Role of Hippo pathway in promoting invasion and metastasis of liver cancer cells by oxygenated polycyclic aromatic hydrocarbons

GAO Rui; WU Xiuyu; CAI Zhihong; SANG Nan

doi:10.7524/j.issn.0254-6108.2023042101

2024 Volume 43 Issue 4

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GAO Rui, WU Xiuyu, CAI Zhihong, SANG Nan. Role of Hippo pathway in promoting invasion and metastasis of liver cancer cells by oxygenated polycyclic aromatic hydrocarbons[J]. Environmental Chemistry, 2024, 43(4): 1118-1126. doi: 10.7524/j.issn.0254-6108.2023042101

Citation:

GAO Rui, WU Xiuyu, CAI Zhihong, SANG Nan. Role of Hippo pathway in promoting invasion and metastasis of liver cancer cells by oxygenated polycyclic aromatic hydrocarbons[J]. Environmental Chemistry, 2024, 43(4): 1118-1126. doi: 10.7524/j.issn.0254-6108.2023042101

Role of Hippo pathway in promoting invasion and metastasis of liver cancer cells by oxygenated polycyclic aromatic hydrocarbons

1.
School of Forensic Medicine, Shanxi Medical University, Jinzhong, 030600, China.
2.
College of Environmental and Resource Sciences, Shanxi University, Taiyuan, 030006, China

Corresponding author: SANG Nan, sangnan@sxu.edu.cn
Received Date: 21/04/2023
Accepted Date: 26/05/2023
Available Online: 27/04/2024

Fund Project: the National Natural Science Foundation of China (22106097), Fundamental Research Program of Shanxi Province (20210302124059) and Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi.

Abstract

Due to the presence of oxygenated functional groups, oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) possess higher polarity, solubility, mobility, and biological reactivity than their parent PAHs, and are more widely distributed in the environment. Although numerous oxy-PAHs exhibit heightened genotoxicity, carcinogenicity, and developmental toxicity, limited research has been conducted to elucidate the underlying mechanisms responsible for their tumorigenic promotion. Notably, the Hippo-YAP pathway has emerged as a pivotal regulatory process governing cancer cell immunity, proliferation, and metastasis. In this study, the tumor promoting effect of oxy-PAHs (specifically 9-Fluorenone) that are commonly found in the environment, as well as the underlying Hippo-YAP regulation mechanism were investigated by using the liver cancer cell exposure model. The results showed that a high concentration of 9-Fluorenone exposure significantly enhanced the invasion, migration, and epithelial-mesenchymal transition (EMT) of HepG2 cells, and the Hippo-YAP pathway was identified as playing a crucial role in the tumor-promoting process. Specifically, 9-Fluorenone exposure can induce dephosphorylation of upstream kinases in the Hippo pathway, such as MST and LATS, and then induce dephosphorylation of YAP. Consequently, YAP is able to bind to TEAD in the nucleus, thereby regulating the occurrence of EMT and ultimately promoting the invasion and metastasis of cancer cells. This study contributes valuable toxicological evidence supporting the tumor-promoting effect of oxy-PAHs and holds significant reference value for the comprehensive assessment of the ecological and health risks associated with PAHs and their derivatives.
- oxygenated polycyclic aromatic hydrocarbons,
- invasion and metastasis,
- Hippo,
- EMT

References

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沈阳化工大学材料科学与工程学院沈阳 110142

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Role of Hippo pathway in promoting invasion and metastasis of liver cancer cells by oxygenated polycyclic aromatic hydrocarbons

Corresponding author: SANG Nan, sangnan@sxu.edu.cn

Received Date: 21/04/2023

Accepted Date: 26/05/2023

Available Online: 27/04/2024

Fund Project: the National Natural Science Foundation of China (22106097), Fundamental Research Program of Shanxi Province (20210302124059) and Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi.

Key words:

oxygenated polycyclic aromatic hydrocarbons /
invasion and metastasis /
Hippo /
EMT

Abstract: Due to the presence of oxygenated functional groups, oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) possess higher polarity, solubility, mobility, and biological reactivity than their parent PAHs, and are more widely distributed in the environment. Although numerous oxy-PAHs exhibit heightened genotoxicity, carcinogenicity, and developmental toxicity, limited research has been conducted to elucidate the underlying mechanisms responsible for their tumorigenic promotion. Notably, the Hippo-YAP pathway has emerged as a pivotal regulatory process governing cancer cell immunity, proliferation, and metastasis. In this study, the tumor promoting effect of oxy-PAHs (specifically 9-Fluorenone) that are commonly found in the environment, as well as the underlying Hippo-YAP regulation mechanism were investigated by using the liver cancer cell exposure model. The results showed that a high concentration of 9-Fluorenone exposure significantly enhanced the invasion, migration, and epithelial-mesenchymal transition (EMT) of HepG2 cells, and the Hippo-YAP pathway was identified as playing a crucial role in the tumor-promoting process. Specifically, 9-Fluorenone exposure can induce dephosphorylation of upstream kinases in the Hippo pathway, such as MST and LATS, and then induce dephosphorylation of YAP. Consequently, YAP is able to bind to TEAD in the nucleus, thereby regulating the occurrence of EMT and ultimately promoting the invasion and metastasis of cancer cells. This study contributes valuable toxicological evidence supporting the tumor-promoting effect of oxy-PAHs and holds significant reference value for the comprehensive assessment of the ecological and health risks associated with PAHs and their derivatives.

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多环芳烃（PAHs）广泛分布于大气、土壤、水、生活垃圾等各种环境中，由于具有遗传毒性和致癌活性，它们在食品和环境中的污染水平受到严格监管^{[1 − 4]}. 含氧多环芳烃（oxy-PAHs）、硝基多环芳烃、烷基多环芳烃等衍生物通常与PAHs共同存在，其排放源与PAHs相似，主要包括化石燃料的不完全燃烧、汽车尾气排放以及多环芳烃的光化学与生物转化作用等^[5]. 由于具有高极性、高水溶性、低蒸汽压等特点，oxy-PAHs在水、土壤、大气颗粒物等环境中的分布比母体PAHs更为广泛^{[6 − 7]}. 且在各种食物以及电子垃圾等环境中也检测到较高浓度的oxy-PAHs^{[8 − 9]}. 最近研究表明，与PAH相比，许多oxy-PAHs具有更高的遗传毒性、促肿瘤活性、发育毒性、心血管毒性和细胞毒性^{[10 − 13]}. 然而，目前有关oxy-PAHs的毒性作用机制研究主要集中于遗传毒性、发育毒性等，对其促肿瘤活性作用机制的研究甚少.

9-芴酮（9-Fluorenone）是大气和水环境中含量最为丰富的oxy-PAHs之一，在多种环境介质中均有检出. 例如，法国南部地区大气环境中9-Fluorenone含量约占oxy-PAHs总浓度的60%^[14]. 汽车尾气颗粒物中9-Fluorenone含量明显高于其他oxy-PAHs^[10]. 我国多个地下水样本中均检出大量9-Fluorenone^[15]. 对宜昌市化工园区的土壤样品有机污染物进行非靶标筛查中发现了9-Fluorenone的存在^[16]. 此外，研究发现9-Fluorenone是矿质土壤和燃烧排放中最丰富的oxy-PAHs之一^[17]. 目前，有关9-Fluorenone的毒性研究主要集中于发育毒性、遗传毒性等^[18]，有关9-Fluorenone的促癌作用研究较少. 本课题组前期研究发现，9-Fluorenone暴露可诱导肺癌细胞EMT的发生进而促进侵袭转移^[19]，但关于9-Fluorenone的促癌作用机制仍不明确.

Hippo-YAP通路被发现是调节癌细胞存活、增殖、转移、免疫、凋亡等的关键因子^{[20 − 21]}. 与大量动物研究结果一致，美国TCGA（The Cancer Genome Atlas，癌症基因组图谱）项目对9125个肿瘤样本进行了多组学分析，结果表明Hippo通路失调发生在多种人类癌症中^{[22 − 23]}. Hippo信号通路是一个激酶级联通路，主要作用是调节Yes相关蛋白（YAP和TAZ）、转录共激活因子和效应靶标分子. Hippo信号通路开始于肿瘤抑制性丝氨酸/苏氨酸激酶（MST1/2）的激活，然后磷酸化并激活大肿瘤抑制因子（LATS1/2），进而磷酸化并抑制YAP（Yes相关蛋白）向细胞核的转移^[24]. 当Hippo信号通路受刺激失活时，MST1/2与LATS1/2的磷酸化激活被抑制，使得未磷酸化的YAP转移至细胞核以调节癌细胞增殖转移等相关靶基因的表达^[25]. Hippo通路异常与肿瘤细胞的迁移和侵袭密切相关，Hippo通路的关键效应分子YAP和TAZ的异常积累和核转移在转移性肿瘤中经常发生^[26]. YAP作为细胞核内TEAD等转录因子的共激活因子，可以调控肿瘤转移相关基因的表达^[27]或者通过激活或抑制MAPK通路、WNT通路等促进肿瘤转移和侵袭^{[28 − 29]}. 此外，Hippo通路失调可以通过诱导EMT和减少细胞之间或细胞与细胞外基质之间的黏附诱导肿瘤侵袭和转移发生^{[30 − 31]}. 本课题组前期研究发现1-硝基芘暴露可以诱导Hippo-YAP通路失活，促进肺癌细胞EMT，诱导转移的发生^[32]. 然而，目前仍不清楚oxy-PAHs的促肿瘤作用是否与Hippo通路有关.

为了解9-Fluorenone的促肿瘤作用是否与Hippo-YAP通路有关，本文采用肝癌细胞暴露模型，探究了9-Fluorenone对细胞侵袭、转移等的影响，检测了MST1/2、LATS1/2、YAP等蛋白磷酸化的发生，结合免疫荧光检测了YAP-TEAD的结合，并采用YAP-TEAD阻断剂处理进一步确证了Hippo-YAP通路在9-Fluorenone促肿瘤发生中的关键作用.

1. 材料与方法（Materials and methods）

1.1. 材料与试剂

HepG2肝癌细胞购买自中国科学院上海细胞库，9-Fluorenone和YAP-TEAD抑制剂verteporfin购买自Sigma公司，DMEM培养基、链霉素、青霉素均购买自Gibco公司，胎牛血清购自于Biological Industries公司，Transwell小室、Matrigel基质胶购买自Corning公司，蛋白一抗（β-actin，bs-0061R；p-MST1/2，bs-3294R；MST1/2，bs-8477R；p-LATS1/2，bs-7913R；YAP，bs-3605R；p-YAP，bs-3475R）购自Bioss公司，免疫荧光一抗（YAP，ab52771；TEAD，ab58310）购自Abcam公司，荧光标记二抗（IRDye800 CW，926-32211）购自LICOR公司，反转录试剂与SYBR扩增酶购自TaKaRa公司.

1.2. 细胞培养与处理

将HepG2肝癌细胞于含10%血清的DMEM培养基（加100 U·mL⁻¹链霉素和青霉素）中进行单层培养. 待HepG2细胞生长至80%时，用胰酶消化，铺细胞，将其随机分为对照组和暴露组，暴露组细胞用不同浓度（1、3、10 μmol·L⁻¹）的9-Fluorenone处理不同时间并进行后续实验，对于YAP-TEAD阻断组，采用1 μmol·L⁻¹ 的verteporfin预处理细胞1 h后再进行9-Fluorenone暴露.

1.3. 划痕实验

将HepG2细胞接种于12孔板中，待细胞生长至90%时，采用无菌移液枪头划出一个宽度区域，PBS洗涤后用不同浓度（1、3、10 μmol·L⁻¹）的9-Fluorenone暴露处理，12 h、24 h和48 h后于光学显微镜下拍照观察细胞迁移情况，计算迁移速率.

1.4. 细胞侵袭实验

将HepG2细胞接种于35 mm细胞培养皿中，待细胞生长至80%时，用不同浓度（1、3、10 μmol·L⁻¹）的9-Fluorenone对细胞暴露48 h，胰酶消化处理后用无血清培养基重悬至5×10⁵个·mL⁻¹. 在Transwell小室内加入50 μL的Matrigel基质胶（1:8稀释），取10⁴个细胞均匀接种到Transwell小室上方，下室中加入诱导剂即完全培养基，孵育24 h. 去掉上层的培养基，用棉签擦去上方未迁移的细胞，迁移到小室下表面的细胞用多聚甲醛固定，结晶紫染色，光学显微镜拍照计数.

1.5. Western Blot实验

收集不同浓度（1、3、10 μmol·L⁻¹）9-Fluorenone处理24 h后的细胞，用RIPA裂解液提取蛋白，用BCA试剂盒测定蛋白浓度. 采用8%的浓缩胶和分离胶进行SDS-PAGE电泳分离实验，之后90 V恒压转移至PVDF膜上，蛋白冻干粉封闭后选用不同的一抗（β-actin、p-MST1/2、MST1/2、p-LATS1/2、YAP、p-YAP）于4 ℃孵育过夜. 第二天用PBST清洗，荧光标记二抗（1:10000稀释，IRDye800 CW）避光孵育1 h，PBST清洗后使用Odyssey红外荧光成像系统对条带进行扫描.

1.6. PCR扩增实验

收集不同浓度（1、3、10 μmol·L⁻¹）9-Fluorenone处理24 h后的细胞，用Trizol提取细胞RNA，用Nanodrop测定RNA浓度. 采用反转录试剂将1 μg的RNA反转为cDNA，6倍稀释后，选用SYBR Premix Ex TaqⅡ进行实时荧光定量PCR. 扩增结果依据ΔΔCt法计算. 引物序列信息见表1.

1.7. 免疫荧光实验

将2 cm×2 cm的细胞玻片进行高压灭菌处理，用10×多聚赖氨酸（Poly-L）预处理后置于60 mm细胞培养皿中. 将HepG2细胞接种于玻片上，分别用10 μmol·L⁻¹的9-Fluorenone与1 μmol·L⁻¹的verteporfin进行暴露处理. 之后用4%多聚甲醛进行固定，0.5%Triton X-100通透处理. 用山羊血清封闭后滴加稀释好的一抗混合液（YAP和TEAD），置于湿盒中过夜. 第二天用荧光二抗混合液（羊抗兔lgG（IRDye800 CW，926-32211）和羊抗鼠lgG（bs-40296G-IRDye8））避光孵育1 h，用含DAPI抗荧光猝灭封片液进行封片，荧光显微镜下观察拍照.

1.8. 数据统计

所有数据为3次独立重复实验的平均值±标准差. 数据采用SPSS单因素方差分析比较组间均值差异，采用GraphPad Prism 5作图.

3. 结论（Conclusion）

本研究通过肝癌细胞暴露模型发现，较高浓度9-Fluorenone暴露可促进肝癌细胞侵袭、迁移及EMT的发生，且Hippo-YAP通路在该过程中有重要作用. 9-Fluorenone暴露可通过诱导Hippo上游激酶MST、LATS以及YAP蛋白的去磷酸化，使得YAP进入细胞核内与TEAD结合，调控EMT的发生，进一步促进肝癌细胞侵袭转移. 本研究结果为含氧多环芳烃的促肿瘤活性研究提供了实验基础，对于全面评估多环芳烃及衍生物的生态和健康风险具有重要参考作用.

Figure (5) Table (1) Reference (44)

Role of Hippo pathway in promoting invasion and metastasis of liver cancer cells by oxygenated polycyclic aromatic hydrocarbons