TY - JOUR A1 - Adam, Christian A1 - Baeurle, Anne A1 - Brodsky, Jeffrey L. A1 - Schrama, David A1 - Wipf, Peter A1 - Becker, Jürgen Christian A1 - Houben, Roland T1 - The HSP70 Modulator MAL3-101 Inhibits Merkel Cell Carcinoma N2 - Merkel Cell Carcinoma (MCC) is a rare and highly aggressive neuroendocrine skin cancer for which no effective treatment is available. MCC represents a human cancer with the best experimental evidence for a causal role of a polyoma virus. Large T antigens (LTA) encoded by polyoma viruses are oncoproteins, which are thought to require support of cellular heat shock protein 70 (HSP70) to exert their transforming activity. Here we evaluated the capability of MAL3-101, a synthetic HSP70 inhibitor, to limit proliferation and survival of various MCC cell lines. Remarkably, MAL3-101 treatment resulted in considerable apoptosis in 5 out of 7 MCC cell lines. While this effect was not associated with the viral status of the MCC cells, quantitative mRNA expression analysis of the known HSP70 isoforms revealed a significant correlation between MAL3-101 sensitivity and HSC70 expression, the most prominent isoform in all cell lines. Moreover, MAL3-101 also exhibited in vivo antitumor activity in an MCC xenograft model suggesting that this substance or related compounds are potential therapeutics for the treatment of MCC in the future. KW - apoptosis KW - cancer treatment KW - cell staining KW - cultured fibroplasts KW - heat shock response KW - membrans proteins KW - polymerase chain reaction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112795 ER - TY - JOUR A1 - Wobser, Marion A1 - Roth, Sabine A1 - Appenzeller, Silke A1 - Houben, Roland A1 - Schrama, David A1 - Goebeler, Matthias A1 - Geissinger, Eva A1 - Rosenwald, Andreas A1 - Maurus, Katja T1 - Targeted deep sequencing of mycosis fungoides reveals intracellular signaling pathways associated with aggressiveness and large cell transformation JF - Cancers N2 - Introduction: Large-cell transformation (LCT) of mycosis fungoides (MF) has been associated with a higher risk of relapse and progression and, consequently, restricted prognosis. Its molecular pathogenesis has not been elucidated yet. Materials and Methods: In order to address molecular mechanisms of LCT, we performed hybrid capture panel-based sequencing of skin biopsies from 10 patients suffering from MF with LCT versus 17 patients without LCT including follow-up biopsies during clinical course, respectively (51 samples in total). The analyzed patients were attributed to three different groups based on the presence of LCT and clinical behavior. Results: While indolent MF cases without LCT did not show pathogenic driver mutations, a high rate of oncogenic alterations was detected in patients with LCT and aggressive clinical courses. Various genes of different oncogenic signaling pathways, including the MAPK and JAK-STAT signaling pathways, as well as epigenetic modifiers were affected. A high inter-individual and distinctive intra-individual mutation diversity was observed. Oncogenic RAS mutations were exclusively detected in patients with LCT. Conclusion: Our data demonstrate that LCT transition of MF is associated with increased frequency of somatic mutations in cancer-associated genes. In particular, the activation of RAS signaling — together with epigenetic dysregulation — may crucially contribute to the molecular pathogenesis of the LCT phenotype, thus conveying its adverse clinical behavior. KW - mycosis fungoides KW - cutaneous T-cell-lymphoma KW - panel sequencing KW - large cell transformation KW - CD30 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250094 SN - 2072-6694 VL - 13 IS - 21 ER - TY - JOUR A1 - Becker, Jürgen C. A1 - Andersen, Mads H. A1 - Hofmeister-Müller, Valeska A1 - Wobser, Marion A1 - Frey, Lidia A1 - Sandig, Christiane A1 - Walter, Steffen A1 - Singh-Jasuja, Harpreet A1 - Kämpgen, Eckhart A1 - Opitz, Andreas A1 - Zapatka, Marc A1 - Bröcker, Eva-B. A1 - thor Straten, Per A1 - Schrama, David A1 - Ugurel, Selma T1 - Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma JF - Cancer Immunology, Immunotherapy N2 - Background Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. Patients and methods This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). Results Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. Conclusion Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma. KW - peptide vaccination KW - therapy KW - survivin T-cell reactivity KW - melanoma Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126215 VL - 61 IS - 11 ER - TY - JOUR A1 - Becker, Jürgen C. A1 - Andersen, Mads H. A1 - Hofmeister-Müller, Valeska A1 - Wobser, Marion A1 - Frey, Lidia A1 - Sandig, Christiane A1 - Walter, Steffen A1 - Singh-Jasuja, Harpreet A1 - Kämpgen, Eckhart A1 - Opitz, Andreas A1 - Zapatka, Marc A1 - Bröcker, Eva-B. A1 - thor Straten, Per A1 - Schrama, David A1 - Ugurel, Selma T1 - Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma JF - Cancer Immunology, Immunotherapy N2 - Background Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. Patients and methods This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). Results Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. Conclusion Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma. KW - peptide vaccination KW - melanoma KW - survivin KW - T-cell reactivity KW - therapy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124830 VL - 61 IS - 11 ER - TY - JOUR A1 - Schrama, David A1 - Ugurel, Selma A1 - Sucker, Antje A1 - Ritter, Cathrin A1 - Zapatka, Marc A1 - Schadendorf, Dirk A1 - Becker, Jürgen Christian T1 - STAT3 Single Nucleotide Polymorphism rs4796793 SNP Does Not Correlate with Response to Adjuvant IFNα Therapy in Stage III Melanoma Patients JF - Frontiers in Medicine N2 - Interferon alpha (IFNα) is approved for adjuvant treatment of stage III melanoma in Europe and the US. Its clinical efficacy, however, is restricted to a subpopulation of patients while side effects occur in most of treated patients. Thus, the identification of predictive biomarkers would be highly beneficial to improve the benefit to risk ratio. In this regard, STAT3 is important for signaling of the IFNα receptor. Moreover, the STAT3 single-nucleotide polymorphism (SNP) rs4796793 has recently been reported to be associated with IFNα sensitivity in metastatic renal cell carcinoma. To translate this notion to melanoma, we scrutinized the impact of rs4796793 functionally and clinically in this cancer. Interestingly, melanoma cells carrying the minor allele of rs4796793 were the most sensitive to IFNα in vitro. However, we did not detect a correlation between SNP genotype and STAT3 mRNA expression for either melanoma cells or for peripheral blood lymphocytes. Next, we analyzed the impact of rs4796793 on the clinical outcome of 259 stage III melanoma patients of which one-third had received adjuvant IFNα treatment. These analyses did not reveal a significant association between the STAT3 rs4796793 SNP and patients' progression free or overall survival when IFNα treated and untreated patients were compared. In conclusion, STAT3 rs4796793 SNP is no predictive marker for the efficacy of adjuvant IFNα treatment in melanoma patients. KW - predictive marker KW - single nucleotide polymorphism KW - melanoma KW - interferon KW - STAT3 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120602 SN - 2296-858X VL - 1 IS - 47 ER - TY - JOUR A1 - Ritter, Cathrin A1 - Fan, Kaiji A1 - Paulson, Kelly G. A1 - Nghiem, Paul A1 - Schrama, David A1 - Becker, Jürgen C. T1 - Reversal of epigenetic silencing of MHC class I chain-related protein A and B improves immune recognition of Merkel cell carcinoma JF - Scientific Reports N2 - Merkel cell carcinoma (MCC) is a virally associated cancer characterized by its aggressive behavior and strong immunogenicity. Both viral infection and malignant transformation induce expression of MHC class I chain-related protein (MIC) A and B, which signal stress to cells of the immune system via Natural Killer group 2D (NKG2D) resulting in elimination of target cells. However, despite transformation and the continued presence of virally-encoded proteins, MICs are only expressed in a minority of MCC tumors in situ and are completely absent on MCC cell lines in vitro. This lack of MIC expression was due to epigenetic silencing via MIC promoter hypo-acetylation; indeed, MIC expression was re-induced by pharmacological inhibition of histone deacetylases (HDACs) both in vitro and in vivo. This re-induction of MICs rendered MCC cells more sensitive to immune-mediated lysis. Thus, epigenetic silencing of MICs is an important immune escape mechanism of MCCs. KW - epigenetic silencing KW - Merkel cell carcinoma KW - MHC class I chain-related protein KW - skin cancer Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167992 IS - 21678 ET - 6 ER - TY - JOUR A1 - Hesbacher, Sonja A1 - Pfitzer, Lisa A1 - Wiedorfer, Katharina A1 - Angermeyer, Sabrina A1 - Borst, Andreas A1 - Haferkamp, Sebastian A1 - Scholz, Claus-Jürgen A1 - Wobser, Marion A1 - Schrama, David A1 - Houben, Roland T1 - RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells JF - Oncotarget N2 - The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells. KW - Merkel cell carcinoma KW - polyomavirus KW - Large T antigen KW - retinoblastoma protein KW - viral carcinogenesis Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177858 VL - 7 IS - 22 ER - TY - JOUR A1 - Houben, Roland A1 - Celikdemir, Büke A1 - Kervarrec, Thibault A1 - Schrama, David T1 - Merkel cell polyomavirus: infection, genome, transcripts and its role in development of Merkel cell carcinoma JF - Cancers N2 - The best characterized polyomavirus family member, i.e., simian virus 40 (SV40), can cause different tumors in hamsters and can transform murine and human cells in vitro. Hence, the SV40 contamination of millions of polio vaccine doses administered from 1955–1963 raised fears that this may cause increased tumor incidence in the vaccinated population. This is, however, not the case. Indeed, up to now, the only polyomavirus family member known to be the most important cause of a specific human tumor entity is Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma (MCC). MCC is a highly deadly form of skin cancer for which the cellular origin is still uncertain, and which appears as two clinically very similar but molecularly highly different variants. While approximately 80% of cases are found to be associated with MCPyV the remaining MCCs carry a high mutational load. Here, we present an overview of the multitude of molecular functions described for the MCPyV encoded oncoproteins and non-coding RNAs, present the available MCC mouse models and discuss the increasing evidence that both, virus-negative and -positive MCC constitute epithelial tumors. KW - Merkel cell carcinoma KW - polyomavirus KW - T antigen Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-305021 SN - 2072-6694 VL - 15 IS - 2 ER - TY - JOUR A1 - Houben, Roland A1 - Ebert, Marlies A1 - Hesbacher, Sonja A1 - Kervarrec, Thibault A1 - Schrama, David T1 - Merkel Cell Polyomavirus Large T Antigen is Dispensable in G2 and M-Phase to Promote Proliferation of Merkel Cell Carcinoma Cells JF - Viruses N2 - Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV), and proliferation of MCPyV-positive MCC tumor cells depends on the expression of a virus-encoded truncated Large T antigen (LT) oncoprotein. Here, we asked in which phases of the cell cycle LT activity is required for MCC cell proliferation. Hence, we generated fusion-proteins of MCPyV-LT and parts of geminin (GMMN) or chromatin licensing and DNA replication factor1 (CDT1). This allowed us to ectopically express an LT, which is degraded either in the G1 or G2 phase of the cell cycle, respectively, in MCC cells with inducible T antigen knockdown. We demonstrate that LT expressed only in G1 is capable of rescuing LT knockdown-induced growth suppression while LT expressed in S and G2/M phases fails to support proliferation of MCC cells. These results suggest that the crucial function of LT, which has been demonstrated to be inactivation of the cellular Retinoblastoma protein 1 (RB1) is only required to initiate S phase entry. KW - Merkel cell polyomavirus KW - large T antigen KW - cell cycle KW - Merkel cell carcinoma Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-218171 SN - 1999-4915 VL - 12 IS - 10 ER - TY - JOUR A1 - Fusi, Lorenza A1 - Paudel, Rupesh A1 - Meder, Katharina A1 - Schlosser, Andreas A1 - Schrama, David A1 - Goebeler, Matthias A1 - Schmidt, Marc T1 - Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis JF - Journal of Biological Chemistry N2 - Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which—depending on the cellular context—can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain–associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27\(^{kip1}\) and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO–TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences. KW - FoxO3 KW - TRRAP KW - transcription factors Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299820 VL - 298 IS - 3 ER - TY - JOUR A1 - Thiem, Alexander A1 - Hesbacher, Sonja A1 - Kneitz, Hermann A1 - di Primio, Teresa A1 - Heppt, Markus V. A1 - Hermanns, Heike M. A1 - Goebeler, Matthias A1 - Meierjohann, Svenja A1 - Houben, Roland A1 - Schrama, David T1 - IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression JF - Journal of Experimental & Clinical Cancer Research N2 - 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 TP53-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. KW - Melanoma KW - PD-L1 KW - CD274 KW - p53 KW - TP53 KW - JAK2 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201016 VL - 38 ER - TY - JOUR A1 - Banicka, Veronika A1 - Martens, Marie Christine A1 - Panzer, Rüdiger A1 - Schrama, David A1 - Emmert, Steffen A1 - Boeckmann, Lars A1 - Thiem, Alexander T1 - Homozygous CRISPR/Cas9 knockout generated a novel functionally active exon 1 skipping XPA variant in melanoma cells JF - International Journal of Molecular Sciences N2 - Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma. KW - DNA repair KW - nucleotide excision repair KW - XPA KW - CRISPR KW - knockout KW - protein variant KW - melanoma KW - A375 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290427 SN - 1422-0067 VL - 23 IS - 19 ER - TY - JOUR A1 - Fan, Kaiji A1 - Zebisch, Armin A1 - Horny, Kai A1 - Schrama, David A1 - Becker, Jürgen C. T1 - Highly expressed miR-375 is not an intracellular oncogene in Merkel cell polyomavirus-associated Merkel cell carcinoma JF - Cancers N2 - miR-375 is a highly abundant miRNA in Merkel cell carcinoma (MCC). In other cancers, it acts as either a tumor suppressor or oncogene. While free-circulating miR-375 serves as a surrogate marker for tumor burden in patients with advanced MCC, its function within MCC cells has not been established. Nearly complete miR-375 knockdown in MCC cell lines was achieved using antagomiRs via nucleofection. The cell viability, growth characteristics, and morphology were not altered by this knockdown. miR-375 target genes and related signaling pathways were determined using Encyclopedia of RNA Interactomes (ENCORI) revealing Hippo signaling and epithelial to mesenchymal transition (EMT)-related genes likely to be regulated. Therefore, their expression was analyzed by multiplexed qRT-PCR after miR-375 knockdown, demonstrating only a limited change in expression. In summary, highly effective miR-375 knockdown in classical MCC cell lines did not significantly change the cell viability, morphology, or oncogenic signaling pathways. These observations render miR-375 an unlikely intracellular oncogene in MCC cells, thus suggesting that likely functions of miR-375 for the intercellular communication of MCC should be addressed. KW - miR-375 KW - antagomiRs KW - Merkel cell carcinoma KW - Hippo signaling KW - focal adhesion Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200678 SN - 2072-6694 VL - 12 IS - 3 ER - TY - JOUR A1 - Houben, Roland A1 - Hesbacher, Sonja A1 - Schmid, Corinna P. A1 - Kauczok, Claudia S. A1 - Flohr, Ulrike A1 - Haferkamp, Sebastian A1 - Müller, Cornelia S. L. A1 - Schrama, David A1 - Wischhusen, Jörg A1 - Becker, Jürgen C. T1 - High-Level Expression of Wild-Type p53 in Melanoma Cells is Frequently Associated with Inactivity in p53 Reporter Gene Assays N2 - Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. Methodology/Principal Findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. Conclusions/Significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level. KW - Krebs KW - Hautkrebs Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69012 ER - TY - JOUR A1 - Wobser, Marion A1 - Weber, Alexandra A1 - Glunz, Amelie A1 - Tauch, Saskia A1 - Seitz, Kristina A1 - Butelmann, Tobias A1 - Hesbacher, Sonja A1 - Goebeler, Matthias A1 - Bartz, René A1 - Kohlhof, Hella A1 - Schrama, David A1 - Houben, Roland T1 - Elucidating the mechanism of action of domatinostat (4SC-202) in cutaneous T cell lymphoma cells JF - Journal of Hematology & Oncology N2 - Background Targeting epigenetic modifiers is effective in cutaneous T cell lymphoma (CTCL). However, there is a need for further improvement of this therapeutic approach. Here, we compared the mode of action of romidepsin (FK228), an established class I histone deacetylase inhibitor, and domatinostat (4SC-202), a novel inhibitor of class I HDACs, which has been reported to also target the lysine-specific histone demethylase 1A (LSD1). Methods We performed MTS assays and flow cytometric analyses of propidium iodide or annexin V-stained cells to assess drug impact on cellular proliferation, cell cycle distribution, and survival. Histone acetylation and methylation as well as caspase activation was analyzed by immunoblot. Gene expression analysis was performed using NanosString technology. Knockdown and knockout of LSD1 was achieved with shRNA and CRISPR/Cas9, respectively, while the CRISPR/Cas9 synergistic activation mediator system was used to induce expression of endogenous HDACs and LSD1. Furthermore, time-lapse fluorescence microscopy and an in vitro tubulin polymerization assay were applied. Results While FK228 as well as 4SC-202 potently induced cell death in six different CTCL cell lines, only in the case of 4SC-202 death was preceded by an accumulation of cells in the G2/M phase of the cell cycle. Surprisingly, apoptosis and accumulation of cells with double DNA content occurred already at 4SC-202 concentrations hardly affecting histone acetylation and methylation, and provoking significantly less changes in gene expression compared to biologically equivalent doses of FK228. Indeed, we provide evidence that the 4SC-202-induced G2/M arrest in CTCL cells is independent of de novo transcription. Furthermore, neither enforced expression of HDAC1 nor knockdown or knockout of LSD1 affected the 4SC-202-induced effects. Since time-lapse microscopy revealed that 4SC-202 could affect mitotic spindle formation, we performed an in vitro tubulin polymerization assay revealing that 4SC-202 can directly inhibit microtubule formation. Conclusions We demonstrate that 4SC-202, a drug currently tested in clinical trials, effectively inhibits growth of CTCL cells. The anti-cancer cell activity of 4SC-202 is however not limited to LSD1-inhibition, modulation of histone modifications, and consecutive alteration of gene expression. Indeed, the compound is also a potent microtubule-destabilizing agent. KW - Cutaneous lymphoma KW - Epigenetic regulation KW - Histone deacetylase KW - HDAC KW - Lysine-specific methylase KW - LSD1 KW - Tubulin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200703 VL - 12 ER - TY - JOUR A1 - Alb, Miriam A1 - Sie, Christopher A1 - Adam, Christian A1 - Chen, Suzie A1 - Becker, Jürgen C. A1 - Schrama, David T1 - Cellular and cytokine-dependent immunosuppressive mechanisms of grm1-transgenic murine melanoma JF - Cancer Immunology, Immunotherapy N2 - Grm1-transgenic mice spontaneously develop cutaneous melanoma. This model allowed us to scrutinize the generic immune responses over the course of melanoma development. To this end, lymphocytes obtained from spleens, unrelated lymph nodes and tumor-draining lymph nodes of mice with no evidence of disease, and low or high tumor burden were analyzed ex vivo and in vitro. Thereby, we could demonstrate an increase in the number of activated CD4\(^+\) and CD8+ lymphocytes in the respective organs with increasing tumor burden. However, mainly CD4\(^+\) T cells, which could constitute both T helper as well as immunosuppressive regulatory T cells, but not CD8\(^+\) T cells, expressed activation markers upon in vitro stimulation when obtained from tumor-bearing mice. Interestingly, these cells from tumor-burdened animals were also functionally hampered in their proliferative response even when subjected to strong in vitro stimulation. Further analyses revealed that the increased frequency of regulatory T cells in tumor-bearing mice is an early event present in all lymphoid organs. Additionally, expression of the immunosuppressive cytokines TGF-β1 and IL-10 became more evident with increased tumor burden. Notably, TGF-β1 is strongly expressed in both the tumor and the tumor-draining lymph node, whereas IL-10 expression is more pronounced in the lymph node, suggesting a more complex regulation of IL-10. Thus, similar to the situation in melanoma patients, both cytokines as well as cellular immune escape mechanisms seem to contribute to the observed immunosuppressed state of tumor-bearing grm1-transgenic mice, suggesting that this model is suitable for preclinical testing of immunomodulatory therapeutics. KW - regulatory T cell KW - melanoma KW - immune suppression KW - tumor-draining lymph node Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125096 VL - 61 IS - 12 ER - TY - JOUR A1 - Sarma, Bhavishya A1 - Willmes, Christoph A1 - Angerer, Laura A1 - Adam, Christian A1 - Becker, Jürgen C. A1 - Kervarrec, Thibault A1 - Schrama, David A1 - Houben, Roland T1 - Artesunate affects T antigen expression and survival of virus-positive Merkel cell carcinoma JF - Cancers N2 - Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer with frequent viral etiology. Indeed, in about 80% of cases, there is an association with Merkel cell polyomavirus (MCPyV); the expression of viral T antigens is crucial for growth of virus-positive tumor cells. Since artesunate — a drug used to treat malaria — has been reported to possess additional anti-tumor as well as anti-viral activity, we sought to evaluate pre-clinically the effect of artesunate on MCC. We found that artesunate repressed growth and survival of MCPyV-positive MCC cells in vitro. This effect was accompanied by reduced large T antigen (LT) expression. Notably, however, it was even more efficient than shRNA-mediated downregulation of LT expression. Interestingly, in one MCC cell line (WaGa), T antigen knockdown rendered cells less sensitive to artesunate, while for two other MCC cell lines, we could not substantiate such a relation. Mechanistically, artesunate predominantly induces ferroptosis in MCPyV-positive MCC cells since known ferroptosis-inhibitors like DFO, BAF-A1, Fer-1 and β-mercaptoethanol reduced artesunate-induced death. Finally, application of artesunate in xenotransplanted mice demonstrated that growth of established MCC tumors can be significantly suppressed in vivo. In conclusion, our results revealed a highly anti-proliferative effect of the approved and generally well-tolerated anti-malaria compound artesunate on MCPyV-positive MCC cells, suggesting its potential usage for MCC therapy. KW - artesunate KW - Merkel cell carcinoma KW - MCC KW - polyomavirus KW - ferroptosis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203851 SN - 2072-6694 VL - 12 IS - 4 ER - TY - JOUR A1 - Esnault, Clara A1 - Schrama, David A1 - Houben, Roland A1 - Guyétant, Serge A1 - Desgranges, Audrey A1 - Martin, Camille A1 - Berthon, Patricia A1 - Viaud-Massuard, Marie-Claude A1 - Touzé, Antoine A1 - Kervarrec, Thibault A1 - Samimi, Mahtab T1 - Antibody–drug conjugates as an emerging therapy in oncodermatology JF - Cancers N2 - Antibody–drug conjugates (ADCs) are an emerging class of therapeutics, with twelve FDA- and EMA-approved drugs for hematological and solid cancers. Such drugs consist in a monoclonal antibody linked to a cytotoxic agent, allowing a specific cytotoxicity to tumor cells. In recent years, tremendous progress has been observed in therapeutic approaches for advanced skin cancer patients. In this regard, targeted therapies (e.g., kinase inhibitors) or immune checkpoint-blocking antibodies outperformed conventional chemotherapy, with proven benefit to survival. Nevertheless, primary and acquired resistances as well as adverse events remain limitations of these therapies. Therefore, ADCs appear as an emerging therapeutic option in oncodermatology. After providing an overview of ADC design and development, the goal of this article is to review the potential ADC indications in the field of oncodermatology. KW - antibody–drug conjugates KW - oncodermatology KW - melanoma KW - skin squamous cell carcinoma KW - cutaneous T-cell lymphoma and Merkel cell carcinoma Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262192 SN - 2072-6694 VL - 14 IS - 3 ER - TY - JOUR A1 - Hafner, Christian A1 - Houben, Roland A1 - Baeurle, Anne A1 - Ritter, Cathrin A1 - Schrama, David A1 - Landthaler, Michael A1 - Becker, Jürgen C. T1 - Activation of the PI3K/AKT Pathway in Merkel Cell Carcinoma JF - PLoS One N2 - Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients. KW - rare KW - T-antigen KW - PIK3CA mutations KW - squamous cell KW - melanoma KW - polymavirus KW - cancer KW - tumors KW - akt KW - expression Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131398 VL - 7 IS - 2 ER - TY - JOUR A1 - Houben, Roland A1 - Alimova, Pamela A1 - Sarma, Bhavishya A1 - Hesbacher, Sonja A1 - Schulte, Carolin A1 - Sarosi, Eva-Maria A1 - Adam, Christian A1 - Kervarrec, Thibault A1 - Schrama, David T1 - 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone inhibits MCPyV T antigen expression in Merkel cell carcinoma independent of Aurora kinase A JF - Cancers N2 - Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV), and MCPyV-positive tumor cells depend on expression of the virus-encoded T antigens (TA). Here, we identify 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone (PHT) — a reported inhibitor of Aurora kinase A — as a compound inhibiting growth of MCC cells by repressing noncoding control region (NCCR)-controlled TA transcription. Surprisingly, we find that TA repression is not caused by inhibition of Aurora kinase A. However, we demonstrate that β-catenin — a transcription factor repressed by active glycogen synthase kinase 3 (GSK3) — is activated by PHT, suggesting that PHT bears a hitherto unreported inhibitory activity against GSK3, a kinase known to function in promoting TA transcription. Indeed, applying an in vitro kinase assay, we demonstrate that PHT directly targets GSK3. Finally, we demonstrate that PHT exhibits in vivo antitumor activity in an MCC xenograft mouse model, suggesting a potential use in future therapeutic settings for MCC. KW - Merkel cell carcinoma KW - polyomavirus KW - large T antigen KW - phthalazinone pyrazole KW - glycogen synthase kinase 3 KW - GSK3 Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313547 SN - 2072-6694 VL - 15 IS - 9 ER -