic flux by upregulating the expression of key glycolytic genes, including the glucose transporters Glut1 and Glut3 (Ha et al, 2012; Kuang et al, 2017), hexokinase (HK2) (Patra et al, 2013; Yang et al, 2018), lactate dehydrogenase (LDH; Faubert et al, 2017), and phosphofructokinase-1 (PFK1; Krall & Christofk, 2017; Peeters et al, 2017). On the other hand, cancer cells can increase the enzymatic activity of some key enzymes to promote the production of metabolic intermediates(Shaul et al, 2014; Haas et al, 2016). Oncogenic viruses have been reported to alter the cellular metabolic pathway in cancer cells (Mesri et al, 2014; Levy et al, 2016; Zhu et al, 2016). Therefore, identification of the metabolic linker between virus infection and cancer cells could present new opportunities to target oncogenic virus-related cancer. Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor with a unique geographic distribution, as it is mainly found in Southern China and South-East Asia (Chan et al, 2002). NPC is etiologically associated with Epstein–Barr virus (EBV) infection (Raab-Traub, 2002; Young & Rickinson, 2004; Tsao et al, 2015). In NPC, EBV mainly expresses latent genes, including latent membrane protein 1 (LMP1), Epstein–Barr nuclear antigen 1 (EBNA1), latent membrane protein 2A (LMP2A), and EBV-encoded small non-polyadenylated RNAs (EBERs; Pathmanathan et al, 1995; Nanbo & Takada, 2002; Arvey et al, 2012; Tsang et al, 2014). In addition to latent gene expression, EBV lytic gene expression has also been demonstrated in NPC tumors in recent studies by transcriptomic sequence analysis (Hu et al, 2016). Previous studies showed that EBV-encoded latent genes, such as LMP1, could reprogram the glucose metabolism of NPC cells, illustrating the significance of metabolic reprogramming in the development of NPC (Xiao et al, 2014; Lo et al, 2015). However, the potential major molecular targets involved in modulating NPC metabolism remain to be identified. Insulin-like peptide 5 (INSL5) is a member of the relaxin/insulin superfamily and presents a tertiary structure similar to that of other insulin family members (Conklin et al, 1999). Mature INSL5 consists of two chains (an A-chain and a B-chain) that are linked by two disulfide bonds with a third intramolecular disulfide within the A-chain (Akhter Hossain et al, 2008; Haugaard-Jonsson et al, 2009; Luo et al, 2010; Belgi et al, 2011). INSL5 functions by binding to its receptor, G protein-coupled receptor 142 (GPCR142/RXFP4). The positively charged B-chain residues (B13Arg and B23Arg) of INSL5 and the negatively charged extracellular residues (Glu100, Asp104, and Glu182) of GPCR142 are important for INSL5-GPCR142 binding and activation (Wang et al, 2014). The primary functions of INSL5 have been suggested to affect glucose metabolism and reproductive physiology in a mouse model, as Insl5−/− mice exhibit impaired male and female fertility due to a marked reduction in sperm motility and irregular length of the estrous cycle, respectively (Burnicka-Turek et al, 2012; Grosse et al, 2014; Lee et al, 2016). In mice experiencing energy deprivation, Isnl5 contributes to appetite promotion and hepatic glucose production (Grosse et al, 2014). A recent report suggested an important role for Isnl5 in the regulation of insulin secretion and β-cell homeostasis (Luo et al, 2015). Other reports identified that colonic Isnl5 expression is reduced by the gut microbiota and energy availability (Lee et al, 2016). In human diseases, INSL5 has been identified recently in colonic tissue and neuroendocrine tumors(Thanasupawat et al, 2013), but its specific functions in tumors remain to be elucidated. In this study, we identified INSL5 was upregulated after EBV infection in nasopharyngeal epithelial cells. INSL5 was overexpressed in NPC and correlated with poor prognosis of the patients. We found that INSL5 could promote glycolytic gene expression and NPC progression by activating the GPCR142/STAT5 axis. Importantly, inhibition of INSL5 function or its downstream factors by various methods reversed INSL5-induced cancer progression in vitro and in vivo, suggesting INSL5 is a potential therapeutic target for NPC. Results INSL5 is highly expressed in NPC tumors Virus infection can skew metabolism toward glycolysis over oxidative phosphorylation (OXPHOS) in a manner similar to the Warburg effect in cancer (Claus & Liebert, 2014; Zhang et al, 2017). To examine whether tumorigenic virus-induced metabolic alterations can contribute to tumor progression, we performed a gene microarray to analyze the metabolism-associated genes differentially expressed between EBV-infected and uninfected cells in a previously established high-efficiency EBV infection model. INSL5 was among the metabolism-associated genes upregulated after EBV infection (Fig EV1A). The upregulation of INSL5 by EBV was verified in two additional EBV-infected NPC cell lines (Fig EV1B). Although INSL5 has been reported to play an important role in glucose homeostasis in mice, there is little knowledge about its roles in virus infection and cancer progression. Thus, we selected INSL5 for further study. Click here to expand this figure. Figure EV1. EBV-induced INSL5 is highly expressed in NPC and associated with poor prognosis A. Heat map of upregulated metabolism-associated genes after EBV infection. B. mRNA expression of INSL5 in EBV-positive and EBV-negative NPC cell lines. C. Quantitative RT–PCR showing the expression of INSL5 mRNA in normal nasopharyngeal tissues (NPN) and tumor tissues (NPC). D. Representative immunohistochemical (IHC) staining of INSL5 in NPC paraffin samples. Scale bars represent 100 μm. E–I. Kaplan–Meier curves showing the impact of INSL5 expression on overall survival for glioma, kidney renal clear cell carcinoma, sarcoma, uterine carcinosarcoma, and uveal melanoma (GEPIA database). Analysis of TCGA data indicates that high expression of INSL5 was correlated with poor prognosis in these cancer types. J–K. The calibration curve of nomogram for predicting overall survival (OS) at 5 years (J) and predicting disease-free survival (DFS) at 5 years (K). Actual OS or DFS is plotted on the y-axis; nomogram predicted probability of OS or DFS is plotted on the x-axis. Data information: In (B), data are presented as mean ± SEM, in (C), data are presented as mean ± SD, from three different experiments, and P-values were determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P-values are specified in Appendix Table S4. Source data are available online for this figure. Download figure Download PowerPoint We then first determine INSL5 protein levels in NPC cell lines, tumor, and plasma samples of NPC patients. We found that INSL5 expression was higher in most of the NPC cell lines than in the three immortalized nasopharyngeal epithelial cells (NPECs) by Western blotting (Fig 1A). By qRT–PCR analysis, INSL5 mRNA levels were significantly increased in a cohort of 62 NPC biopsy samples than the cohort of 48 normal control samples (NPN; Fig EV1C). In consistent, we found INSL5 expression was mainly detected in cancer cells rather than in stromal cells or normal epithelial cells in 18 NPC tumor samples by immunohistochemistry (Fig EV1D). Collectively, these data indicated that INSL5 was highly expressed in NPC tumor cell lines and tumor tissues. Figure 1. INSL5 is elevated in NPC tissue and plasma and is a potential diagnostic biomarker A. Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control. B. ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n = 3; for NPC, n = 7. C. The concentration of plasma INSL5 in healthy controls (normal EBV (−)), non-tumor individuals with VCA-IgA positive (normal EBV (+)), and NPC patients, in training cohort. D. ROC of the diagnostic prediction model with plasma INSL5 level in the training cohort. E. The concentration of plasma INSL5 in healthy controls (normal EBV (−)), non-tumor individuals with VCA-IgA positive (normal EBV (+)), and NPC patients, in validation cohort 1(GZ). F. The concentration of plasma INSL5 in healthy controls (normal EBV (−)), non-tumor individuals with VCA-IgA positive (normal EBV (+)), and NPC patients, in validation cohort 2(ZS). G. Confusion table of binary results of the diagnostic prediction model in the training cohort, validation cohorts, and the whole cohort. H. ROC of the diagnostic prediction model with plasma INSL5 level in the whole cohort. I. ROC of the diagnostic prediction model with plasma INSL5 level in individuals with VCA-IgA-negative plasma in all three cohorts. J. Confusion table of binary results of the diagnostic prediction model in VCA-IgA-negative individuals in the training cohort, validation cohorts, and the whole cohort. Data information: In (B), data are presented as mean ± SD, in (C, E and F), data are presented as mean ± SEM, and P-values were determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P-values are specified in Appendix Table S4. Source data are available online for this figure. Source Data for Figure 1 [emmm202012050-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Plasma INSL5 is a diagnostic biomarker for NPC patients As INSL5 is a secreted protein, we established an ELISA kit to detect secreted INSL5 in cell culture supernatants and the plasma of NPC patients. We found that the NPC cell lines secreted higher INSL5 levels in the culture supernatants than the immortalized NPECs did (Fig 1B). Importantly, NPC patients (n = 159) presented higher plasma INSL5 concentration (median, 2.50 ng/ml, quartile, 1.47–3.52) than the VCA-IgA-positive (n = 54, median, 0.66 ng/ml; quartile, 0.31–1.26) or VCA-negative (n = 44, median, 0.49 ng/ml; quartile, 0.33–0.91) control groups (Fig 1C) in the training cohort. Compared with the VCA-IgA-negative control group, the VCA-IgA-positive control group presented a significantly elevated INSL5 level (Fig 1C). The plasma INSL5 level could have significant diagnostic potential in NPC (AUC = 0.881, 95% CI: 0.835–0.926) (Fig 1D). Additionally, similar results were confirmed in two validation cohorts, validation cohort 1 (group EBV (−): median 0.47 ng/ml, quartile 0.36–0.70; group EBV (+): median 2.23 ng/ml, quartile 2.10–2.83; group NPC: median 4.13 ng/ml, quartile 3.13–5.06; Fig 1E) and validation cohort 2 (group EBV (−): median 0.27 ng/ml, quartile 0.18–0.58; group EBV (+): median 0.55 ng/ml, quartile 0.35–0.92; group NPC: median 1.64 ng/ml, quartile 1.13–2.31) (Fig 1F). Applying the cut-off value (1.12 ng/ml) of the training cohort to the validation cohorts for NPC diagnosis yielded sensitivities of 97.1% and 76.1% and specificities of 73.4% and 87.0%, respectively (Fig 1G). Combining all the samples, we found that this cut-off value could differentiate NPC patients from normal controls with a sensitivity of 89.6% and a specificity of 77.7% (Fig 1G), and the INSL5 level demonstrated impressive sensitivity and specificity for NPC diagnosis (AUC 0.914, 95% CI: 0.897–0.931) (Fig 1H), which is comparable to VCA-IgA for NPC diagnosis. Plasma INSL5 is a diagnostic biomarker to distinguish EBV seronegative NPC patients from normal controls Although VCA-IgA is the widely used marker for NPC diagnosis, there are still 4–24% of patients with VCA-IgA undetectable. We found that INSL5 had especially high diagnostic efficiency (training cohort: AUC = 0.829, 95% CI 0.695–0.963; validation cohort 1: AUC = 0.961, 95% CI 0.919–1.00; and validation cohort 2: AUC = 0.967, 95% CI 0.924–1.00) in individuals with VCA-IgA-negative plasma in all three cohorts (Fig 1I). Applying the same cut-off value (1.12 ng/ml) to all VCA-IgA-negative individuals yielded a sensitivity of 90.2% and a specificity of 85.9% for NPC diagnosis (Fig 1J). In summary, plasma INSL5 could be a novel diagnostic marker for NPC patients, especially to assist for the diagnosis of VCA-IgA-negative patients. Plasma INSL5 is a prognostic marker for NPC patients To determine whether plasma INSL5 could be a prognostic marker for NPC patients, we preformed receiver operating characteristic (ROC) curve analysis to identify the optimum cut-off value (3.73 ng/ml) for plasma INSL5 level for prognosis analysis and used the Kaplan–Meier test to analyze the correlation between the concentration of INSL5 and patient survival in GZ cohort which is the only one cohort with complete clinical outcome information. The characteristics of the three cohorts’ patients with NPC are listed in Appendix Table S1. As shown, the patients with high INSL5 concentrations had shorter overall survival (OS) (P = 0.005; Fig 2A) and disease-free survival (DFS) (P = 0.028) than those with low INSL5 concentrations (Fig 2D). In addition, as the traditional biomarker for progression prediction (Li et al, 2017), a higher EBV DNA copy number also indicated shorter OS (P = 0.038; Fig 2B) and DFS (P = 0.004) than a lower copy number (Fig 2E). Furthermore, when we combined the INSL5 level and EBV DNA copy number, we found that the patients with high INSL5 levels and EBV DNA copy numbers had significantly shorter OS (P = 0.003) (Fig 2C) and DFS (P = 0.002) than the other patients (Fig 2F). Additionally, the profiles from TCGA database also showed that high INSL5 expression was correlated with poor overall survival in multiple tumors, especially in glioma, kidney renal clear cell carcinoma, sarcoma, uterine carcinosarcoma, and uveal melanoma patients (Fig EV1E–I). Figure 2. Higher plasma INSL5 is associated with poor prognosis of NPC patients A–C. Kaplan–Meier curv
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