Roland Houben

University of Wuerzburg, Würzburg, Bavaria, Germany

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Publications (77)362.22 Total impact

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    ABSTRACT: Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LTS220A demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches. This article is protected by copyright. All rights reserved. © 2014 Wiley Periodicals, Inc.
    No preview · Article · Sep 2015 · International Journal of Cancer

  • No preview · Article · Aug 2015 · Journal der Deutschen Dermatologischen Gesellschaft
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    ABSTRACT: The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release.
    Full-text · Article · Jun 2015 · PLoS ONE
  • A. Vaman · H. Poppe · R. Houben · M. Goebeler · E. Butt

    No preview · Conference Paper · Mar 2015
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    ABSTRACT: Merkel cell carcinoma (MCC) is an aggressive, virus-associated, neuroendocrine tumor of the skin mainly affecting immunocompromised patients. Higher intratumoral infiltration with CD3 and CD8 positive T-cells is associated with a better prognosis, highlighting the relevance of the immune system for MCC development and progression. In this study 21 primary MCCs were stained with immune cell markers including CD3, CD4, CD8, CD68, CD20, and S100. Furthermore, tumor-infiltrating neutrophils, tertiary lymphoid structures and PD-L1 expression were analyzed and correlated with overall and recurrence free survival. All MCCs were Merkel Cell Polyomavirus positive. Overall and recurrence-free survival did not correlate with intra- and peritumoral CD3 and CD8 T-cell infiltration. In addition, no significant association regarding prognosis was found for tumor-associated neutrophils, tumor-associated macrophages or PD-L1 positivity in MCCs. Interestingly, the presence of tertiary lymphoid structures (TLS) in the tumor microenvironment significantly correlated with recurrence-free survival (P=0.025). In addition, TLS were significantly associated with a higher CD8/CD4 ratio in the tumor periphery (P=0.032), but not in the center of the tumor (P > 0.999). These results demonstrate for the first time that TLS, easily assessed in paraffin-embedded tissue in the tumor periphery of MCCs, may be a valuable prognostic factor indicating prolonged recurrence free survival.
    Full-text · Article · Dec 2014 · International journal of clinical and experimental pathology
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    ABSTRACT: Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. This RB-binding domain in MCPyV-LT is - in contrast to other polyomavirus LTs (e.g. SV40) - embedded between two large MCPyV unique regions (MUR1 and MUR2). In order to identify elements of the MCPyV-LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for MCPyV-LT nuclear accumulation nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTO - a second protein encoded by an alternative reading frame in the MCPyV-LT mRNA - are completely dispensable for MCPyV-driven tumor cell proliferation. Notably, even MCPyV-LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying that - while binding to Vam6p is not essential - HSC-70 interaction is significantly involved in mediating MCPyV-LT function in MCC cells including growth promotion and induction of E2F target genes. © 2014 Wiley Periodicals, Inc.
    No preview · Article · Sep 2014 · International Journal of Cancer
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    ABSTRACT: Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-β are well established, we wondered whether TGF-β could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-β promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-β up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-β type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-β signaling.
    Full-text · Article · Jul 2014 · Journal of Leukocyte Biology
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    ABSTRACT: 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.
    Full-text · Article · Apr 2014 · PLoS ONE

  • No preview · Conference Paper · Mar 2014
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    ABSTRACT: p53 is a central tumor suppressor protein and its inhibition is believed to be a prerequisite for cancer development. In approximately 50% of all malignancies this is achieved by inactivating mutations in the p53 gene. However, in several cancer entities, including melanoma, p53 mutations are rare. It has been recently proposed that tyrosinase related protein 2 (TRP2), a protein involved in melanin synthesis, may act as suppressor of the p53 pathway in melanoma. To scrutinize this notion we analyzed p53 and TRP2 expression by immunohistochemistry in 172 melanoma tissues and did not find any correlation. Furthermore, we applied three different TRP2 shRNAs to five melanoma cell lines and could not observe a target specific effect of the TRP2 knockdown on either p53 expression nor p53 reporter gene activity. Likewise, ectopic expression of TRP2 in a TRP2 negative melanoma cell line had no impact on p53 expression. In conclusion our data suggest that p53 repression critically controlled by TRP2 is not a general event in melanoma.
    Full-text · Article · Jan 2014 · PLoS ONE
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    ABSTRACT: Abbreviations: LT, large T antigen; MCC, Merkel cell carcinoma; MCV, Merkel cell polyoma virus; shRNA, small hairpin RNA; sT, small T antigen
    Preview · Article · Nov 2013 · Journal of Investigative Dermatology
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    ABSTRACT: Merkel cell carcinoma (MCC) is a rare, but highly malignant tumor of the skin. In case of systemic disease, possible therapeutic options include irradiation or chemotherapy. The aim of this study was to evaluate whether the flavonoid resveratrol enhances the effect of radiotherapy or chemotherapy in MCC cell lines. The two MCC cell lines MCC13 and MCC26 were treated with increasing doses of resveratrol. Combination experiments were conducted with cisplatin and etoposide. Colony forming assays were performed after sequential irradiation with 1, 2, 3, 4, 6, and 8 Gy and apoptosis was assessed with flow cytometry. Expression of cancer drug targets was analyzed by real-time PCR array. Resveratrol is cytotoxic in MCC cell lines. Cell growth is inhibited by induction of apoptosis. The combination with cisplatin and etoposide resulted in a partially synergistic inhibition of cell proliferation. Resveratrol and irradiation led to a synergistic reduction in colony formation compared to irradiation alone. Evaluation of gene expression did not show significant difference between the cell lines. Due to its radiosensitizing effect, resveratrol seems to be a promising agent in combination with radiation therapy. The amount of chemosensitizing depends on the cell lines tested.
    No preview · Article · Nov 2013 · Strahlentherapie und Onkologie

  • No preview · Article · Jun 2013 · International Journal of Cancer
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    ABSTRACT: A proangiogenic micromilieu is associated with a worse prognosis in systemic lymphoma. Hence, targeting the tumour microenvironment and its vasculature has evolved as a promising novel treatment strategy. The role of tumour neoangiogenesis in cutaneous B-cell lymphoma, however, has not yet been elucidated. Therefore, we examined the expression of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2, as well as microvessel density by immunohistochemistry in paraffin-embedded specimens of different subtypes of primary cutaneous B-cell lymphomas, systemic diffuse large B-cell lymphoma, and cutaneous B-cell pseudolymphoma. Primary cutaneous large B-cell lymphoma (PCLBCL) were characterized by significantly higher intratumoral expression levels of VEGF and its receptors in comparison with the indolent lymphoma subtypes. Moreover, PCLBCL exhibited significantly higher intratumoral microvessel counts. Our study provides evidence that the most aggressive subtype of cutaneous B-cell lymphoma, PCLBCL, is characterized by a proangiogenic micromilieu.
    Preview · Article · Apr 2013 · Acta Dermato-Venereologica
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    ABSTRACT: Merkel cell carcinoma (MCC) is a rare and very aggressive skin cancer with viral etiology. The tumor associated Merkel cell polyoma virus (MCV) belongs to a group of viruses encoding T antigens (TA) that can induce tumorigenesis by interfering with cellular tumor suppressor proteins like p53. To explore possible modes of p53 inactivation in MCC p53 sequencing, expression analysis and reporter gene assays for functional analyses were performed in a set of MCC lines. In one MCV-negative and in one MCV-positive cell line, p53 inactivating mutations were found. In the majority of MCC lines, however, wild type p53 is expressed and displays some transcriptional activity, which is yet not sufficient to effectively restrict cellular survival or growth in these cell cultures. Interestingly, the MCV TAs are not responsible for this critical lack in p53 activity, as TA knockdown in MCV-positive MCC cells does not induce p53 activity. In contrast, inhibition of the ubiquitin ligase HDM-2 by Nutlin-3a leads to p53 activation and p53 dependent apoptosis or cell cycle arrest in five out of seven p53 wild type MCC lines highlighting p53 as potential target for future therapies of this aggressive tumor.Journal of Investigative Dermatology accepted article preview online, 5 April 2013; doi:10.1038/jid.2013.169.
    Full-text · Article · Apr 2013 · Journal of Investigative Dermatology
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    ABSTRACT: Increasing evidence suggests that Merkel cell carcinoma (MCC) is caused by the Merkel cell polyoma virus (MCV). The viral sequence encodes for two potential oncoproteins, i.e. the small T antigen (sT) and the Large T antigen (LT). Indeed, sT has recently been shown to bear transforming activity. Here, we confirm this observation by demonstrating focus formation upon expression of MCV sT in NIH3T3 fibroblasts. On the other hand, however, we provide evidence that established MCC cells do not require sT for growth and survival. Silencing of sT protein expression by two different sT specific shRNAs leads to variable degrees of growth retardation in MCV-positive MCC cell lines. However, these effects are not sT-specific since proliferation of MCV-negative cell lines is similarly affected by these sT shRNAs. Furthermore, ectopic expression of shRNA-insensitive sT does not revert the growth inhibition implicated by sT silencing. Finally, the unambiguous and specific growth inhibition induced by means of an shRNA targeting both T antigens, can be completely rescued by ectopic expression of LT alone, thus demonstrating a dispensable role of sT. Altogether, our results indicate that MCV LT is more relevant in maintaining the proliferation and survival of established MCC cell lines.Journal of Investigative Dermatology accepted article preview online, 25 February 2013; doi:10.1038/jid.2013.82.
    No preview · Article · Feb 2013 · Journal of Investigative Dermatology
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    ABSTRACT: A large proportion of human melanomas harbor a mutation leading to permanent activation of the serine/threonine kinase BRAF, and as a consequence, they have developed dependence on BRAF/MAPK signaling. Accordingly, BRAF inhibitors such as Vemurafenib show a good anti-tumorigenic effect on metastases with the BRAF(V600E) mutation. Although an initial period of sustained tumor regression is usually observed after Vemurafenib treatment, tumors often relapse at the same site, and apoptosis induction of melanoma cells in vitro is incomplete. Here, we demonstrate, using a large panel of melanoma cell lines, that Vemurafenib induces features of stress-induced senescence in addition to apoptosis. This senescence phenotype is characterized by heterochromatin formation, changes in cell shape and increased senescence-associated β-galactosidase activity. Importantly, senescence features induced by BRAF(V600E) inhibition was also detected in human melanoma cells xenografted into nude mice. Our observations provide a possible explanation for the lack of complete and durable pro-apoptotic effect of Vemurafenib in patients.Journal of Investigative Dermatology accepted article preview online, 15 January 2013; doi:10.1038/jid.2013.6.
    Full-text · Article · Jan 2013 · Journal of Investigative Dermatology
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    ABSTRACT: Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer associated with the Merkel cell polyomavirus (MCV). As MCC cell lines show oncogene addiction to the MCV T antigens, pharmacologic interference of the large T antigen (LTA) may represent an effective therapeutic approach for this deadly cancer. In this study, we investigated the effects of IFNs on MCC cell lines, especially on MCV-positive (MCV(+)) lines. Type I IFNs (i.e., Multiferon, a mix of different IFN-α subtypes, and IFN-β) strongly inhibited the cellular viability. Cell-cycle analysis showed increased sub-G fractions for these cells upon IFN treatment indicating apoptotic cell death; these effects were less pronounced for IFN-γ. Notably, this inhibitory effect of type I IFNs on MCV(+) MCC cell lines was associated with a reduced expression of the MCV LTA as well as an increased expression of promyelocytic leukemia (PML) protein, which is known to interfere with the function of the LTA. In addition, the intratumoral application of Multiferon resulted in a regression of MCV(+) but not MCV(-) MCCs in vivo. Together, our findings show that type I IFNs have a strong antitumor effect, which is at least in part explained by modulation of the virally encoded LTA.
    Full-text · Article · Mar 2012 · Cancer Research

  • No preview · Conference Paper · Mar 2012
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    ABSTRACT: 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.
    Full-text · Article · Feb 2012 · PLoS ONE

Publication Stats

2k Citations
362.22 Total Impact Points

Institutions

  • 1997-2015
    • University of Wuerzburg
      • • Institute of Clinical Biochemistry and Pathobiochemistry
      • • Department of Dermatology, Venereology and Allergology
      • • Department of Internal Medicine I
      • • Institute for Medical Radiation and Cell Research
      Würzburg, Bavaria, Germany
  • 2010
    • University of Pittsburgh
      Pittsburgh, Pennsylvania, United States
    • Medical University of Graz
      Gratz, Styria, Austria