PO.ET01.06 · 实验与分子治疗
CDK4/6抑制性和抗缺氧miRNA-6883脂质纳米颗粒与铁死亡诱导剂或MEK抑制剂联合应用于乳腺癌和结直肠癌临床前模型
Combination of CDK4/6 inhibitory and anti-hypoxia miRNA-6883 lipid nanoparticles with ferroptosis inducers or MEK inhibitors in preclinical breast and colorectal cancer models
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摘要 Abstract
中文摘要
引言
MicroRNA 6883-5p（miR-6883）通过沉默细胞周期蛋白依赖性激酶CDK4/6，诱导下游缺氧诱导因子1alpha（HIF1alpha）降解，从而抑制癌症增殖和缺氧信号。先前的研究表明，CDK4/6抑制（CDK4/6i）通过E3泛素连接酶SMURF2以VHL非依赖性机制诱导HIF1alpha降解。值得注意的是，多项临床前研究报道了CDK4/6i与MEK抑制（MEKi）之间的协同作用。另有研究报道CDK4/6i使癌细胞对铁死亡诱导剂敏感，尽管这一观察结果尚未得到明确支持，且可能具有背景依赖性。在此，我们使用体外实验和体内小鼠异种移植模型，研究miR-6883脂质纳米颗粒（LNP）与MEKi或铁死亡诱导剂的联合应用。
方法
将缺氧反应元件（HRE）驱动的报告基因（Addgene #118706）转导入HCT116结直肠癌和SK-BR-3 HER2+乳腺癌（BC）细胞。在体内实验中，将200万个HCT116-HRE细胞皮下注射至裸鼠，对肿瘤≥150mm3者给予0、1或10μg LNP包封的miRNA治疗。在活力实验中，每孔接种4,000个细胞并使其贴壁过夜。治疗后48-72小时，通过CellTiter-Glo®评估活力。体外缺氧实验在0.5% O2缺氧工作站中进行。
结果
作为单一药物，miR-6883 LNP在HCT116-HRE异种移植模型中显著减弱HIF信号，与基线相比，1μg miR-6883治疗后3天观察到发光减少94.3%。10μg剂量显著降低Ki67，支持miR-6883的抗增殖活性。在体外，SK-BR-3-HRE模型在0.5% O2下经miR-6883治疗后HIF活性显著降低，提示miR-6883的抗缺氧效应可延伸至BC。体外活力实验在HCT116（结直肠）、SK-BR-3（HER2+）、MCF-7（ER+）、MDA-MB-231（三阴性）和MDA-MB-468（三阴性）中进行。小分子CDK4/6i与铁死亡诱导剂（RSL3或erastin）或MEKi（trametinib）联合应用显示出大体上为正的协同评分。相应地，miR-6883 LNP使细胞对单药无效剂量的RSL3、erastin或trametinib敏感。
结论
我们的数据支持miR-6883的抗缺氧和抗增殖活性，并为其与铁死亡诱导剂或MEKi的联合治疗提供了依据。未来的实验将采用BC异种移植模型，并评估联合治疗对肿瘤生长的长期抑制作用。
查看英文原文 English abstract
Introduction
MicroRNA 6883-5p (miR-6883) inhibits cancer proliferation and hypoxia signaling by silencing the cyclin-dependent kinases CDK4/6, inducing downstream degradation of Hypoxia-Inducible Factor 1alpha (HIF1alpha). Prior work demonstrated that CDK4/6 inhibition (CDK4/6i) induces HIF1alpha degradation through a VHL-independent mechanism via the E3 ubiquitin ligase SMURF2. Notably, several preclinical studies have reported synergy between CDK4/6i and MEK inhibition (MEKi). Others have reported that CDK4/6i sensitizes cancer cells to ferroptosis inducing agents, though this observation is not unequivocally supported and may be context-dependent. Here, we investigate combinations of miR-6883 lipid nanoparticles (LNPs) with MEKi or ferroptosis inducers using in vitro assays and in vivo murine xenograft models.
Methods
A hypoxia response element (HRE)-driven reporter (Addgene #118706) was transduced into HCT116 colorectal carcinoma and SK-BR-3 HER2+ breast cancer (BC) cells. For in vivo experiments, 2 million HCT116-HRE cells were injected subcutaneously into nude mice and tumors ≥150mm3 were treated with 0, 1, or 10µg LNP-encapsulated miRNA. For viability assays, 4,000 cells/well were plated and allowed to adhere overnight. 48-72h after treatment, viability was assessed by CellTiter-Glo®. In vitro hypoxia experiments were conducted in a 0.5% O2 hypoxia workstation.
Results
As a single agent, miR-6883 LNPs significantly attenuated HIF signal in the HCT116-HRE xenograft model, with a 94.3% reduction in luminescence observed 3 days post-treatment with 1µg miR-6883 compared to baseline. Ki67 was significantly reduced with a 10µg dose, supporting the antiproliferative activity of miR-6883. In vitro, the SK-BR-3-HRE model demonstrated a significant reduction in HIF activity with miR-6883 treatment under 0.5% O 2 , suggesting that the anti-hypoxia effect of miR-6883 extends to BC. In vitro viability assays were conducted in HCT116 (colorectal), SK-BR-3 (HER2+), MCF-7 (ER+), MDA-MB-231 (triple-negative), and MDA-MB-468 (triple-negative). Combination of small-molecule CDK4/6i and ferroptosis inducers (RSL3 or erastin) or MEKi (trametinib) revealed largely positive synergy scores. Accordingly, miR-6883 LNPs sensitized cells to doses of RSL3, erastin, or trametinib that were ineffective as monotherapies.
Conclusions
Our data support the anti-hypoxia and antiproliferative activity of miR-6883, and provide a basis for combination therapies with ferroptosis inducers or MEKi. Future experiments will employ BC xenografts and assess long-term tumor growth inhibition of combination therapies.
利益披露 Disclosure
C. Purcell, None..
S. Holmes-Farley, None..
A. Sidonia, None..
A. Raissi, None..
M. Barrette, None.
E. Youssef,
SMURF Therapeutics g., Board of Directors, non-salaried role).
Kapadi g., Board of Directors, non-salaried role).
T. M. Raimondo, None.
W. S. El-Deiry,
Oncoceutics/Chimerix Stock.
p53 Therapeutics Stock.
Caris Life Sciences g., Board of Directors, non-salaried role).
Ocean Biomedical g., Board of Directors, non-salaried role).
Jazz Pharmaceuticals Stock.
Global Cancer Technology Stock.
ACS BrightEdge g., Board of Directors, non-salaried role).
Resurrect Therapeutics g., Board of Directors, non-salaried role).
WIN Consortium g., Board of Directors, non-salaried role).
SMURF Therapeutics g., Board of Directors, non-salaried role), Stock.