Cellular Inhibitor Of Apoptosis Research Articles

Rsf‑1 regulates malignant melanoma cell viability and chemoresistance via NF‑κB/Bcl‑2 signaling.

Remodeling and spacing factor 1 (Rsf-1) has been reported as overexpressed in numerous cancers; however, its expression, biological functions and mechanisms in malignant melanoma remain unknown. In the present study, the expression of Rsf-1 was investigated in 50 cases of malignant melanoma samples using immunohistochemistry. The results revealed that Rsf-1 expression was elevated in 38% of specimens. MTT, colony formation, Transwell and flow cytometry assays were performed to investigate the functions of Rsf-1. Knockdown of Rsf-1 in the MV3 and A375 melanoma cell lines decreased the viability, invasion and cell cycle transition of cells. Conversely, overexpression of Rsf-1 in M14 cells with low endogenous Rsf-1 expression induced opposing effects. Further analysis revealed that Rsf-1 knockdown decreased matrix metalloproteinase-2, cyclin E and phosphorylated-IκB expression. Additionally, Rsf-1 depletion reduced cisplatin resistance and significantly increased the cisplatin-associated apoptotic rate, whereas Rsf-1 overexpression exhibited opposing effects. Rsf-1 also maintained the mitochondrial membrane potential following cisplatin treatment. Analysis of apoptosis-associated proteins revealed that Rsf-1 positively regulated B-cell lymphoma 2 (Bcl-2), cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2, and downregulated Bcl-2-associated X protein expression. Nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB) inhibition reversed the effects of Rsf-1 on Bcl-2. In conclusion, Rsf-1 was overexpressed in malignant melanoma and may contribute to the malignant behaviors of melanoma cells, possibly via the regulation of NF-κB signaling. Therefore, Rsf-1 may be a potential therapeutic target in the treatment of malignant melanoma.

SM‑164 enhances the antitumor activity of adriamycin in human U2‑OS cells via downregulation of X‑linked inhibitor of apoptosis protein.

The antitumor effects of SM-164 and adriamycin (ADM) on human osteosarcoma U2-OS cells, the underlying mechanism are yet to be investigated. In the present study, U2-OS cells were divided into control, ADM, SM-164, and ADM + SM-164 groups. In addition, cells treated with both SM-164 and ADM were further divided into three subgroups: SM-164 + ADM, SM-164 + ADM + vector and SM-164 + ADM + X-linked inhibitor of apoptosis protein (XIAP) silencing groups. XIAP expression was achieved via transfection with shRNA lentiviral vectors. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the expression of caspases-7, −9, and −3, poly ADP-ribose polymerase (PARP), XIAP, cellular inhibitor of apoptosis protein-1 (cIAP-1) and survivin. Cell viability and apoptosis were evaluated using MTT and flow cytometry assays, respectively. Compared with the control group, cell viability decreased, while apoptosis was increased in the ADM and SM-164-treatment group. ADM and SM-164 treatment promoted the expression of caspases-7, −9 and −3, and PARP, but reduced the expression of XIAP, survivin and cIAP-1. Compared with ADM + SM-164 group, XIAP silencing with ADM + SM-164 treatment further reduced cell viability, promoted apoptosis, increased caspase-7, −9 and −3, and PARP expression; however the expression of survivin and cIAP-1 were reduced. Combined ADM and SM-164 treatment may be considered as potential therapeutic agent in the treatment of osteosarcoma, possibly via reductions XIAP expression.

Smac mimetic suppresses tunicamycin-induced apoptosis via resolution of ER stress

Since Inhibitor of Apoptosis (IAP) proteins have been implicated in cellular adaptation to endoplasmic reticulum (ER) stress, we investigated the regulation of ER stress-induced apoptosis by small-molecule second mitochondria-derived activator of caspase (Smac) mimetics that antagonize IAP proteins. Here, we discover that Smac mimetic suppresses tunicamycin (TM)-induced apoptosis via resolution of the unfolded protein response (UPR) and ER stress. Smac mimetics such as BV6 selectively inhibit apoptosis triggered by pharmacological or genetic inhibition of protein N-glycosylation using TM or knockdown of DPAGT1, the enzyme that catalyzes the first step of protein N-glycosylation. In contrast, BV6 does not rescue cell death induced by other typical ER stressors (i.e., thapsigargin (TG), dithiothreitol, brefeldin A, bortezomib, or 2-deoxyglucose). The protection from TM-triggered apoptosis is found for structurally different Smac mimetics and for genetic knockdown of cellular IAP (cIAP) proteins in several cancer types, underlining the broader relevance. Interestingly, lectin microarray profiling reveals that BV6 counteracts TM-imposed inhibition of protein glycosylation. BV6 consistently abolishes TM-stimulated accumulation of ER stress markers such as glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) and reduces protein kinase RNA-like ER kinase (PERK) phosphorylation and X box-binding protein 1 (XBP1) splicing upon TM treatment. BV6-stimulated activation of nuclear factor-κB (NF-κB) contributes to the resolution of ER stress, since NF-κB inhibition by overexpression of dominant-negative IκBα superrepressor counteracts the suppression of TM-stimulated transcriptional activation of CHOP and GRP78 by BV6. Thus, our study is the first to show that Smac mimetic protects from TM-triggered apoptosis by resolving the UPR and ER stress. This provides new insights into the regulation of cellular stress responses by Smac mimetics.

E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding.

The cellular inhibitor of apoptosis 1 (cIAP1) is an E3-ubiquitin ligase that regulates cell signaling pathways involved in fundamental cellular processes including cell death, cell proliferation, cell differentiation and inflammation. It recruits ubiquitination substrates thanks to the presence of three baculoviral IAP repeat (BIR) domains at its N-terminal extremity. We previously demonstrated that cIAP1 promoted the ubiquitination of the E2 factor 1 (E2F1) transcription factor. Moreover, we showed that cIAP1 was required for E2F1 stabilization during the S phase of cell cycle and in response to DNA damage. Here, we report that E2F1 binds within the cIAP1 BIR3 domain. The BIR3 contains a surface hydrophobic groove that specifically anchors a conserved IAP binding motif (IBM) found in a number of intracellular proteins including Smac. The Smac N-7 peptide that includes the IBM, as well as a Smac mimetic, competed with E2F1 for interaction with cIAP1 demonstrating the importance of the BIR surface hydrophobic groove. We demonstrated that the first alpha-helix of BIR3 was required for E2F1 binding, as well as for the binding of Smac and Smac mimetics. Overexpression of cIAP1 modified the ubiquitination profile of E2F1, increasing the ratio of E2F1 conjugated with K11- and K63-linked ubiquitin chains, and decreasing the proportion of E2F1 modified by K48-linked ubiquitin chains. ChIP-seq analysis demonstrated that cIAP1 was required for the recruitment of E2F1 onto chromatin. Lastly, we identified an E2F-binding site on the cIAP1-encoding birc2 gene promoter, suggesting a retro-control regulation loop.

Ubiquitin-proteasome dependent regulation of Profilin2 (Pfn2) by a cellular inhibitor of apoptotic protein 1 (cIAP1)

The two major isoforms of the profilin (Pfn) family of proteins in mammals are Pfn1 and Pfn2. Pfn1 is a universal actin cytoskeletal regulator, while Pfn2 is an actin binding protein and mediator of synapse architecture, specific to neural tissues. However, it has recently been suggested that Pfn2 is also widely distributed in various tissues and involved in numerous cellular events as well as cytoskeletal regulation. In our previous study, we showed that Pfn1 is regulated by carboxyl terminus of Hsc70-Interacting Protein (CHIP) via an ubiquitin (Ub) proteasome system; although, the mechanism of regulation of Pfn2 is unknown. In this report, we demonstrate that Pfn2 is heavily ubiquitinated via differential Ub-linkages for degradation or as a regulatory signal. We also show that cellular inhibitor of apoptosis 1 (cIAP1) rather than CHIP, functions as an E3 ligase that targets Pfn2 for proteasomal degradation. Finally, we observed that Pfn2 levels, regulated by cIAP1, affected intracellular levels of reactive oxygen species. These results may provide a regulatory mechanism for cellular function of Pfn2 in various tissues.

Sodium selenite induces apoptosis via ROS-mediated NF-κB signaling and activation of the Bax-caspase-9-caspase-3 axis in 4T1 cells.

Sodium selenite (SSE), a source of inorganic selenium, has been widely used as a clinical cancer treatment, but the precise molecular mechanisms of SSE remain to be elucidated. Our in vitro experiments have confirmed that SSE treatment causes a transient increase in intracellular reactive oxygen species (ROS) levels, resulting in the inhibition of nuclear transcription factor-κB (NF-κB) signaling and p65 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha phosphorylation levels in 4T1 cells. The inhibition of NF-κB subsequently increased the expression of the apoptosis gene B-cell lymphoma-2-associated X (Bax) and downregulated the transcription of antiapoptosis genes, such as B-cell lymphoma-2, cellular inhibitor of apoptosis 1, and X-linked inhibitor of apoptosis. Additionally, the accumulation of ROS caused mitochondrial dysfunction, leading to the activation of caspase-9 and -3, thereby resulting in apoptosis. However, modulation of the ROS level by the chemical inhibitor N-acetyl-cysteine reversed these events. Similarly, in vitro murine syngeneic breast tumor models showed that SSE inhibits tumor growth by promoting apoptosis. These results indicate that SSE induces apoptosis via ROS-mediated inhibition of NF-κB signaling and activation of the Bax-caspase-9-caspase-3 axis.

Release of c-FLIP brake selectively sensitizes human cancer cells to TLR3-mediated apoptosis

Toll-like receptor 3 (TLR3) mediates innate immune responses by sensing viral dsRNA, but also induces apoptosis selectively in cancer cells. Our analysis by immunohistochemistry revealed that TLR3 is frequently overexpressed in 130 non-small cell lung cancer (NSCLC) patients’ samples compared with normal bronchial epithelium (P < 0.0001, Mann–Whitney test), supporting the therapeutic potential of TLR3 ligand for this type of cancer. However, a proportion of TLR3-expressing cancer cell lines, including NSCLC, remain resistant to TLR3-mediated apoptosis, and the underlying mechanism of resistance remains unclear. We here investigated the molecular basis conferring resistance to non-transformed vs. transformed cells against TLR3-mediated cell death. In non-transformed epithelial cells cellular FLICE-like inhibitory protein (c-FLIP) and cellular Inhibitor of APoptosis (cIAPs) ubiquitin ligases exerted an efficient double brake on apoptosis signaling. In contrast, releasing only one of these two brakes was sufficient to overcome the resistance of 8/8 cancer cell lines tested. Remarkably, the release of the c-FLIP, but not cIAPs, brake only results in the sensitization of all human cancer cells to TLR3-mediated apoptosis. Taking advantage of the difference between transformed and non-transformed cells, we developed a rational strategy by combining the chemotherapeutic agent paclitaxel, which decreases c-FLIP expression, with TLR3 ligand. This combination was highly synergistic for triggering apoptosis in cancer cells but not in non-transformed cells. In vivo, the combination of paclitaxel with dsRNA delayed tumor growth and prolonged survival in a mouse xenograft lung tumor model. In conclusion, combining the release of the c-FLIP brake with TLR3 ligand synergizes to selectively kill cancer cells, and could represent an efficient and safe therapy against TLR3-expressing cancers such as NSCLC.

CIAP1/2-TRAF2-SHP-1-Src-MyD88 Complex Regulates Lipopolysaccharide-Induced IL-27 Production through NF-κB Activation in Human Macrophages.

The inhibitors of apoptosis (IAP) proteins, initially described in the context of apoptosis regulation as promoting cell survival, have recently emerged as key regulators of innate immune signaling. As a result, downregulation of IAP via Smac mimetics (SMM) has both survival and immunoregulatory effects. IAPs modulate cytokine production in murine models either as a single agent or in response to LPS. However, the role of SMM and the involvement of IAPs in primary human cells and in particular macrophages with respect to cytokine production and innate immune responses remain largely unknown. IL-27, a member of the IL-12 cytokine family produced by APCs such as macrophages, has broad immunoregulatory properties in both innate and adaptive immune responses. Herein, we show that cellular IAPs (cIAPs) positively regulate LPS-induced IL-27 production in both primary human monocytes and macrophages. Investigations for the signaling mechanism of cIAPs involvement in IL-27 production in human macrophages revealed that LPS-induced IL-27 production is regulated by a novel signaling complex comprising cIAP1/2, TNFR-associated factor 2 (TRAF2), SHP-1, Src, and MyD88 leading to p38, c-Jun N-terminal kinases (JNK) and Akt activation and NF-κB signaling. In cancer cells, SMM induce the production of cytokines by activating the noncanonical alternate NF-κB pathway. However, in human macrophages, SMM do not induce the production of TNF-α and other cytokines while inhibiting LPS-induced IL-27 production by inhibiting the classical NF-κB pathway. These signaling pathways may constitute novel therapeutic avenues for immune modulation of IL-27 and provide insight into the modulatory immune effects of SMM.

Proteasome inhibition blocks necroptosis by attenuating death complex aggregation

Proteasome inhibitors have achieved clinical success because they trigger intrinsic and extrinsic cell death to eliminate susceptible human cancers. The ubiquitin-proteasome protein degradation system regulates signaling pathways by controlling levels of components such as cellular inhibitor of apoptosis (cIAP)1 and cIAP2 in TNF-mediated cell death. Here, we sought to evaluate the contribution of necroptosis to the cell death pattern induced by the specific proteasome inhibitor Carfilzomib (Cf). Proteasome inhibitor-sensitive multiple myeloma cell lines die in response to Cf by apoptosis in combination with serine protease-dependent death, without any contribution of RIPK3-dependent necroptosis. Proteasome inhibition leads to the induction of apoptotic markers such as activated caspase-3 rather than necroptotic markers such as phosphorylated-MLKL in all cell lines tested. In HT-29 cells, Cf attenuates the late RIPK1 interaction with TNFR1 during TNF-induced necroptosis without altering the sensitivity of cIAP antagonists. Cf treatment results in decreased translocation of death signaling components RIPK1, FADD, caspase-8, cFLIP, and RIPK3 to detergent insoluble fractions. Our results show that proteasome inhibition with Cf impairs necroptosis and favors apoptosis even in cells with intact necroptotic machinery. Following the induction of TNFR1-mediated necroptosis, proteasome activity stabilizes effective aggregation and activation of ripoptosome/necrosome complexes.

Smac mimetics and type II interferon synergistically induce necroptosis in various cancer cell lines

Since cancer cells often evade apoptosis, induction of necroptosis as another mode of programmed cell death is considered a promising therapeutic alternative. Here, we identify a novel synergistic interaction of Smac mimetics that antagonize x-linked Inhibitor of Apoptosis (XIAP), cellular Inhibitor of Apoptosis (cIAP) 1 and 2 with interferon (IFN)γ to induce necroptosis in apoptosis-resistant cancer cells in which caspase activation is blocked. This synergism is confirmed by calculation of combination indices (CIs) and found in both solid and hematological cancer cell lines as well as for different Smac mimetics (i.e. BV6, Birinapant), pointing to a broader relevance. Importantly, individual genetic knockdown of key components of necroptosis signaling, i.e. receptor-interacting protein (RIP) 1, RIP3 or mixed lineage kinase domain-like pseudokinase (MLKL), significantly protects from BV6/IFNγ-induced cell death. Similarly, pharmacological inhibitors of RIP1 (necrostatin-1(Nec-1)), RIP3 (GSK′872) or MLKL (necrosulfonamide (NSA)) significantly reduce BV6/IFNγ-stimulated cell death. Of note, IFN-regulatory factor (IRF)1 is required for BV6/IFNγ-mediated necroptosis, as IRF1 silencing provides protection from cell death. By comparison, antibodies blocking tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand (TRAIL) or CD95 ligand fail to inhibit BV6/IFNγ-induced cell death, pointing to a mechanism independently of death receptor ligands. This is the first report showing that Smac mimetics synergize with IFNγ to trigger necroptosis in apoptosis-resistant cancer cells with important implications for Smac mimetic-based strategies for the treatment of cancer.

The cytoplasmic nuclear receptor RAR\u03b3 controls RIP1 initiated cell death when cIAP activity is inhibited

Tumor necrosis factor (TNF) has a critical role in diverse cellular events including inflammation, apoptosis and necroptosis through different signaling complexes. However, little is known about how the transition from inflammatory signaling to the engagement of death pathways is modulated. Here we report that the cytoplasmic retinoic acid receptor gamma (RARγ) controls receptor-interacting protein kinase 1 (RIP1)-initiated cell death when cellular inhibitor of apoptosis (cIAP) activity is blocked. Through screening a short hairpin RNA library, we found that RARγ was essential for TNF-induced RIP1-initiated apoptosis and necroptosis. Our data suggests that RARγ initiates the formation of death signaling complexes by mediating RIP1 dissociation from TNF receptor 1. We demonstrate that RARγ is released from the nucleus to orchestrate the formation of the cytosolic death complexes. In addition, we demonstrate that RARγ has a similar role in TNF-induced necroptosis in vivo. Thus, our study suggests that nuclear receptor RARγ provides a key checkpoint for the transition from life to death.

CIAP2 promotes gallbladder cancer invasion and lymphangiogenesis by activating the NF-κB pathway.

Several studies have produced contradictory findings about the prognostic implications for inhibitor of apoptosis proteins (IAP) in different types of cancer. Cellular inhibitor of apoptosis 2 (cIAP2/BIRC) is one of the most extensively characterized human IAP. To date, no studies have focused on the expression level of cIAP2 in human gallbladder cancer (GBC), and the mechanism of cIAP2 in GBC invasion and lymphangiogenesis remains unclear. Therefore, in the present study, cIAP2 expression in GBC was detected using quantitative real‐time polymerase chain reaction and immunohistochemistry, and the relationship between cIAP2 levels in cancer tissues and the clinicopathological characteristics of patients was analyzed. The biological effect of cIAP2 in GBC cells was tested using the Cell Counting Kit‐8 Assay, Transwell assays and the ability of human dermal lymphatic endothelial cells (HDLEC) to undergo tube formation. The role of cIAP2 in activating the NF‐κB pathway was determined using a dual‐luciferase reporter assay, immunofluorescence staining, western blotting and ELISA. Finally, an animal model was used to further confirm the role of cIAP2 in lymphangiogenesis. We showed that cIAP2 expression was elevated in human GBC tissues and correlated with a negative prognosis for patients. Moreover, cIAP2 was identified as a lymphangiogenic factor of GBC cells and, thus, promoted lymph node metastasis in GBC cells. Our study is the first to suggest that cIAP2 can promote GBC invasion and lymphangiogenesis by activating the NF‐κB pathway.

Abstract B034: Smac mimetics synergistically improve the efficacy of cancer immunotherapies including immune checkpoint blockade in preclinical models

Abstract Aim: To demonstrate the immune potentiating effects of small-molecule inhibitor-of-apoptosis (IAP) antagonists, known as Smac mimetics, with current standard-of-care cancer immunotherapies. Background: Smac mimetic compounds (SMCs) are synthetic small-molecule antagonists of the cellular inhibitor of apoptosis, cIAP1 and cIAP2, proteins. The IAPs act at a critical nexus in the cancer cell, capable of suppressing intrinsic cell death and avoiding immune-mediated killing of the target cancer cell. The cIAPs are essential mediators of TNF cytokine superfamily signaling which is responsible for the activation of classical and alternative NF-κB survival pathways. SMCs cause the rapid loss of cIAP1/2 proteins by ubiquitin-induced and proteasomal-mediated degradation. SMCs sensitize tumor cells to TNFα-mediated killing by blocking the formation of the RIP1 signalosome and by generating RIP1 death-inducing complexes, the ripoptosome and the necrosome. In addition, SMCs by depleting cIAPs in immune cells promote anti-tumor immunity by inducing T-cell co-stimulation, resulting in a multi-pronged cytolytic attack against tumor cells. SMCs are in early stage clinical trials for cancer and have proven safe as single agents or in combination with chemotherapy. Methodology: We tested various combination immunotherapies in vitro and in vivo in multiple different orthotopic models of cancer, including glioblastoma, in immunocompetent mice to ascertain tumor responses. In addition, we elucidated the role of cytokines and immune cells in the efficacy observed. Results: SMCs dramatically enhanced the anti-tumor effects of Toll-like receptor agonists, recombinant type-1 interferon, BCG vaccine or oncolytic rhabdoviruses in models of cancer. Importantly, we observed for the first time remarkable synergy between SMCs and immune checkpoint inhibitor biologics (e.g. anti-PD1, anti-CTLA-4 monoclonal antibodies) in brain tumor models for which neither single agent had any significant activity. We now demonstrate that SMCs induce long-term cancer immunity that is dependent on cytotoxic T-cell activity. Furthermore, SMCs cooperate with anti-PD1 immune checkpoint inhibitors to induce durable cures in aggressive tumor. We demonstrate that both the innate and adaptive immune response are responsible for the anti-tumor responses and the generation of durable cures in these models. Cytokine neutralization and immune-cell depletion experiments point to key roles for TNFα, interferons, macrophages and CD8-positive killer T-cells in the combination immunotherapy effects observed. Conclusions: SMCs can make use of both innate and adaptive immunity to eradicate cancers in mice. Smac mimetic small-molecules represent a novel and universal form of cancer immunotherapy that greatly compliments immune checkpoint blockade. This highly effective combination approach is readily translatable to the clinic. (Acknowledgements: This project was supported by an Impact grant co-funded by CCSRI and Brain Canada, and by operating grants from CIHR). Citation Format: Eric LaCasse, Shawn Beug, Cristin Healey, Caroline Beauregard, Tarun Sanda, Tommy Alain, Robert Korneluk. Smac mimetics synergistically improve the efficacy of cancer immunotherapies including immune checkpoint blockade in preclinical models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B034.

Expression and Critical Role of Interleukin Enhancer Binding Factor 2 in Hepatocellular Carcinoma

Interleukin enhancer binding factor 2 (ILF2), a transcription factor, regulates cell growth by inhibiting the stabilization of mRNA. Currently, its role has gained recognition as a factor in the tumorigenic process. However, until now, little has been known about the detailed role ILF2 plays in hepatocellular carcinoma (HCC). In this study, we investigated the expression levels of ILF2 in HCC tissue with Western blot and immunohistochemical assays. To examine the effect of ILF2 on liver cancer cell growth and apoptosis, small interfering RNAs (siRNAs) targeting ILF2 were recombined to create lentiviral overexpression vectors. Our results showed higher expression levels of ILF2 mRNA and ILF2 protein in HCC tissue compared with matched peritumoral tissue. Expression of ILF2 may regulate cell growth and apoptosis in liver cancer cells via regulation of B-cell lymphoma 2 (Bcl-2), Bcl-2 related ovarian killer (Bok), Bcl-2-associated X protein (BAX), and cellular inhibitor of apoptosis 1 (cIAP1). Moreover, we inoculated nude mice with liver cancer cells to investigate the effect of ILF2 on tumorigenesis in vivo. As expected, a rapid growth was observed in cancer cells inoculated with a lentiviral vector coding Flag-ILF2 (Lenti-ILF2) compared with the control cells. Hence, these results promote a better understanding of ILF2’s potential role as a therapeutic target in HCC.

Blockade of Inhibitors of Apoptosis Proteins in Combination with Conventional Chemotherapy Leads to Synergistic Antitumor Activity in Medulloblastoma and Cancer Stem-Like Cells.

BackgroundMedulloblastoma (MB) is the most common pediatric primary malignant brain tumor. Approximately one-third of MB patients succumb to treatment failure and some survivors suffer detrimental side effects. Hence, the purpose of this study is to explore new therapeutic regimens to overcome chemotherapeutic agent resistance or reduce chemotherapy-induced toxicity.MethodsWe detected the expression of inhibitors of apoptosis proteins (IAPs) in MB and CD133+ MB cell lines and MB tissues using immunoblotting and immunohistochemical staining. The antitumor effects of inhibitors against IAPs on MB or CD133+ MB cells were evaluated by MTT assay, Annexin V/PI analysis, and caspase-3/7 activity. Autophagy was assessed by the conversion of light chain (LC) 3-I to LC3-II and Cyto-ID autophagy detection kit.ResultsMB cells showed higher expression of IAPs compared to normal astrocytes and normal brain tissues. Conventional chemotherapeutic agents combined with small-molecule IAP inhibitors (LCL161 or LBW242) showed a synergistic effect in MB cells. Combined treatments triggered apoptosis in MB cells through activation of caspase-3/7 and autophagic flux simultaneously. In addition, we found that CD133+ MB cells with features of cancer stem cells displayed higher levels of X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 1/2 (cIAP1/2), and were hypersensitive to treatment with IAP inhibitors.ConclusionsThese results shed light on the biological effects of combination therapy on MB cells and illustrate that IAP inhibitors are more effective for CD133+ stem-like MB cells.

Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis.

Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non-small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977-89. ©2016 AACR.

Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells

Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that small-molecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2′-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as Receptor-Interacting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK′872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies for ALL.

Triptolide sensitizes human breast cancer cells to tumor necrosis factor‑α‑induced apoptosis by inhibiting activation of the nuclear factor‑κB pathway.

Tumor necrosis factor‑α (TNF‑α) can act as either a tumor promoter, linking inflammation with carcinogenesis, or a tumor inhibitor, inducing cancer cell death. However, several types of cancer, including breast cancer, are resistant to TNF‑α therapy. Triptolide, a diterpene triepoxide, has been reported to exert anti‑inflammatory and antiproliferative effects, associated with the inhibition of nuclear factor‑κB (NF‑κB). The present study investigated the effects of triptolide sensitization on human breast cancer cells to TNF‑α‑induced apoptosis by inhibiting activation of the NF‑κB pathway. Human breast cancer MDA‑MB‑231 cells and MCF‑7 cells were treated with different concentrations of triptolide, with or without 10 ng/ml TNF‑α, for different durations, followed by measurement of cell proliferation using a 3‑[4,5‑dimethyltiazol‑2‑yl]‑2.5‑diphenyl‑tetrazolium bromide assay, apoptosis induction, through determination of caspase‑3 activity and poly (ADP‑ribose) polymerase (PARP) cleavage, and NF‑κB pathway activation, through determination of inhibitor of NF‑κB (IκB) and the NF‑κB downstream genes, X‑linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein1/2 (cIAP1/2)] using Western blot and reverse transcription‑quantitative polymerase chain reaction analyses. TNF‑α, when combined with triptolide, was observed to inhibit the activation of IκBα, increase the level of cleaved PARP, and further activate caspase‑3 in the breast cancer cells. Triptolide also inhibited the expression levels of the downstream anti‑apoptotic genes of NF‑κB activation, XIAP and cIAP1/2. The results of the present study demonstrated that triptolide sensitized human breast cancer cells to TNF‑α‑induced apoptosis, which may provide a promising combination strategy for human breast cancer therapeutics.

MiR-145 promotes TNF-α-induced apoptosis by facilitating the formation of RIP1-FADDcaspase-8 complex in triple-negative breast cancer.

Researches indicate that the dysregulation of microRNA (miRNA) is involved in tumorigenesis. Among such dysregulated miRNAs in cancer, miR-145 is reported to be downregulated in multiple cancers. In this study, we demonstrated the downregulation of miR-145 in triple-negative breast cancer (TNBC) tissues and TNBC cell lines by quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Furthermore, we found that the tumor necrosis factor-alpha (TNF-α)-induced apoptosis was expanded by the transfection of miR-145 in MDA-MB-231 which belongs to the TNBC cell lines. We then indicated that the mechanism by which miR-145 promotes the TNF-α-induced apoptosis is dependent on the formation of RIP1-FADD-caspase-8 complex. The cellular inhibitor of apoptosis (cIAP1), which is the inhibitor of apoptosis protein, was found to be a target of miR-145 in MDA-MB-231 cells. As a result of cIAP1 overexpression, the promotion of miR-145 on TNF-α-induced apoptosis was inhibited in MDA-MB-231 cells. Therefore, our results indicate that miR-145 acts as a tumor suppressor in TNBC, suggesting that the miR-145-cIAP1 axis might be a potential therapeutic target for TNBC.

Inhibitor of apoptosis proteins as intracellular signaling intermediates

Inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c-IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c-IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c-IAPs illustrate their importance as intracellular signaling intermediates.