Proteins cIAP1 Research Articles

189 (PB177): Downregulation of microRNA-214-3p by long non-coding RNA DNAJC3-As1 promotes carboplatin resistance in esophageal cancer cells through the overexpression of the anti-apoptotic protein c-IAP1

189 (PB177): Downregulation of microRNA-214-3p by long non-coding RNA DNAJC3-As1 promotes carboplatin resistance in esophageal cancer cells through the overexpression of the anti-apoptotic protein c-IAP1

CIAP-2 protein is upregulated by human papillomavirus in oropharyngeal cancers: role in radioresistance in vitro

BackgroundHigh-risk human papillomaviruses are the causal agents of a subset of head and neck cancers. A previous transcriptomic analysis showed that cIAP2 protein, involved in cell survival and apoptosis, is upregulated in OKF6 oral cells that express HPV16 E6/E7. In addition, cIAP2 promotes radioresistance, a very important concern in HNC treatment. However, cIAP2 increase has not yet been evaluated in oropharyngeal carcinomas (OPCs), nor has been the role of cIAP2 in HNC radioresistance.MethodsWe carried out a descriptive-analytical retrospective study in 49 OPCs from Chilean patients. We determined the expression of cIAP2 at transcript and proteins levels using reverse-transcriptase -polymerase chain reaction and immunohistochemistry, respectively. HPV and p16 expression were previously analyzed in these specimens. In addition, SCC-143 HNC cells ectopically expressing HPV16 E6/E7 were analyzed for cIAP2 expression and after transfection with a siRNA for HPV16 E6/E7 knocking down.ResultsWe found a statistically significant association between HPV presence and cIAP2 expression (p = 0.0032 and p = 0.0061, respectively). An association between p16 and cIAP2 levels was also found (p = 0.038). When SCC-143 cells were transfected with a construct expressing HPV16 E6/E7, the levels of cIAP2 were significantly increased (p = 0.0383 and p = 0.0115, respectively). Conversely, HPV16 E6 and E7 knocking down resulted in a decrease of cIAP2 levels (p = 0.0161 and p = 0.006, respectively). Finally, cIAP2 knocking down in HPV16 E6/E7 cells resulted in increased apoptosis after exposure to radiation at 4 and 8 Gy (p = 0.0187 and p = 0.0061, respectively).ConclusionThis study demonstrated for the first time a positive relationship between HPV presence and cIAP2 levels in OPCs. Additionally, cIAP2 knocking down sensitizes HNC cells to apoptosis promoted by radiation. Therefore, cIAP2 is a potential therapeutic target for radiation in HPV-driven HNC.

Enhancing cell death in B-cell malignancies through targeted inhibition of Bcl-3

The t(14;19)(q32;q13) is a rare recurring translocation found in B-cell lymphoproliferative malignancies, involving the Bcl-3 gene. This chromosomal translocation is often found in patients under the age of 50 and causes a more progressive disease. The Bcl-3 gene encodes a protein belonging to the IκB family of proteins, which tightly regulates NFκB signaling by acting as an activator or repressor of transcription. Previously, we developed a second-generation Bcl-3 inhibitor that could directly interfere with Bcl-3 signaling pathway, resulting in reduced melanoma cell proliferation, invasion, and migration. The present study aimed to investigate the effect of a Bcl-3 inhibitor on B-cell lymphoma and leukemia cells. It was found that treatment of cells with this inhibitor caused a decrease in cell proliferation and cell survival. Furthermore, Bcl-3 inhibition in B-cell malignant cells resulted in the loss of mitochondrial membrane potential and functionality, as well as the increased expression of cleaved caspase 3, indicating that cell death occurs through the intrinsic apoptotic pathway. This observation is further supported by reduced expression of cIAP1 protein 1 (cIAP1) upon treatment of cancer cells. Given the current lack of clinical advancements targeting Bcl-3 in oncology, this opens a novel avenue for the development and investigation of highly specific therapeutic interventions against B-cell malignancies.

Fisetin reduces the resistance of MOLT-4 and K562 cells to TRAIL-induced apoptosis through upregulation of TRAIL receptors.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that is capable of apoptosis induction selectively in tumor cells. Although TRAIL has been harnessed in numerous clinical trials, resistance to TRAIL-induced apoptosis is a major challenge ahead of this therapy in various cancer models as well as in leukemia. Since histone deacetylases (HDACs) are known to affect drug resistance in malignant cells, the present study aimed to evaluate the potential of fisetin for sensitization of MOLT-4 and K-562 leukemic cells to TRAIL-induced apoptosis. The MOLT-4 and K-562 cells were treated with increasing concentrations of fisetin and its impact on the growth inhibition and apoptosis induction of TRAIL were evaluated by MTT and Annexin V/7-AAD assays. The impact of fisetin on the mRNA and protein expression levels of apoptosis regulatory genes such as BIRC2/c-IAP1, CFLAR/cFLIP, CASP3, CASP7, CASPP9, TNFRSF10A/DR4, TNFRSF10B/DR5, and BID were examined by PCR array, qRT-PCR, and flow cytometry. Pre-treatment of MOLT-4 and K-562 cells with fisetin reduced the IC50 of TRAIL in growth inhibition along with an improvement in apoptosis induction by TRAIL. The expression of the BIRC2 gene encoding antiapoptotic protein c-IAP1 downregulated in the fisetin-treated cells while the expressions of TNFRSF10A and TNFRSF10B encoding TRAIL death receptors increased. Fisetin demonstrated a potential for alleviating the TRAIL resistance by modulating the apoptosis regulatory factors and improving the expressions of TRAIL receptors that could facilitate the application of TRAIL in cancer therapies.

Abstract 5088: Evaluating TNF-receptor associated factor 2 (TRAF2) as a targetable driver of immune resistance in LKB1 deficient non-small cell lung cancer

Abstract STK11 is a serine-threonine kinase and tumor suppressor lost in about 25% of NSCLC. It potently regulates a network of downstream effector kinases in the AMPK family, thereby exerting major effects on cell signaling, metabolic activity, and epigenetic patterning. Clinically loss of STK11 in NSCLC is associated with poor response to immune checkpoint blockade. Several mechanisms of immune resistance have been identified involving both tumor intrinsic pathways as well as changes in the immune microenvironment. Dysregulation of cellular inhibitor of apoptosis proteins, specifically cIAP1 has been identified as a specific resistance mechanism, which was shown to also influence the STING pathway in the context of STK11 loss. However, there is a gap in understanding how loss of STK11 influences cIAP1 signaling.We evaluated cIAP1, its TRAF family member binding partners, and downstream NF-kB and immune signaling using a multiomic approach. Using CPTAC multiomic data of 110 lung adenocarcinomas, we observe discordance between protein vs mRNA expression for cIAP1, which shows no significant association with STK11 loss in mRNA, but is significantly over-expressed at the protein level (P<1e-4). Conversely, we identify TRAF2 as the only TRAF member whose expression is increased in STK11-deficient NSCLC, with concordant increase in both mRNA (P<1e-8) and protein (P<1e-10). Further, on the protein level TRAF2 and cIAP1 levels were highly correlated (P<1e-12), strongly suggesting that TRAF2 is the proximal factor driving dysregulation of cIAP1 signaling in these tumors. We also observe a correlated pattern of phosphorylation sites within cIAP1, TRAF2, and other NF-kB signaling members that we putatively link to STK11 loss. We confirm that TRAF2 mRNA is consistently over-expressed in STK11 deficient NSCLC across multiple additional transcriptomic datasets together comprising >2000 tumors. Across this large dataset we observe that among STK11-deficient tumors, TRAF2 is anti-correlated with NF-kB signaling and immune infiltration signatures, a relationship that is strikingly absent among the STK11-WT subset. Using GEMM cell lines (KRAS, TP53, STK11 GEMM; Winslow lab, Stanford), we derived STK11 mutant vs WT isogenic pairs and performed T-cell co-culture experiments using ovalbumin antigen and OT-I CD8 T-cells. This showed that inhibition of cIAP1 using birinapant was synergistic to CD8 cytotoxicity, as previously known, but the synergy was much more pronounced in STK11 mutant vs STK11-WT context (Bliss index 38, P<1e-10). Together, these data demonstrate that the link between STK11-loss and cIAP1 dysregulation is likely through over-expression of TRAF2, and support the strategy of targeting TRAF2/cIAP1 to overcome immune resistance STK11-deficient NSCLC. Citation Format: Bahareh Nourmohammadi, Joseph M. Amann, Rahul Shivahare, Qin Ma, Zihai Li, David P. Carbone, Jacob Kaufman. Evaluating TNF-receptor associated factor 2 (TRAF2) as a targetable driver of immune resistance in LKB1 deficient non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5088.

Abstract A035: Radiation-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting matrix metalloproteinases, chemokines, cytokines, and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells as well as promoted tumor cell proliferation and radioresistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and publicly available data from the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells, like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also observed in PDX models of GBM. Using these PDX models, we are currently validating novel senolytic-based therapeutic approaches to mitigate tumor recurrence after radiotherapy. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Ben R. Jordan, Nozomi Tomimatsu, Luis Macedo Di Cristofaro, Suman Kanji, Bipasha Mukherjee, Sandeep Burma. Radiation-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A035.

Abstract A009: DNA damage-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A009.

Abstract PR001: DNA damage-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR001.

New insights into the cytotoxic effects of Thymus vulgaris essential oil on the human triple-negative breast cancer cell line MDA-MB-231

Essential oils (EOs) are natural products that have gained wide interest due to their biological activities and anticancer properties through various mechanisms. The present study aimed to test the cytotoxicity of Thymus vulgaris L. (thyme) EO of Italian origin, rich in thymol (49.6%) and p-cymene (18.8%), towards the triple-negative breast cancer cell line MDA-MB-231 and to investigate the biochemical mechanisms underlying its antitumor activity. Thyme EO reduced cancer cell viability in a dose-dependent manner after 24 h treatment, with an IC50 value equal to 75.1 ± 15.2 μg/ml; simultaneously, the inhibition of cancer cell migration and colony formation capacity was evidenced. Thyme EO antiproliferative effects were related to the induction of apoptosis as demonstrated by the increased expression of the pro-apoptotic proteins Bax, cleaved caspase-3, phospho-p53, and SMAC/Diablo and by the reduction of the anti-apoptotic proteins Bcl-2, cIAP-1, cIAP-2, HIF-1α, survivin, and XIAP. Thyme EO administration led to the early formation of intracellular ROS, followed by the increment of MDA as an index of lipid peroxidation and by the decreased expression of the antioxidant enzymes catalase and PON2. The upregulation of Nrf2 mRNA expression and the strong induction of HO-1 sustained the activation of the Nrf2 pathway by thyme EO. These data showed that the EO from Thymus vulgaris L. might inhibit the malignant phenotype of MDA-MB-231, thus suggesting potential benefits against human triple-negative breast cancer.

Dual-targeting peptides@PMO, a mimetic to the pro-apoptotic protein Smac/DIABLO for selective activation of apoptosis in cancer cells.

The refractoriness of tumor cells to apoptosis represents the main mechanism of resistance to chemotherapy. Smac/DIABLO mimetics proved to be effective in overcoming cancer-acquired resistance to apoptosis as a consequence of overexpression of the anti-apoptotic proteins XIAP, cIAP1, and cIAP2. In this work, we describe a dual-targeting peptide capable of selectively activating apoptosis in cancer cells. The complex consists of a fluorescent periodic mesoporous organosilica nanoparticle that carries the short sequences of Smac/DIABLO bound to the αvβ3-integrin ligand. The dual-targeting peptide @PMO shows significantly higher toxicity in αvβ3-positive HeLa cells with respect to αvβ3-negative Ht29 cells. @PMO exhibited synergistic effects in combination with oxaliplatin in a panel of αvβ3-positive cancer cells, while its toxicity is overcome by XIAP overexpression or integrin β3 silencing. The successful uptake of the molecule by αvβ3-positive cells makes @PMO promising for the re-sensitization to apoptosis of many cancer types.

Expression and clinical significance of Capase3 and C-IAP1/2 in ectopic endometrium of patients with endometriosis.

To analyze the expression of capase3 and C-IAP1/2 in endometriosis and their relationship with clinical practice. Ectopic and eutopic endometrial tissues were collected from women with endometriosis, and the control tissues were obtained from regular patients without endometriosis. The expression level of capase3 and C-IAP1/2 was detected by immunohistochemistry; in addition, the relationship between C-IAP1/2 expression and the clinical stage of endometriosis was analyzed. The expression of Caspase-3 in ectopic and eutopic endometrium was significantly lower than that in the control group (P < 0.005). The expression of C-IAP1/2 in ectopic and situ endometrium was significantly higher than in the control group (P < 0.005). The expression of C-IAP1/2 in stage III-IV was higher than in stage I-II endometriosis. The positive expression of c-IAP1 protein in ectopic endometrial tissue was negatively correlated with the positive expression of Caspase-3 protein in ectopic endometrial tissue (P<0.001). The low expression of Caspase-3 and the high expression of C-IAP1/2 in the ectopic endometrium and eutopic endometrial tissues compared with the controls. The results indicated that the apoptotic ability of eutopic and ectopic endometrium is weakened, which is involved in the pathogenesis of endometriosis. It is expected to be a target for the diagnosis and therapy of this disease. At the same time, the expression level of C-IAP1/2 is related to the stage of endometriosis, which can guide clinical treatment.

The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP

The poor prognosis of HNSCC is partly due to treatment resistance. The SMAC mimetic Xevinapant is a promising new approach to targeted cancer therapy. Xevinapant inhibits cIAP1/2 and XIAP, leading to apoptosis, necroptosis and inhibition of prosurvival signaling. Combining Xevinapant with IR could improve therapeutic potential. The effect of Xevinapant in combination with IR on HNSCC and healthy tissue cells was investigated. Cell growth, cell death, clonogenic survival and DNA double-strand breaks (DSBs) were studied, and intracellular cIAP1 and XIAP levels were evaluated. Xevinapant had cytostatic and cytotoxic, as well as radiosensitizing, effects on the malignant cells, while healthy tissue cells were less affected. Apoptotic and necrotic cell death was particularly affected, but the increase in residual DSBs and the reduced survival implied an additional effect of Xevinapant on DNA damage repair and other cell inactivation mechanisms. cIAP1 and XIAP levels varied for each cell line and were affected by Xevinapant and IR treatment. There was an association between higher IAP levels and increased cell death. Xevinapant appears to be a potent new drug for HNSCC therapy, especially in combination with IR. IAP levels could be an indicator for impaired DNA damage repair and increased susceptibility to cellular stress.

Novel Tetrahydro-[1,2,4]triazolo[3,4-a]isoquinoline Chalcones Suppress Breast Carcinoma through Cell Cycle Arrests and Apoptosis.

Chalcones are interesting anticancer drug candidates which have attracted much interest due to their unique structure and their extensive biological activity. Various functional modifications in chalcones have been reported, along with their pharmacological properties. In the current study, novel chalcone derivatives with the chemical base of tetrahydro-[1,2,4]triazolo[3,4-a]isoquinolin-3-yl)-3-arylprop-2-en-1-one were synthesized, and the structure of their molecules was confirmed through NMR spectroscopy. The antitumor activity of these newly synthesized chalcone derivatives was tested on mouse (Luc-4T1) and human (MDA-MB-231) breast cancer cell lines. The antiproliferative effect was evaluated through SRB screening and the MTT assay after 48 h of treatment at different concentrations. Interestingly, among the tested chalcone derivatives, chalcone analogues with a methoxy group were found to have significant anticancer activity and displayed gradient-dependent inhibition against breast cancer cell proliferation. The anticancer properties of these unique analogues were examined further by cytometric analysis of the cell cycle, quantitative PCR, and the caspases-Glo 3/7 assay. Chalcone methoxy derivatives showed the capability of cell cycle arrest and increased Bax/Bcl2 mRNA ratios as well as caspases 3/7 activity. The molecular docking analysis suggests that these chalcone methoxy derivatives may inhibit anti-apoptotic proteins, particularly cIAP1, BCL2, and EGFRK proteins. In conclusion, our findings confirm that chalcone methoxy derivatives could be considered to be potent drug candidates against breast cancer.

Inhibitor of Apoptosis Proteins Antagonist Induces T-cell Proliferation after Cross-Presentation by Dendritic Cells.

Cross-presentation of tumor antigens by dendritic cells (DC) is crucial to prime, stimulate and restimulate CD8+ T cells. This process is important in initiating and maintaining an antitumor response. Here, we show that the presence of conventional type 1 DCs (cDC1), a DC subtype that excels in cross-presentation, in the tumor correlated with response to neoadjuvant immune checkpoint blockade (ICB) in melanoma. This led us to hypothesize that patients failing to respond to ICB could benefit from enhanced cross-presentation of tumor antigens. We therefore established a cross-presentation assay to screen over 5,500 compounds for enhancers of DC cross-presentation using induced T-cell proliferation as the readout. We identified 145 enhancers, including AZD5582, an antagonist of inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2, and XIAP. AZD5582 treatment led to DC activation of the noncanonical NF-kB pathway, enhanced antigen import from endolysosomes into the cytosol, and increased expression of genes involved in cross-presentation. Furthermore, it upregulated expression of CD80, CD86, MHC class II, CD70 and secretion of TNF by DCs. This enhanced DC activation and maturation program was observed also in tumor-bearing mice upon AZD5582 treatment, culminating in an increased frequency of systemic tumor antigen-specific CD8+ T cells. Our results merit further exploration of AZD5582 to increase antigen cross-presentation for improving the clinical benefit of ICB in patients who are unlikely to respond to ICB.

Self-synthesized second mitochondria-derived activator of caspase (SMAC) mimetic TP-WY-1345 enhances the radiosensitivity of NSCLC cells H1299 by targeting anti-apoptotic protein cIAP1

ObjectiveTo explore the toxicity and cellular uptake of the self-synthesized second mitochondria-derived activator of caspase (SMAC) mimetic TP-WY-1345 polypeptide and its radiosensitizing effect on non-small cell lung cancer (NSCLC). MethodsA fluorescence microscope was used to observe the uptake efficiency of TP-WY-1345 by human NSCLC cells H1299. Toxicity of the TP-WY-1345 peptide in normal cells was examined in human embryonic lung fibroblasts MRC5. CCK-8 and clone formation experiments were performed to detect the radiosensitizing effect of TP-WY-1345 in the H1299 ​cells. The AutoDock Vina simulation software was employed to predict the binding efficiency of TP-WY-1345 to the inhibitor of apoptosis proteins (IAPs). Moreover, Western blot and qPCR experiments were carried out to determine the protein and gene expressions, and the flow cytometry (FCM) technique was used to detect the apoptosis level under different conditions. ResultsTP-WY-1345 can be self-synthesized, and significant green fluorescence was observed in H1299 2 ​h and 6 ​h after incubation with TP-WY-1345. The cell counting and CCK-8 results showed that 10 ​μmol/L and 20 ​μmol/L TP-WY-1345 did not produce a significant toxic effect on the MRC5 cells (P ​> ​0.05). Compared with the single ionizing radiation group, the cell viability and clone formation of H1299 ​cells were significantly inhibited after treatment with TP-WY-1345 at a concentration of 50 ​μmol/L (P ​< 0.05). The sensitivity enhancement ratio (SER) was calculated to be 1.14. Moreover, the binding efficiency of TP-WY-1345 to cIAP1 protein was predicted to be −7.3, and this strong binding force was demonstrated by Western blot. TP-WY-1345 inhibited the protein and mRNA expressions of cIAP1 and further increased the apoptosis level of the H1299 ​cells at 24 ​h ​(P ​< ​0.01) and 48 ​h after radiation (P ​< ​0.05). ConclusionSMAC mimetic TP-WY-1345 can enter the H1299 ​cells and produce a radiosensitizing effect by increasing the level of radiation-induced apoptosis of the cells. There, TP-WY-1345 is expected to become a new generation of radiosensitizing drugs.

TRIM56 promotes malignant progression of glioblastoma by stabilizing cIAP1 protein

BackgroundThe tripartite motif (TRIM) family of proteins plays a key role in the developmental growth and therapeutic resistance of many tumors. However, the regulatory mechanisms and biological functions of TRIM proteins in human glioblastoma (GBM) are not yet fully understood. In this study, we focused on TRIM56, which emerged as the most differentially expressed TRIM family member with increased expression in GBM.MethodsWestern blot, real-time quantitative PCR (qRT-PCR), immunofluorescence (IF) and immunohistochemistry (IHC) were used to study the expression levels of TRIM56 and cIAP1 in GBM cell lines. Co-immunoprecipitation (co-IP) was used to explore the specific binding between target proteins and TRIM56. A xenograft animal model was used to verify the tumor promoting effect of TRIM56 on glioma in vivo.ResultsWe observed elevated expression of TRIM56 in malignant gliomas and revealed that TRIM56 promoted glioma progression in vitro and in a GBM xenograft model in nude mice. Analysis of the Human Ubiquitin Array and co-IPs showed that cIAP1 is a protein downstream of TRIM56. TRIM56 deubiquitinated cIAP1, mainly through the zinc finger domain (amino acids 21–205) of TRIM56, thereby reducing the degradation of cIAP1 and thus increasing its expression. TRIM56 also showed prognostic significance in overall survival of glioma patients.ConclusionsTRIM56-regulated post-translational modifications may contribute to glioma development through stabilization of cIAP1. Furthermore, TRIM56 may serve as a novel prognostic indicator and therapeutic molecular target for GBM.

Abstract 15005: Tlr4/py14-Caveolin-1-Mediated Endothelial C-iap2 Expression During Schistosomiasis-Associated Pulmonary Arterial Hypertension

Introduction: During schistosomiasis-associated pulmonary arterial hypertension (Sch-PAH), hyperproliferation of abnormal endothelial cells (ECs) contributes to development of severe vascular lesions within the lungs; a life-threatening phenomenon that remains with no target therapy. Hypothesis: Here, we found that antigenic molecules from the major etiological agent of Sch-PAH, i.e., S. mansoni , amplify the secretion of the antiapoptotic protein c-IAP2 via TLR4-pY14Cav-1 signaling pathway, which we hypothesized to contribute to generation of an EC memory of survival and severity of PAH. Methods/Results: Cav-1 deficiency via shedding of extracellular vesicles (EVs) leads to survival of an apoptosis-resistant EC phenotype during idiopathic PAH. Similarly, S. mansoni egg antigens also reduced lung EC-Cav-1 expression, suggesting that Cav-1 also plays a role during pathogen-associated PAH. Cav-1 depletion via EVs precedes its phosphorylation at residue Tyr14 (pY14Cav-1), which in turn, contributes to EV loading. Among apoptotic regulators, IAPs have been found as a cargo of EVs in different conditions, including in TLR4-activated cells. Accordingly, our data indicates that S. mansoni egg antigen p40 also activated TLR4/NFkB-mediated signaling, inducing c-IAP2 expression and a time-dependent phosphorylation of Cav-1 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p&lt;0.05; n=4). Moreover, although Sm-p40 alone was unable to induce shedding of EVs, combination with NFkB-associated proinflammatory mediators (TNF-α, IL-6, and ATP), significantly increased EV shedding by HMVEC-L, suggesting that S. mansoni may epigenetically prime ECs, but co-factors are required to induce EV shedding. Finally, Sch-PAH model using EC- Cav1 -/- mice ( End-scl.Cre ERT2 ;Rosa mt/mg ;Cav1 fl/fl ) revealed that Cav-1 absence led to accumulation of eggs within the lungs and loss of eGFP+ cells, indicating that EC-Cav-1 phosphorylation may be required to development of Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced pY14Cav-1 is associated to expression of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.

BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins

The present study aimed to investigate the inhibitory role of second mitochondria determined activator of caspases mimetic on inhibitor of apoptosis proteins (IAPs) and regulation of caspases in nonsmall cell lung cancer cell line. Dimethyl sulfoxide and 3-(4, 5-dimethyl thizol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was done to determine the IC50 of BV6 using NCI-H23 cell line. The levels of mRNA of X-linked IAP (XIAP), cellular IAP (cIAP-1), cIAP-2, caspase-6, and caspase-7 in H23 cell line were evaluated by a quantitative real-time polymerase chain reaction, while their protein expressions were tested using western blotting. Two doses of BV6 dependently downregulated the expression of mRNA of XIAP (P = 0.002, P= 0.0003 vs. untreated), cIAP-1 (P = 0.05, P = 0.005 vs. untreated), and cIAP-2 (P = 0.001, P = 0.0002 vs. untreated), respectively, while the compound upregulated the mRNA expression of caspase-6 (P = 0.001, P < 0.0001 vs. untreated) and caspase-7 (P = 0.001, P = 0.0004 vs. untreated), respectively. Dose dependent of BV6 treatment significantly decreased the protein level of XIAP (P = 0.003, P = 0.007 vs. untreated), cIAP-1 (P = 0.02, P = 0.01 vs. untreated), and cIAP-2 (P = 0.008,P = 0.008 vs. untreated), respectively. However, the compound increased the protein level of caspase-6 and caspase-7 when compared to untreated control (P = 0.006,P = 0.001) and (P = 0.01, P = 0.001), respectively. The result showed that BV6 treatment reduced the level of mRNA of XIAP, cIAP-1, and cIAP-2 and increased the gene expression of caspase-6 and caspase-7 in NCI-H23 cell line. Therefore, the study revealed that BV6 could be used in future as additional therapeutics in lung cancer.

DIFFERENTIAL EXPRESSION AND STABILITY OF XIAP AND CIAP-1 MAY INFLUENCE THE APOPTOSIS-RESISTANT PHENOTYPE OF LUNG FIBROBLASTS IN IDIOPATHIC PULMONARY FIBROSIS

DIFFERENTIAL EXPRESSION AND STABILITY OF XIAP AND CIAP-1 MAY INFLUENCE THE APOPTOSIS-RESISTANT PHENOTYPE OF LUNG FIBROBLASTS IN IDIOPATHIC PULMONARY FIBROSIS

Abstract 388: Role Of The Antiapoptotic C-iap2 On The Generation Of An Endothelial Memory In Schistosomiasis-associated Pulmonary Arterial Hypertension

Introduction: Schistosomiasis-associated Pulmonary Arterial Hypertension (Sch-PAH) is a life-threatening complication of chronic S. mansoni infection, which can evolve to heart failure and death. During PAH, hyperproliferation of apoptosis-resistant endothelial cells (ECs) has been extensively reported, but therapeutic approaches to reverse this pathological phenotype remain clinically challenging. Hypothesis: Thus, our hypothesis is that antigenic molecules from S. mansoni eggs amplify the secretion of inhibitor of apoptosis protein 2 (c-IAP2), leading to generation of an abnormal EC memory of survival and the development of PAH. Methods/Results: Previously, we showed that depletion of the anti-apoptotic protein Caveolin-1 (Cav-1) via shedding of extracellular vesicles (EVs) leads to the survival of an abnormal EC phenotype through an unclear mechanism. Analysis of survival-associated genes in isolated murine lung ECs from Flk1 +/GFP ;Cav1 -/- mice, revealed high expression of the anti-apoptotic genes BIRC3 and BIRC5 (which encodes cIAP2 and survivin, respectively) when compared to control ECs. Interesting, exposure to 1 μg/mL of the major S. mansoni egg antigen Sm-p40 induced time-dependent phosphorylation of Cav-1 at residue Tyr14 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p&lt;0.05; n=4), culminating in c-IAP2 expression with no difference on the constitutive expression of c-IAP1. Furthermore, Sm-p40 treatment of HMVEC-L in the presence of pro-inflammatory mediators IL-6, TNF-α, and ATP induced a significant release of EVs containing c-IAP2. Finally, in vivo analysis of lungs from S. mansoni -infected mice confirmed a significant decrease in Cav-1 expression unique to the egg-dependent granuloma area, indicating that local inflammatory response due to egg-derived antigenic molecules is essential for reprogramming and survival of an abnormal EC phenotype during Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced Cav-1 depletion promotes the secretion of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.