PO.ET03.06 · 实验与分子治疗
TROP2靶向抗体-药物偶联物(ADC)的抗癌效应可能因癌细胞中胆固醇代谢失调而减弱
The anticancer effect of TROP2-targeted antibody-drug conjugates (ADC) is potentially attenuated by dysregulated cholesterol metabolism in cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：Trop-2是一种在多种不同肿瘤上广泛表达的糖蛋白。Datopotamab deruxtecan(Dato-DXd)是一种Trop-2靶向的ADC，已在EGFR突变的非小细胞肺癌(NSCLC)患者中显示出抗癌疗效。然而，其疗效并不一致。在癌细胞中，胆固醇合成被促进，胆固醇定位于细胞膜，在细胞膜上各种受体的内吞作用中发挥关键作用。本研究旨在使用EGFR突变的NSCLC细胞探索Dato-DXd疗效有限的潜在机制。
材料与方法：我们通过将EGFR突变的NSCLC PC9细胞长期暴露于Dato-DXd，建立了Dato-DXd耐药的PC9DR细胞系。进行集落形成实验以评估抗癌效应。免疫学方法测定细胞膜上的Trop-2表达。使用pH-Rhod方法测定Dato-DXd的细胞内摄取。使用Transcriptome Analysis Console软件分析微阵列数据，以检查差异表达基因及相关通路。
结果：根据集落形成实验，1 µg/mL Dato-DXd抑制了PC9细胞89%的集落形成，但仅抑制了PC9DR细胞5%的集落形成。在PC9DR细胞中，细胞表面Trop-2表达高于PC9细胞（每细胞434,338对400,722个分子，p = 0.0012）。然而，与PC9细胞相比，PC9DR细胞中Dato-DXd的细胞内摄取显著减少（双因素ANOVA，p < 0.0001）。这些结果提示，细胞膜上Trop-2蛋白内化的减少可能与对Dato-DXd的耐药相关。其次，通路分析显示，与PC9细胞相比，胆固醇代谢通路在PC9DR细胞中表现出最显著的变化。具体而言，与PC9细胞相比，PC9DR细胞显示胆固醇合成相关酶（包括HMGCR、MVD和SQLE）的表达显著降低，而相反地表现出胆固醇转运体ABCA1的表达增加（ANOVA p < 0.001，变化超过两倍）。因此，我们假设胆固醇代谢受损导致对Dato-DXd的耐药。最后，我们将包括PC9和HCC827在内的Dato-DXd敏感细胞暴露于HMGCoA还原酶抑制剂1 µM mevastatin，并使用集落形成实验评估1 µg/mL Dato-DXd的抗癌效应。在PC9细胞中，Dato-DXd有力地抑制了集落形成（相对集落形成面积，对照组：100%，Dato-DXd：3.6%）。然而，在用mevastatin处理的PC9细胞中，Dato-DXd对集落形成的抑制极小（对照组：100%，Dato-DXd：77.4%）。在HCC827细胞中观察到类似趋势。
结论：这些结果提示，胆固醇代谢受损可能导致EGFR突变NSCLC中对Dato-DXd的耐药。
查看英文原文 English abstract
Background: Trop-2 is a widely expressed glycoprotein on a variety of different tumors. Datopotamab deruxtecan (Dato-DXd) is a Trop-2-targeted ADC that has demonstrated anticancer efficacy in patients with EGFR -mutated non-small cell lung cancer (NSCLC). However, their efficacy is inconsistent. In cancer cells, cholesterol synthesis is promoted, and cholesterol localizes to the cell membrane, playing a crucial role in the endocytosis of various receptors on the cell membrane. The current study aimed to explore the mechanisms underlying the limited efficacy of Dato-DXd using EGFR -mutated NSCLC cells.
Materials and methods: We established a Dato-DXd-resistant PC9DR cell line by long-term exposure of EGFR -mutated NSCLC PC9 cells to Dato-DXd. A colony formation assay was performed to evaluate anticancer effects. Trop-2 expression on the cell membrane was measured immunologically. Intracellular uptake of Dato-DXd was measured using a pH-Rhod method. Microarray data were analyzed using Transcriptome Analysis Console Software to examine differentially expressed genes and related-pathways.
Result: According to the colony formation assay, 1µg/mL Dato-DXd inhibited 89% colony formation in PC9 cells, but inhibited 5% colony formation in PC9DR cells. In PC9DR cells, cell surface Trop-2 expression was greater than PC9 cells (434,338 vs 400,722 molecules per cell, p = 0.0012). However, intracellular uptake of Dato-DXd was significantly reduced in PC9DR cells compared to PC9 cells (two-way ANOVA, p < 0.0001). These results suggested that reduced internalization of Trop-2 protein on the cell membrane is hypothetically associated with resistance to Dato-DXd. Second, pathway analysis revealed that the cholesterol metabolism pathway showed the most significant change in PC9DR cells compared to PC9 cells. Specifically, compared to PC9 cells, PC9DR cells showed a marked decrease in the expression of cholesterol synthesis-related enzymes, including HMGCR, MVD, and SQLE, while conversely exhibiting increased expression of the cholesterol transporter ABCA1 (ANOVA p<0.001, greater than two-fold change). Therefore, we hypothesized that impaired cholesterol metabolism cause resistance to Dato-DXd. Finally, we exposed the Dato-DXd-sensitive cells including PC9 and HCC827 to the HMGCoA reductase inhibitor, 1µM mevastatin, and evaluated anticancer effect of 1µg/mL Dato-DXd using a colony formation assay. In PC9 cells, Dato-DXd potently inhibited colony formation (relative colony formation area, Control; 100%, Dato-DXd; 3.6%). However, PC9 cells treated with mevastatin, Dato-DXd minimally inhibited colony formation (Control; 100%, Dato-DXd; 77.4%). Similar trend was observed in HCC827 cells.
Conclusion: These results suggest that impaired cholesterol metabolism may cause resistance to Dato-DXd in EGFR -mutated NSCLC.
利益披露 Disclosure
K. Yonesaka,
Daiichi-Sankyo Patent.
Y. Kawanaka, None.
T. Kurosaki,
Boehringer Ingelheim ).
Chugai Pharmaceutical Co., Ltd. ).
S. Watanabe, None..
J. Tanizaki, None..
K. Sakai, None..
T. Teramura, None.
K. Nishio,
Nippon Boehringer Ingelheim ).
West Japan Oncology Group ).
Nichirei Biosciences Inc. ).
Eli Lilly Japan ).
Hitachi ).
Sysmex ).
Otsuka Pharmaceutical ).
Thoracic Oncology Research Group ).
Okayama University ).
Japan Breast Cancer Research Group ).
H. Hayashi,
Boehringer Ingelheim ).
IQVIA Service JAPAN KK ).
Eisai Co., Ltd. ).
SYNEOS HEALTH CLINICAL KK ).
Shionogi & Co. Ltd. ).
EP-CRSU Co., Ltd ).
Nippon Kayaku Co.,Ltd. ).
EPS Corporation ).
GlaxoSmithKline KK ).
Otsuka Pharmaceutical ).
Chugai ).
MSD KK ).
Bristol Myers Squibb Company ).
Amgen Inc. ).
SRL Mediserch Inc. ).
Chugai Pharmaceutical Co. Ltd. ).
PRA Health Sciences Inc. ).
Nippon BOehringer Ingelheim CO.,Ltd. ).
CMIC CO., Ltd ).
Janssen Pharmaceutical KK ).