Explore the vast range of titles available.

The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H2O2 or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.

Background Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274 ) expression is associated with better clinical response and increased survival to anti-PD-1 therapy . Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53 ) in the regulation of PD-L1 expression in melanoma. Methods We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53 -mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP 53-wt or a TP53 -knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. Results For TP53 -mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53 -wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53 -mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53 L22Q,W23S , a transcriptionally-impaired variant, in TP53 -wt cells. Accordingly, expression of p53 L22Q,W23S in a TP53 -knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. Conclusions While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR‐activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN ‐amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc − . The identification of LRP8 as a specific vulnerability of MYCN ‐amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet‐unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high‐risk neuroblastoma and potentially other MYCN ‐amplified entities. Synopsis The low‐density lipoprotein receptor (LRP8) was identified as a critical suppressor of ferroptosis in MYCN‐amplified neuroblastoma. Blocking selenium/selenocysteine uptake mechanisms via LRP8 offers a selective strategy to induce ferroptosis and disrupt GPX4 function. Ferroptosis, a cell death modality, is gaining interest as a therapeutic approach against challenging tumors. GPX4 is crucial for suppressing ferroptosis, but suitable in vivo inhibitors are lacking, limiting translation to cancer therapies. Genome‐wide and single‐cell CRISPR‐activation screens reveal LRP8 as a critical ferroptosis suppressor in MYCN‐amplified neuroblastoma. Blocking selenium/selenocysteine uptake via LRP8 disrupts GPX4 function and selectively induces ferroptotic cell death. LRP8 dependency emerges as the result of the low system Xc − activity suggesting that targeting LRP8 could be explore in other entities such as AML and lymphoma. Graphical Abstract The low‐density lipoprotein receptor (LRP8) was identified as a critical suppressor of ferroptosis in MYCN‐amplified neuroblastoma. Blocking selenium/selenocysteine uptake mechanisms via LRP8 offers a selective strategy to induce ferroptosis and disrupt GPX4 function.

Receptor tyrosine kinases (RTK) are rarely mutated in cutaneous melanoma, but the expression and activation of several RTK family members are associated with a proinvasive phenotype and therapy resistance. Epidermal growth factor receptor (EGFR) is a member of the RTK family and is only expressed in a subgroup of melanomas with poor prognosis. The insight into regulators of EGFR expression and activation is important for the understanding of the development of this malignant melanoma phenotype. Here, we describe that the transcription factor NRF2, the master regulator of the oxidative and electrophilic stress response, mediates the expression and activation of EGFR in melanoma by elevating the levels of EGFR as well as its ligands EGF and TGFα. ChIP sequencing data show that NRF2 directly binds to the promoter of EGF, which contains a canonical antioxidant response element. Accordingly, EGF is induced by oxidative stress and is also increased in lung adenocarcinoma and head and neck carcinoma with mutationally activated NRF2. In contrast, regulation of EGFR and TGFA occurs by an indirect mechanism, which is enabled by the ability of NRF2 to block the activity of the melanocytic lineage factor MITF in melanoma. MITF effectively suppresses EGFR and TGFA expression and therefore serves as link between NRF2 and EGFR. As EGFR was previously described to stimulate NRF2 activity, the mutual activation of NRF2 and EGFR pathways was investigated. The presence of NRF2 was necessary for full EGFR pathway activation, as NRF2-knockout cells showed reduced AKT activation in response to EGF stimulation compared to controls. Conversely, EGF led to the nuclear localization and activation of NRF2, thereby demonstrating that NRF2 and EGFR are connected in a positive feedback loop in melanoma. In summary, our data show that the EGFR-positive melanoma phenotype is strongly supported by NRF2, thus revealing a novel maintenance mechanism for this clinically challenging melanoma subpopulation.

Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin‐dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor‐suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)‐specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA‐I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non‐canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA‐specific T cell responses by decreasing both absolute and relative MDA‐peptide presentation in melanoma. Melanoma cells elude detection and killing by melanoma differentiation antigen (MDA)‐specific T cells via downregulation of p14. Reduction of p14 leads to a decrease of melanoma differentiation proteins via Wnt5a signaling and increases HLA class‐I expression in melanoma cells. Diminished MDA surface density prevents effective engagement of cognate T cells.

Inhibition of the BRAF/MAPK pathway belongs to the standard therapies for patients with activating BRAF V600E/K mutations. However, even in well-responding tumors, anti-tumorigenic effect and clinical benefit are only transient, and the original tumors often relapse. This demonstrates that there are remaining residual tumors, which have withstood therapy-induced apoptosis and which have the potential to resume growth. Although BRAF mutant melanoma cells seem to depend on BRAF/MAPK signaling, the inhibition of this pathway triggers several events, which modulate the tumor as well as the tumor niche. After a certain adaptation period, this can turn out to be beneficial for tumor growth and metastasis—even in cases of good initial tumor response. This review sheds light on the biology of BRAF/MEK inhibitor-sensitive melanoma cells, which survive targeted therapy and will address the crosstalk signaling events occurring in BRAF mutant melanomas when the BRAF/MAPK pathway is fully blocked. The knowledge of these events is important for potential future drug combinations, which enhance the inhibitory effect of BRAF/MEK inhibition, particularly in patients not eligible for immune therapy.

Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.

Background Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.

Background Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk ( Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 ( Fosl1 ), early growth response 1 ( Egr1 ), osteopontin ( Opn ), insulin-like growth factor binding protein 3 ( Igfbp3 ), dual-specificity phosphatase 4 ( Dusp4 ), and tumor-associated antigen L6 ( Taal6 ). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1 , OPN , IGFBP3 , DUSP4 , and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.