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

Abstract

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. Gene expression dataset from TCGA, GTEX and GEO databases was analysed 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. HIP1 depleted A549 cells were analysed for deregulated global proteome using label-free LC-MS and this data is available via ProteomeXchange with identifier PXD054307. Various functional assays such as matrigel based invasion, trans-well migration, soft agar colony and angiogenesis tube formation were 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 significantly reverses invasion of HIP1 depleted 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 and anchorage-independent growth in HIP1 depleted subsets of NSCLC cells is via upregulation of NRF2 and can be reversed by its inhibitor.

Keywords: HIP1; Invasion; NRF2; NSCLC.