Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models

Paula Olaizola,Rui E Castro,Matthias Evert,Patricia Aspichueta,Diego F Calvisi,Jesper B Andersen,Massimiliano Cadamuro,Francisco J Caballero-Camino,Mikel Azkargorta,Pedro M Rodrigues,Irene Olaizola,Luca Fabris,Colm J O’Rourke,Rocı́o I.r Macı́as ,José J.g Marin ,Marina Serrano‐Maciá ,Pui Yuen Lee ,Maite G Fernández‐Barrena ,Maria Luz Martínez‐Chantar ,Félix Elortza ,María J Perugorria ,Laura Álvarez ,Matı́as A Avila ,Luís Bujanda ,Jesús M Bañales

doi:10.1016/j.jhep.2022.02.007

Abstract

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated invitro, invivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation invitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA invivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells invitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.

Highlights

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignancies emerging at every point of the biliary tree.[1]
The NEDDylation machinery is upregulated in CCA To evaluate whether NEDDylation was dysregulated in CCA, the expression of the activating enzyme NAE1 and the ligand NEDD8, was analyzed in human resected CCA and control tissues [i.e., surrounding liver or normal bile ducts (NBD)] in 5 different cohorts of patients (i.e., Copenhagen, The Cancer Genome Atlas (TCGA), TIGER-LC, Job and Regensburg)
Laser microdissection studies on CCA tumor samples from the Copenhagen cohort revealed increased NAE1 expression in CCA epithelia compared to matched tumor stroma [mainly formed by cancer-associated fibroblast (CAF), immune cells such as tumor-associated macrophages (TAMs), and endothelial cells] (Figure 1C)

Summary

Introduction

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignancies emerging at every point of the biliary tree.[1]. Journal Pre-proof at advanced phases when symptoms associated with biliary obstruction arise This situation severely compromises the access to potential curative options, mainly based on surgery.[1] the high risk of tumor recurrence and the elevated chemoresistance of CCAs contribute to patients’ poor prognosis. Journal Pre-proof (NEDDylation inhibitor) or CRISPR/Cas against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation impairment in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, hampering CAF proliferation and migration.

Methods

Results

Conclusion