PO.MCB07.04 · 分子与细胞生物学
癌细胞系中敲除NRG1可降低其侵袭性
NRG1 knock out in cancer cell lines decreases their aggressiveness
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言 神经调节蛋白-1（NRG1）是一种生长因子，可结合ErbB/人表皮生长因子受体（HERs），尤其是HER3，并激活下游信号通路。NRG1基因有可能与多种基因伴侣形成致癌性基因融合，从而导致癌症发生。目前存在若干或多或少直接靶向NRG1信号通路的治疗方法，但患者往往对治疗反应不佳或迅速产生耐药。更好地理解NRG1依赖性和NRG1融合依赖性的细胞生物学，可使我们为这些患者提出新的治疗选择。在此，我们的目的是研究多种癌细胞系（有或无NRG1基因改变）中NRG1依赖性的细胞生物学参数。
方法 在高表达野生型NRG1的A549和SW1573非小细胞肺癌（NSCLC）细胞系、具有NRG1扩增的HCC95 NSCLC细胞系，以及携带复杂PPP6R3-TENM4-NRG1融合的MDA-MB-175乳腺癌细胞系中，通过基于CRISPR-Cas9的方法获得NRG1缺失。将修饰后的细胞系培养为多克隆和/或单克隆群体，并使用经典的细胞生物学技术进行表征：如western blot和流式细胞术监测蛋白表达，融合度实验和Cell-Trace CFSE研究增殖能力，划痕愈合实验和Boyden小室监测迁移能力，以及软琼脂和克隆形成实验研究干性特征。
结果 NRG1缺陷型A549克隆（n=5）聚为两个不同的行为组。其中一组表现出HER3表达丧失、对afatinib的敏感性增加3倍，以及迁移能力下降最多达2倍。而另一组则出现间充质标志物的诱导表达和FGFR1表达丧失。对于其他参数，所有A549克隆的表现与对照克隆相似。NRG1缺陷型HCC95（n=2）的初步结果提示其增殖能力略有下降，而NRG1缺陷型MDA-MB-175（n=2）对包括afatinib在内的多种治疗的敏感性升高2倍。SW1573细胞系的所有克隆（n=4）在所有实验中均未表现出差异。
结论 总体而言，NRG1缺陷以细胞特异性的方式降低了癌细胞的侵袭性。即将进行的对我们不同模型的转录组学实验将使我们更好地洞察与NRG1相关的分子改变。对所有模型的进一步分析正在进行中（MDA-MB-175和HCC95的克隆、3D生物打印实验、体内肿瘤生长⋯），结果将纳入海报中。
查看英文原文 English abstract
Introduction Neuregulin-1 (NRG1) is a growth factor which can bind to the ErbB/human epidermal growth factor receptors (HERs), especially to HER3, and activate the downstream signalling pathways. The NRG1 gene has the potential to form oncogenic gene fusions with diverse gene partners leading to the emergence of cancer. Several therapeutic approaches targeting more or less directly NRG1 's signalling pathway exist, but patients tend to respond poorly to treatments or rapidly develop resistances. A better understanding of the NRG1- and NRG1 -fusion-dependent cell biology could allow us to propose new treatment options to these patients. Here our objective is to study NRG1-dependent cell biology parameters in various cancer cell lines, with or without genetic alterations of NRG1.
Methods A CRISPR-Cas9-based deletion of NRG1 was obtained in the A549 and SW1573 Non-Small Cell Lung Cancer (NSCLC) cell lines which highly express wild type NRG1, in the HCC95 NSCLC cell line which has an amplification of NRG1 , and in the MDA-MB-175 breast cancer cell line which bears a complex PPP6R3-TENM4-NRG1 fusion. The modified cell lines were grown as either or both polyclonal and monoclonal populations and were characterized using classic cell biology techniques such as western blot and flow cytometry to monitor protein expression, confluence assay and Cell-Trace CFSE to study proliferative capacities, scratch wound assay and Boyden chamber to monitor migratory capacities, and soft agar and clonogenic assay to study stemness characteristics.
Results The NRG1-deficient A549 clones (n=5) clustered into two distinct behavioural groups. One of them showed a loss of HER3 expression, a 3-fold increased sensitivity to afatinib, and up to 2-fold decreased migratory capacities. As for the other group, there was an induced expression of mesenchymal markers and a loss of FGFR1 expression. For other parameters, all A549 clones behaved in a similar manner as control clones. Preliminary results in NRG1-deficient HCC95 (n=2) suggest they have slightly reduced proliferative capacities and NRG1-deficient MDA-MB-175 (n=2) have a 2-fold higher sensitivity to several treatments including afatinib. All clones for the SW1573 cell line (n=4) exhibited no difference within all performed experiments.
Conclusion Overall, NRG1-deficiency decreases cancer cells' aggressiveness, although in a cell-specific manner. Upcoming transcriptomic assay of our different models will give us a better insight in NRG1-related molecular modifications. Further analyses of all models are ongoing (clones of MDA-MB-175 and HCC95, 3D bioprinting experiments, in vivo tumour growth⋯) and results will be included in the poster.
利益披露 Disclosure
M. Issenmann, None..
C. Thiollier-Schmitt, None..
M. Barre, None..
E. Cros-Perrial, None.
M. Duruisseaux,
Pfizer g., Board of Directors, non-salaried role), ), Other, Remuneration for participation in scientific meetings.
Merus ).
Takeda g., Board of Directors, non-salaried role), ).
Guardant g., Board of Directors, non-salaried role), ), Other, Remuneration for participation in scientific meetings.
Eli Lilly ).
Boehringer Ingelheim g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Astra Zeneca g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Roche g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
BMS g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Abbvie g., Board of Directors, non-salaried role).
Novartis g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
GSK g., Board of Directors, non-salaried role).
Sanofi g., Board of Directors, non-salaried role).
Amgen g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Regeneron g., Board of Directors, non-salaried role).
Revolution Medecine g., Board of Directors, non-salaried role).
Novocure g., Board of Directors, non-salaried role).
MSD g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
L. Jordheim, None.