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.

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.

Liu J ，Edagawa M ，Goshima H ，Inoue M ，Yagita H ，Liu Z ，Kitajima S ... -  《-》

Role of activating transcription factor 3 (ATF3) in endoplasmic reticulum (ER) stress-induced sensitization of p53-deficient human colon cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through up-re

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers cell death upon binding to its ligand, TNF-related apoptosis-inducing ligand (TRAIL), and a combination of TRAIL and agents that increase the expression of DR5 is expected to be a novel anticancer therapy. In this report, we demonstrate that the stress response gene ATF3 is required for endoplasmic reticulum stress-mediated DR5 induction upon zerumbone (ZER) and celecoxib (CCB) in human p53-deficient colorectal cancer cells. Both agents activated PERK-eIF2α kinases and induced the expression of activating transcription factor 4 (ATF4)-CCAAT enhancer-binding protein (C/EBP) homologous protein, which were remarkably suppressed by reactive oxygen species scavengers. In the absence of ATF3, the induction of DR5 mRNA and protein was abrogated significantly, and this was associated with reduced cell death by cotreatment of TRAIL with ZER or CCB. By contrast, exogenous expression of ATF3 caused a more rapid and elevated expression of DR5, resulting in enhanced sensitivity to apoptotic cell death by TRAIL/ZER or TRAIL/CCB. A reporter assay demonstrated that at least two ATF/cAMP response element motifs as well as C/EBP homologous protein motif at the proximal region of the human DR5 gene promoter were required for ZER-induced DR5 gene transcription. Taken together, our results provide novel insights into the role of ATF3 as an essential transcription factor for p53-independent DR5 induction upon both ZER and CCB treatment, and this may be a useful biomarker for TRAIL-based anticancer therapy.

Edagawa M ，Kawauchi J ，Hirata M ，Goshima H ，Inoue M ，Okamoto T ，Murakami A ，Maehara Y ，Kitajima S ... -  《-》

Salermide up-regulates death receptor 5 expression through the ATF4-ATF3-CHOP axis and leads to apoptosis in human cancer cells.

Sirtuins (a class III histone deacetylase) have emerged as novel targets for cancer therapy. Salermide, a reverse amide compound that inhibits Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2), has been shown to induce apoptosis in human cancer cells. The mechanism underlying cellular apoptotic signalling by salermide remains unclear. In this study, we show that salermide up-regulates the expression of death receptor 5 (DR5) in human non-small cell lung cancer (NSCLC) cells. Blocking DR5 expression by gene silencing technology results in a decrease in activated forms of several pro-apoptotic proteins (caspase-8, caspase-9, caspase-3, PARP). Increasing DR5 protein expression correlates with salermide-induced apoptosis in human NSCLC cells. We discovered that IRE-1α, Bip, activating transcription factor 3 (ATF4), activating transcription factor 3 (ATF3) and C/EBP homologous protein (CHOP) are induced by salermide, which suggests that DR5-dependent apoptosis is induced by endoplasmic reticulum stress. Moreover, knockdown of Sirt1 and Sirt2 expression resulted in up-regulation of ATF4, CHOP and DR5. Transfected NSCLC cells with ATF4, ATF3 or CHOP siRNA results in a decline in pro-apoptotic proteins (such as caspase-8, caspase-9, caspase-3 and PARP) despite salermide treatment. We demonstrate that salermide induces expression of ATF4, and ATF4 up-regulates ATF3 and subsequently modulates CHOP. This suggests that DR5 is modulated by the ATF4-ATF3-CHOP axis in NSCLC after Sirt1/2 inhibition or salermide treatment. This study highlights the importance of DR5 up-regulation in apoptosis induced by Sirt1/2 inhibition and elucidates the underlying mechanism in human NSCLC cells.

Liu G ，Su L ，Hao X ，Zhong N ，Zhong D ，Singhal S ，Liu X ... -  《-》

PKCδ regulates death receptor 5 expression induced by PS-341 through ATF4-ATF3/CHOP axis in human lung cancer cells.

PS-341 (bortezomib), a proteasome inhibitor, has been approved for the treatment of multiple myeloma. Our previous work has shown that PS-341 induces death receptor 5 (DR5)-dependent apoptosis and enhances the TNF-related apoptosis-inducing ligand-induced apoptosis in human non-small cell lung cancer cells. However, the definite mechanism remains undefined. In the present study, we reveal that PKCδ and RSK2 mediate PS-341-induced DR5 upregulation, involving coactivation of endoplasmic reticulum (ER) stress. We discovered that PS-341 activated ER stress through elevating the expression of BiP, p-eIF2α, IRE1α, ATF4, ATF3, and CCAAT/enhancer-binding protein homologous protein (CHOP). Further study showed that DR5 upregulation was dependent on ATF4, ATF3, and CHOP expression. Silencing either one of the ATF4, ATF3, and CHOP expression decreased DR5 upregulation and subsequent apoptosis. We determined that ATF4 regulated ATF3 and CHOP expression. Thereafter, ATF3 and CHOP formed a complex and regulated DR5 expression. In addition, we discovered that the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and RSK2 were elevated after PS-341 treatment and inhibition of their phosphorylation using MAP-ERK kinase 1/2 inhibitor decreased the DR5 level, indicating that ERK/RSK2 signaling is involved in DR5 upregulation. Furthermore, we detected the cleavage of PKCδ, and the blockage of PKCδ expression cut down DR5 upregulation and apoptosis. Importantly, knockdown of PKCδ expression decreased the induction of ER stress and the phosphorylation of ERK1/2 and RSK2, suggesting that PKCδ regulates DR5 expression through ERK/RSK2 signaling and ATF4-CHOP/ATF3 axis. Collectively, we show that PS-341 induces PKCδ-dependent DR5 expression through activation of ERK/RSK2 and ER stress signaling pathway.

Xu L ，Su L ，Liu X 《-》

Key role of ATF3 in p53-dependent DR5 induction upon DNA damage of human colon cancer cells.

Taketani K ，Kawauchi J ，Tanaka-Okamoto M ，Ishizaki H ，Tanaka Y ，Sakai T ，Miyoshi J ，Maehara Y ，Kitajima S ... -  《-》