脂肪因子chemerin通过上调PD-L1的表达促进卵巢癌细胞增殖和迁移

Chemerin promotes proliferation and migration of ovarian cancer cells by upregulating expression of PD-L1 脂肪因子chemerin通过上调PD-L1的表达促进卵巢癌细胞增殖和迁移 1 first-author 1 2 2 corresp corresp Yi ZHANG, syzi@163.com Yi ZHANG, syzi@163.com Yin LI, yinli@bjmu.edu.cn Yin LI, yinli@bjmu.edu.cn yinli@bjmu.edu.cn https://orcid.org/0000-0002-4019-3391 1 corresp Yi ZHANG, syzi@163.com Yi ZHANG, syzi@163.com syzi@163.com https://orcid.org/0000-0001-5875-6108 Department of Gynecology, the First Affiliated Hospital of China Medical University, Shenyang 110001, China Department of Gynecology, the First Affiliated Hospital of China Medical University, Shenyang 110001, China Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing 100191, China Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing 100191, China 目的： 2 探究腹腔肿瘤微环境内脂肪因子趋化素（chemerin）与程序性死亡配体-1（PD-L1）是否存在关联，及其对卵巢癌细胞系HO8910细胞生物学功能的影响。 创新点： 2 首次发现了chemerin可以上调卵巢癌细胞PD-L1的表达，促进肿瘤增殖和迁移。这对进一步研究免疫抑制剂在卵巢癌治疗中的应用、卵巢癌的早期诊断，以及血清和腹水中chemerin的检测具有一定的指导意义。此外，chemerin结合其他脂肪因子是否能成为卵巢癌诊断的标志物，需进一步的研究证实。 方法： 2 使用酶联免疫吸附剂测定（ELISA）检测卵巢癌患者腹水和血清中chemerin的含量；使用免疫组化检测卵巢癌肿瘤组织中chemerin和PD-L1的表达；使用免疫印迹法检测卵巢癌组织中PD-L1的表达及在HO8910和HO891PM细胞中chemerin表达的差异；利用浓度分别为0、10、50和100 ng/mL外源性chemerin刺激HO8910和HO8910PM细胞；使用水溶性四唑（WST）检测细胞增殖；使用划痕实验检测细胞迁移功能；利用鬼笔环肽荧光染色在共聚焦显微镜下观察PD-L1敲除前后的HO8910和HO8910PM的细胞骨架变化；使用免疫印迹法检测细胞骨架蛋白的表达情况。 结论： 2 Chemerin可以上调卵巢癌细胞PD-L1的表达，对肿瘤增殖和迁移具有促进作用。 卵巢癌 腹腔肿瘤微环境 脂肪因子 程序性死亡配体-1（PD-L1） Ovarian cancer is the third-most-common malignant reproductive tumor in women. According to the American Cancer Society, it has the highest mortality rate of gynecological tumors. The five-year survival rate was only 29% during the period from 1975 to 2008 ( Reid et al., 2017 Reid et al., 2017 ). In recent decades, the five-year survival rate of ovarian cancer has remained around 30% despite continuous improvements in surgery, chemotherapy, radiotherapy, and other therapeutic methods. However, because of the particularity of the volume and location of ovarian tissue, the early symptoms of ovarian cancer are hidden, and there is a lack of highly sensitive and specific screening methods. Most patients have advanced metastasis, including abdominal metastasis, when they are diagnosed ( Reid et al., 2017 Reid et al., 2017 ). Therefore, exploring the mechanism of ovarian cancer metastasis and finding early preventive measures are key to improving the survival rate and reducing mortality caused by ovarian cancer. Epithelial ovarian cancer is the most important type. Barbolina (2018) Barbolina (2018) found that metastasis and diffusion of epithelial ovarian cancer to the abdominal cavity tended to be directed toward the greater omentum. Paget (1889) Paget (1889) once proposed the theory of "seed and soil," which states that tumor cell metastasis only occurs in environments conducive to the survival and growth of tumor cells. The greater omentum microenvironment is surrounded by a monolayer of mesothelial cells, mainly fat cells. Adipocytes in the omentum can promote the metastasis of ovarian cancer cells ( Nieman et al., 2011 Nieman et al., 2011 ). Fat cells can secrete a large number of bioactive substances called fat factors, including lipids, hormones, proinflammatory cytokines, and a series of fat-derived signaling molecules ( Chi et al., 2018 Chi et al., 2018 ). Meanwhile, leptin is positively correlated with breast cancer risk, and its receptor is overexpressed in high-grade breast cancer ( Ishikawa et al., 2004 Ishikawa et al., 2004 ). The level of omentum-1 is higher in colon and prostate cancers, and it has an antiproliferative effect ( Rennier et al., 2020 Rennier et al., 2020 ). In addition, expression of adipokines is increased in pleural stromal tumors as well as lung, colorectal, and prostate cancers. Chemerin (also known as retinoic acid receptor responder 2, cluster of differenciation 247 (CD247)) is an adipokine and multifunctional chemotactic protein which exerts key functions in inflammation, adipogenesis, and glucose homeostasis. Elevated levels of serum chemerin have been linked to gastric, breast, and pancreatic cancers ( Goralski et al., 2019 Goralski et al., 2019 ). In ovarian cancer, differential expression of chemerin may have a biological effect on different ovarian cell types ( Reverchon et al., 2012 Reverchon et al., 2012 ; Hoffmann et al., 2018 Hoffmann et al., 2018 ; Treeck et al., 2019 Treeck et al., 2019 ), but a more specific role in this type of tumor has not been found. In recent years, immunosuppressants have been widely used in tumor treatment. Programmed death ligand 1 (PD-L1), a member of the B7-CD28 family, was discovered in 2000 ( Freeman et al., 2000 Freeman et al., 2000 ). PD-L1 is expressed on many cancer cell surfaces, such as gastric, lung, liver and breast cancer cells, oral squamous cell carcinoma, and ovarian cancer cells. After combination of programmed death protein 1 (PD-1) and PD-L1, antitumor immune response is weakened through inhibition of lymphocytes ( Ghebeh et al., 2006 Ghebeh et al., 2006 ). The relationship between PD-L1 and tumor prognosis is still unclear, and there are conflicting research results. It has been found that synthetic leptin OB3 inhibits expression of PD-L1 and reduces proliferation of hepatocarcinoma cells ( Ho et al., 2019 Ho et al., 2019 ), while obesity leads to immune aging and tumor progression, and affects the function of T cells mediated by PD-1. Obesity is also related to the change in efficacy of PD-1/PD-L1 blockers ( Wang et al., 2019 Wang et al., 2019 ). Therefore, we discussed whether the adipokine chemerin affects the expression of PD-L1 in cells, and thus affects the biological function of epithelial ovarian cancer cells. It was reported that chemerin reactivates phosphatase and tensin homolog deleted on chromosome ten ( PTEN ) and suppresses PD-L1 in prostate cancer cells via modulation of a novel chemokine-like receptor-1 (CMKLR1)‍-mediated signaling cascade ( Wise et al., 2017 Wise et al., 2017 ). Therefore, we discussed whether the adipokine chemerin affects the expression of PD-L1 in cells, and thereby affects the biological function of epithelial ovarian cancer cells. Our first task was to detect the expression of chemerin and PD-L1 in clinical specimens and ovarian cancer cells (see "Materials and methods" for details on the specimens). Assessments of the ascites and serum samples of 12 ovarian cancer patients showed that the concentration of chemerin in ascites was significantly higher than that in serum ( P < 0.05; Fig. 1 Fig. 1 a). Expression of chemerin in ovarian carcinoma cell line HO8910 cells was significantly lower than that in HO8910PM cells (the highly metastatic ovarian carcinoma cell line) ( Fig. 1 Fig. 1 b). Positive staining of chemerin and PD-L1 in ovarian cancer tissues was significantly higher than that in adjacent normal tissues (Figs. 1c 1c and 1d 1d ). Using the Gene Expression Profiling Interactive Analysis (GEPIA), we found the PD-L1 gene in stages II–IV ovarian cancers but not in stage I ( Fig. 1 Fig. 1 e). Therefore, the expression levels of chemerin and PD-L1 were higher in the clinical specimens and ovarian cancer cell lines. We then explored whether chemerin promoted proliferation and migration of ovarian cancer cells. Exogenous chemerin at the concentrations of 10, 50, and 100 ng/mL was used to stimulate HO8910 and HO8910PM cells, and a scratch-wound assay showed that under 10 ng/mL exogenous chemerin stimulation, cell healing did not change significantly. After stimulation with 50 and 100 ng/mL of exogenous chemerin, cell healing was apparent at 24 h and was fastest with the 100 ng/mL chemerin treatment ( Fig. 2 Fig. 2 a). A water-soluble tetrazolium (WST) assay showed that the cell proliferation rate exhibited an increasing trend after chemerin stimulation in HO8910 cells ( Fig. 2 Fig. 2 b). Therefore, it is clear that chemerin promotes proliferation and migration of ovarian cancer cells. We then investigated whether chemerin affected expression of PD-L1 in ovarian cancer cells. After stimulation of HO8910 and HO8910PM cells with 10, 50, and 100 ng/mL of exogenous chemerin, expression of PD-L1 in ovarian cancer cells increased; the highest expression was found in the 100 ng/mL group ( Fig. 3 Fig. 3 a). Flow cytometry detection showed that after stimulating ovarian cancer cells with 100 ng/mL of chemerin, expression of PD-L1 reached a peak at 12 h ( Fig. 3 Fig. 3 b). Therefore, chemerin upregulates expression of PD-L1 in ovarian cancer cells. Next, we looked at whether chemerin promoted proliferation and migration of ovarian cancer cells through upregulating expression of PD-L1. Small interfering RNA (siRNA) was transfected to knock down PD-L1 expression ( Fig. 4 Fig. 4 a). After chemerin stimulation, the cell proliferation rate was significantly higher than that of untreated cells, and PD-L1 knockdown significantly abolished chemerin-stimulated cell proliferation ( Fig. 4 Fig. 4 b). A scratch-wound assay also showed that in HO8910PM cells, the chemerin-stimulated cell healing rate was significantly abolished by PD-L1 knockdown ( Fig. 4 Fig. 4 c). We also found that the microfilament cytoskeleton protein of PD-L1 knockdown cells changed, and the filiform pseudopodia and migration process decreased, suggesting that PD-L1 can affect cell migration by altering the cytoskeleton ( Fig. 4 Fig. 4 d). Our main goal in the present study was to explore the relationship between chemerin and PD-L1 in ovarian cancer. We found that chemerin was expressed in the serum and ascites of ovarian cancer patients, and its content in ascites was significantly higher than that in serum. In addition, the expression of chemerin in ovarian cancer tissue was significantly higher than that in normal ovarian tissue, and the expression of chemerin in HO8910 cells was significantly lower than that in the high metastatic cell line HO8910PM. These results indicate that chemerin can promote metastasis of ovarian cancer cells. A previous study has found that increased serum chemerin concentration is related to the clinical stage and classification of gastric cancer ( Kumar et al., 2019 Kumar et al., 2019 ). Chemerin increases the invasiveness of gastric cancer, inducing p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and upregulation of vascular endothelial growth factor (VEGF), matix metalloproteinase-7 (MMP-7), and interleukin-6 (IL-6). A review of the tumor microenvironment of ovarian cancer ( Drakes and Stiff, 2018 Drakes and Stiff, 2018 ) suggested that ascites are a common concomitant symptom of ovarian cancer, and are conducive to the adhesion of cancer cells to the omentum. Adipocytes of the omentum secrete IL-6, IL-8, chemokine (C-C motif) ligand 2 (CCL2), and metformin to promote ovarian cancer metastasis. The microenvironment of intraperitoneal tumors is important for the growth and diffusion of tumor cells. Ovarian cancer is often accompanied by omental metastasis. According to the seed and soil theory proposed by Paget (1889) Paget (1889) , tumor cell metastasis only occurs in an environment conducive to the survival and growth of tumor cells. We believe that the adipokine chemerin secreted by adipocytes is involved in the formation of the intraperitoneal tumor microenvironment, which promotes metastasis of ovarian cancer cells to the omentum. Our study found for the first time that, in HO8910 and HO8910PM ovarian cancer cells, PD-L1 is upregulated by chemerin, and chemerin promotes the proliferation and migration of ovarian cancer cells. In HO8910 cells, the proliferation and scratch healing rate reached a peak at 24 h under treatment with 100 ng/mL chemerin. In HO8910PM, the effect of chemerin was not obvious, which may have been because the chemerin concentration in metastatic ovarian cancer cell lines was higher than the optimal. We knocked down PD-L1 in ovarian cancer cells and found that cell proliferation and migration were inhibited. A previous study has found that expression of PD-L1 in tumor cells is induced by interferon ( Garcia-Diaz et al., 2017 Garcia-Diaz et al., 2017 ). By combining with PD-1 expressed on T cells, PD-L1 expression is promoted, and apoptosis and tolerance are induced, which inhibits immunity. PD-L1 also affects tumors in several ways, by increasing survival and proliferation of cancer cells, regulating the glucose utilization rate of tumors, and inhibiting autophagy ( Chang et al., 2015 Chang et al., 2015 ). PD-L1 is expressed on the surface of cancer cells, and its increased expression is related to poor prognosis in ovarian cancer ( Hamanishi et al., 2007 Hamanishi et al., 2007 ; Maine et al., 2014 Maine et al., 2014 ). Chemerin may upregulate expression of IL-6 by upregulation of p38 and ERK1/2 phosphorylation, which further affects expression of PD-L1 and promotes proliferation, migration, and metastasis of tumor cells ( Wang et al., 2014 Wang et al., 2014 ; Clark et al., 2016 Clark et al., 2016 ). In addition, we found that the microfilament cytoskeleton protein changed after PD-L1 knockdown, and the filiform pseudopodia and migration process decreased, suggesting that PD-L1 can affect cell migration by changing the cytoskeleton. The tumor microenvironment is a comprehensive system composed of tumor cells, adipocytes, and other stromal cells. Nontumor tissues adjacent to tumor cells often play an important role in tumor development. Epidemiological studies showed that obese people have a higher risk of cancer, with faster tumor growth and deterioration and higher mortality ( Steele et al., 2017 Steele et al., 2017 ). Therefore, in recent years, scientists have conducted a large number of studies on the role of fat cells near tumors, and eventually discovered that fat cells in the tumor microenvironment can affect the development and deterioration of tumor tissue through their own pathological expansion ( Ibrahim, 2010 Ibrahim, 2010 ). The "immune escape" of tumor cells is the main mechanism of invasion and metastasis of cancer cells. Immune cells play an important role in the process of achieving immune tolerance of cancer cells, which can help cancer cells escape monitoring by the immune system by inhibiting various types of immune responses. At present, immunotherapy with T cells has been applied to the treatment of multiple types of cancer. Recent studies have shown that chemerin plays a key role in the development of a variety of malignant tumors. It has been proved that chemerin is highly expressed in oral and esophageal cancers ( Kumar et al., 2016 Kumar et al., 2016 ; Ghallab and Shaker, 2017 Ghallab and Shaker, 2017 ). There is a positive correlation between the concentration of chemerin in serum and colorectal cancer and gastric adenocarcinoma ( Zhang et al., 2014 Zhang et al., 2014 ; Alkady et al., 2018 Alkady et al., 2018 ). In breast cancer, compared with nonmetastatic malignant tissues, the expression of chemerin protein in cancer tissues, adjacent healthy tissues, or metastatic lymph nodes is higher ( El-Sagheer et al., 2018 El-Sagheer et al., 2018 ), but in skin and liver cancers, chemerin has been found to have an anticancer effect ( Pachynski et al., 2012 Pachynski et al., 2012 ; Li et al., 2018 Li et al., 2018 ). In lung and gastric cancers, chemerin is expected to become a diagnostic marker ( Goralski et al., 2019 Goralski et al., 2019 ). More specific roles of chemerin in tumors have not been reported. Our study suggests that chemerin upregulates expression of PD-L1 in ovarian cancer cells and promotes tumor proliferation and migration. The mechanism by which chemerin upregulates PD-L1 remains to be studied. Although existing studies have suggested that chemerin activates a variety of downstream pathways, including the phosphorylation of p42‍–‍p44 mitogen-activated protein kinases (MAPKs) ( Shin and Pachynski, 2018 Shin and Pachynski, 2018 ), and recruits β‍-arrestin 1 and 2 to CMKLR1 or G protein-coupled receptor 1 (GPR1) ( Rennier et al., 2020 Rennier et al., 2020 ), the mechanism by which chemerin upregulates PD-L1 requires further investigation. In summary, our study found for the first time that chemerin can upregulate the expression of PD-L1 in ovarian cancer cells, and thereby promote tumor proliferation and migration. It may be helpful to study further the application of immunosuppressive agents in ovarian cancer. Detection of chemerin in serum and ascites may be important for the early diagnosis of ovarian cancer. Chemerin combined with other adipokines may become a diagnostic target for ovarian cancer. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. Author contributioauthor contribution statement 1 Chenxi GAO, Jinming SHI, and Jingxin ZHANG performed the experimental research and data analysis, and wrote the manuscript. Yin LI and Yi ZHANG contributed to the study design, data analysis, and writing and editing of the manuscript. 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Peptides , 61 : 7 - 11 . https://doi.org/10.1016/j.peptides.2014.08.007 https://doi.org/10.1016/j.peptides.2014.08.007 Prognostic significance of plasma chemerin levels in patients with gastric cancer 10.1631/jzus.B2100392 1673-1581（2022）02-0164-07 This work was supported by the Special Project for Central Guidance of Local Science and Technology Development of Liaoning Province (No. 2019JH6/10400006), China. 2021-04-25 2021-08-30 2022-02-15 2022-04-19 2022-01-04 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 Chenxi GAO, Jinming SHI, Jingxin ZHANG, Yin LI, and Yi ZHANG declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 2 23