Absence of RIPK3 predicts necroptosis resistance in malignant melanoma.

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.

TRAF2 inhibits TRAIL- and CD95L-induced apoptosis and necroptosis.

The relevance of the adaptor protein TNF receptor-associated factor 2 (TRAF2) for signal transduction of the death receptor tumour necrosis factor receptor1 (TNFR1) is well-established. The role of TRAF2 for signalling by CD95 and the TNF-related apoptosis inducing ligand (TRAIL) DRs, however, is only poorly understood. Here, we observed that knockdown (KD) of TRAF2 sensitised keratinocytes for TRAIL- and CD95L-induced apoptosis. Interestingly, while cell death was fully blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) in control cells, TRAF2-depleted keratinocytes were only partly rescued from TRAIL- and CD95L-induced cell death. In line with the idea the only partially protective effect of zVAD-fmk on TRAIL- and CD95L-treated TRAF2-depleted keratinocytes is due to the induction of necroptosis, combined treatment with zVAD-fmk and the receptor interacting protein 1 (RIP1) inhibitor necrostatin-1 [corrected] fully rescued these cells. To better understand the impact of TRAF2 levels on RIP1- and RIP3-dependent necroptosis and RIP3-independent apoptosis, we performed experiments in HeLa cells that lack endogenous RIP3 and HeLa cells stably transfected with RIP3. HeLa cells, in which necroptosis has no role, were markedly sensitised to TRAIL-induced caspase-dependent apoptosis by TRAF2 KD. In RIP3-expressing HeLa transfectants, however, KD of TRAF2 also strongly sensitised for TRAIL-induced necroptosis. Noteworthy, priming of keratinocytes with soluble TWEAK, which depletes the cytosolic pool of TRAF2-containing protein complexes, resulted in strong sensitisation for TRAIL-induced necroptosis but had only a very limited effect on TRAIL-induced apoptosis. The necroptotic TRAIL response was not dependent on endogenously produced TNF and TNFR signalling, since blocking TNF by TNFR2-Fc or anti-TNFα had no effect on necroptosis induction. Taken together, we identified TRAF2 not only as a negative regulator of DR-induced apoptosis but in particular also as an antagonist of TRAIL- and CD95L-induced necroptosis.

The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin.

Tumour progression and therapy resistance in squamous cell carcinoma of the skin (SCC) is strongly associated with resistance to intrinsic mitochondrial apoptosis. We thus investigated the role of various anti-apoptotic Bcl-2 proteins for apoptosis protection in SCC using the BH3 agonist ABT737 that can overcome multidomain Bcl-2 protein protection. Sensitive SCC cells underwent rapid loss of mitochondrial membrane potential (MMP), subsequent apoptosis concomitant with caspase-3 activation and an early release of mitochondria-derived cytochrome c and smac/DIABLO. In contrast, ABT737 resistance in subsets of SCC cells was not explained by XIAP, important for protection from DR-induced apoptosis in SCC. Of note, ABT737 did not prime SCC cells to DR-induced apoptosis. Interestingly, the ratio of Mcl-1 and Noxa determined sensitivity to ABT737: loss of Mcl-1 rendered resistant cells sensitive to ABT737, whereas loss of Noxa promoted resistance in sensitive cells. In line, suppression of Mcl-1 by the pan-Bcl-2 inhibitor Obatoclax or overexpression of Noxa rendered resistant SCC cells sensitive to BH3 mimetics. Our data indicate that targeting of the Mcl-1/Noxa axis is important to overcome resistance to mitochondrial apoptosis in SCC. Therefore, combination treatment of ABT737 or derivatives with Mcl-1 inhibitors, or inducers of Noxa, may represent a novel option of targeted therapy in metastatic SCC of the skin.

Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy.

Histone deacetylase (HDAC) inhibitors (HDACis) are well-characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome-associated myeloid leukemia (DS-AMKL) blasts. Investigating the antileukemic function of HDACis revealed their transcriptional and post-translational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mammalian target of rapamycin (mTOR) activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.

Systems analysis of apoptosis protein expression allows the case-specific prediction of cell death responsiveness of melanoma cells.

Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein-protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to personalizing anti-cancer treatments in the future.

Cutaneous side effects of inhibitors of the RAS/RAF/MEK/ERK signalling pathway and their management.

Mutations in genes encoding for proteins along the RAS-RAF-MEK-ERK pathway have been detected in a variety of tumor entities, including malignant melanoma, thyroid, colonic and ovarian carcinomas, and some sarcomas. Thus, a number of inhibitors of this pathway have been developed, whose antitumor potential is currently being assessed in different clinical trials. Up to now one drug of this category (vemurafenib) has been approved by the FDA and the European Commission for late-stage melanoma. Although these new targeted anticancer therapies are generally considered to be safe and well tolerated, certain toxicities have been attributed to them, with cutaneous side effects being perhaps the most frequent amongst them. Based on results of clinical trials and on case series, a distinct profile of cutaneous toxicity has been observed, which is similar to that of EGFR and multikinase inhibitors. As exanthema, palmar-plantar erythrodysesthesia syndrome, hyperkeratosis, xerosis, pruritus, photosensitivity, and paronychia, can be controlled in most cases with common conservative modalities, special attention should be given to the early detection of epithelial skin tumors (mainly keratoakanthomas) that can be induced during therapy with these agents. This report reviews all current published data on cutaneous side effects of RAS-RAF-MEK-ERK pathway inhibitors, and attempts to provide the clinician with clear hints for their management.

Glycogen synthase kinase-3ß is a crucial mediator of signal-induced RelB degradation.

The immediate early transcription factor nuclear factor (IκBs) kappa B (NF-κB) is crucially involved in the regulation of numerous physiological or pathophysiological processes such as inflammation and tumourigenesis. Therefore, the control of NF-κB activity, which is mainly regulated by signal-induced degradation of cytoplasmic inhibitors of NF-κB (IκBs), is of high relevance. One known alternative pathway of NF-κB regulation is the stimulus-induced proteasomal degradation of RelB, a component of the NF-κB dimer. Here, we identified the serine/threonine protein kinase glycogen synthase kinase-3ß (GSK-3ß) as a critical signalling component leading to RelB degradation. In Jurkat leukaemic T cells as well as in primary human T cells, tetradecanoylphorbolacetate/ionomycin- and CD3/CD28-induced RelB degradation were impaired by a GSK-3ß-specific pharmacological inhibitor, an ectopically expressed dominant-negative GSK-3ß mutant and by small-interfering RNA-mediated silencing of GSK-3ß expression. Furthermore, a physical interaction between RelB and GSK-3ß was shown by co-immunoprecipitation, which was already notable in unstimulated cells. Most importantly, as demonstrated by in vitro kinase assays, human RelB is inducibly phosphorylated by GSK-3ß, indicating a direct substrate-enzyme relationship. The serine residue 552 is a target of GSK-3ß-mediated phosphorylation in vitro and in vivo. We conclude that GSK-3ß is a crucial regulator of RelB degradation, stressing the relevant linkage between the NF-κB system and GSK-3ß.

Proapoptotic signalling through Toll-like receptor-3 involves TRIF-dependent activation of caspase-8 and is under the control of inhibitor of apoptosis proteins in melanoma cells.

Toll-like receptor-3 (TLR3), a member of an immune recognition receptor family, is widely expressed in tumour cells and has been shown previously to have the capacity to not only activate immune signalling pathways, but also to exert proapoptotic activity in some cells. We show here that HaCaT human keratinocytes are susceptible to apoptosis induction by the TLR3 ligand poly I:C, and use these cells as a model to analyse the apoptotic signalling pathway. Although the BH3-only protein Noxa was transcriptionally induced by poly I:C and translocated to mitochondria, RNAi experiments showed that the BH3-only proteins Noxa, Bim and Puma were individually dispensable for poly I:C-induced apoptosis. Instead, poly I:C-induced activation of caspase-8 via TLR3 and its adapter TRIF was required for apoptosis. In human melanoma cell lines poly I:C failed to induce apoptosis unless protein synthesis was blocked. Significantly, sensitisation towards poly I:C-dependent caspase-8 activation and apoptosis in melanoma cells was also achieved by the synthetic Smac mimetic/inhibitor of apoptosis protein (IAP) antagonist, LBW242, or by specific downregulation of cIAP1 by siRNA. Inactivation of caspase-8 by CrmA overexpression reduced poly I:C/LBW242-induced apoptosis. These results indicate that the proapoptotic activity of TLR3/TRIF/caspase-8 in melanoma cells is under the control of IAPs, and the use of novel Smac mimetics might be a feasible approach to target melanoma.

UV-induced squamous cell carcinoma--a role for antiapoptotic signalling pathways.

The incidence of nonmelanoma skin cancer including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) has dramatically increased in the last decades, and chronic sun exposure was identified as a main etiologic agent. UV radiation may produce DNA damage either directly or through reactive oxygen species (ROS). As mutations caused by UV may lead to skin cancer due to oncogene activation and tumor suppressor gene inactivation, efficient safeguard mechanisms have been developed during evolution. These enclose induction of apoptosis and formation sunburn cells aiming at the removal of premalignant cells. The keratinocyte apoptotic machinery in response to UV consists of both intrinsic/mitochondrial and extrinsic/death receptor-mediated cell-death pathways, which are particularly regulated by mitogen-activated protein kinases (MAPKs, JNK and p38) and the tumor-suppressor protein p53. For development of skin cancer, it appears that critical steps in apoptosis control are dysregulated leading to resistance both to death ligand-mediated and intrinsic proapoptotic pathways. These particularly include inactivation of p53, as well as activation of EGFR, COX-2 and MAPKs, which result in specific regulation of Bcl-2 proteins, death ligands and death receptors. The final unravelling of apoptosis regulation in epithelial skin cancer may allow the development of new targeted therapeutic strategies.

[Livid polycyclic plaques of the lower extremities]. / Livide polyzyklisch begrenzte Plaques an den unteren Extremitäten.

In 10%-55% of patients, leukemia cutis (LC) manifest as a symptom of acute myelomonocytic leukemia and is associated with a poor overall prognosis. Disseminated bluish-violet or red-brownish papules and plaques, nodules and also hemorrhagic ulcers may dominate the initial clinical picture. Importantly, nonspecific infiltrates (leukemids) must be differentiated from specific infiltrates of malignant cells in patients presenting with dermatoses and concomitant underlying hematopoietic neoplasms. The role of the dermatologist is the rapid clinical and dermatohistopathological diagnosis in order to allow immediate, adequate treatment of the patient's underlying systemic disease.

Metastatic squamous cell carcinoma of the ankle in long-standing untreated acrodermatitis chronica atrophicans.

Acrodermatitis chronica atrophicans (ACA) represents the persistent late stage of borreliosis in which Borrelia species may survive for decades. Occasionally, B-cell lymphoma may develop in these patients, and additional neoplastic complications such as basal cell carcinoma or squamous cell carcinoma (SCC) have been reported once each over the past 60 years. Here we describe, to the best of our knowledge, the first case of metastatic SCC in a European patient with long-standing ACA caused by Borrelia burgdorferi sensu stricto. Our case highlights a potential pathophysiological connection of untreated Borrelia infection with the initiation or progression of SCC and should alert dermatologists to this rare complication.

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis.

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.

Bullous delayed pressure urticaria: pathogenic role for eosinophilic granulocytes?

Bullous delayed pressure urticaria (DPU) is a rare variant of DPU. Treatment of DPU is difficult and the underlying pathogenic mechanism of DPU remains elusive. We report a 72-year-old man with DPU and associated chronic urticaria as well as delayed urticarial dermographism. Pressure challenge gave rise to a deep weal covered by multiple vesicles and bullae after 24 h. Histological examination of a skin biopsy specimen obtained 24 h after pressure challenge demonstrated intraepidermal bullae filled with eosinophils accompanied by a dense, predominantly eosinophilic infiltrate in the dermis. Whereas the numbers and morphology of mast cells were unaltered, the extracellular deposition of eosinophil cationic protein revealed evidence for eosinophil activation. Concomitantly, both CD4+ and CD8+ T lymphocytes were present in the infiltrate and expressed interleukin 5. As bullous DPU may represent the maximal variant of DPU, the investigation of the cellular infiltrate and the chemokines/cytokines released may reveal potential pathogenic mechanisms. A possible effector role of eosinophilic granulocytes, T-cell subsets and mast cells is discussed.

[Photoprotection: effects and side effects]. / Lichtschutzmittel: Wirkungen und Nebenwirkungen.

Sunlight is required for life on Earth. Nevertheless it may also be hazardous and can lead to the development of skin cancer and photosensitivity diseases. After a short summary of current knowledge of photocarcinogenesis, the article emphasizes the role of topical sunscreens, their mode of action and potential side effects. The current recommendations for photoprotection for the general public are outlined.