Targeted inhibition of NRF2 reduces the invasive and metastatic ability of HIP1 depleted lung cancer cells.

Background: Non-Small Cell Lung Cancer (NSCLC) presents as a highly metastatic disease with Kras and P53 as prevalent oncogenic driver mutations. Endocytosis, through its role in receptor recycling and enrichment, is important for cancer cell proliferation and metastasis. Huntingtin Interacting Protein 1 (HIP1) is a clathrin mediated endocytic adapter protein found overexpressed in different cancers. However, conflicting roles both as a tumour promoter and suppressor are reported. HIP1 expression is found repressed at advanced stages and some HIP1-ALK fusions are reported in NSCLC patients. However, the molecular mechanisms and implications of HIP1 depletion are not completely understood. Methods: HIP1 depletion was performed using siRNA transient transfection and validated using immunoblotting for each experiment. HIP1 depleted A549 cells were analysed for deregulated global proteome using label-free LC-MS. Gene expression dataset was analysed using TCGA, GTeX and GEO to explore HIP1 expression in Lung cancer patients. Kaplan–Meier Plotter database was used to analyse the survival correlation between HIP1 mRNA expression in lung cancer patients. Various functional assays such as matrigel based invasion, trans-well migration, soft agar colony, tube formation are performed after HIP1 depletion. NRF2 inhibitor was used after HIP1 knockdown to assess its effect on invasion and soft agar colony formation. Results: In silico analysis of HIP1 transcript expression reveals that it is reduced in high-grade and metastatic lung cancer patients correlating with poor survival. Global proteome profiling reveals that HIP1 depleted A549 cells are enriched in pathways associated with metabolism, proliferation and survival. Molecular and functional analysis indicate higher invasive ability of HIP1 depleted cells. The secretome from HIP1 depleted cells also increases the angiogenic potential of HUVEC cells. NRF2 inhibition in HIP1 depleted cells reduces invasion of NSCLC cells with different driver mutations.  Conclusion: Our study shows that HIP1 depletion leads to activation of various molecular pathways responsible for cell proliferation and survival. Additionally, enhancement of invasion in HIP1 depleted subsets of NSCLC cells is via upregulation of NRF2 and can be reversed by its inhibitor.

背景：非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）是一类高转移性疾病，KRAS与P53是其常见的致癌驱动突变。内吞作用通过调控受体回收与富集过程，在癌细胞增殖与转移中发挥关键作用。亨廷顿互作蛋白1（Huntingtin Interacting Protein 1, HIP1）是一种网格蛋白介导的内吞衔接蛋白，在多种癌症中呈过表达状态，但现有研究对其兼具肿瘤促进与肿瘤抑制的双重功能存在争议。HIP1的表达在癌症晚期会被抑制，且有研究在NSCLC患者中报道了部分HIP1-ALK融合基因病例。然而，HIP1缺失的分子机制与生物学意义尚未完全阐明。
方法：本研究通过siRNA瞬时转染构建HIP1敲低模型，并通过免疫印迹对每一次实验进行验证。采用非标记液相色谱-质谱联用（label-free LC-MS）技术，分析HIP1敲低的A549细胞的全局差异蛋白质组表达谱。利用癌症基因组图谱（The Cancer Genome Atlas, TCGA）、基因型-组织表达数据库（Genotype-Tissue Expression, GTeX）及基因表达综合数据库（Gene Expression Omnibus, GEO）的基因表达数据集，探究HIP1在肺癌患者中的表达模式。通过Kaplan-Meier绘图仪数据库，分析肺癌患者HIP1 mRNA表达与生存预后的相关性。在HIP1敲低后，开展多项功能实验，包括基质胶侵袭实验、Transwell迁移实验、软琼脂集落形成实验及管形成实验。使用核因子红细胞2相关因子2（Nuclear factor erythroid 2-related factor 2, NRF2）抑制剂处理HIP1敲低细胞，评估其对癌细胞侵袭及软琼脂集落形成能力的影响。
结果：对HIP1转录表达的生物信息学分析显示，其在高级别与转移性肺癌患者中表达下调，且与不良生存预后显著相关。全局蛋白质组分析结果表明，HIP1敲低的A549细胞富集于与细胞代谢、增殖及存活相关的通路。分子与功能实验证实，HIP1敲低的癌细胞侵袭能力显著增强。HIP1敲低细胞的分泌组可增强人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVEC）的血管生成潜能。在HIP1敲低的、携带不同驱动突变的非小细胞肺癌细胞中抑制NRF2，可降低其侵袭能力。
结论：本研究表明，HIP1缺失可激活多种调控细胞增殖与存活的分子通路。此外，非小细胞肺癌细胞中HIP1缺失介导的侵袭能力增强，依赖于NRF2的上调，且该效应可被NRF2抑制剂逆转。