X-linked Inhibitor Of Apoptosis Protein Protein Research Articles

Understanding the molecular mechanism of pathogenic variants of BIR2 domain in XIAP-deficient inflammatory bowel disease

X-linked inhibitor of apoptosis protein (XIAP) deficiency causes refractory inflammatory bowel disease. The XIAP protein plays a pivotal role in the pro-inflammatory response through the nucleotide-binding oligomerization domain-containing signaling pathway that is important in mucosal homeostasis. We analyzed the molecular mechanism of non-synonymous pathogenic variants (PVs) of XIAP BIR2 domain. We generated N-terminally green fluorescent protein-tagged XIAP constructs of representative non-synonymous PVs. Co-immunoprecipitation and fluorescence cross-correlation spectroscopy showed that wild-type XIAP and RIP2 preferentially interacted in live cells, whereas all non-synonymous PV XIAPs failed to interact properly with RIP2. Structural analysis showed that various structural changes by mutations, such as hydrophobic core collapse, Zn-finger loss, and spatial rearrangement, destabilized the two loop structures (174–182 and 205–215) that critically interact with RIP2. Subsequently, it caused a failure of RIP2 ubiquitination and loss of protein deficiency by the auto-ubiquitination of all XIAP mutants. These findings could enhance our understanding of the role of XIAP mutations in XIAP-deficient inflammatory bowel disease and may benefit future therapeutic strategies.

TP induces hepatic intolerance to FasL-mediated hepatocyte apoptosis by inhibiting XIAP

Triptolide (TP) is extracted from the traditional Chinese medicine Tripterygium wilfordii Hook. F. (TWHF). Its severe toxic side effects, especially hepatotoxicity, have limited the clinical application of TP-related drugs. In this study, we investigated the mechanism of the hepatotoxic effects of TP from the perspective that TP inhibited the expression of the pro-survival protein X-linked inhibitor of apoptosis protein (XIAP) and enhanced FasL-mediated apoptosis of hepatocytes. TP and CD95/Fas antibody (Jo-2) were administered by gavage to C57BL/6 mice for 7 consecutive days. After co-administration of TP and Jo-2, mouse livers showed large areas of necrosis and apoptosis and significantly increased Caspase-3 activity. KEGG pathway enrichment analysis indicated that TP may cause the development of liver injury through the apoptotic signaling pathway. Proteinprotein interaction networks showed that XIAP played an essential role in this process. TP reduced the protein expression of XIAP after combination treatment with Jo-2/FasL in vivo/in vitro. TP and FasL co-stimulation significantly increased microRNA-137 (miR-137) levels in AML12 cells, while inhibition of miR-137 expression induced a rebound in XIAP protein expression. In conclusion, TP presensitizes hepatocytes and enhances the sensitivity of hepatocytes to the Fas/FasL pathway by inhibiting the protein expression of XIAP, leading to hepatocyte apoptosis.

X-linked inhibitor of apoptosis protein (XIAP) expression level analyzed by immunohistochemistry (IHC) as it relates to clinical and pathologic characteristics in prostate cancer.

e17012 Background: XIAP acts in both the extrinsic and intrinsic apoptotic pathways as an inhibitor of cell death, protecting cells from a range of triggers. Regulation due to apoptosis resistance may represent a targetable factor for therapeutic intervention in prostate cancer. This study focuses on XIAP as a biomarker and its expression in correlation with disease aggression. Methods: Expression levels of XIAP was analyzed by immunohistochemistry (IHC), graded 1 through 3 according to signal intensity, in radical prostatectomy samples from 90 patients with prostate cancer with a range of phenotypes including: indolent (A), locally advanced (B), progressive to metastatic (C) and de novo metastatic (D). Prognostic data was collected and Fisher’s exact statistical analysis was performed. Secondary analyses included a Fisher’s exact test with a pairwise comparison between individual disease grades and Gleason scores. Results: Higher XIAP protein expression levels on IHC correlated with disease grade and with total Gleason score (p = 0.0008 and p = 0.0002 respectively) by Fisher’s exact test analysis, indicating more aggressive disease phenotypes. Secondary results looked at a pairwise Fisher’s exact analysis comparing XIAP expression levels among disease grades (A-D) and Gleason scores. XIAP expression only significantly differed between those with grade A and D as well as between individuals with grades B and D (measured by Bonferroni-adjusted p-value of 0.003 and 0.018 respectively). Statistical significance was also achieved in XIAP expression levels when comparing among Gleason scores 6 and 8 (p = 0.025), 6 and 9 (p = 0.0125), as well as 7 and 9 (p = 0.030). Conclusions: This study demonstrates a correlation between XIAP expression levels by IHC and aggressiveness of disease, demonstrating clinical significance of XIAP as a prostate cancer biomarker. [Table: see text]

Synthesis and biological evaluation of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold

Overexpression of both human murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) is detected in tumor cells from several cancer types, including childhood acute leukemia lymphoma (ALL), neuroblastoma (NB), and prostate cancer, and is associated with disease progression and treatment resistance. In this report, we described the design and syntheses of a series of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold from our previously reported lead compound JW-2-107 and tested their cytotoxicity in a panel of human cancer cell lines. The best compound identified in this study is compound 3e. Western blot analyses demonstrated that treatments with 3e decreased MDM2 and XIAP protein levels and increased expression of p53, resulting in cancer cell growth inhibition and cell death. Furthermore, compound 3e effectively inhibited tumor growth in vivo when tested using a human 22Rv1 prostate cancer xenograft model. Collectively, results in this study strongly suggest that the tetrahydroquinoline scaffold, represented by 3e and our earlier lead compound JW-2-107, has abilities to dual target MDM2 and XIAP and is promising for further preclinical development.

Effect of Butylphthalide Capsules on Smac and XIAP Expression in Rats after Ischemia Reperfusion

IntroductionLittle is known about the protective effects of butylphthalide on cerebral ischemia-reperfusion injury. This study aims to investigate the impact on the second mitochondrial-derived activator of Caspases (Smac) and X-linked inhibitor of apoptosis protein (XIAP) expression in the ischemic semidark area using a rat model of carotid artery stenosis. MethodsThirty Sprague–Dawley rats were randomly divided into the sham-operated group, carotid stenosis model controls, low-dose (20 mg/kg), medium-dose (40 mg/kg), and high-dose (80 mg/kg) butylphthalide groups. The neurological function was scored by the balance beam test (BBT). The morphological changes of brain tissue were detected by Hematoxylin-eosin (HE) staining, with apoptosis detected by Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) staining. Smac and XIAP protein expression were detected by immunohistochemistry (IHC). The expressions of Smac and XIAP mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR). ResultsHE showed that neuronal loss, nuclear consolidation, and vacuolar degeneration were significantly reduced in the medium and high-dose butylphthalide groups compared with the model controls. The BBT scores and apoptotic index were significantly lower in the medium and high doses of butylphthalide compared with the model controls. RT-qPCR and IHC showed that Smac, XIAP mRNA and protein expressions in the ischemic hemispheric region were significantly reduced in low, medium, and high doses of butylphthalide compared with the model controls (P < 0.05), showing some concentration effect. ConclusionsButylphthalide can significantly reduce Smac and XIAP mRNA and protein expression, inhibit neuronal apoptosis induced by ischemia-reperfusion injury in rats with carotid stenosis, and exert neuroprotective effects.

Abstract P1-04-07: Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) &amp; gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P&amp;lt; 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P&amp;lt; 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P&amp;lt;0.001). Furthermore, we discovered a strong association between XIAP protein and cellular stress response- &amp; inflammation-related gene sets. Finally, in the CIBERSORT data, we observed the strongest correlation between XIAP expression and M2 macrophages &amp; activated dendritic cells. CONCLUSION: This is the first study showing high XIAP expressing IBC tumors have increased infiltration of immunosuppressive TAMs, which co-localized near tumor and cytotoxic T cells. We also demonstrated an association between XIAP protein expression and inflammation related &amp; cellular stress response gene signatures. This is relevant as XIAP function depends on translational upregulation during cellular stress which leads to immunosuppressive signaling and an aggressive breast cancer phenotype. XIAP antagonists are in clinical trials and these data suggest potential benefit in targeting XIAP to enhance immunotherapeutic efficacy in IBC therapy. FUNDING: DoDs-W81XWH-17-1-0297,W81XWH-20-1-0153, ACS Mission Boost Grants(GRD) Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-07.

Identification of Germline Non-coding Deletions in XIAP Gene Causing XIAP Deficiency Reveals a Key Promoter Sequence

X-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants inXIAPincluding missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons ofXIAPwas identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression. Targeted next-generation sequencing (NGS) of the entire locus ofXIAPwas performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells. NGS ofXIAPidentified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839bp that encompassed the first non-coding exon ofXIAPand contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression. These results identify a key promoter sequence contained in the first non-coding exon ofXIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

Expression of X-Linked Inhibitor of Apoptosis Protein (XIAP) in Breast Cancer Is Associated with Shorter Survival and Resistance to Chemotherapy.

Simple SummaryThe X-linked inhibitor of apoptosis protein (XIAP) is considered the most potent inhibitor of cell death, and it is well established that XIAP promotes resistance to chemotherapy, radiation, and anti-cancer immune responses. This study evaluates the correlations between XIAP expression and clinicopathological features, including disease-free survival (DFS) and pathological complete response (pCR) to chemotherapy, in more than 2300 invasive primary breast cancer samples. We found a significant association of XIAP expression with younger patients’ age (≤50 years), pathological ductal type, lower tumor grade, node-positive status, and PAM50 luminal B subtype. Analysis of molecular subtypes revealed a stronger prognostic value in HR+/HER2− tumors. Higher XIAP expression was associated with shorter DFS and lower pCR rate to chemotherapy in both uni- and multivariate analyses. All these correlations were observed at both the RNA and protein level, indicating the potential of XIAP as a promising therapeutic target in primary invasive breast cancer.XIAP, the most potent inhibitor of cell death pathways, is linked to chemotherapy resistance and tumor aggressiveness. Currently, multiple XIAP-targeting agents are in clinical trials. However, the characterization of XIAP expression in relation to clinicopathological variables in large clinical series of breast cancer is lacking. We retrospectively analyzed non-metastatic, non-inflammatory, primary, invasive breast cancer samples for XIAP mRNA (n = 2341) and protein (n = 367) expression. XIAP expression was analyzed as a continuous value and correlated with clinicopathological variables. XIAP mRNA expression was heterogeneous across samples and significantly associated with younger patients’ age (≤50 years), pathological ductal type, lower tumor grade, node-positive status, HR+/HER2− status, and PAM50 luminal B subtype. Higher XIAP expression was associated with shorter DFS in uni- and multivariate analyses in 909 informative patients. Very similar correlations were observed at the protein level. This prognostic impact was significant in the HR+/HER2− but not in the TN subtype. Finally, XIAP mRNA expression was associated with lower pCR rate to anthracycline-based neoadjuvant chemotherapy in both uni- and multivariate analyses in 1203 informative patients. Higher XIAP expression in invasive breast cancer is independently associated with poorer prognosis and resistance to chemotherapy, suggesting the potential therapeutic benefit of targeting XIAP.

XIAP inhibits gentamicin-induced hair cell damage and ototoxicity through the caspase-3/9 pathway

Gentamicin (GM), an aminoglycoside antibiotic, is one commonly used clinical drugs with ototoxic side effects. One of the most principal mechanisms of its ototoxicity is that GM can activate caspase-mediated cell death pathways in the cochlea. Since the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) has been reported to directly bind to activated caspase protein and inhibit their activities, we hypothesized that it might protect cochlea hair cells from GM ototoxicity. To evaluate this hypothesis, postnatal day 2–3 (P2-3) transgenic (TG) mice, in which XIAP gene is over-expressed under a pure C57BL/6J genetic background was constructed. We first extracted the cochlea tissue of normal mice and treated them with different concentrations of GM, and the number of hair cells were observed to determine the concentration of GM used in subsequent experiments. Next, we used Western Blot experiment to examine the effect of GM on XIAP protein expression in normal mouse cochlea, and then Western Blot and RT-PCR experiments were used to identify the transgenic mice. Finally, immunofluorescence assays were used to detect the effect of GM on the expression of caspase protein and verify the protective effect of XIAP. We found that GM at a concentration of 0.5 mM significantly affected the function of cochlea hair cells, up-regulating the expression of cleaved-caspase-3 and cleaved-caspase-9 protein but down-regulating XIAP protein. In the cochlea tissues of TG mice, this effect of GM was suppressed, and the destruction of hair cells was significantly reduced, and the cleaved-caspase-3 and cleaved-caspase-9 proteins were significantly suppressed. These results suggested that XIAP reduces GM-induced ototoxicity and caspase-3/9 pathway is associated with this process.

Structure based pharmacophore modeling, virtual screening, molecular docking and ADMET approaches for identification of natural anti-cancer agents targeting XIAP protein

X-linked inhibitor of apoptosis protein (XIAP) is a member of inhibitor of apoptosis protein (IAP) family responsible for neutralizing the caspases-3, caspases-7, and caspases-9. Overexpression of the protein decreased the apoptosis process in the cell and resulting development of cancer. Different types of XIAP antagonists are generally used to repair the defective apoptosis process that can eliminate carcinoma from living bodies. The chemically synthesis compounds discovered till now as XIAP inhibitors exhibiting side effects, which is making difficulties during the treatment of chemotherapy. So, the study has design to identifying new natural compounds that are able to induce apoptosis by freeing up caspases and will be low toxic. To identify natural compound, a structure-based pharmacophore model to the protein active site cavity was generated following by virtual screening, molecular docking and molecular dynamics (MD) simulation. Initially, seven hit compounds were retrieved and based on molecular docking approach four compounds has chosen for further evaluation. To confirm stability of the selected drug candidate to the target protein the MD simulation approach were employed, which confirmed stability of the three compounds. Based on the finding, three newly obtained compounds namely Caucasicoside A (ZINC77257307), Polygalaxanthone III (ZINC247950187), and MCULE-9896837409 (ZINC107434573) may serve as lead compounds to fight against the treatment of XIAP related cancer, although further evaluation through wet lab is necessary to measure the efficacy of the compounds.

Identification of hub genes associated with RNAi-induced silencing of XIAP through targeted proteomics approach in MCF7 cells

BackgroundThe X-linked inhibitor of apoptosis protein (XIAP) is the most potent caspase inhibitor of the IAP family in apoptosis pathway. This study aims to identify the molecular targets of XIAP in human breast cancer cells exposed to XIAP siRNA by proteomics screening. The expression of XIAP was reduced in MCF-7 breast cancer cells by siRNA. Cell viability and the mRNA expression level of this gene were evaluated by MTS and quantitative real-time PCR procedures, respectively. Subsequently, the XIAP protein level was visualized by Western blotting and analyzed by two-dimensional (2D) electrophoresis and LC–ESI–MS/MS.ResultsFollowing XIAP silencing, cell proliferation was reduced in XIAP siRNA transfected cells. The mRNA transcription and protein expression of XIAP were decreased in cells exposed to XIAP siRNA than si-NEG. We identified 30 proteins that were regulated by XIAP, of which 27 down-regulated and 3 up-regulated. The most down-regulated proteins belonged to the Heat Shock Proteins family. They participate in cancer related processes including apoptosis and MAPK signaling pathway. Reduced expression of HSP90B1 was associated with apoptosis induction by androgen receptor and prostate specific antigen. Suppression of XIAP resulted in the enhancement of GDIB, ENO1, and CH60 proteins expression. The network analysis of XIAP-regulated proteins identified HSPA8, HSP90AA1, ENO1, and HSPA9 as key nodes in terms of degree and betweenness centrality methods.ConclusionsThese results suggested that XIAP may have a number of biological functions in a diverse set of non-apoptotic signaling pathways and may provide an insight into the biomedical significance of XIAP over-expression in MCF-7 cells.

In vivo Study of siRNA Silencing XIAP Gene to Reverse Taxol-resistance in Human Ovarian Cancer Cells

To study the relationship between down-regulated expression of X linked inhibitor of apoptosis protein (XIAP) gene and the reversal effect of taxol-resistance by using siRNA interference technology in the taxol-resistant ovarian cancer. Randomly assigned the nude mice into six groups (6 in each group) . Group A: normal saline; Group B: taxol; Group C: siRNA-NC+normal saline; Group D: siRNA-NC+taxol; Group E: siRNA XIAP+normal saline; Group F: siRNA XIAP+taxol. Each group was dealt with the corresponding processing depending on the agreed protocol and the transplanted tumors had a multi-point injection with reagents related siRNA, one time every 3 days, 9 times (27 d) in total. Taxol (2 mg/kg) was used in the intraperitoneal injection, 0.2 mL every time, once a week, for four weeks. After 27 d of siRNA treatment, xenograft volumes and qualities were measured and the inhibitory rate was calculated; RNA expression levels and protein levels of XIAP gene in xenografts were detected respectively by real-time fluorescent quantitative PCR and Western blot. Apoptosis of the transplanted tumor cells was examined by TUNEL method. Among the six groups, the proliferation of transplanted tumor in Group F was the slowest, and the tumor inhibition rate was the highest compared with control Group A, followed by Group E, and the tumor inhibition rate was the lowest in Group C. Group F and E expressed the lowest XIAP mRNA and protein expressions ( P<0.05, vs. the other 4 groups) .The apoptosis rate was highest in Group F, followed by Group E, and lowest in Group A and C ( P<0.05). XIAP siRNA has synergy with taxol in taxol-resistant ovarian cancer cells.

MicroRNA-106b-5p participates in lead (Pb2+)-induced cell viability inhibition by targeting XIAP in HT-22 and PC12 cells

Previous studies reported perturbed expressing of X-linked inhibitor of apoptosis protein (XIAP) under lead (Pb) exposure. However, researches on XIAP expression mainly focused on its transcriptional and post-translational regulation, rarely involving post-transcriptional mechanism manipulated by certain indispensable microRNAs (miRNAs). Interestingly, we unveiled that miR-106b-5p, a widely expressed miRNA in various tissues, is up-regulated by Pb2+-induced stress. Moreover, we found a binding site for miR-106b-5p in the 3’-UTR of xiap mRNA using bioinformatics analysis, and provided the evidences that miR-106b-5p can interact and function with this regulatory region via luciferase reporter assay. Our results further showed that miR-106b-5p down-regulates XIAP protein level, and suppression of miR-106b-5p reverses the decrease in both XIAP level and cell viability in Pb2+-treated HT-22 and PC12 cells. In brief, we identified a novel function of miR-106b-5p in the post-transcriptional regulation of XIAP expression associated with Pb neurotoxicity.

ATM Regulated PTEN Degradation Is XIAP E3 Ubiquitin Ligase Mediated in p85α Deficient Cancer Cells and Influence Platinum Sensitivity.

Ataxia-telegiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), and p85α are key tumour suppressors. Whether ATM regulates PTEN expression and influence platinum sensitivity is unknown. We generated ATM knockdowns (KD) and CRISPR knock outs (KO) in glioblastoma (LN18, LN229) and ovarian cancer cells (OVCAR3, OVCAR4). Doxycycline inducible PTEN expression was generated in LN18 and LN229 cells. Transient KD of p85α, CK2, and XIAP was accomplished using siRNAs. Stable p85α knock-in was isolated in LN18 cells. Molecular biology assays included proteasome activity assays, PCR, flow cytometry analysis (cell cycle, double strand break accumulation, apoptosis), immunofluorescence, co-immunoprecipitation, clonogenic, invasion, migration, and 3D neurosphere assays. The clinicopathological significance of ATM, PTEN, p85α, and XIAP (X-linked inhibitor of apoptosis protein) was evaluated in 525 human ovarian cancers using immunohistochemistry. ATM regulated PTEN is p85α dependant. ATM also controls CK2α level which in turn phosphorylates and stabilizes PTEN. In addition, p85α physically interacts with CK2α and protects CK2α from ATM regulated degradation. ATM deficiency resulted in accumulation of XIAP/p-XIAP levels which ubiquitinated PTEN and CK2α thereby directing them to degradation. ATM depletion in the context of p85α deficiency impaired cancer cell migration and invasion reduced 3D-neurosphere formation and increased toxicity to cisplatin chemotherapy. Increased sensitivity to platinum was associated with DNA double strand breaks accumulation, cell cycle arrest, and induction of autophagy. In ovarian cancer patients, ATM, PTEN, p85α, and XIAP protein levels predicted better progression free survival after platinum therapy. We unravel a previously unknown function of ATM in the regulation of PTEN throμgh XIAP mediated proteasome degradation.

CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP

Cytochrome P450 1B1 (CYP1B1) is a key enzyme that catalyzes the metabolism of 17β-estradiol (E2) into catechol estrogens, such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). CYP1B1 is related to tumor formation and is over-expressed in a variety of cancer cells. In particular, CYP1B1 is highly expressed in hormone-related cancers such as breast cancer, ovarian cancer, or prostate cancer compared to other cancers. However, the detailed mechanisms involving this protein remain unclear. In this study, we demonstrate that CYP1B1 affects X-linked inhibitor of apoptosis protein (XIAP) expression. When CYP1B1 was over-expressed in cells, there was significant increase in the XIAP protein level, whereas the XIAP mRNA level was not affected by CYP1B1 expression. Treatment with 4-OHE2, mainly formed by CYP1B1 activity, also increased XIAP protein levels, whereas treatment with 2-OHE2 did not have a significant effect. Treatment with 4-OHE2 significantly prevented proteasome-mediated XIAP degradation. In addition, phosphorylation of XIAP on serine 87, which is known to stabilize XIAP, was up-regulated by 4-OHE2, indicating that 4-OHE2 affects XIAP stability through XIAP phosphorylation. We also found that phosphorylation of protein kinase C (PKC)ε, which is required for XIAP phosphorylation, increased when cells were treated with 4-OHE2. In summary, our data show that CYP1B1 may play an important role in preventing ubiquitin-proteasome-mediated XIAP degradation through the activation of PKCε signaling in cancer cells.

The reduction of XIAP is associated with inflammasome activation in RPE: implications for AMD pathogenesis

BackgroundAge-related macular degeneration (AMD) is a multifactorial chronic disease of the eye. Several candidate pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others suggests inflammasome activity as a mechanism associated with retinal pigment epithelial (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptosis factor, has recently been shown to regulate inflammasome activity in non-ocular cells. The purpose of this study is to characterize XIAP’s regulatory role in RPE.MethodsProtein lysates of eye tissues from rats (vinpocetine- or aurin tricarboxylic acid complex-treated, ATAC, vs naïve) and mice (wild type vs Caspase-4−/−) were utilized to analyze XIAP protein levels. Immunohistochemistry was used to detect NLRP3 levels in the RPE layer. In vitro inflammasome activation on RPE cells was achieved with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation. Levels of XIAP mRNA and 18S RNA were quantified by RT-PCR. Cell culture supernatants were tested directly for secreted IL-1β by ELISA or concentrated for the detection of secreted IL-18 by western blot. Protein lysates from RPE in cell culture were collected for the measurement of cleaved caspase-1 p20, XIAP, and GAPDH. Data are presented as Mean ± SD. p < 0.05 is considered statistically significant.ResultsThe XIAP protein level was significantly increased when the inflammasome was inhibited at the “activation” step by ATAC, but not the “priming” step, in vivo. Concomitantly, NLRP3 immunoreactivity was lower in the RPE layer of animals fed with ATAC. In mice where caspase-1 cleavage was impaired by the genetic deficiency in caspase-4, the XIAP protein level increased in eye tissues. In RPE cell culture, Leu-Leu-OMe stimulation led to caspase-1 cleavage, cytokine secretion, and XIAP reduction, which can be abolished by Z-YVAD-FMK. When XIAP siRNA was given as a pre-treatment to RPE in vitro, Leu-Leu-OMe induced IL-1β/IL-18 secretion was enhanced, whereas overexpressing XIAP reduced IL-1β secretion under inflammasome activation, both compared to controls cells.ConclusionsTogether, these data suggest XIAP-mediated inhibition of inflammasome activity in RPE may provide insights into the biological consequences of inflammasome activation in RPE and reveals the caspase-1/XIAP/IL-1β/IL-18 axis as a target for broader applications in AMD biology and treatment design.

Immunity to X-linked inhibitor of apoptosis protein (XIAP) in malignant melanoma and check-point blockade.

Expression of inhibitors of apoptosis protein (IAP) family members is associated with poor prognosis in cancer patients. Immunity to ML-IAP (livin) and survivin has been well studied in patients with a variety of tumors. XIAP, the most potent inhibitor of apoptosis, is widely expressed in melanoma. To better define its potential role as an immunogenic target, cellular and humoral responses to XIAP were investigated in patients with advanced melanoma. An overlapping peptide library covering the full length of the XIAP protein was used to screen T cell responses of peripheral blood mononuclear cells (PBMC) from stage-IV melanoma patients treated with or without anti-CTLA4 (ipilimumab). The screen identified an array of peptides that predominantly induced CD4+ T cell responses. XIAP epitope-specific CD4+ T cells revealed proliferative responses to melanoma cells that express XIAP. Humoral responses to XIAP were also explored. Cellular and humoral responses to XIAP were associated with beneficial clinical outcomes after ipilimumab-based treatment, supporting XIAP as a potential therapeutic target.

Abstract 723: MX69 induces apoptosis by inhibiting XIAP in both rituximab sensitive and resistant lymphomas

Abstract Background: X-linked inhibitor of apoptosis protein (XIAP) has been implicated in development of resistance to chemotherapy via its direct inhibition of caspases 3,7 and 9. It is overexpressed in several lymphoma cell lines including rituximab-resistant cell lines (RRCL), Raji4RH and RL4RH, produced by our group. We have previously demonstrated that XIAP is critical for chemotherapy-resistance and survival in RRCL by knocking down XIAP using siRNA interference which demonstrated that XIAP is critical for chemotherapy-sensitivity and survival in RRCL. MDM2 inhibition activates p53 transcription and inhibits translation of XIAP, thus promoting apoptosis of cancer cells. Here, we targeted XIAP at a translational level by inhibiting it with MX69, a dual inhibitor of MDM2 and XIAP. Materials and Methods: Cell lines representing Burkitt’s lymphoma (BL), activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL, germinal center B-cell like DLBCL (GCB-DLBCL), mantle cell lymphoma (MCL) and B-cell lymphoblastic leukemia were exposed to MX69 as a single agent (0-80 uM) over 24, 48 and 72 hrs and IC50 concentrations were calculated. Subsequently, Raji, Raji4RH, RL, RL4RH, HBL-2, TMD-8, Daudi and Reh cell lines were exposed to MX69 (0-80 uM), in combination with doxorubicin (0-1 uM), cytarabine (0-50 uM), vincristine (0-10 nM), etoposide (0-50 uM), carboplatin (0-10 uM), ixazomib (0-1.5 uM), ibrutinib (0-10 uM) and venetoclax (0-10 uM) for 48 hours. Cell viability was determined by Cell Titer-Glo. Coefficient of synergy was calculated using CalcuSyn. Induction of apoptosis was evaluated by flow cytometry (Annexin V/PI stain). MDM2, p53, XIAP and PARP protein expression was determined by Western blotting. Results: MX69 induced cell death in a dose- and time-dependent manner in all cell lines. Annexin/PI staining showed caspase-dependent apoptosis with MX69 in all cell lines. Western blotting confirmed significant inhibition of MDM2, XIAP and changes in p53 and PARP following exposure to MX69. MX69 demonstrated significant synergistic activity when combined with doxorubicin, ixazomib, ibrutinib or venetoclax; synergy was strongest for the MX69- venetoclax combination. Conclusion: Our data suggests that in vitro exposure to MX69 resulted in anti-tumor activity in a wide variety of B-cell lymphoma cells lines (including BL, DLBCL, MCL). Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior siRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models have been planned. Targeting MDM2 and XIAP can be an attractive therapeutic strategy in patients with rituximab-sensitive or -resistant B-cell lymphoma. Citation Format: Sumera Khan, Cory Mavis, Ahmad Hanif, Juan Gu, Pallawi Torka, Francisco Hernandez-Ilizaliturri. MX69 induces apoptosis by inhibiting XIAP in both rituximab sensitive and resistant lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 723.

Abstract 1866: Sea cucumber (Stichopus japonicas) F2 enhanced TRAIL-induced apoptosis via XIAP ubiquitination-ER stress in colorectal cancer cells

Abstract Natural products have shown great promise in sensitizing cells to TNF-related apoptosis-inducing ligand (TRAIL) therapy. Sea cucumber (SC) extracts possess antitumor activity, and hence their potential to sensitize CRC cells to TRAIL therapy was evaluated. This study used Western blotting to evaluate the combination effects of SC and TRAIL in CRC and determined the molecular mechanism underlying these effects. SC fractions and TRAIL alone had no effect on apoptosis; however, combined treatment dramatically induced apoptosis of CRC cells, but not normal colon cells. Combined treatment induced expression of apoptotic proteins (poly (ADP-ribose) polymerase (PARP), caspase 3, and 8), and this effect was markedly inhibited by ubiquitination of X-linked inhibitor of apoptosis protein (XIAP). SC did not affect the mRNA levels, but increased proteasomal degradation and ubiquitination of XIAP protein. Furthermore, SC induced reactive oxygen species (ROS) production, thereby activating c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress-related apoptotic pathways in CRC. Taken together, our results demonstrate that the SC fraction may sensitize CRC cells to TRAIL-induced apoptosis through XIAP ubiquitination-ER stress. Citation Format: Jung lim Kim, Seong Hye Park, Soyeon Jeong, Bo Ram Kim, Yoo Jin Na, Min Jee Jo, Yoon A Jeong, Hye Kyeong Yun, Dae Yeong Kim, Bu Gyeom Kim, Sang Cheul Oh, Dae-Hee Lee. Sea cucumber (Stichopus japonicas) F2 enhanced TRAIL-induced apoptosis via XIAP ubiquitination-ER stress in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1866.

Sea Cucumber (Stichopus japonicas) F2 Enhanced TRAIL-Induced Apoptosis via XIAP Ubiquitination and ER Stress in Colorectal Cancer Cells.

Natural products have shown great promise in sensitizing cells to TNF-related apoptosis-inducing ligand (TRAIL) therapy. Sea cucumber (SC) extracts possess antitumor activity, and hence their potential to sensitize colorectal cancer (CRC) cells to TRAIL therapy was evaluated. This study used Western blotting to evaluate the combination effects of SC and TRAIL in CRC, and determined the molecular mechanism underlying these effects. SC fractions and TRAIL alone did not affect apoptosis; however, combined treatment dramatically induced the apoptosis of CRC cells, but not of normal colon cells. Combined treatment induced the expression of apoptotic proteins (poly (ADP-ribose) polymerase (PARP), caspase 3, and 8), and this effect was markedly inhibited by the ubiquitination of X-linked inhibitor of apoptosis protein (XIAP). SC did not affect the mRNA levels, but it increased proteasomal degradation and ubiquitination of the XIAP protein. Furthermore, SC induced reactive oxygen species (ROS) production, thereby activating c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress-related apoptotic pathways in CRC. Altogether, our results demonstrate that the SC F2 fraction may sensitize CRC cells to TRAIL-induced apoptosis through XIAP ubiquitination and ER stress.