TRAIL-receptor 2-a novel negative regulator of p53.

TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors.

Prognostic significance of high mobility group A2 (HMGA2) in pancreatic ductal adenocarcinoma: malignant functions of cytoplasmic HMGA2 expression.

PURPOSE: HMGA2 has frequently been found in benign as well as malignant tumors and a significant association between HMGA2 overexpression and poor survival in different malignancies was described. In pancreatic ductal adenocarcinoma (PDAC), nuclear HMGA2 expression is associated with tumor dedifferentiation and presence of lymph node metastasis. Nevertheless, the impact of HMGA2 occurrence in other cell compartments is unknown. METHODS: Intracellular distribution of HMGA2 was analyzed in PDAC (n = 106) and peritumoral, non-malignant ducts (n = 28) by immunohistochemistry. Findings were correlated with clinico-pathological data. Additionally, intracellular HMGA2 presence was studied by Western blotting of cytoplasmic and nuclear fractions of cultured cells. RESULTS: HMGA2 was found in the cytoplasm and in the nucleus of cultured cells. In human tumor tissue, HMGA2 was also frequently found in the cytoplasm and the nucleus of tumor cells, however, nuclear staining was generally stronger. Direct comparison from tumor tissue with corresponding non-neoplastic peritumoral tissue revealed significantly stronger expression in tumors (p = 0.003). Of note, the nuclear staining was significantly stronger in lymph node metastatic cell nuclei compared to primary tumor cell nuclei (p = 0.049). Interestingly, cytoplasmic staining positively correlated with lymph vessel (p = 0.004) and venous invasion (p = 0.046). CONCLUSION: HMGA2 is a prognostic marker in PDAC. Firstly, we found a positive correlation for cytoplasmic HMGA2 expression with lympho-vascular invasion and, secondly, we found a significantly stronger nuclear expression of HMGA2 in cancer-positive lymph node nuclei compared to primary tumor cell nuclei. So far, the role of cytoplasmic HMGA2 is nearly unknown, however, our data lend support to the hypothesis that cytoplasmic HMGA2 expression is involved in nodal spread.

TRAIL Induces Nuclear Translocation and Chromatin Localization of TRAIL Death Receptors.

Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.

Impact of p53 status on TRAIL-mediated apoptotic and non-apoptotic signaling in cancer cells.

Due to their ability to preferentially induce cell death in tumor cells, while sparing healthy cells, TNF-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL-R1 or anti-TRAIL-R2-specific antibodies are under clinical investigations for cancer-treatment. However, TRAIL-Rs may also induce signaling pathways, which result in malignant progression. TRAIL receptors are transcriptionally upregulated via wild-type p53 following radio- or chemotherapy. Nevertheless, the impact of p53 status on the expression and signaling of TRAIL-Rs is not fully understood. Therefore, we analyzed side by side apoptotic and non-apoptotic signaling induced by TRAIL or the agonistic TRAIL-R-specific antibodies Mapatumumab (anti-TRAIL-R1) and Lexatumumab (anti-TRAIL-R2) in the two isogenic colon carcinoma cell lines HCT116 p53+/+ and p53-/-. We found that HCT116 p53+/+ cells were significantly more sensitive to TRAIL-R-triggering than p53-/- cells. Similarly, A549 lung cancer cells expressing wild-type p53 were more sensitive to TRAIL-R-mediated cell death than their derivatives with knockdown of p53. Our data demonstrate that the contribution of p53 in regulating TRAIL-R-induced apoptosis does not correlate to the levels of TRAIL-Rs at the plasma membrane, but rather to p53-mediated upregulation of Bax, favouring the mitochondrial amplification loop. Consistently, stronger caspase-9 and caspase-3 activation as well as PARP-cleavage was observed following TRAIL-R-triggering in HCT116 p53+/+ compared to HCT116 p53-/- cells. Interestingly, HCT116 p53+/+ cells showed also a more potent activation of non-canonical TRAIL-R-induced signal transduction pathways like JNK, p38 and ERK1/ERK2 than p53-/- cells. Likewise, these cells induced IL-8 expression in response to TRAIL, Mapatumumab or Lexatumumab significantly stronger than p53-/- cells. We obtained similar results in A549 cells with or without p53-knockdown and in the two isogenic colon cancer cell lines RKO p53+/+ and p53-/-. In both cellular systems, we could clearly demonstrate the potentiating effects of p53 on TRAIL-R-mediated IL-8 induction. In conclusion, we found that wild-type p53 increases TRAIL-R-mediated apoptosis but simultaneously augments non-apoptotic signaling.

The novel TRAIL-receptor agonist APG350 exerts superior therapeutic activity in pancreatic cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has raised attention as a novel anticancer therapeutic as it induces apoptosis preferentially in tumor cells. However, first-generation TRAIL-receptor agonists (TRAs), comprising recombinant TRAIL and agonistic receptor-specific antibodies, have not demonstrated anticancer activity in clinical studies. In fact, cancer cells are often resistant to conventional TRAs. Therefore, in addition to TRAIL-sensitizing strategies, next-generation TRAs with superior apoptotic activity are warranted. APG350 is a novel, highly potent TRAIL-receptor agonist with a hexavalent binding mode allowing the clustering of six TRAIL-receptors per drug molecule. Here we report on preclinical in vitro and in vivo studies testing the activity of APG350 on pancreatic ductal adenocarcinoma (PDAC) cells. We found that APG350 potently induced apoptosis of Colo357, PancTuI and Panc89 cells in vitro. In addition, APG350 treatment activated non-canonical TRAIL signaling pathways (MAPK, p38, JNK, ERK1/ERK2 and NF-κB) and induced the secretion of IL-8. Stable overexpression of Bcl-xL inhibited APG350-induced cell death and augmented activation of non-canonical pathways. Intriguingly, pre-treatment of Bcl-xL-overexpressing cells with the BH3-mimic Navitoclax restored their sensitivity to APG350. To study the effects of APG350 on PDAC cells in vivo, we applied two different orthotopic xenotransplantation mouse models, with and without primary tumor resection, representing adjuvant and palliative treatment regimes, respectively. APG350 treatment of established tumors (palliative treatment) significantly reduced tumor burden. These effects, however, were not seen in tumors with enforced overexpression of Bcl-xL. Upon primary tumor resection and subsequent APG350 treatment (adjuvant therapy), APG350 limited recurrent tumor growth and metastases. Importantly, therapeutic efficacy of APG350 treatment was more effective compared with treatment with soluble TRAIL in both models. In conclusion, APG350 represents a promising next-generation TRA for the treatment of PDAC. Moreover, our results suggest that combining APG350 with Navitoclax might be a succesfull strategy for cancers harboring mitochondrial apoptosis resistance.

Defects in Mitochondrial Iron-Sulfur Cluster Assembly Induce Cysteine S-Polythiolation on Iron-Sulfur Apoproteins.

AIMS: Mitochondria play a central role in the maturation of proteins with iron-sulfur (Fe/S) clusters. During their biogenesis, the apoforms of Fe/S proteins expose unprotected Fe/S cluster-coordinating cysteine side chains, rendering them vulnerable to oxidative modifications that interfere with subsequent Fe/S cluster insertion. Whether and how cells protect these delicate cysteine residues are unknown. RESULTS: In this study, we show that sulfhydryl groups of Fe/S cluster-coordinating cysteine residues of mitochondrial Fe/S apoproteins acquire cyclic S-polythiol modifications. These adducts are the result of persulfide addition, followed by a subsequent oxidation step. These modifications not only accumulate upon defects in the early stages of the mitochondrial Fe/S cluster assembly system but are also found in wild-type cells under normal growth conditions. They are, however, not found on Fe/S apoproteins in the cytosol. INNOVATION AND CONCLUSION: Our work describes a novel in vivo chemical modification of cysteine side chains in mitochondrial Fe/S apoproteins. These cyclic S-polythiolation adducts are resistant to oxidation, yet can be removed by reductive cleavage, suggesting that they serve as a reversible protection device for cysteine ligands sensitive to oxidative modification. Antioxid. Redox Signal. 25, 28-40.