A Bosserhoff

Loss of E-cadherin leads to upregulation of NFkappaB activity in malignant melanoma.

Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here, we show that loss of E-cadherin leads to induction of nuclear factor kappa B (NFκB) activity in melanoma cell lines. Melanoma cells show constitutively active NFκB, whereas no activity is found in primary melanocytes. After re-expression of E-cadherin in melanoma cells, strong downregulation of NFκB activity was found. Consistently, NFκB activity was induced in primary human melanocytes after inhibition of E-cadherin activity by functionally blocking anti-E-cadherin antibodies. Interestingly, re-expression of E-cadherin-blocked p38 MAPK activity and the p38 MAPK inhibitors SB203580 and SB202190 almost completely prevented NFκB activation in melanoma cells. Furthermore, cytoplasmatic β-catenin induced p38 and NFκB activation in malignant melanoma. To our knowledge, this is the first report suggesting a correlation between E-cadherin and NFκB activity in melanocytes and melanoma cells. In summary, we conclude that loss of E-cadherin and cytoplasmatic β-catenin induces p38-mediated NFκB activation, potentially revealing an important mechanism of tumorigenesis in malignant melanomas.

MicroRNA miR-125b controls melanoma progression by direct regulation of c-Jun protein expression.

A fundamental event in the development and progression of malignant melanoma is the deregulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of tumor progression in melanoma and thus the most important member of the AP-1 transcription factor family for this disease. Interestingly, we revealed that c-Jun expression was regulated on the post-transcriptional level and therefore speculated that miRNAs could be involved in c-Jun regulation. We determined seed sequences for miR-125b and miR-527 in the coding region of c-Jun mRNA that hints at the direct involvement of miRNA-dependent regulation on the protein level. We found that the expression of miR-125b was significantly reduced in malignant melanoma cell lines and tissue samples compared with melanocytes, whereas miR-527 remained unchanged. In further functional experiments, treatment of melanoma cells with pre-miR-125b resulted in strong suppression of cellular proliferation and migration, supporting the role of miR-125b in melanoma. In addition, transfection of pre-miR-125b led to strong downregulation of c-Jun protein but not mRNA expression in melanoma cells. Luciferase assays using reporter plasmids containing the miR-125b seed sequence in the luciferase coding region confirmed the direct interaction with miR-125b. Furthermore, immunoprecipitation of Ago-2 revealed that c-Jun mRNA accumulated in the RNA-induced silencing complex after pre-miR-125b transfection in melanoma cells. In summary, we identified an important role for miR-125b in malignant melanoma. Moreover, we demonstrated post-transcriptional regulation of c-Jun by this miRNA and showed that c-Jun is a main mediator of the effects of miR-125b on melanoma cells.

Interim analysis of toxicity and response in phase 1 trial of systemic targeted alpha therapy for metastatic melanoma

Purpose. The aim is to assess toxicity and response of systemic alpha therapy for metastatic melanoma. Experimental design. This is an open-labelled Phase 1 dose escalation study to establish the effective dose of the alpha-immunoconjugate 213Bi-cDTPA-9.2.27 mAb (AIC). Tools used to investigate the effects were physical examination; imaging of tumours; pathology; GFR; CT and changes in tumour marker. Responses were assessed using RECIST criteria. Results and Discussion. 22 patients with stage IV melanoma/ in-transit metastasis were treated with activities of 55-947 MBq. Using RECIST criteria 50% showed stable disease and 14% showed partial response. One patient (6%) showed near complete response and was retreated because of an excellent response to the initial treatment. Another patient showed response in his tumour on mandible and reduction in lung lesions. Overall 30% showed progressive disease. The tumour marker melanoma inhibitory activity protein (MIA) showed reductions over 8 weeks in most of the patients. The disparity of dose with responders is discussed. No toxicity was observed over the range of administered activities. Conclusion. Observation of responses without any toxicity indicates that targeted alpha therapy has the potential to be a safe and effective therapeutic approach for metastatic melanoma.

MicroRNAs in melanocyte and melanoma biology

The importance of microRNAs as key molecular components of cellular processes is now being recognized. Recent reports have shown that microRNAs regulate processes as diverse as protein expression and nuclear functions inside cells and are able to signal extracellularly, delivered via exosomes, to influence cell fate at a distance. The versatility of microRNAs as molecular tools inspires the design of novel strategies to control gene expression, protein stability, DNA repair and chromatin accessibility that may prove very useful for therapeutic approaches due to the extensive manageability of these small molecules. However, we still lack a comprehensive understanding of the microRNA network and its interactions with the other layers of regulatory elements in cellular and extracellular functions. This knowledge may be necessary before we exploit microRNA versatility in therapeutic settings. To identify rules of interactions between microRNAs and other regulatory systems, we begin by reviewing microRNA activities in a single cell type: the melanocyte, from development to disease.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

IntroductionThe transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e−/−) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e−/− mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice.MethodsMurine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e−/− mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels.ResultsOnly minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e−/− mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e−/− cartilage explants. OA progression was significantly enhanced in the Tfap2e−/− mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e−/− articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT.ConclusionsWe reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

ERK activation and autophagy impairment are central mediators of irinotecan-induced steatohepatitis.

Objective Preoperative chemotherapy with irinotecan is associated with the development of steatohepatitis, which increases the risk of perioperative morbidity and mortality for liver surgery. The molecular mechanisms of this chemotherapeutic complication are widely unknown. Design Mechanisms of irinotecan-induced steatohepatitis were studied in primary human hepatocytes in vitro, in mice treated with irinotecan and in liver specimens from irinotecan-treated compared with control patients. Results Irinotecan dose-dependently induced lipid accumulation and pro-inflammatory gene expression in hepatocytes. This was accompanied by an impairment of mitochondrial function with reduced expression of carnitine palmitoyltransferase I and an induction of acyl-coenzyme A oxidase-1 (ACOX1), oxidative stress and extracellular signal-regulated kinase (ERK) activation. ERK inhibition prevented irinotecan-induced pro-inflammatory gene expression but had only a slight effect on lipid accumulation. However, irinotecan also induced an impairment of the autophagic flux mediated by alkalisation of lysosomal pH. Re-acidification of lysosomal pH abolished irinotecan-induced autophagy impairment and lipid accumulation. Also in mice, irinotecan treatment induced hepatic ACOX1 expression, ERK phosphorylation and inflammation, as well as impairment of autophagy and significant steatosis. Furthermore, irinotecan-treated patients revealed higher hepatic ERK activity, expression of pro-inflammatory genes and markers indicative for a shift to peroxisomal fatty acid oxidation and an impaired autophagic flux. Pretreatment with the multityrosine kinase inhibitor sorafenib did not affect autophagy impairment and steatosis but significantly reduced ERK phosphorylation and inflammatory response in irinotecan-treated hepatocytes and murine livers. Conclusions Irinotecan induces hepatic steatosis via autophagy impairment and inflammation via ERK activation. Sorafenib appears as a novel therapeutic option for the prevention and treatment of irinotecan-induced inflammation.

Wild-type KRAS is a novel therapeutic target for melanoma contributing to primary and acquired resistance to BRAF inhibition

Malignant melanoma reveals rapidly increasing incidence and mortality rates worldwide. By now, BRAF inhibition is the standard therapy for advanced melanoma in patients carrying BRAF mutations. However, only approximately 50% of melanoma patients harbor therapeutically attackable BRAF mutations, and overall survival after treatment with BRAF inhibitors is modest. KRAS (Kirsten Rat sarcoma) proteins are acting upstream of BRAF and have a major role in human cancer. Recent approaches awaken the hope to use KRAS inhibition (KRASi) as a clinical tool. In this study, we identified wild-type KRAS as a novel therapeutic target in melanoma. KRASi functions synergistically with BRAF inhibition to reduce melanoma proliferation and to induce apoptosis independently of BRAF mutational status. Moreover, acquired resistance to BRAF inhibitors in melanoma is dependent on dynamic regulation of KRAS expression with subsequent AKT and extracellular-signal regulated kinase activation and can be overcome by KRASi. This suggests KRASi as novel approach in melanoma—alone or in combination with other therapeutic regimes.

Specific c-Jun target genes in malignant melanoma

ABSTRACT A fundamental event in the development and progression of malignant melanoma is the de-regulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of melanoma progression and, thus, is the most important member of the AP-1 transcription factor family in this disease. Surprisingly, no cancer-related specific c-Jun target genes in melanoma were described in the literature, so far. Therefore, we focused on pre-existing ChIP-Seq data (Encyclopedia of DNA Elements) of 3 different non-melanoma cell lines to screen direct c-Jun target genes. Here, a specific c-Jun antibody to immunoprecipitate the associated promoter DNA was used. Consequently, we identified 44 direct c-Jun targets and a detailed analysis of 6 selected genes confirmed their deregulation in malignant melanoma. The identified genes were differentially regulated comparing 4 melanoma cell lines and normal human melanocytes and we confirmed their c-Jun dependency. Direct interaction between c-Jun and the promoter/enhancer regions of the identified genes was confirmed by us via ChIP experiments. Interestingly, we revealed that the direct regulation of target gene expression via c-Jun can be independent of the existence of the classical AP-1 (5´-TGA(C/G)TCA-3´) consensus sequence allowing for the subsequent down- or up-regulation of the expression of these cancer-relevant genes. In summary, the results of this study indicate that c-Jun plays a crucial role in the development and progression of malignant melanoma via direct regulation of cancer-relevant target genes and that inhibition of direct c-Jun targets through inhibition of c-Jun is a potential novel therapeutic option for treatment of malignant melanoma.

Can checkpoint inhibitor therapy improve response to chemotherapy

The emergence of novel therapies has substantially changed the treatment landscape in advanced melanoma. Disrupting the MAPK pathway in BRAF mutant patients with targeted therapies and enhancing antitumor immunity with immune checkpoint inhibitors have shown unique clinical benefit and superseded the era of chemotherapy in melanoma. Randomized clinical trials showed response rates of 12–19% for the anti-cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) antibody ipilimumab, 33–44% for antiprogrammed death 1 (PD-1) antibodies, and 57–62% for combined ipilimumab and nivolumab treatment (Larkin et al. 2015; Robert et al. 2015) with only rare responses to combined checkpoint inhibitor therapy after progression upon sequential antibody monotherapy (Kirchberger et al. 2016). Serum biomarkers can be used to monitor clinical response and enrich for populations with clinical benefit. However, predictive markers to guide therapy decisions are still missing in melanoma. Despite these results only a subset of patients achieves long-term stable disease. One factor that is associated with clinical response is the abundance of CD8+T cells in the tumor microenvironment. Thus, transforming immunologically “cold” tumors into cytotoxic T-cell rich “hot” tumors is assumed to have the potential to boost tumor response rates. In murine lung adenocarcinoma models, the combination of oxaliplatin and cyclophosphamide induced CD8+T cell infiltration and thus sensitized tumors to anti-PD-1 and anti-CTLA-4 checkpoint inhibitor treatment (Pfirschke et al. 2016). Our clinical experience indicates that patients could benefit from chemotherapy after failure of checkpoint inhibitor therapy and the response rates of second-line chemotherapy need further investigation. A total of 17 patients with histologically confirmed, unresectable melanoma (AJCC 2009, stage IV) from three German skin cancer centers (Erlangen, Essen, and Regensburg), who had progressed under anti-CTLA4 and anti-PD1 antibodies were subsequently treated with chemotherapy. Tumor control, defined as complete response, partial response, mixed response or stable disease, was achieved in 53% (n = 9) of the patients (Table 1). The median overall survival (mOS) was 25 months from stage IV disease and 8 months from chemotherapy with 29% (n = 5) of the patients being still alive. A median of 5.5 cycles (range 2–10) was applied for anti-PD-1 antibodies and 4 cycles (range 2–8) for ipilimumab. In this small cohort, the choice of chemotherapy did not seem to influence the overall survival or tumor control rate. The patient with the most pronounced partial response under polychemotherapy with carboplatin and paclitaxel had previously shown severe autoimmune hypophysitis and progressive disease under combined checkpoint inhibition. All BRAF-mutated patients had been pre-treated with BRAF/MEK inhibitors. None of the patients had an NRAS mutation, 71% had an elevated LDH, and 41% of the patients had brain metastases. * Lucie Heinzerling Lucie.Heinzerling@uk-erlangen.de

Immunometabolic Determinants of Chemoradiotherapy Response and Survival in Head and Neck Squamous Cell Carcinoma

Tumor immune microenvironment and tumor metabolism are major determinants of chemoradiotherapy response. The interdependency and prognostic significance of specific immune and metabolic phenotypes in head and neck squamous cell carcinoma (HNSCC) were assessed and changes in reactive oxygen species were evaluated as a mechanism of treatment response in tumor spheroid/immunocyte co-cultures. Pretreatment tumor biopsies were immunohistochemically characterized in 73 HNSCC patients treated by definitive chemoradiotherapy and correlated with survival. The prognostic significance of CD8A, GLUT1, and COX5B gene expression was analyzed within The Cancer Genome Atlas database. HNSCC spheroids were co-cultured inxa0vitro with peripheral blood mononuclear cells (PBMCs) in the presence of the glycolysis inhibitor 2-deoxyglucose and radiation treatment followed by PBMC chemotaxis determination via fluorescence microscopy. In the chemoradiotherapy-treated HNSCC cohort, mitochondrial-rich (COX5B) metabolism correlated with increased and glucose-dependent (GLUT1) metabolism with decreased intratumoral CD8/CD4 ratios. High CD8/CD4, together with mitochondrial-rich or glucose-independent metabolism, was associated with improved short-term survival. The Cancer Genome Atlas analysis confirmed that patients with a favorable immune and metabolic gene signature (high CD8A, high COX5B, low GLUT1) had improved short- and long-term survival. Inxa0vitro, 2-deoxyglucose and radiation synergistically up-regulated reactive oxygen species-dependent PBMC chemotaxis to HNSCC spheroids. These results suggest that glucose-independent tumor metabolism is associated with CD8-dominant antitumor immune infiltrate, and together, these contribute to improved chemoradiotherapy response in HNSCC.