Role of ATF3 in synergistic cancer cell killing by a combination of HDAC inhibitors and agonistic anti-DR5 antibody through ER stress in human colon cancer cells

Highlights

• Six different HDACIs upregulate DR5 by ER stress/UPR pathway in colon cancer cells.

• Co-treatment with HDACIs and DJR2-2 sensitizes cancer cells to apoptosis.

• Pro-apoptotic genes ATF3 and CHOP are required to activate DR5 transcription.

• ATF3 plays role in TRAIL-based cell death by HDACIs.

Abstract

Histone deacetylase inhibitors (HDACIs) are promising agents for cancer therapy. However, the mechanism(s) responsible for the efficacy of HDACIs have not yet to be fully elucidated. Death receptor 5 (DR5) is a transmembrane receptor containing death domain that triggers cell death upon binding to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or agonistic anti-DR5 monoclonal antibody, and the combination of TRAIL/agonistic anti-DR5 monoclonal antibody and agents that increase the expression of DR5 is expected as a novel anticancer therapeutic strategy. Here we report that six different HDACIs activated endoplasmic reticulum (ER) stress sensor PERK and eIF2α and induced the ATF4/ATF3/CHOP pathway in p53-deficient human colon cancer cells. This resulted in an increased expression of DR5 on the cell surface and sensitized cells to apoptosis by agonistic anti-DR5 monoclonal antibody. Stress response gene ATF3 was required for efficient DR5 induction by HDACIs, and DR5 reporter assay showed that ATF3 play crucial role for the HDACIs-induced activation of DR5 gene transcription. These provide important mechanistic insight into how HDACIs exhibit pro-apoptotic activity in clinical anti-cancer treatments when they are used in combination with other therapeutic strategies.

Introduction

Histone deacetylase inhibitors (HDACIs) are a class of targeted therapeutics with promising anti-tumor efficacy that reverse aberrant epigenetic changes associated with cancer. HDACIs induce hyper-acetylation of DNA-bound histones or sequence-specific transcription factors, which can either increase or decrease the transcription of cancer-related genes [1], [2], [3]. It has also been shown that the treatment with HDACIs results in hyper-acetylation of chaperones such as HSP90, HSP70 and HSP40, which impairs the chaperone activity and leads to the accumulation of unfolded or misfolded proteins in endoplasmic reticulum (ER) [3], [4], [5]. This causes the activation of a specific cellular process called the unfolded protein response (UPR), which is mediated through at least three major pathways initiated by IRE1, PERK or ATF6 respectively, coordinating a temporal shut-down of protein translation and a complex gene transcriptional program to safeguard cell survival. Upon excessive or prolonged UPR, however, apoptosis is activated to eliminate faulty cells [6], [7].

To date, several structurally distinct HDACIs have been developed and are classified into six groups, including short-chain fatty acids, hydroxamic acids, cyclic peptides, benzamides, electrophilic ketones, and hybrid molecules [8]_. At present, an increasing number of HDACIs have been reported as candidate for stand-alone therapeutics or combination therapy with other agents. For instance, suberoylanilide hydroxamic acid (SAHA, also called Vorinostat) is a broad set and non-selective inhibitor of HDACs. After its approval for the treatment of cutaneous T cell lymphoma [9], SAHA is trying to find its value for solid tumors including thyroid cancer [10], breast cancer [11] and mesothelioma [12]. In colon cancer, it is reported that SAHA can induce sub-G1 arrest and apoptosis [13], [14].

Death receptor 5 (DR5) belongs to the TNF receptor family. Upon binding of its ligand TRAIL or agonistic antibody, DR5 mediates TRAIL-induced apoptosis through the formation of a death-inducing signaling complex [15]. DR5 is expressed more strongly in cancer cells than in normal cells [16], and mediates apoptosis selectively in cancer cells both in vitro and in vivo, with little or no effect on normal cells. However, clinical trials have shown that small therapeutic effects are observed when TRAIL or TRAIL agonistic monoclonal antibodies specific for DR5, such as DJR2-2 [17], [18] are used as monotherapy. Thus, it is required to maximize the therapeutic efficacy through drug combination that synergize with TRAIL-based agents. It has been shown that some HDACIs such as Butyrate may increase the expression of DR5 through Sp1 in colon cancer cells [19]. However, it is unclear whether there are other mechanisms at work as well.

Stress response gene ATF3 is a member of the ATF/CREB family. Its messenger RNA (mRNA) level is low or undetectable, but is greatly induced upon exposure of cells to a variety of stress signals including DNA damage, oxidative stress, and cytokines and growth factors [20]. We have previously shown that ATF3 plays a role in p53-dependent DR5 induction upon DNA damage of human colorectal cancer cells [21]. More recently, Wilson et al. have reported that HDACIs sensitized colon cancer cells to apoptosis by a Sp1/Sp3-dependent transcriptional program [22]. However, it still remains elusive whether ATF3 plays role in the transcriptional networks or cell death provoked by HDACIs in human colon cancer cells.

The present study reports that at least six structurally different HDAC inhibitors caused ER stress and activated the PERK-eIF2α pathway of the UPR, leading to up-regulation of DR5 expression on the cell surface of p53-deficient human colon cancer cells. Combined treatment with HDACIs and anti-DR5 monoclonal antibody synergistically sensitized cancer cells to apoptotic cell death. Functional role of ATF3 in HDACIs-induced DR5 gene transcription and cell death was investigated.