Key necroptotic proteins are required for Smac mimetic-mediated sensitization of cholangiocarcinoma cells to TNF-α and chemotherapeutic gemcitabine-induced necroptosis

Abstract

Cholangiocarcinoma (CCA), a malignant tumor originating in the biliary tract, is well known to be associated with adverse clinical outcomes and high mortality rates due to the lack of effective therapy. Evasion of apoptosis is considered a key contributor to therapeutic success and chemotherapy resistance in CCA, highlighting the need for novel therapeutic strategies. In this study, we demonstrated that the induction of necroptosis, a novel regulated form of necrosis, could potentially serve as a novel therapeutic approach for CCA patients. The RNA sequencing data in The Cancer Genome Atlas (TCGA) database were analyzed and revealed that both receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), two essential mediators of necroptosis, were upregulated in CCA tissues when compared with the levels in normal bile ducts. We demonstrated in a panel of CCA cell lines that RIPK3 was differentially expressed in CCA cell lines, while MLKL was more highly expressed in CCA cell lines than in nontumor cholangiocytes. We therefore showed that treatment with both tumor necrosis factor-α (TNF-α) and Smac mimetic, an inhibitor of apoptosis protein (IAP) antagonist, induced RIPK1/RIPK3/MLKLdependent necroptosis in CCA cells when caspases were blocked. The necroptotic induction in a panel of CCA cells was correlated with RIPK3 expression. Intriguingly, we demonstrated that Smac mimetic sensitized CCA cells to a low dose of standard chemotherapy, gemcitabine, and induced necroptosis in an RIPK1/RIPK3/MLKL-dependent manner upon caspase inhibition but not in nontumor cholangiocytes. We further demonstrated that Smac mimetic and gemcitabine synergistically induced an increase in TNF-α mRNA levels and that Smac mimetic reversed gemcitabine-induced cell cycle arrest, leading to cell killing. Collectively, our present study demonstrated that TNF-α and gemcitabine induced RIPK1/ RIPK3/MLKL-dependent necroptosis upon IAP depletion and caspase inhibition; therefore,

Necroptosis in Cholangiocarcinoma

“…Only two studies have investigated the efficacy of different drugs in limiting CCA development by inducing necroptosis in CCA cell lines. Akara-amornthum et al demonstrated that treatment with both TNFα and a Smac mimetic induced RIPK1/RIPK3/MLKL-dependent necroptosis when caspase was blocked, evidenced by an increased expression of RIPK3 and MLKL in CCA cell lines after treatment [122]. They also proved that the administration of a Smac mimetic could sensitize CCA cells to a low dose of standard chemotherapy with gemcitabine, by increasing TNFα mRNA levels and reversing gemcitabine-induced cell cycle arrest, leading to CD [122].…”

“…A widely adopted approach for killing cancer cells is to activate apoptosis, and evasion from this type of CD is considered a key step to therapeutic failure and chemotherapy resistance in CCA [122], as reported above. A switch to a non-apoptotic CD, such as necroptosis, can function as a backup system in apoptosis-resistant cells, so antitumor drugs inducing non-apoptotic CD are now considered a new approach for overcoming such a big obstacle in CCA treatment [0].…”

“…Akara-amornthum et al demonstrated that treatment with both TNFα and a Smac mimetic induced RIPK1/RIPK3/MLKL-dependent necroptosis when caspase was blocked, evidenced by an increased expression of RIPK3 and MLKL in CCA cell lines after treatment [122]. They also proved that the administration of a Smac mimetic could sensitize CCA cells to a low dose of standard chemotherapy with gemcitabine, by increasing TNFα mRNA levels and reversing gemcitabine-induced cell cycle arrest, leading to CD [122]. Xu et al reported, for the first time, that matrine, an alkaloid isolated from traditional Chinese medicine Sophora flavescens, induced necroptosis in CCA cell lines, differing from its classical role of inducing apoptosis in many other types of cancer cells [123].…”

A role for TNF-α in alveolar macrophage damage-associated molecular pattern release

“…Second mitochondria-derived activator of caspase (Smac) is a mitochondrial protein that inhibits cellular inhibitors of apoptotic proteins if released into the cytosol, leading to activation of apoptotic caspases, activation of RIP1K, and initiation of apoptosis (39). When caspase activity is inhibited, as occurs in certain infections or tumor cells, Smac and Smac mimetics promote necroptosis (40)(41)(42). We tested whether the Smac mimetic CUDC-427 induced necroptosis of AMs in the presence of the pancaspase inhibitor Z-VAD-FMK (SZ).…”

Receptor-interacting protein kinase 1 is a key mediator in TLR3 ligand and Smac mimetic-induced cell death and suppresses TLR3 ligand-promoted invasion in cholangiocarcinoma

“…Necroptosis has been reported to enhance anti-tumor immunity in colon cancer and melanoma [ 41 , 42 ] and RIPK3 expression status is proposed to influence the clinical outcome of TLR3-based cancer immunotherapy [ 45 ]. The loss of key necroptotic proteins in cancers has become a major hindrance for necroptosis-based therapy [ 73 , 74 ] but results of our recent studies in CCA patients demonstrated that RIPK3 and MLKL were both expressed in a great majority of CCA patients, allowing for the possible development of necroptosis-based therapeutic approaches [ 75 ]. Collectively, these results provide a potential for development of a novel therapeutic approach targeting TLR3 by TLR3 ligands in combination with Smac mimetic that can trigger both apoptosis and necroptosis in a RIPK1-dependent manner.…”