研究背景与目的

胰腺癌作为消化系统侵袭性最强的恶性肿瘤之一，其生物学特性以早期转移倾向显著、化疗耐药性频发及多模态治疗响应率低为特征。临床数据显示，患者5年总体生存率仅为13%，这一严峻现状与肿瘤微环境高度异质性、早期诊断标志物匮乏及预后预测模型不精准等因素密切相关。近年来，肿瘤代谢重编程被证实是恶性肿瘤的重要标志性特征之一，其参与到了包括胰腺癌在内的多种肿瘤的恶性生物学行为中。近年来的研究揭示，脂质代谢重编程作为肿瘤代谢异质性的关键特征，已成为肿瘤生物学领域的研究热点。然而，作为脂质稳态核心组分的胆固醇代谢重塑在胰腺癌发生、转移及耐药性演化等方面中的分子机制仍未阐明。本研究旨在探索并验证胰腺癌生长过程中的关键胆固醇代谢调控者，并探究其作用与具体分子机制。

研究方法

通过分析公共数据库，筛选胰腺癌中的关键胆固醇代谢调控基因。通过对胰腺癌组织芯片进行免疫组化染色，验证角鲨烯环氧化酶（Squalene epoxidase, SQLE）在组织水平的表达，并分析其与患者预后的相关性。利用慢病毒载体构建稳定敲低及过表达SQLE的胰腺癌细胞系。在体外进行一系列功能实验包括CCK-8细胞增殖实验、EdU细胞增殖检测、克隆形成实验、细胞周期检测及细胞凋亡实验检测SQLE对胰腺癌细胞的生长能力的影响。利用小鼠皮下移植瘤模型研究SQLE对胰腺癌细胞在体内成瘤能力的影响。通过BODIPY荧光染色及内质网应激相关蛋白的检测探索干预SQLE后对胰腺癌细胞内脂滴含量及内质网应激的影响。通过分析对照及SQLE敲低的胰腺癌细胞系的RNA-seq数据及利用Western blot验证，探究SQLE引起下游信号通路的改变。利用胆固醇含量检测试剂盒和Filipin III染色验证干预SQLE表达后胰腺癌细胞内胆固醇含量的变化。利用PI3K/Akt通路的抑制剂及激动剂验证SQLE通过影响PI3K/Akt信号来介导对细胞增殖的调控。利用非靶向代谢组学检测影响SQLE表达后细胞内有意义代谢物的差异。通过对脂筏标记染色、消除脂筏及外源性补充胆固醇等方式验证SQLE对脂筏的调控。通过公共数据库分析、Co-IP实验、多重免疫组织化学染色及Western blot验证等证明SQLE对Src/PI3K/Akt信号通路的影响。使用SQLE抑制剂特比萘芬和NB-598验证其对胰腺癌细胞恶性表型、内质网应激及Src/PI3K/Akt信号通路的影响。利用小鼠皮下移植瘤模型验证SQLE抑制剂对胰腺癌细胞在体内成瘤能力的抑制作用。利用多重免疫组织化学实验探索SQLE差异表达的胰腺癌组织内多种免疫细胞亚群的组成差异。

研究结果

在本研究中，我们首先分析了多个胰腺癌公共数据集，发现在胆固醇代谢调控基因中，SQLE是其中唯一表达显著上调的调控分子。通过对组织芯片染色，我们发现SQLE在胰腺癌组织中显著上调，并且SQLE的高表达预示着患者的不良预后。功能实验表明，高表达SQLE的胰腺癌细胞表现出更强的恶性生物学特征，表现为在体外能够促进细胞增殖，诱导细胞周期进展及抑制细胞凋亡。同时，动物实验的结果表明，SQLE高表达的肿瘤细胞促进了小鼠成瘤能力。在机制层面，一方面，SQLE抑制能够导致其上游底物角鲨烯积累，这激活了以GRP78-PERK-eIF2α-ATF4信号为主导的内质网应激，从而触发了CHOP介导的凋亡级联反应；另一方面，SQLE过表达增加了胰腺癌细胞内的胆固醇从头合成，维持了脂筏结构的稳定性，进而激活了下游Src/PI3K/Akt信号通路，促进了细胞增殖。使用SQLE抑制剂能够抑制胰腺癌细胞的多种恶性生物学行为，并减缓小鼠皮下移植瘤模型的生长。SQLE高表达的肿瘤组织内存在抑制性的免疫微环境。

研究结论

本研究首次阐明了SQLE作为关键的胆固醇代谢调控分子在胰腺癌中的异常表达及其临床意义，为胰腺癌代谢分型提供了新标志物。本研究通过一系列细胞功能实验、胆固醇相关代谢物检测及动物实验等方法证明出SQLE通过脂质积累引起的内质网应激与脂筏所介导的信号通路这两方面因素协同调控胰腺癌生长。靶向SQLE有望成为胰腺癌的潜在治疗方式。

Background and Objective

Pancreatic cancer, as one of the most aggressive malignant tumors in the digestive system, is biologically characterized by a significant tendency of early metastasis, frequent chemoresistance and low response rate to multimodal therapy. Clinical data show that the 5-year overall survival rate of patients is only 13%, which is closely related to the highly heterogeneous tumor microenvironment, the lack of early diagnostic markers and the imprecise prognostic model. In recent years, tumor metabolic reprogramming has been confirmed to be one of the important hallmark features of malignant tumors, which is involved in the malignant biological behaviors of many tumors, including pancreatic cancer. Recent studies have revealed that lipid metabolic reprogramming, as a key feature of tumor metabolic heterogeneity, has become a hot research topic in tumor biology. However, the molecular mechanism underlying the remodeling of cholesterol metabolism, as a core component of lipid homeostasis, in pancreatic carcinogenesis, metastasis, and drug resistance evolution remains to be elucidated. The present study aims to explore and validate the key cholesterol metabolism regulators during pancreatic cancer growth, and to investigate their roles and specific molecular mechanisms.

Methods

Key cholesterol metabolism regulatory genes in pancreatic cancer were screened by analyzing public databases. Immunohistochemical staining of pancreatic cancer tissue microarrays was performed to verify the expression of squalene epoxidase (SQLE) at the tissue level and to analyze its correlation with patient prognosis. Stable knockdown and overexpression of SQLE in pancreatic cancer cell lines were constructed using lentiviral vectors. A series of functional assays were performed in vitro to detect the effect of SQLE on the growth ability of pancreatic cancer cells, including CCK-8 cell proliferation assay, EdU cell proliferation assay, colony formation assay, cell cycle assay and apoptosis assay. The effect of SQLE on the tumorigenic capacity of pancreatic cancer cells in vivo was investigated using a mouse subcutaneous transplantation tumor model. The effects on intracellular lipid droplet content and endoplasmic reticulum stress in pancreatic cancer cells after intervention of SQLE were explored by BODIPY fluorescence staining and endoplasmic reticulum stress-related protein detection. The alteration of downstream signaling pathways induced by SQLE was explored by analyzing RNA-seq data of control and SQLE knockdown pancreatic cancer cell lines and validated using Western blot. Changes in cholesterol content in pancreatic cancer cells after intervention with SQLE expression were verified using a cholesterol content assay kit and Filipin III staining. Inhibitor and agonist of the PI3K/Akt pathway were utilized to validate that SQLE mediates the regulation of cell proliferation by affecting PI3K/Akt signaling. Detection of intracellular differences in meaningful metabolites after affecting SQLE expression was performed by untargeted metabolomics. Regulation of lipid rafts by SQLE was verified by labeling staining of lipid rafts, elimination of lipid rafts, and exogenous cholesterol supplementation. The effect of SQLE on the Src/PI3K/Akt signaling pathway was demonstrated by public database analysis, Co-IP experiments, multiplex immunohistochemical staining and Western blot validation. The SQLE inhibitor terbinafine and NB-598 were used to verify their effects on the malignant phenotype, endoplasmic reticulum stress and Src/PI3K/Akt signaling pathway of pancreatic cancer cells. The inhibitory effect of SQLE inhibitor on the tumorigenic ability of pancreatic cancer cells in vivo was verified using a mouse subcutaneous transplantation tumor model. The multiplex immunohistochemistry was employed to investigate the compositional differences of diverse immune cell subsets within pancreatic cancer tissues exhibiting differential SQLE expression.

Results

In this study, we first analyzed multiple pancreatic cancer public datasets and found that among the cholesterol metabolism regulatory genes, SQLE was the only regulatory molecule among them whose expression was significantly upregulated. By staining tissue microarrays, we found that SQLE was significantly upregulated in pancreatic cancer tissues and that high expression of SQLE predicted a poor prognosis for patients. Functional experiments showed that pancreatic cancer cells with high expression of SQLE exhibited stronger malignant biological characteristics, as evidenced by the ability to promote cell proliferation, induce cell cycle progression and inhibit apoptosis in vitro. Meanwhile, the results of animal experiments indicated that tumor cells with high SQLE expression promoted tumorigenicity in mice. Mechanistically, on the one hand, SQLE inhibition was able to lead to the accumulation of its upstream substrate squalene, which activated endoplasmic reticulum stress dominated by GRP78-PERK-eIF2α-ATF4 signaling, thereby triggering a CHOP-mediated apoptotic cascade response; on the other hand, SQLE overexpression increased de novo cholesterol synthesis in pancreatic cancer cells and maintained the stability of lipid raft structure, which in turn activated the downstream Src/PI3K/Akt signaling pathway and promoted cell proliferation. The use of SQLE inhibitors suppressed multiple malignant biological behaviors of pancreatic cancer cells and slowed the growth of subcutaneous xenograft tumor models in mice. The tumor tissues with the high expression of SQLE harbored an immunosuppressive microenvironment.

Conclusion

This study elucidated for the first time the aberrant expression of SQLE as a key cholesterol metabolism regulatory molecule in pancreatic cancer and its clinical significance, and provided a new marker for metabolic typing of pancreatic cancer. Through a series of cell functional experiments, cholesterol-related metabolite assays and animal experiments, this study demonstrated that SQLE regulates pancreatic cancer growth through both endoplasmic reticulum stress induced by lipid accumulation and signaling pathways mediated by lipid rafts. Targeting SQLE is expected to be a potential treatment modality for pancreatic cancer.