ELF3 plays an important role in defining TRAIL sensitivity in breast cancer by modulating the expression of decoy receptor 2 (DCR2).

BACKGROUND: TRAIL protein on binding to its cognate death receptors (DR) can induce apoptosis specifically in breast tumor cells sparing normal cells. However, TRAIL also binds to decoy receptors (DCR) thereby inhibiting the apoptotic pathways thus causing TRAIL resistance. Also, one of the barriers due to which TRAIL-based therapy could not become FDA-approved might be because of resistance to therapy. Therefore, in the current study we wanted to explore the role of transcription factors in TRAIL resistance with respect to breast cancer. METHODS: Microarray data from TRAIL-sensitive (TS) and TRAIL-resistant (TR) MDA-MB-231 cells were reanalyzed followed by validation of the candidate genes using quantitative PCR (qPCR), immunoblotting and immunofluorescence technique. Overexpression of the candidate gene was performed in MDA-MB-231 and MCF7 cells followed by cell viability assay and immunoblotting for cleaved caspase-3. Additionally, immunoblotting for DCR2 was carried out. TCGA breast cancer patient survival was used for Kaplan-Meier (KM) plot. RESULTS: Validation of the candidate gene i.e. ELF3 using qPCR and immunoblotting revealed it to be downregulated in TR cells compared to TS cells. ELF3 overexpression in MDA-MB-231 and MCF7 cells caused reversal of TRAIL resistance as observed using cell viability assay and cleaved caspase-3 immunoblotting. ELF3 overexpression also resulted in DCR2 downregulation in the MDA-MB-231 and MCF7 cells. Furthermore, KM analysis found high ELF3 and low DCR2 expression to show better patient survival in the presence of TRAIL. CONCLUSION: Our study shows ELF3 to be an important factor that can influence TRAIL-mediated apoptosis in breast cancer. Also, ELF3 and DCR2 expression status should be taken into consideration while designing strategies for successful TRAIL-based therapy.

Integrated analysis of mRNA and miRNA profiles revealed the role of miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer.

BACKGROUND: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. METHODS: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. RESULTS: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. CONCLUSIONS: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA.

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.

Circadian rhythms of factors involved in luteal regression are modified in p55 tumour necrosis factor receptor (TNFRp55)-deficient mice.

The rhythm of factors involved in luteal regression is crucial in determining the physiological duration of the oestrous cycle. Given the role of tumour necrosis factor (TNF)-α in luteal function and circadian regulation and that most of the effects of TNF-α are mediated by p55 TNF receptor (TNFRp55), the aims of the present study were to analyse the following during the luteal regression phase in the ovary of mice: (1) whether the pattern of expression of progesterone (P4) and the enzymes involved in the synthesis and degradation of P4 is circadian and endogenous (the rhythm persists in constant conditions, (i.e., constant darkness) with a period of about 24 hours); (2) circadian oscillations in clock gene expression; (3) whether there are daily variations in the expression of key genes involved in apoptosis and antioxidant mechanisms; and (4) the consequences of TNFRp55 deficiency. P4 was found to oscillate circadianally following endogenous rhythms of clock factors. Of note, TNFRp55 deficiency modified the circadian oscillation in P4 concentrations and its enzymes involved in the synthesis and degradation of P4, probably as a consequence of changes in the circadian oscillations of brain and muscle ARNT-Like protein 1 (Bmal1) and Cryptochrome 1 (Cry1). Furthermore, TNFRp55 deficiency modified the circadian rhythms of apoptosis genes, as well as antioxidant enzymes and peroxidation levels in the ovary in dioestrus. The findings of the present study strengthen the hypothesis that dysregulation of TNF-α signalling may be a potential cause for altered circadian and menstrual cycling in some gynaecological diseases.

Epigenetic inactivation of TRAIL decoy receptors at 8p12-21.3 commonly deleted region confers sensitivity to Apo2L/trail-Cisplatin combination therapy in cervical cancer.

Multiple chromosomal regions are affected by deletions in cervical cancer (CC) genomes, but their consequence and target gene involvement remains unknown. Our single nucleotide polymorphism (SNP) array identified 8p copy number losses localized to an 8.4 Mb minimal deleted region (MDR) in 36% of CC. The 8p MDR was associated with tumor size, treatment outcome, and with multiple HPV infections. Genetic, epigenetic, and expression analyses of candidate genes at MDR identified promoter hypermethylation and/or inactivation of decoy receptors TNFRSF10C and TNFRSF10D in the majority of CC patients. TNFRSF10C methylation was also detected in precancerous lesions suggesting that this change is an early event in cervical tumorigenesis. We further demonstrate here that CC cell lines exhibiting downregulated expression of TNFRSF10C and/or TNFRSF10D effectively respond to TRAIL-induced apoptosis and this affect was synergistic in combination with DNA damaging chemotherapeutic drugs. We show that the CC cell lines harboring epigenetic inactivation of TRAIL decoy receptors effectively activate downstream caspases suggesting a critical role of inactivation of these genes in efficient execution of extrinsic apoptotic pathway and therapy response. Therefore, these findings shed new light on the role of genetic/epigenetic defects in TRAIL decoy receptor genes in the pathogenesis of CC and provide an opportunity to explore strategies to test decoy receptor gene inactivation as a biomarker of response to Apo2L/TRAIL-combination therapy.

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors.

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin ß. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin ß. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs.

Cooperative role of lymphotoxin ß receptor and tumor necrosis factor receptor p55 in murine liver regeneration.

BACKGROUND & AIMS: The liver exhibits a unique capacity for regeneration in response to injury. Lymphotoxin-ß receptor (LTßR), a core member of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily is known to play an important role in this process. However, the function of LTßR during pathophysiological alterations and its molecular mechanisms during liver regeneration are so far ill-characterized. METHODS: LTßR(-/-) mice were subjected to 70% hepatectomy and liver regeneration capacity, bile acid profiles, and transcriptome analysis were performed. RESULTS: LTßR(-/-) deficient mice suffered from increased and prolonged liver tissue damage after 70% hepatectomy, accompanied by deregulated bile acid homeostasis. Pronounced differences in the expression patterns of genes relevant for bile acid synthesis and recirculation were observed. LTßR and TNFRp55 share downstream signalling elements. Therefore, LTßR(-/-) mice were treated with etanercept to create mice functionally deficient in both signalling pathways. Strikingly, the combined blockade of TNFRp55 and LTßR signalling leads to complete failure of liver regeneration resulting in death within 24 to 48h after PHx. Transcriptome analysis revealed a marked disparity in gene expression programs in livers of LTßR(-/-) and etanercept-treated LTßR(-/-) vs. wild-type animals after PHx. Murinoglobulin 2 was identified as a significantly differentially regulated gene. CONCLUSIONS: LTßR is essential for efficient liver regeneration and cooperates with TNFRp55 in this process. Differences in survival kinetics strongly suggest distinct functions for these two cytokine receptors in liver regeneration. Failure of TNFR and LTßR signalling renders liver regeneration impossible.

Interleukin-6 receptor in spindle-shaped stromal cells, a prognostic determinant of early breast cancer.

Spindle-shaped stromal cells, like carcinoma-associated fibroblasts and mesenchymal stem cells, influence tumor behavior and can serve as parameters in the clinical diagnosis, therapy, and prognosis of early breast cancer. Therefore, the aim of this study is to explore the clinicopathological significance of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) receptors (Rs) 2 and 4 (TRAIL-R2 and R4), and interleukin-6 R (IL-6R) in spindle-shaped stromal cells, not associated with the vasculature, as prognostic determinants of early breast cancer patients. Receptors are able to trigger the migratory activity, among other functions, of these stromal cells. We conducted immunohistochemical analysis for the expression of these receptors in spindle-shaped stromal cells, not associated with the vasculature, of primary tumors from early invasive breast cancer patients, and analyzed their association with clinicopathological characteristics. Here, we demonstrate that the elevated levels of TRAIL-R2, TRAIL-R4, and IL-6R in these stromal cells were significantly associated with a higher risk of metastatic occurrence (p = 0.034, 0.026, and 0.006; respectively). Moreover, high expression of TRAIL-R4 was associated with shorter disease-free survival and metastasis-free survival (p = 0.013 and 0.019; respectively). Also, high expression of IL-6R was associated with shorter disease-free survival, metastasis-free survival, and overall survival (p = 0.003, 0.001, and 0.003; respectively). Multivariate analysis showed that IL-6R expression was an independent prognostic factor for disease-free survival and metastasis-free survival (p = 0.035). This study is the first to demonstrate that high levels of IL-6R expression in spindle-shaped stromal cells, not associated with the vasculature, could be used to identify early breast cancer patients with poor outcomes.

HSV-mediated p55TNFSR reduces neuropathic pain induced by HIV gp120 in rats through CXCR4 activity.

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite extensive research, the detailed neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. In this study, we investigated the role of proinflammatory molecules, tumor necrosis factor-α (TNFα), CXCR4 and stromal-derived factor-1 α (SDF1α), in the L4/5 dorsal root ganglia (DRG) and the spinal dorsal horn in HIV gp120 protein-mediated neuropathic pain. Our results showed that the application of HIV gp120 to the sciatic nerve induced upregulation of TNFα, CXCR4 and SDF1α in both the DRG and the lumbar spinal dorsal horn. Non-replicating herpes simplex virus (HSV) vector encoding the p55TNFSR gene and producing a TNF-soluble receptor (TNFSR) to block bioactivity of TNFα reversed mechanical allodynia. Intrathecal AMD3100 (CXCR4 antagonist) increased mechanical threshold. The HSV vectors expressing p55TNFSR reversed upregulation of TNFα, CXCR4 and SDF1α induced by gp120 in the DRG and the spinal dorsal horn. These studies suggest that proinflammatory TNFα to the CXCR4/SDF1 pathway has an important role in the HIV-related neuropathic pain state and that blocking the proinflammatory cytokines or chemokines is able to reduce neuropathic pain. This work provides a novel gene therapy proof-of-concept for HIV-associated neuropathic pain.

Decrease in neuroimmune activation by HSV-mediated gene transfer of TNFα soluble receptor alleviates pain in rats with diabetic neuropathy.

The mechanisms of diabetic painful neuropathy are complicated and comprise of peripheral and central pathophysiological phenomena. A number of proinflammatory cytokines are involved in this process. Tumor necrosis factor α (TNF-α) is considered to be one of the major contributors of neuropathic pain. In order to explore the potential role of inflammation in the peripheral nervous system of Type 1 diabetic animals with painful neuropathy, we investigated whether TNF-α is a key inflammatory mediator to the diabetic neuropathic pain and whether continuous delivery of TNFα soluble receptor from damaged axons achieved by HSV vector mediated transduction of DRG would block or alter the pain perception in animals with diabetic neuropathy. Diabetic animals exhibited changes in threshold of mechanical and thermal pain perception compared to control rats and also demonstrated increases in TNFα in the DRG, spinal cord dorsal horn, sciatic nerve and in the foot skin, 6 weeks after the onset of diabetes. Therapeutic approaches by HSV mediated expression of p55 TNF soluble receptor significantly attenuated the diabetes-induced hyperalgesia and decreased the expression of TNFα with reduction in the phosphorylation of p38MAPK in the spinal cord dorsal horn and DRG. The overall outcome of this study suggests that neuroinflammatory activation in the peripheral nervous system may be involved in the pathogenesis of painful neuropathy in Type 1 diabetes which can be alleviated by local expression of HSV vector expressing p55 TNF soluble receptor.

Association of TNFRSF10D DNA-methylation with the survival of melanoma patients.

In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012). In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 (95% CI: 2.0-11.0; p < 0.001), RR of relapse, 7.2 (95% CI: 2.8-18.3; p < 0.001)). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.

[Effect of tumor necrosis factor-related apoptosis-inducing ligand on developing human oligodendrocytes in culture].

Accumulating evidence suggests that proinflammatory cytokines play an important role in white matter injury in preterm infants, a condition in which oligodendrocyte (OL) progenitor cells are preferentially injured. We investigated the role of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its death (TRAIL-R1, TRAIL-R2) and decoy (TRAIL-R3, TRAIL-R4) receptors in periventricular white matter injury (PWMI). We hypothesized that the maturation-dependent vulnerability of OLs to TRAIL is due to differential TRAIL receptor expression. We previously investigated TRAIL/TRAIL receptor expression levels in rat OLs in vivo in the context of PWMI. We found that during different developmental stages, human OLs differentially express TRAIL receptors; there is a progressive loss of sensitivity to TRAIL as OLs proceed through the maturation process. Our results show that both TRAIL-R1 and -R2 are highly expressed on human OL progenitors and pre-OLs, while TRAIL-R3 and TRAIL-R4 are mainly expressed on immature and mature human OLs. Our results suggest that TRAIL-R1 and TRAIL-R2 might mediate the death signal in human OL precursor cells and pre-OLs.

Sunitinib induces cellular senescence via p53/Dec1 activation in renal cell carcinoma cells.

Although multitargeted tyrosine kinase inhibitor sunitinib has been used as first-line therapeutic agent against metastatic renal cell carcinoma (mRCC), the molecular mechanism and functional role per se for its therapeutic performance remains obscure. Our present study revealed that sunitinib-treated RCC cells exhibit senescence characteristics including increased SA-ß-gal activity, DcR2 and Dec1 expression, and senescence-associated secretary phenotype (SASP) such as proinflammatory cytokines interleukin (IL)-1α, IL-6 and IL-8 secretion. Moreover, sunitinib administration also led to cell growth inhibition, G1-S cell cycle arrest and DNA damage response in RCC cells, suggesting therapeutic significance of sunitinib-induced RCC cellular senescence. Mechanistic investigations indicated that therapy-induced senescence (TIS) following sunitinib treatment mainly attributed to p53/Dec1 signaling activation mediated by Raf-1/NF-κB inhibition in vitro. Importantly, in vivo study showed tumor growth inhibition and prolonged overall survival were associated with increased p53 and Dec1 expression, decreased Raf-1 and Ki67 staining, and upregulated SA-ß-gal activity after sunitinib treatment. Immunohistochemistry analysis of tumor tissues from RCC patients receiving sunitinib neoadjuvant therapy confirmed the similar treating phenotype. Taken together, our findings suggested that sunitinib treatment performance could be attributable to TIS, depending on p53/Dec1 activation via inhibited Raf-1/nuclear factor (NF)-κB activity. These data indicated potential insights into therapeutic improvement with reinforcing TIS-related performance or overcoming SASP-induced resistance.

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma.

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.

The roles of lipid oxidation products and receptor activator of nuclear factor-κB signaling in atherosclerotic calcification.

This review focuses on the roles of oxylipids and receptor activator of nuclear factor-κB ligand signaling in calcific cardiovascular disease. Both intimal and valvular calcifications are closely associated with atherosclerosis, leading investigators to study the role of atherogenic oxidatively modified lipids (oxylipids). Results have identified the molecular signaling through which oxylipids induce osteogenic differentiation and calcification in vascular cells. A surprising concomitant finding was that, in bona fide osteoblasts from skeletal bone, oxylipids have the opposite effect, ie, inhibiting osteoblastic maturation. This is the basis for the lipid hypothesis of osteoporosis. Oxylipids also induce resorptive osteoclastic cells within the bone environment, raising the question of whether resorptive osteoclasts can be harnessed in the vascular context for cell-based therapy to remove artery wall mineral deposits. The challenge is that vascular cells produce antiosteoclastogenic factors, including the soluble decoy receptor for receptor activator of nuclear factor-κB ligand, possibly accounting for the paucity of resorptive cells and the dominance of mineral in atherosclerotic plaque. These factors may have therapeutic use in osteoclastogenic removal of mineral deposits from arteries.

Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-induced apoptosis through p53-independent up-regulation of death receptors: evidence for the role of reactive oxygen species and JNK.

Discovery of the molecular targets of traditional medicine and its chemical footprints can validate the use of such medicine. In the present report, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the mechanism, we found that UA down-regulated cell survival proteins and induced the cell surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of receptors by UA occurred independently of cell type. Gene silencing of either receptor by small interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was independent of p53 because UA induced DR4 and DR5 in HCT116 p53(-/-) cells. Induction of DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic inhibition abolished the induction of the receptors. The down-regulation of survival proteins and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS, and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In addition, UA was also found to induce the expression of DRs, down-regulate cell survival proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of DcR2 and cell survival proteins.

TRAIL-R3-related apoptosis: epigenetic and expression analyses in women with ovarian neoplasia.

OBJECTIVE: To assess the expression of TRAIL-R3 and the methylation of a CpG island within the TRAIL-R3 promoter both in cystadenoma tumors and primary and metastatic epithelial ovarian carcinoma (EOC). METHODS: RNA was obtained from women with normal ovarian (NO) tissues (n=18), ovarian serous cystadenoma tumors (n=11) and EOC (n=16) using Trizol. Quantitative PCR (qRT-PCR) was performed to quantify the relative levels of TRAIL-R3. The methylation frequency of the CpG island in the TRAIL-R3 promoter was assessed using the methylation-specific PCR (MSP) assay after DNA bisulfite conversion. The differences between the groups were evaluated using the chi-square, Student's t, ANOVA, Mann-Whitney U, Wilcoxon or Kruskal-Wallis tests as indicated. The survival rates were calculated using the Kaplan-Meier method. RESULTS: Cystadenoma and metastatic EOC tumors expressed significantly more TRAIL-R3 mRNA than primary EOC tumors. Methylation of the TRAIL-R3 promoter was absent in NO tissues, while hemimethylation of the TRAIL-R3 promoter was frequently found in the neoplasia samples with 45.4% of the cystadenoma tumors, 8.3% of the primary EOC samples and 11.1% of the metastatic EOC samples showing at least partial methylation (p=0.018). Neither the expression of TRAIL-R3 nor alterations in the methylation profile were associated to cumulative progression-free survival or the overall survival in EOC patients. CONCLUSIONS: Primary EOC is associated to a lower TRAIL-R3 expression, which leads to a better understanding of the complex disease and highlighting potential therapeutic targets. Promoter DNA methylation was not related to this finding, suggesting the presence of other mechanisms to transcriptional control.

Enhanced extrinsic apoptosis of therapy-induced senescent cancer cells using a death receptor 5 (DR5) selective agonist.

Genotoxic agents are widely used anti-cancer therapies because of their ability to interfere with highly proliferative cells. An important outcome of these interventions is the induction of a state of permanent arrest also known as cellular senescence. However, senescent cancer cells are characterized by genomic instability and are at risk of escaping the growth arrest to eventually facilitate cancer relapse. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via Death Receptors (DR) 4 and 5, while Decoy Receptors (DcR) 1 and 2, and Osteoprotegerin (OPG) are homologous to death receptors but incapable of transducing an apoptotic signal. The use of recombinant TRAIL as an anti-cancer strategy in combination with chemotherapy is currently in development, and a major question remains whether senescent cancer cells respond to TRAIL. Here, we show variable sensitivity of cancer cells to TRAIL after senescence induction, and upregulation of both pro-apoptotic and anti-apoptotic receptors in therapy-induced senescent cancer cells. A DR5-selective TRAIL variant (DHER), unable to bind to DcR1 or OPG, was more effective in inducing apoptosis of senescent cancer cells compared to wild-type TRAIL. Importantly, no apoptosis induction was observed in non-cancerous cells, even at the highest concentrations tested. Our results suggest that targeting DR5 can serve as a novel therapeutic strategy for the elimination of therapy-induced senescent cancer cells.

Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis.

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.

TNFR1-dependent regulation of myeloid cell function in experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis is an autoimmune disease induced in mice, which involves the infiltration of CD11b(+) macrophages and CD4(+) T cells into the normally immune-privileged retina. Damage is produced in the target organ following the activation of Th1 and Th17 T cells and by the release of cytotoxic mediators such as NO by activated macrophages. The majority of immune cells infiltrating into the retina are CD11b(+) myeloid cells, but, despite the presence of these APCs, relatively limited numbers of T cells are observed in the retina during the disease course. These T cells do not proliferate when leukocytes are isolated from the retina and restimulated in vitro, although they do produce both IFN-gamma and IL-17. T cell proliferation was restored by depleting the myeloid cells from the cultures and furthermore those isolated myeloid cells were able to regulate the proliferation of other T cells. The ability of macrophages to regulate proliferation depends on activation by T cell-produced IFN-gamma and autocrine TNF-alpha signaling in the myeloid cells via TNFR1. In the absence of TNFR1 signaling, relative T cell expansion in the retina is increased, indicating that regulatory myeloid cells may also act in vivo. However, TNFR1 signaling is also required for macrophages, but not T cells, to migrate into the target organ. Thus, in TNFR1 knock out mice, the amplification of autoimmunity is limited, leading to resistance to experimental autoimmune uveoretinitis induction.