X-linked Ectodermal Dysplasia Research Articles

Abstract 5651: Characterization the mechanistic role of the proapoptotic receptor XEDAR and its therapeutic potential in TNBC

Abstract Breast cancer is one of the most common cancers and a leading cause of death in women worldwide. Among these subtypes, triple negative breast cancer (TNBC) has the worst prognosis, with a dismal outcome especially in metastatic disease. Metastatic TNBC treated with conventional chemotherapy has a median overall survival (OS) of ~ 18 months. The addition of checkpoint inhibitors extend the median OS to more than 2 years in PD-L1 positive patients, but ultimately most patients succumb to the disease. Currently TNBC still poses as a clinical unmet need, and further understanding of the cancer biology may shed light in improving therapeutics against this deadly disease. Apoptosis, or programmed cell death, is a crucial function for cell homeostasis. The ability to evade or circumvent apoptosis is an important hallmark of cancer cells. X-linked ectodermal dysplasia receptor (XEDAR), a cell membrane receptor that binds to the pro-apoptotic ligand EDA-A2, is a member of the TNF receptor family and has been reported to be downregulated in breast cancer cells. XEDAR has been shown to induce downstream apoptosis by binding to EDA-A2, by a mechanism similar to extrinsic apoptotic pathway. By interrogating clinical databases and cross comparison using TCGA, CCLE, and other breast cancer datasets, we discover XEDAR as substantially downregulated in TNBC. Transcription profile analysis between different XEDAR expressing profiles uncovered novel pathways that could be potentially targeted for TNBC treatment. RNA-seq profiling in XEDAR overexpressing TNBC cells were cross compared with aforementioned discovered pathways. Vulnerable pathways including angiogenesis were discovered as possible therapeutic targets. As a proof of concept, we combined angiogenesis inhibitors with EDA-A2 ligand treatment in XEDAR overexpression TNBC cells and observed synergistic effects for combination. Our study uncover XEDAR as a potential target for further treatment in TNBC, which remains to be further validated in preclinical and clinical studies. Citation Format: Chih-Yi Lin, Chung-Jen Yu, Chih-Yang Wang, Kai-Jun Liu, Jiun-I Lai. Characterization the mechanistic role of the proapoptotic receptor XEDAR and its therapeutic potential in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5651.

Ectodysplasin A/Ectodysplasin A Receptor System and Their Roles in Multiple Diseases

Ectodysplasin A (EDA) is a member of the tumor necrosis factor (TNF) family of ligands that was initially reported to induce the formation of various ectodermal derivatives during normal prenatal development. EDA exerts its biological activity as two splice variants, namely, EDA-A1 and EDA-A2. The former binds to the EDA receptor (EDAR), resulting in the recruitment of the intracellular EDAR-associated death domain (EDARADD) adapter protein and the activation of the NF-κB signaling pathway, while the latter binds to a different receptor, EDA2R, also known as X-linked ectodermal dysplasia receptor (XEDAR). Inactivation mutation of the EDA gene or the genes coding for its receptors can result in hypohidrosis ectodermal dysplasia (HED), a condition that is characterized by oligotrichosis, edentulosis or oligodontia, and oligohidrosis or anhidrosis. Recently, as a new liver factor, EDA is gradually known and endowed with some new functions. EDA levels were observed to be upregulated in several metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance. In addition, EDA and its receptors have been implicated in tumor pathogenesis through the regulation of tumor cell proliferation, apoptosis, differentiation, and migration. Here, we first review the role of EDA and its two-receptor system in various signaling pathways and then discuss the physiological and pathological roles of EDA and its receptors.

Tumor Suppressor Gene XEDAR Promotes Differentiation and Suppresses Proliferation and Migration of Gastric Cancer Cells Through Upregulating the RELA/LXRα Axis and Deactivating the Wnt/β-Catenin Pathway

X-linked ectodermal dysplasia receptor (XEDAR) is a new member of the tumor necrosis factor receptor (TNFR) family that induces cell death. The purpose of this study is to determine the tumor-suppressive potential of XEDAR in the development and differentiation of gastric cancer (GC). XEDAR levels were analyzed in human GC tissues and adjacent normal tissues by immunohistochemistry (IHC), quantitative real-time reverse transcription PCR (RT-qPCR), and Western blot analysis. We found that XEDAR expression was significantly downregulated in GC tissues and further decreased in low differentiated GC tissues. Overexpression of XEDAR in MKN45 and MGC803 cells suppressed the ability of cell proliferation and migration, whereas silencing XEDAR showed the opposite effect. Additionally, XEDAR silencing resulted in the upregulation of the differentiation molecular markers β-catenin, CD44 and Cyclin D1 at the protein levels, whereas XEDAR overexpression showed the opposite effect. Notably, XEDAR positively regulated the expression of liver X receptor alpha (LXRα) through upregulating the RELA gene that was characterized as a transcription factor of LXRα in this study. Inhibition of LXRα by GSK2033 or activation of the Wnt/β-catenin pathway by Wnt agonist 1 impaired the effect of XEDAR overexpression on differentiation of MKN45 cells. Moreover, inhibition of RELA mediated by siRNA could promote cell proliferation/migration and rescue the effect of XEDAR overexpression on cell behaviors and expression of genes. Subsequently, overexpression of XEDAR suppressed the growth of GC cells in vivo. Taken together, our findings showed that XEDAR could promote differentiation and suppress proliferation and invasion of GC cells.

Prenatal diagnosis of HNF1b mutation allows recognition of neonatal dysglycemia

Maturity onset diabetes of the young (MODY) is the most common form of monogenic diabetes in Europe, affecting between 1 and 6% of diabetic patients. It comprises a group of heterogeneous genetic disorders characterized by early onset of diabetes (commonly before age 25), absence of autoimmunity, and beta-cell dysfunction. So far, mutations in 14 different genes involved in glucose homeostasis and pancreatic development [1] have been associated with this disease. Although it is an autosomal dominant disorder, de novo mutations should be taken into consideration in patients without a family history of diabetes. Most cases of MODY are due to mutations in GCK, HNF1a, HNF4a and HNF1b, previously known as MODY2, MODY3, MODY1, and MODY5, respectively. Among these, HNF1b is an active transcription factor that forms homodimers or heterodimers with HNF1a and plays a fundamental role in kidney development, nephron differentiation, and pancreatic growth and differentiation. Mutations in this gene lead to congenital anomalies of the kidney and urinary tract, genital malformations, pancreatic atrophy with endocrine and exocrine deficiency [2]. Diabetes usually onsets in early adulthood and frequently requires insulin treatment. We present an unusual case of a prenatal diagnosis that revealed a mutation in the HNF1b gene responsible for neonatal hyperglycemia in a pregnant woman affected by X-linked ectodermal dysplasia.

XEDAR inhibits the proliferation and induces apoptosis of gastric cancer cells by regulating JNK signaling pathway.

X-linked ectodermal dysplasia receptor (XEDAR) has been widely studied in epidermal morphogenesis, but few studies have been conducted on tumorigenesis and development, including gastric cancer. In the present research, we aimed to investigate the effect of XEDAR on gastric cancer and further explore the molecular mechanisms involved. The differential expression of XEDAR in 90 tissue specimens (30 gastric cancer tissues, 30 adjacent tissues and 30 normal tissues) was detected by real-time PCR (RT-PCR) and Western blot. Cell proliferation and apoptosis were explored using MTT and Annexin-V/propidium iodide (PI) assays, respectively. The results revealed that the expression of XEDAR was decreased in gastric cancer tissues and in gastric cancer cell lines, and its expression is regulated by p53 in BGC-823 cells. Furthermore, overexpression of XEDAR inhibited cell proliferation and induced apoptosis in BGC-823 cells. XEDAR moreover inhibited proliferation and induced apoptosis in gastric cancer cells by regulating the JNK signaling pathway. Collectively, the results of the present study suggested that XEDAR inhibits cell proliferation and induces apoptosis by participating in p53-mediated signaling pathway and inhibiting the downstream JNK signaling pathway in gastric cancer.

TNF receptor signalling in autoinflammatory diseases.

Autoinflammatory syndromes are a group of disorders characterized by recurring episodes of inflammation as a result of specific defects in the innate immune system. Patients with autoinflammatory disease present with recurrent outbreaks of chronic systemic inflammation that are mediated by innate immune cells, for the most part. A number of these diseases arise from defects in the tumour necrosis factor receptor (TNFR) signalling pathway leading to elevated levels of inflammatory cytokines. Elucidation of the molecular mechanisms of these recently defined autoinflammatory diseases has led to a greater understanding of the mechanisms of action of key molecules involved in TNFR signalling, particularly those involved in ubiquitination, as found in haploinsufficiency of A20 (HA20), otulipenia/OTULIN-related autoinflammatory syndrome (ORAS) and linear ubiquitin chain assembly complex (LUBAC) deficiency. In this review, we also address other TNFR signalling disorders such as TNFR-associated periodic syndrome (TRAPS), RELA haploinsufficiency, RIPK1-associated immunodeficiency and autoinflammation, X-linked ectodermal dysplasia and immunodeficiency (X-EDA-ID) and we review the most recent advances surrounding these diseases and therapeutic approaches currently used to target these diseases. Finally, we explore therapeutic advances in TNF-related immune-based therapies and explore new approaches to target disease-specific modulation of autoinflammatory diseases.

Hematopoietic stem cell transplantation for RelB deficiency

Hematopoietic stem cell transplantation for RelB deficiency

Hematopoietic stem cell transplantations for primary immune deficiencies associated with NFκB mutations: A review of the literature

The nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) family of transcription factors plays an instrumental role in human immunity and lymphoid organ development. Inherited defects affecting these factors or their regulation are associated with increased susceptibility to infections, as well as non-immune abnormalities. Hematopoietic stem cell transplantations (HSCT) have been shown to correct the immune abnormalities in a few patients with NFκB pathway defects. Here we review the pre-HSCT characteristics, as well as the HSCT and outcome of 35 patients who received HSCT for NFκB defects. Twenty-three patients (65.7%) were reported to have survived HSCT. Survival was higher among patients with X-linked ectodermal dysplasia and immunodeficiency (XL-EDA-ID), and those with CARD11-BCL10-MALT1 (CBM) complex defects, in comparison to patients with autosomal dominant ectodermal dysplasia and immunodeficiency (AD-EDA-ID) and IKBKB defects. Survival following myeloablative conditioning was simi...

Recruitment of A20 by the C-terminal domain of NEMO suppresses NF-κB activation and autoinflammatory disease

Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease.

X-linked ectodermal dysplasia receptor (XEDAR) gene silencing prevents caspase-3-mediated apoptosis in Sjögren's syndrome.

Despite recent advancements in the knowledge of the etiology and pathogenic mechanisms, treatment of the autoimmune disease Sjögren's syndrome (SS) remains mostly empiric and symptom-based, indicating the need for novel therapeutic approaches. Ectodysplasin-A2 (EDA-A2) is a recently isolated member of the tumor necrosis factor superfamily that binds to X-linked ectodermal dysplasia receptor (XEDAR). In this report, we have analyzed the expression and the biological activity of EDA-A2 in human salivary gland epithelial cells (SGEC) from primary Sjögren's syndrome (pSS) patients. We report that EDA-A2 and its receptor XEDAR are overexpressed in pSS SGEC in comparison with healthy individuals and that the EDA-A2/XEDAR system in these cells is involved in the induction of apoptosis via caspases activation. Collectively, our results suggest that EDA-A2/XEDAR system may be a promising agent for the gene therapy of pSS.

A nutritional nonalcoholic steatohepatitis minipig model

Background and AimsThe objective of this study was to elucidate whether a Western diet was associated with nonalcoholic steatohepatitis (NASH), and the relationship between NASH, autophagy and endoplasmic reticulum (ER) stress. MethodsFour-month-old Lee–Sung minipigs were randomly assigned to two groups: control diet (C) and Western diet (W), for a 5-month experimental period. ResultsFeeding a Western diet produced a body composition with more fat, less lean and a greater liver weight. Compared with C pigs, W pigs also exhibited an elevated level of plasma insulin and free fatty acid. The W pigs displayed glucose intolerance, lower circulation antioxidant capacity and greater hepatic oxidative stress. Furthermore, pig fed the W diets had increased collagen accumulation in the liver and elevated systemic inflammation [tumor necrosis factor α and interleukin (IL)-6]. Compared with C pigs, W pigs had higher hepatic ER stress-related protein expression of GRP94, CHOP and caspase-12. The W pigs also had greater hepatic autophagy-related protein expression of p62 and LC3II. In an obesity antibody array analysis, W pigs had higher type 2 diabetes mellitus- (insulin-like growth factor 1, osteoprotegerin and resistin), atherosclerosis- (vascular endothelial growth factor, platelet-derived growth factor-AA and plasminogen activator inhibitor-I) and inflammation- [IL-1, macrophage-stimulating protein alpha, X-linked ectodermal dysplasia receptor and serum amyloid A (SAA)] related protein expressions. In addition, W pigs had greater plasma SAA concentration than C pigs and plasma SAA level was highly associated with IL-6. ConclusionsWe successfully established a NASH pig model, and our findings suggested an association of NASH with ER stress and autophagy. The SAA has potential as a novel plasma biomarker for nonalcoholic fatty liver disease pigs.

XEDAR activates the non-canonical NF-κB pathway

Members of the tumor necrosis factor receptor (TNFR) superfamily are involved in a number of physiological and pathological responses by activating a wide variety of intracellular signaling pathways. The X-linked ectodermal dysplasia receptor (XEDAR; also known as EDA2R or TNFRSF27) is a member of the TNFR superfamily that is highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2), a member of the TNF family that is encoded by the anhidrotic ectodermal dysplasia (EDA) gene. Although XEDAR was first described in the year 2000, its function and molecular mechanism of action is still largely unclear. XEDAR has been reported to activate canonical nuclear factor κB (NF-κB) signaling and mitogen-activated protein (MAP) kinases. Here we report that XEDAR is also able to trigger the non-canonical NF-κB pathway, characterized by the processing of p100 (NF-κB2) into p52, followed by nuclear translocation of p52 and RelB. We provide evidence that XEDAR-induced p100 processing relies on the binding of XEDAR to TRAF3 and TRAF6, and requires the kinase activity of NIK and IKKα. We also show that XEDAR stimulation results in NIK accumulation and that p100 processing is negatively regulated by TRAF3, cIAP1 and A20.

Clinical and histological characterization of hair coat and glandular tissue of Chinese crested dogs

Two varieties exist in the Chinese crested dog breed, namely hairless Chinese crested dogs presenting with hypotrichosis and dentition abnormalities, and the coated powderpuffs. Hairless Chinese crested dogs are obligate heterozygotes for a FOXI3 mutation, and this phenotype is classified as a form of canine ectodermal dysplasia. We provide a detailed histological description of hair follicles and their density for the three subphenotypes (true hairless, semi-coated and powderpuffs) of Chinese crested dogs. Apocrine and exocrine glands of the skin and other tissues were compared with findings reported from dogs with X-linked ectodermal dysplasia. Skin biopsies were collected from 22 Chinese crested dogs. Additionally, the glands of the skin and other tissues were examined from another two dogs available for postmortem examination. Skin biopsies and tissues were processed, stained and evaluated in a blinded fashion. Hair follicular anomalies decreased with increasing number of hairs in the different phenotypes. The FOXI3 mutants had only simple primary hair follicles, whereas the nonmutant powderpuffs had compound follicles identical to other dog breeds. All Chinese crested dogs had an anagen-dominated hair cycle. Furthermore, apocrine glands in the skin and respiratory mucous glands of the mutant Chinese crested dogs were present and normal. We have identified striking histopathological differences between the three subphenotypes of Chinese crested dogs. We clearly demonstrated distinct differences between the canine ectodermal dysplasia in Chinese crested dogs and dogs with X-linked ectodermal dysplasia.

Decreased Expression in Nuclear Factor-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes X-linked Ectodermal Dysplasia with Immunodeficiency

X-linked ectodermal dysplasia with immunodeficiency (XL-ED-ID) is caused by hypomorphic mutations in NEMO, which encodes nuclear factor-kappaB (NF-κB) essential modulator. We identified a novel mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in NEMO in a Japanese patient with XL-ED-ID. Although various abnormally spliced NEMO messenger RNAs (mRNAs) were observed, a small amount of wild-type (WT) mRNA was also identified. Decreased NEMO protein expression was detected in various lineages of leukocytes. Although one abnormally spliced NEMO protein showed residual NF-κB transcription activity, it did not seem to exert a dominant-negative effect against WT-NEMO activity. CD4(+) T cell proliferation was impaired in response to measles and mumps, but not rubella. These results were consistent with the clinical and laboratory findings of the patient, suggesting the functional importance of NEMO against specific viral infections. The 769-1 G>C mutation is responsible for decreased WT-NEMO protein expression, resulting in the development of XL-ED-ID.

Dendritic cells from humans with hypomorphic mutations in IKBKG/NEMO have impaired mitogen-activated protein kinase activity

The covalent attachment of lysine 63-linked polyubiquitin to the zinc-finger domain of IKBKG/NEMO (also known as IKKγ) is necessary for full activation of NF-κB. Impairments of this biochemical mechanism explain the deleterious effects of hypomorphic NEMO mutations on NF-κB signaling function in humans suffering from X-linked ectodermal dysplasia and immunodeficiency. Nevertheless, the biological function of the NEMO zinc-finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity is poorly understood. Here we show that dendritic cells from patients with EDI caused by a C-terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc-finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation.

Decreased Expression In NF-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes Ectodermal Dysplasia with Immunodeficiency

AbstractAbstract 1732 <Introduction>X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the nuclear factor κB (NF-κB) essential modulator (NEMO) gene, also called IKBKG, and these mutations impair but do not abolish NF-κB signaling, resulting in distinct clinical and immunologic phenotypes. We presented the patient with XL-EDA-ID showing a novel splice-site mutation. In this report we precisely study the involvement of this mutation in the molecular pathogenesis of XL-EDA-ID. <Patient>The patient is 12 years old Japanese boy. He had conical-shaped teeth and hypodontia. He had suffered from recurrent bacterial infections and these pathogenic bacteria were mostly streptococcus pneumonia. The laboratory examination revealed that the number of white blood cell counts, the classification of lymphocytes, and the serum immunoglobulin levels were within normal range. However the specific antibodies against measles and streptococcus pneumonia were negative in spite of these infections. <Results>We identified a novel hemizygous mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in the IKBKG gene. In order to clarify the effect of this mutation on mRNA, we performed a cloning analysis. Although various abnormal spliced NEMO (mutant NEMO) mRNAs were observed, a small amount of wild-type NEMO (WT NEMO) mRNA was also identified. The rate of WT and mutant was variable on the time of blood collection. Further we performed FACS and immunoblot analysis in order to evaluate the effect of this mutation at protein level. Two major bands which were presumed to be derived from WT and one mutant were detected in immunoblots using EBV transfected B cells, however the expression levels of these bands were markedly decreased compared to those of the healthy controls. The NEMO protein expression was confirmed to be decreased in various lineages of leukocytes. We generated WT and two representative mutant NEMO constructs and measured their NF-κB transcriptional activity using reporter assay. One mutant NEMO abolished NF-κB transcriptional activity, the other showed weak activity. However, neither of mutant NENO showed a dominant-negative effect against WT NEMO activity. CD14+ cells from the patient produced a lower level of TNF-α in response to IFN-γ stimulation, on the other hand CD3+ cells produced very little IFN-γ in response to IL-12. CD4+ T-cell proliferation was impaired in response to measles and mumps, but not rubella. <Conclusion>The hemizygous 769-1 G>C mutation was shown to cause the decreased expression of WT NEMO protein, resulting in the decrease in NF-κB activation and the development of XL-EDA-ID. Disclosures:No relevant conflicts of interest to declare.

Immune deficiency caused by impaired expression of nuclear factor-κB essential modifier (NEMO) because of a mutation in the 5′ untranslated region of the NEMO gene

Nuclear factor-kappaB (NF-kappaB) is a key transcription factor that regulates both innate and adaptive immunity as well as ectodermal development. Mutations in the coding region of the IkappaB kinase gamma/NF-kappaB essential modifier (NEMO) gene cause X-linked ectodermal dysplasia with immunodeficiency. To determine the genetic cause of recurrent sinopulmonary infections and dysgammaglobulinemia in a patient with a normal NEMO coding sequence and his affected brother. TNF-alpha and IFN-alpha production in response to Toll-like receptor (TLR) stimulation was analyzed by ELISA, NEMO mRNA levels were measured by quantitative PCR, and NEMO protein expression was measured by Western blotting. NF-kappaB activation was assessed by nuclear translocation of p65 and luciferase reporter gene assays. TLR-induced TNF-alpha and IFN-alpha production by PBMCs was impaired in the patient and his brother. Sequencing of the patient's NEMO gene revealed a novel mutation in the 5' untranslated region, which was also present in the brother, resulting in abnormally spliced transcripts and a 4-fold reduction in mRNA levels. NEMO protein levels in EBV transformed B cells and fibroblasts from the index patient were 8-fold lower than normal controls. NF-kappaB p65 nuclear translocation in the patient's EBV B cells after TLR7 ligation was defective. NF-kappaB-dependent luciferase gene expression in IL-1-stimulated fibroblasts from the patient was impaired. This is the first description of immune deficiency resulting from low expression of a normal NEMO protein.

Crosstalk of EDA-A2/XEDAR in the p53 Signaling Pathway

We recently identified X-linked ectodermal dysplasia receptor (XEDAR, also known as TNFRSF27 or EDA2R) as a direct p53 target that was frequently downregulated in colorectal cancer tissues due to its epigenetic alterations or through the p53 gene mutations. However, the role of the posttranslational regulation of XEDAR protein in colorectal carcinogenesis was not well clarified thus far. Here, we report that the extracellular NH(2) terminus of XEDAR protein was cleaved by a metalloproteinase and released into culture media. The remaining COOH-terminal membrane-anchored fragment was rapidly degraded through the ubiquitin-proteasome pathway. Interestingly, ectopic p53 expression also transactivated an XEDAR ligand, EDA-A2, together with XEDAR. Moreover, EDA-A2 blocked the cleavage of XEDAR and subsequently inhibited cell growth. We also found a missense mutation of the XEDAR gene in NCI-H716 colorectal cancer cells, which caused the translocation of XEDAR protein from cell membrane to cytoplasm. This mutation attenuated the growth-suppressive effect of XEDAR, indicating that membrane localization is critical for physiologic XEDAR function. Thus, our findings clearly revealed the crucial role of EDA-A2/XEDAR interaction in the p53-signaling pathway.

X-Linked Ectodermal Dysplasia Receptor Is Downregulated in Breast Cancer via Promoter Methylation

The X-linked ectodermal dysplasia receptor (XEDAR) is a novel receptor of the tumor necrosis factor receptor family that binds to ectodysplasin-A2 (EDA-A2) and induces cell death. The purpose of this study was to determine the tumor-suppressive potential of XEDAR in the development of breast cancer. We analyzed the expression of XEDAR in breast cancer cell lines and tumor samples using quantitative real-time PCR analysis and immunoblotting. We analyzed the human XEDAR gene promoter for the presence of any CpG island and examined its methylation status using methylation-specific real-time PCR. We examined the effect of 5-aza-2'-deoxycytidine on the expression of XEDAR and sensitivity to EDA-A2-induced apoptosis in breast cancer cell lines. Expression of XEDAR, but not EDA-A2, was downregulated in most tumorigenic breast cancer cell lines and tumor samples. Loss of XEDAR expression correlated with the hypermethylation of its promoter. Ectopic expression of XEDAR in MDA-MB-231 cells resulted in significant induction of apoptosis and reduction in colony formation. Treatment with 5-aza-2'-deoxycytidine restored XEDAR expression in breast cancer cell lines with methylated XEDAR promoter and sensitized them to EDA-A2-induced cell death. Our results suggest that XEDAR expression is downregulated in most breast cancers via promoter methylation, which may contribute to accelerated tumor development by blocking EDA-A2-induced cell death. XEDAR may represent a novel breast tumor suppressor gene, and restoration of its expression by treatment with DNA demethylating agents may represent an attractive approach for the treatment of breast cancer.

A Tangled Web of Ubiquitin Chains: Breaking News in TNF-R1 Signaling

A flurry of recent revelations is challenging the current dogma on how ubiquitin-dependent processes culminate in the activation of NF-kappaB by TNF. Here, we integrate these findings into a model for TNF-R1 signaling-and underscore the importance of individual components, including linear ubiquitin chains-which allows for the remarkable versatility of the ubiquitin system.