Knockdown of Atg7 Induces Nuclear-LC3 Dependent Apoptosis and Augments Chemotherapy in Colorectal Cancer Cells.

Scherr Scherr,Gdynia Gdynia,Jassowicz Jassowicz,Goeppert Goeppert,Jäger Jäger,Köhler Köhler,Pató Pató,Elssner Elssner,Hoffmeister Hoffmeister,Ismail Ismail,Schmitt Schmitt,Neukirch Neukirch,Schulze-Bergkamen Schulze-Bergkamen

doi:10.3390/ijms21031099

Abstract

Autophagy is a catabolic process that enables cells to degrade obsolete content and refuel energy depots. In colorectal cancer (CRC) autophagy has been shown to promote tumorigenesis through energy delivery in the condition of uncontrolled proliferation. With this study, we aimed at evaluating whether autophagy sustains CRC cell viability and if it impacts therapy resistance. Initially, a colorectal cancer tissue micro array, containing mucosa (n = 10), adenoma (n = 18) and adenocarcinoma (n = 49) spots, was stained for expression of essential autophagy proteins LC3b, Atg7, p62 and Beclin-1. Subsequently, central autophagy proteins were downregulated in CRC cells using siRNA technology. Viability assays, flow cytometry and immunoblotting were performed and three-dimensional cell culture was utilized to study autophagy in a tissue mimicking environment. In our study we found an upregulation of Atg7 in CRC. Furthermore, we identified Atg7 as crucial factor within the autophagy network for CRC cell viability. Its disruption induced cell death via triggering apoptosis and in combination with conventional chemotherapy it exerted synergistic effects in inducing CRC cell death. Cell death was strictly dependent on nuclear LC3b, since simultaneous knockdown of Atg7 and LC3b completely restored viability. This study unravels a novel cell death preventing function of Atg7 in interaction with LC3b, thereby unmasking a promising therapeutic target in CRC.

Highlights

Colorectal cancer (CRC) is the third most common malignant neoplasia in humans [1,2]
In the tissue microarray (TMA), Atg7 expression was found to be significantly upregulated (p < 0.01; Figure 1a), whereas Beclin-1 expression was significantly decreased in adenocarcinomas compared to normal mucosa (p < 0.001, Figure 1a)
In order to evaluate whether the expression levels of key autophagic proteins correlate with the amount of Atg7, tissue spots were assigned to three groups (Atg7 low: ≤4; medium: ≤8; high: >8), based on their IHC score

Summary

Introduction

Colorectal cancer (CRC) is the third most common malignant neoplasia in humans [1,2]. Despite tremendous progress in therapeutic approaches, patients with distant organ metastases still face a poor prognosis. Autophagy is an evolutionary highly conserved process by which cells refuel energy [4]. In situations of energy deprivation, autophagy enables cells to digest unnecessary organelles and misfolded proteins in order to maintain their essential metabolism. It has been shown that autophagy contributes to the onset and progression of a variety of diseases, including cancer [5]. There is growing evidence for the dependency of cancer cells on autophagy for outgrowth and metastatic spread, indicating a tumor promoting role [6,7]. In the context of CRC, autophagy enhances the resistance towards nutrient deprivation, thereby driving tumorigenicity [8]

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