Jürgen Eberle

Prevention of non-melanoma skin cancer in organ transplant patients by regular use of a sunscreen: a 24 months, prospective, case-control study

Background  Skin cancers represent a major challenge within the ever growing group of long time surviving organ transplant recipients (OTR) world wide. Especially UV‐induced non‐melanoma skin cancers (NMSC) like invasive squamous cell carcinomas (SCC) and actinic keratoses (AK), and basal cell carcinoma (BCC), outnumber every other form of cancer in organ transplant recipients. Despite encouraging reports of protective effects of broad‐spectrum sunscreens in immunocompetent patients, evidence for the prevention of NMSC in immunocompromised patients is still missing.

Sphingosine kinase activity counteracts ceramide-mediated cell death in human melanoma cells: role of Bcl-2 expression

While most of the pharmacological therapies for melanoma utilize the apoptotic machinery of the cells, the available therapeutic options are limited due to the ability of melanoma cells to resist programmed cell death. Human melanoma cell lines A-375 and M186 are sensitive to ceramide- and Fas-induced cell death, while Mel-2a and M221 are resistant. We have now found that Mel-2a and M221 cells have a significantly higher ceramide/sphingosine-1-phosphate (S1P) ratio than A-375 and M186 cells. As sphingosine kinase (SphK) type 1 plays a critical role in determining the dynamic balance between the proapoptotic sphingolipid metabolite ceramide and the prosurvival S1P, we examined its role in apoptosis of melanoma cells. Increasing SphK1 expression reduced the sensitivity of A-375 melanoma cells to Fas- and ceramide-mediated apoptosis. Conversely, downregulation of SphK1 with small interfering RNA decreased the resistance of Mel-2a cells to apoptosis. Importantly, overexpression of the prosurvival protein Bcl-2 in A-375 cells markedly stimulated SphK1 expression and activity, while downregulation of Bcl-2 reduced SphK1 expression. This link between Bcl-2 and SphK1 might be an additional clue to chemotherapy resistance of malignant melanoma.

Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention as a novel anticancer agent. However, its efficiency may be diminished by occurring resistance in cancer cells. The mechanisms of TRAIL resistance in melanoma are still unsolved. Here we show for the first time that TRAIL-induced activation of NF-κB occurs in apoptosis-sensitive melanoma cell lines through TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4), whereas TRAIL failed to activate nuclear factor kappa B (NF-κB) in melanoma cells positive only for TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5). However, activation of NF-κB by TRAIL was not associated with enhanced expression of antiapoptotic factors: cellular FLICE-inhibitory protein (c-FLIP), Bcl-xL, X-linked inhibitor of apoptosis protein (XIAP), Survivin, Livin. Rather in one of the cell lines, TRAIL induced the downregulation of DR4. In an established cell culture model for TRAIL resistance and regained TRAIL sensitivity, resistance was neither associated with increased NF-κB activity by TRAIL nor by an increased expression of antiapoptotic proteins. However, significant downregulation of caspase-8, caspase-10 and of DR4 was characteristic for TRAIL-resistant, DR4-positive melanoma cells, and regained TRAIL sensitivity coincided with re-expression of these factors. Sensitivity was also largely retained after their exogenous overexpression. Thus, initiator caspases and DR4 rather than NF-κB may control melanoma cell sensitivity to TRAIL, and strategies, which result in their upregulation, may be useful for enhancement of TRAIL sensitivity.

Vemurafenib Potently Induces Endoplasmic Reticulum Stress–Mediated Apoptosis in BRAFV600E Melanoma Cells

Drug resistance in melanomas may be overcome by therapies that trigger endoplasmic reticulum stress. Stressing Out Resistance Many melanomas harbor a form of the kinase BRAF with an amino acid substitution (V600E) that renders the protein constitutively active. The mutant BRAF drives cancer growth by activating extracellular signal–regulated kinase (ERK), which promotes cell proliferation and survival. The BRAFV600E kinase inhibitor vemurafenib is initially an effective therapy for melanoma but loses its efficacy because the tumor cells become drug-resistant. Beck et al. found that vemurafenib inhibited survival signaling mediated by ERK and induced endoplasmic reticulum (ER) stress, a form of cellular stress that can culminate in apoptosis. Combined application of vemurafenib with the ER stress inducer thapsigargin to BRAFV600E melanoma cell lines that were resistant to vemurafenib resulted in an enhanced ER stress response and apoptosis. Their findings indicate a potential strategy to overcome drug resistance in BRAF-mutated melanoma. The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of antiapoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress–induced response, vemurafenib decreased the abundance of the ER chaperone protein glucose-regulated protein 78, increased the abundance of the spliced isoform of the transcription factor X-box binding protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress–related genes. Knockdown of the ER stress response protein activating transcription factor 4 (ATF4) significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.

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.

Occludin Is Involved in Adhesion, Apoptosis, Differentiation and Ca2+-Homeostasis of Human Keratinocytes: Implications for Tumorigenesis

Tight junction (TJ) proteins are involved in a number of cellular functions, including paracellular barrier formation, cell polarization, differentiation, and proliferation. Altered expression of TJ proteins was reported in various epithelial tumors. Here, we used tissue samples of human cutaneous squamous cell carcinoma (SCC), its precursor tumors, as well as sun-exposed and non-sun-exposed skin as a model system to investigate TJ protein alteration at various stages of tumorigenesis. We identified that a broader localization of zonula occludens protein (ZO)-1 and claudin-4 (Cldn-4) as well as downregulation of Cldn-1 in deeper epidermal layers is a frequent event in all the tumor entities as well as in sun-exposed skin, suggesting that these changes result from chronic UV irradiation. In contrast, SCC could be distinguished from the precursor tumors and sun-exposed skin by a frequent complete loss of occludin (Ocln). To elucidate the impact of down-regulation of Ocln, we performed Ocln siRNA experiments in human keratinocytes and uncovered that Ocln downregulation results in decreased epithelial cell-cell adhesion and reduced susceptibility to apoptosis induction by UVB or TNF-related apoptosis-inducing ligand (TRAIL), cellular characteristics for tumorigenesis. Furthermore, an influence on epidermal differentiation was observed, while there was no change of E-cadherin and vimentin, markers for epithelial-mesenchymal transition. Ocln knock-down altered Ca2+-homeostasis which may contribute to alterations of cell-cell adhesion and differentiation. As downregulation of Ocln is also seen in SCC derived from other tissues, as well as in other carcinomas, we suggest this as a common principle in tumor pathogenesis, which may be used as a target for therapeutic intervention.

Apoptosis pathways and oncolytic adenoviral vectors: promising targets and tools to overcome therapy resistance of malignant melanoma

Abstract: In the last decades melanoma incidence has been increasing worldwide, while mortality remained on a high level. Until now, there is no suitable therapy for metastasized melanoma, which could lead to a significant increase in overall survival. Apoptosis deficiency is supposed to be a critical factor for therapy resistance, and previous work has characterized the basic mechanisms of apoptosis regulation in melanoma. Genes and strategies suitable for efficient induction of apoptosis in melanoma cells were identified, which are based on proapoptotic Bcl‐2 proteins (Bcl‐xS, Bcl‐xAK, Bik/Nbk and Bax) as well as on tumor necrosis factor (TNF)‐related death ligands (CD95L/Fas ligand and TNF‐related apoptosis‐inducing ligand, TRAIL). Proapoptotic genes may be employed in improved gene therapeutic strategies, based on conditional oncolytic adenoviral vectors.

General Sensitization of melanoma cells for TRAIL-induced apoptosis by the potassium channel inhibitor TRAM-34 depends on release of SMAC.

The death ligand TRAIL represents a promising therapeutic strategy for metastatic melanoma, however prevalent and inducible resistance limit its applicability. A new approach is presented here for sensitization to TRAIL. It is based on inhibition of the membrane potassium channel KCa3.1 (IK1), which serves fundamental cellular functions related to membrane potential. The selective inhibitor TRAM-34 did not induce apoptosis by itself but synergistically enhanced TRAIL sensitivity and overrode TRAIL resistance in a large panel of melanoma cell lines. Expression of IK1 was also found in mitochondria, and its inhibition resulted in mitochondrial membrane hyperpolarization and an early activation of Bax. The combination of TRAM-34 and TRAIL resulted in massive release of mitochondrial factors, cytochrome c, AIF and SMAC/DIABLO. Bax knockdown and Bcl-2 overexpression abolished apoptosis. Overexpression of XIAP diminished apoptosis by two-fold, and SMAC knockdown almost completely abolished apoptosis. These data uncover the existence of a rheostat in melanoma cells, consisting of inhibitor of apoptosis proteins and SMAC, which regulates TRAIL sensitivity. Thus, a new strategy is described based on mitochondrial membrane channels, which correspond to Bax activation. As both TRAIL and IK1 inhibitors had shown only minor side effects in clinical trials, a clinical application of this combination is conceivable.

New caspase-independent but ROS-dependent apoptosis pathways are targeted in melanoma cells by an iron-containing cytosine analogue

Chemotherapy resistance and related defects in apoptotic signaling are crucial for the high mortality of melanoma. Effective drugs are lacking, also due to the fact that apoptosis regulation in this tumor is essentially not understood. The cytosine analogue ferropoptoside (N69), which contains an iron carbonyl complex, resulted in strong induction of apoptosis in melanoma cells starting already after 2h, whereas cytotoxicity remained at a low level. Surprisingly, there was no indication for any caspase activation at early times, although cytochrome c was released from mitochondria. Indicative for new proapoptotic pathways was the production of reactive oxygen species (ROS) as an early effect, and the inhibition of apoptosis by the antioxidant vitamin E. Apoptosis was also blocked by exogenous Bcl-2 overexpression and by the pan-protease inhibitor zVAD. However, only zVAD also prevented ROS production, for which Bcl-2 remained without an effect. Thus, new proapoptotic pathways are described here for melanoma cells clearly related to ROS production. A cascade enclosing enhanced levels of intracellular iron, which lead to enhanced ROS production in a Fenton reaction, appears as suggestive. Whereas off-target effects of zVAD appear as upstream, Bcl-2 may exert its inhibitory activity downstream of ROS. New proapoptotic pathways are of particular interest for melanoma as they may open new options for targeting this highly therapy-refractory tumor.

Enhanced Death Ligand-Induced Apoptosis in Cutaneous SCC Cells by Treatment with Diclofenac/Hyaluronic Acid Correlates with Downregulation of c-FLIP

Actinic keratosis (AK) occurs on sun-exposed skin and may progress to invasive squamous cell carcinoma (SCC). As for its topical treatment, diclofenac/hyaluronic acid (HA) has been recently approved. The NSAID diclofenac is an inhibitor of COX-2; however, its mode of action in cutaneous epithelial cancer cells is largely unknown. Here, the effects of diclofenac/HA were investigated in relation to death ligand-mediated apoptosis (TNF-alpha, TRAIL, and CD95 activation). Whereas diclofenac/HA only moderately induced apoptosis by itself, it resulted in pronounced enhancement of death ligand-mediated apoptosis in sensitive SCC cell lines (3/4). Apoptosis was associated with activation of initiator caspases of the extrinsic pathway (caspase-8/caspase-10). Furthermore, death ligand and diclofenac/HA-mediated apoptosis were blocked by the same caspase inhibitors, indicating related pathways. The proapoptotic effects of diclofenac/HA appeared independent of the p53 pathway. Also, upregulation of death receptors appeared less important; however, strong downregulation of c-FLIP isoforms was seen after diclofenac/HA treatment. The crucial role of c-FLIP was proven through overexpression and knockdown experiments. Thus, induction of apoptosis appears to be highly characteristic of the mode of action of diclofenac/HA, and the therapeutic effect may be related to sensitization of neoplastic keratinocytes for death ligand-induced apoptosis.