The miR-27a-calreticulin axis affects drug-induced immunogenic cell death in human colorectal cancer cells

T Colangelo,G Mazzoccoli,M Rita Milone,G Matarese,M Galgani,M Santopaolo,V Colantuoni,L Sabatino,B Pucci,L Muccillo,A Budillon,G Polcaro,P Ziccardi

doi:10.1038/cddis.2016.29

Abstract

Immunogenic cell death (ICD) evoked by chemotherapeutic agents implies emission of selected damage-associated molecular patterns (DAMP) such as cell surface exposure of calreticulin, secretion of ATP and HMGB1. We sought to verify whether miR-27a is implicated in ICD, having demonstrated that it directly targets calreticulin. To this goal, we exposed colorectal cancer cell lines, genetically modified to express high or low miR-27a levels, to two bona fide ICD inducers (mitoxantrone and oxaliplatin). Low miR-27a-expressing cells displayed more ecto-calreticulin on the cell surface and increased ATP and HMGB1 secretion than high miR-27a-expressing ones in time-course experiments upon drug exposure. A calreticulin target protector counteracted the miR-27a effects while specific siRNAs mimicked them, confirming the results reported. In addition, miR-27a negatively influenced the PERK-mediated route and the late PI3K-dependent secretory step of the unfolded protein response to endoplasmic reticulum stress, suggesting that miR-27a modulates the entire ICD program. Interestingly, upon chemotherapeutic exposure, low miR-27a levels associated with an earlier and stronger induction of apoptosis and with morphological and molecular features of autophagy. Remarkably, in ex vivo setting, under the same chemotherapeutic induction, the conditioned media from high miR-27a-expressing cells impeded dendritic cell maturation while increased the secretion of specific cytokines (interleukin (IL)-4, IL-6, IL-8) and negatively influenced CD4+ T-cell interferon γ production and proliferation, all markers of a tumor immunoevasion strategy. In conclusion, we provide the first evidence that miR-27a impairs the cell response to drug-induced ICD through the regulatory axis with calreticulin.

Highlights

We have identified miR-27a as one of the upregulated miRNAs in Colorectal cancer (CRC) and proved that it acts as an oncomiRNA as it is already overexpressed in adenomas and further increases during tumor progression.[16] miR-27a is a member of the miR-23a, miR-27a and miR-24-2 cluster and is acquiring a growing regulatory role because it is implicated in multiple processes including tumorigenesis.[17,18]
Having shown that calreticulin is a target of miR-27a16 and given its pivotal role in Immunogenic cell death (ICD), we assessed the effects of miR-27a on ecto-calreticulin, ATP and HMGB1 release (i.e., damage-associated molecular patterns (DAMP) emission) from dying cells undergoing anticancer drug-induced ICD.[4,6,10,19]
This conceptual change stems from the observation that apoptotic cancer cells treated with these chemotherapeutics or radiotherapy release a series of DAMPs that could provoke an immune response.[8,9,10,34]

Summary

Introduction

Received 22.12.15; accepted 05.1.16; Edited by G Melino miR-27a influences immunogenic cell death T Colangelo et al calreticulin is gaining interest because of its pleiotropic functions: it acts, as an ER chaperone, is implicated in the protein-loading complex to assemble the mature MHC class I molecules on the cell surface, activates the apoptotic pathway and, upon ICD inducer administration, translocates to the cell surface where it acts as an ‘eat me’ signal to mount an efficient immune response.

Methods

Results

Conclusion