Member Of TNF Receptor Family Research Articles

Antibody-based targeting of TNF-ligands for cancer therapy

The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamily constitute an important immunoregulatory axis pivotal for the correct execution of immune responses. TNF ligand and receptor family members among others are involved in induction of cell death in malignant cells as well as in providing co-stimulatory signals that help mount effective anti-cancer immune responses. This diverse and important regulatory role in immunity has sparked great interest in the development of TNFL/TNFR-targeted cancer immunotherapeutics. Here, I will discuss our cancer immunotherapeutic drug discovery and development program using selected examples of the TNF-ligand superfamily.

AR-regulated TWEAK-FN14 pathway promotes prostate cancer bone metastasis.

The recurrence of prostate cancer metastases to bone after androgen deprivation therapy is a major clinical challenge. We identified FN14 (TNFRSF12A), a TNF receptor family member, as a factor that promotes prostate cancer bone metastasis. In experimental models, depletion of FN14 inhibited bone metastasis, and FN14 could be functionally reconstituted with IKKβ-dependent, NFκB signaling activation. In human prostate cancer, upregulated FN14 expression was observed in more than half of metastatic samples. In addition, FN14 expression was correlated inversely with androgen receptor (AR) signaling output in clinical samples. Consistent with this, AR binding to the FN14 enhancer decreased expression. We show here that FN14 may be a survival factor in low AR output prostate cancer cells. Our results define one upstream mechanism, via FN14 signaling, through which the NFκB pathway contributes to prostate cancer metastasis and suggest FN14 as a candidate therapeutic and imaging target for castrate-resistant prostate cancers.

Transcriptome profile of the murine macrophage cell response to Candida parapsilosis

Candida parapsilosis is a human fungal pathogen with increasing global significance. Understanding how macrophages respond to C. parapsilosis at the molecular level will facilitate the development of novel therapeutic paradigms. The complex response of murine macrophages to infection with C. parapsilosis was investigated at the level of gene expression using an Agilent mouse microarray. We identified 155 and 511 differentially regulated genes at 3 and 8h post-infection, respectively. Most of the upregulated genes encoded molecules involved in immune response and inflammation, transcription, signaling, apoptosis, cell cycle, electron transport and cell adhesion. Typical of the classically activated macrophages, there was significant upregulation of genes coordinating the production of inflammatory cytokines such as TNF, IL-1 and IL-15. Further, we used both primary murine macrophages and macrophages differentiated from human peripheral mononuclear cells to confirm the upregulation of the TNF-receptor family member TNFRSF9 that is associated with Th1 T-helper cell responses. Additionally, the microarray data indicate significant differences between the response to C. parapsilosis infection and that of C. albicans.

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

Nuclear Factor κB is Required for Tumor Growth Inhibition Mediated by Enavatuzumab (PDL192), a Humanized Monoclonal Antibody to TweakR

TweakR is a TNF receptor family member, whose natural ligand is the multifunctional cytokine TWEAK. The growth inhibitory activity observed following TweakR stimulation in certain cancer cell lines and the overexpression of TweakR in many solid tumor types led to the development of enavatuzumab (PDL192), a humanized IgG1 monoclonal antibody to TweakR. The purpose of this study was to determine the mechanism of action of enavatuzumab’s tumor growth inhibition and to provide insight into the biology behind TweakR as a cancer therapeutic target. A panel of 105 cancer lines was treated with enavatuzumab in vitro; and 29 cell lines of varying solid tumor backgrounds had >25% growth inhibition in response to the antibody. Treatment of sensitive cell lines with enavatuzumab resulted in the in vitro and in vivo (xenograft) activation of both classical (p50, p65) and non-classical (p52, RelB) NFκB pathways. Using NFκB DNA binding functional ELISAs and microarray analysis, we observed increased activation of NFκB subunits and NFκB-regulated genes in sensitive cells over that observed in resistant cell lines. Inhibiting NFκB subunits (p50, p65, RelB, p52) and upstream kinases (IKK1, IKK2) with siRNA and chemical inhibitors consistently blocked enavatuzumab’s activity. Furthermore, enavatuzumab treatment resulted in NFκB-dependent reduction in cell division as seen by the activation of the cell cycle inhibitor p21 both in vitro and in vivo. The finding that NFκB drives the growth inhibitory activity of enavatuzumab suggests that targeting TweakR with enavatuzumab may represent a novel cancer treatment strategy.

Transcriptional Profiling of Melanoma Sentinel Nodes Identify Patients with Poor Outcome and Reveal an Association of CD30+ T Lymphocytes with Progression

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30(+) lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30(+) lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4(+)Foxp3(+) or PD1(+) subpopulations and CD4(-)CD8(-) T cells. CD30(+) T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30(+) lymphocytes at those sites associate positively with melanoma progression.

Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy.

With the success of ipilimumab and promise of programmed death-1 pathway-targeted agents, the field of tumor immunotherapy is expanding rapidly. Newer targets for clinical development include select members of the tumor necrosis factor receptor (TNFR) family. Agonist antibodies to these co-stimulatory molecules target both T and B cells, modulating T-cell activation and enhancing immune responses. In vitro and in vivo preclinical data have provided the basis for continued development of 4-1BB, OX40, glucocorticoid-induced TNFR-related gene, herpes virus entry mediator, and CD27 as potential therapies for patients with cancer. In this review, we summarize the immune response to tumors, consider preclinical and early clinical data on select TNFR family members, discuss potential translational challenges and suggest possible combination therapies with the aim of inducing durable antitumor responses.

Autophagy chews Fap to promote apoptosis

Macroautophagy is a key regulator of cellular integrity and viability, but how the process facilitates apoptosis has remained poorly defined. It has now become clear that autophagy degrades the Fap-1 protein phosphatase, a critical negative regulator of apoptotic cell death signalled by the TNF receptor family member, Fas.

Abstract B110: Characterization of the role of TRAF2 and TNFRSF14 in breast cancer

Abstract Human breast cancer patients display significant diversity in terms of their survival, recurrence, metastasis as well as response to treatment. Our group recruited a multi-centre prospective cohort of over 1900 women with ANN breast cancer and followed them clinically. Tumors from 135 women in this group were selected for gene expression analysis on human cDNA microarrays to examine the molecular alterations in ANN patients that could be predictive of recurrence. From genes that demonstrated a statistically significant difference in expression in tumors from patients who have experienced an early recurrence versus who have remained disease free for over 10 years, TRAF2 (tumor necrosis factor (TNF) receptor-associated factor 2) was found to be highly expressed in early recurrence. TRAF2 is involved in immune system cytokine signaling pathways and interacts with many receptors in the TNF receptor superfamily and mediates the survival effects of these receptors including NF-κB, LTβR, JNK and MAPKs kinases. TRAF2 has been reported to be overexpressed or constitutively phosphorylated in some cancers, including lymphoma and pancreatic cancers. The tumor necrosis factor receptor TNFRSF14, also called LIGHTR or HVEM is located on the short arm of chromosome 1p36. Deletion of 1p36 is especially frequent in breast tumors and is associated with progression and lymph node metastasis, poor prognosis, higher rate of recurrence, larger tumor size, and DNA aneuploidy7. Cheung and al. (2010) have described somatic TNFRSF14 mutations in Follicular Lymphoma (FL) which are associated with worse prognosis. They identified 46 cases (18.3%) with non-synonymous mutations affecting TNFRSF14 in a cohort of 251 FL patients. The significance of such acquired mutations on the structure-function relationship of TNFRSF14 is still unknown, but it appears that TNFRSF14 can be considered as a potential candidate gene that might contribute to FL development. TNFRSF14 belongs to the TNF-receptor superfamily and contains multiple copies of a cysteine-rich motif in the extracellular region that is known to provide the ligand recognition motifs. Activation of TNF-receptor family members is induced by their cognate, trivalent, TNF-related ligand, clustering multiple receptors, which in turn recruits the cytosolic TNF receptor-associated factors (TRAFs) adaptors leading to serine kinase activation of the NFκB system. TNFRSF14 associated with TRAF2 and other members of the TRAF family and activates JNK as well as NF κB and AP-1, which control expression of multiple immune, inflammatory and acute phase action genes in response to infection or cellular stress. The short cytoplasmic regions are frequently capable of stimulating the transcription factors activator protein 1 (AP1) and NFKB1. This particular region of the TNFRSF14 gene interacts with members of the TNFR-associated factor (TRAF) family and mediates the signal transduction pathways that activate the immune response. Since TNFRSF14 interacts with TRAF2 and/or other TRAFs to mediate the NF-κB activation and TRAF2 is highly expressed in breast cancer early recurrence, we hypothesized that altered TRAF2 and TNFRSF14 could contribute to recurrence in ANN breast cancer. Single-strand conformation polymorphism (SSCP) and DNA Sequencing were performed to detect any small alteration in PCR-amplified product from 130 ANN breast cancer tumor samples and 48 germline breast cancer tumor samples. Known polymorphisms were excluded and direct sequencing revealed one new coding SNP (Pro262Leu; C>T) located at exon 8. The genotypes CC were observed in 177 of the 178 samples in total. Only one ANN breast cancer tumor sample (936A) revealed a heterozygote genotype (CT). According to algorithms ‘SIFT’ and PolyPhen-2, the Pro262Leu amino acid exchange alters the secondary structure of TNFRSF14 and is predicted to affect the function of the protein. Due to the impact of TNFRSF14 mutations associated with poor prognosis during carcinogenesis, this variant may play a role in the therapy outcome in breast cancer. Quantitative Real-time RT-PCR was performed to quantify TRAF2 expression in a subset of cell lines and tumor samples. Current studies include immunohistochemistry of tissue microarrays containing 888 ANN tumors to detect TRAF2 protein levels and localization and investigation of the association of TRAF2 expression with clinical outcome. In addition, the characterization of the importance of alterations in TRAF2 gene and its associated pathways is being performed using a combination of bioinformatics, to identify potential. Furthermore, the TNFRSF14 Pro262Leu_C>T SNP is being analysed using TaqMan allelic discrimination to investigate a putative association of this variant with breast cancer risk or recurrence. These studies could lead to the discovery of new prognostic markers that may improve our understanding of the molecular pathways responsible for breast cancer development and progression and identify novel targets for breast cancer therapeutics. Citation Format: Sandrine Tchatchou, Lucine Bosnoyan-Collins, Dushanthi Pinnaduwage, Shelley B. Bull, Irene L. Andrulis. Characterization of the role of TRAF2 and TNFRSF14 in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B110.

The contextual role of TNFR family members in CD8+ T‐cell control of viral infections

Immunity to viruses must be tightly controlled to avoid pathology. Receptors and ligands of the tumor necrosis factor (TNF) family play important roles in controlling lymphocyte activation and survival during an immune response. The role of specific TNF receptor (TNFR) family members in antiviral immunity depends on the stage of the immune response and can vary with the virus type and its virulence. Here, we focus on five members of the TNFR family that are prominently expressed on CD8(+) T cells during viral infections, namely, 4-1BB (CD137), CD27, OX40 (CD134), GITR, and TNFR2. 4-1BB, CD27, OX40, and GITR have primarily prosurvival roles for CD8(+) T cells during viral infection, although under some circumstances 4-1BB, GITR, or CD27 signals can limit immunity. Although TNFR2 can be costimulatory under some circumstances, its main role in CD8(+) T-cell responses during viral infection appears to be in contraction of the response. Several TNF family ligands are being explored as adjuvants for viral vaccines, and agonistic antibodies to TNFR family members are being investigated for immunotherapy of chronic viral infection alone and in combination with checkpoint blockade. Such therapies will require thorough and specific optimization to avoid pathology induced by hyperstimulation of these pathways.

Of Chimeric Antigen Receptors and Antibodies: OX40 and 41BB Costimulation Sharpen Up T Cell-Based Immunotherapy of Cancer

Recent success in immunotherapy of malignant diseases has generated a considerable level of interest in controlling cancer by the immune system in the long term. To activate T cells in the immune ‑ suppressive tumor environment and to maintain an antitumor response, adequate costimulation is required along with a strong T cell receptor-peptide MHC interaction. For this purpose, stimulation by TNF receptor family members, OX40 and 41BB, is currently evaluated in early ‑ phase trials, mediated by agonistic anti‑ bodies or, alternatively, delivered to engineered T cells by a recombinant chimeric antigen recep‑ tor. We here briefly discuss both strategies and their clinical perspectives in the immunotherapy of cancer.

THU0108 Associations Between TRAF1-C5 Gene Polymorphisms and Rheumatoid Arthritis: A Meta-Analysis

BackgroundThe tumor necrosis factor receptor-associated factor 1 (TRAF1) gene encodes TRAF1, which acts in association with TNF receptor family members and regulates cytokine signaling networks. Complement 5 (C5) is a...

Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells

AimThe aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. Main methodsFas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. Key findingsCurcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. SignificanceOur results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.

Expression of CD137 on Hodgkin and Reed–Sternberg Cells Inhibits T-cell Activation by Eliminating CD137 Ligand Expression

Hodgkin lymphoma is caused by a minority population of malignant Hodgkin and Reed-Sternberg (HRS) cells that recruit an abundance of inflammatory cells. The long-term survival of HRS cells among the vast majority of immune cells indicates that they have developed potent immune escape mechanisms. We report that the TNF receptor family member CD137 (TNFRSF9) is expressed on HRS cells, while normal B cells, from which HRS cells are most often derived, do not express CD137. In 48 of 53 cases of classical Hodgkin lymphoma, CD137 was detected on HRS cells. Ectopically expressed CD137 transferred by trogocytosis from HRS cells to neighboring HRS and antigen-presenting cells, which constitutively express the CD137 ligand (CD137L and TNFSF9), became associated with CD137L and the CD137-CD137L complex was internalized. Disappearance of CD137L from the surface of HRS and antigen-presenting cells led to reduced costimulation of T cells through CD137, reducing IFN-γ release and proliferation. Our results reveal a new regulatory mechanism for CD137L expression that mediates immune escape by HRS cells, and they identify CD137 as a candidate target for immunotherapy of Hodgkin lymphoma.

514 The Autoimmune Lymphoproliferative Syndrome: A Case Report

BackgroundThe Autoimmune Lymphoproliferative Syndrome (ALPS) is an impairment of lymphocyte apoptosis expressed by generalized non-malignant lymphoproliferation, lymphadenopathy and/or splenomegaly. Majority of patients with ALPS harbor heterozygous germline mutations in the...

CD40 pathway activation reveals dual function for macrophages in human endometrial cancer cell survival and invasion.

Reproductive malignancies are a major cause of cancer death in women worldwide. CD40 is a TNF receptor family member, which upon activation may mediate tumor regression. However, despite the great potential of CD40 agonists, their use as a therapeutic option for reproductive cancers has never been investigated. Because CD40 ligation is a potent pathway of macrophage activation, an in vitro model of pro-inflammatory type-1 (Mϕ-1) and anti-inflammatory type-2 (Mϕ-2) macrophages was developed to determine whether and how macrophage CD40 pathway activation might influence endometrial tumor cell behavior. Analysis of tumor growth kinetic in the endometrial cancer xenograft model indicates that, when injected once into the growing tumors, CD40-activated Mϕ-1 greatly reduced, while CD40-activated Mϕ-2 increased tumor size when compared to control isotype-activated Mϕ-1 and Mϕ-2, respectively. In vitro assays indicated that CD40-activated Mϕ-2 increased cell viability but failed to promote cell invasion. CD40-activated Mϕ-1, in contrast, decreased cell survival but greatly increased cell invasion in tumor cells less susceptible to cell death by apoptosis; they also induced the expression of some pro-inflammatory genes, such as IL-6, LIF, and TNF-α, known to be involved in tumor promotion and metastasis. The presence of IFN-γ is minimally required for CD40-activated Mϕ-1 to promote tumor cell invasion, a process that is mediated in part through the activation of the PI3K/Akt2 signaling pathway in tumor cells. From these results, we speculate that some functions of CD40 in tumor-associated Mϕs might limit the therapeutic development of CD40 agonists in endometrial cancer malignancies.

Selective Activation of Oxygen-Deprived Tumor-Infiltrating Lymphocytes through Local Intratumoral Delivery of CD137 Monoclonal Antibodies

Hypoxia-inducing transcription factor-1α (HIF-1α) in hypoxic tumors induces the TNF receptor family member CD137 on tumor-infiltrating lymphocytes. This can be exploited for intratumoral low-dose injection of effective systemic immunotherapy with agonist CD137-specific monoclonal antibodies that induce circulation of systemic tumor-specific effector T cells capable of eradicating distant metastases.

TNFα regulates the localization of CD40 in lipid rafts of glioma cells

Resistance of glioblastoma multiforme (GBM) to TNFα induced apoptosis is attributed to NFκB activation. As TNF-receptor family member CD40 regulates NFκB activation, we investigated the role of CD40 in NFκB activation in GBM. We observed elevated CD40 levels in human glioma samples as compared to the surrounding normal tissue. Treatment with TNFα elevated CD40 levels in glioma cells and inhibition of CD40 signaling failed to abrogate TNFα induced NFκΒ activity. While TNFα increased the interaction between TRAF2/6, IκBα, IKKα/β in the CD40 signalosome, the level of CD40 in the signalosome remained unaffected upon TNFα treatment. Interestingly, TNFα decreased the spatial localization of CD40 and increased TRAF2/6 co-localization with lipid raft marker Caveolin. As localization of CD40 signalosome in lipid raft is crucial for NFκB activation, TNFα mediated decreased clustering of CD40 in lipid rafts could have possibly contributed to its non-involvement in NFκB activation.

Skeletal and extraskeletal actions of denosumab

Osteoclasts and osteoblasts define skeletal mass, structure and strength through their respective actions in resorbing and forming bone. This remodeling process is orchestrated by the actions of hormones and growth factors, which regulate a cytokine system comprising the receptor activator of nuclear factor κB ligand (RANKL), its receptor RANK and the soluble decoy receptor osteoprotegerin (OPG). Bone resorption depends on RANKL, which determines osteoclast formation, activity and survival. Importantly, cells of the osteoblastic lineage mainly provide RANKL and therefore, are central in the regulation of osteoclast functions. Catabolic effects of RANKL are inhibited by OPG, a TNF receptor family member that binds RANKL, thereby preventing the activation of its receptor RANK, which is expressed by osteoclast precursors. Because this cytokine network is pivotal for the regulation of bone mass in health and diseases, including osteoporosis, rheumatoid arthritis and malignant bone conditions, it has been successfully used for the generation of a targeted therapy to block osteoclast actions. The clinical approval of denosumab, a fully monoclonal antibody against RANKL, provides a novel option to treat bone diseases with a potent, targeted and reversible inhibitor of bone resorption. Although RANKL is also expressed by endothelial cells, T lymphocytes, synovial fibroblasts and various tumor cells, no meaningful clinical extraskeletal effects have been reported after administration of denosumab. This article summarizes the molecular and cellular basis of the RANKL/RANK/OPG system and presents preclinical and clinical studies on the skeletal actions of denosumab.

Antibody Discovery Ex Vivo Accelerated by the LacO/LacI Regulatory Network

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.