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Prof. Dr. Melanie Kappelmann-Fenzl

Faculty of Computer Science

Professors

Professor

K 008

0991/3615-473

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Journal article

B. Spangler
Melanie Kappelmann-Fenzl
B. Schittek
S. Meierjohann
L. Vardimon
A.-K. Bosserhoff
S. Kuphal

ETS‐1/RhoC signaling regulates the transcription factor c‐Jun in melanoma.

In: International Journal of Cancer vol. 130 pg. 2801-2811

(2012)

DOI: 10.1002/ijc.26277

show abstract

Recently, we discovered that the loss of E‐cadherin induces c‐Jun protein expression, which is a member of the AP‐1 transcription factor family and a key player in the processes of cell proliferation and tumor development and also found in elevated levels in melanomas. Notably, the mRNA level of c‐Jun was not affected, suggesting that c‐Jun is regulated at post‐transcriptional level. Here, we present data that suggest that the dynamic cytoskeletal network, linked to E‐cadherin, is involved in the regulation of the c‐Jun protein and transcriptional activity. In a signaling cascade, the loss of E‐cadherin activates the transcriptional regulator ETS‐1 and consequently leads to the induction of RhoC expression that stabilizes c‐Jun in melanoma. The link between RhoC and c‐Jun seems to be indirect via the cytoskeleton. We conclude that the loss of E‐cadherin mediated cell‐adhesion induces c‐Jun protein expression in a multistep process, offering several possibilities for therapeutic intervention.

Journal article

Melanie Kappelmann-Fenzl
S. Kuphal
G. Meister
L. Vardimon
A.-K. Bosserhoff

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

In: Oncogene vol. 32 pg. 2984-2991

(2013)

DOI: 10.1038/onc.2012.307

show abstract

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.

Thesis

Melanie Kappelmann-Fenzl

Regulation und Funktion des Trankriptionsfaktors c-Jun im malignen Melanom.

Universität Regensburg Regensburg Naturwissenschaftliche Fakultät für Biologie und vorklinische Medizin

2014 (2014)

Journal article

S. Hagemann
L. Stöger
Melanie Kappelmann-Fenzl
I. Hassl
A. Ellinger
B. Velimirov

DNA-bearing membrane vesicles produced by Ahrensia kielensis and Pseudoalteromonas marina.

In: Journal of Basic Microbiology vol. 54 pg. 1062-1072

(2014)

DOI: 10.1002/jobm.201300376

show abstract

Outer membrane vesicles (OMVs) derived from the alphaproteobacterium Ahrensia kielensis and from Pseudoalteromonas marina, a gammaproteobacterium, were sampled from liquid cultures in order to extract the MV-associated DNA, establish a shotgun library, and sequence randomly chosen clones to determine the origins of their DNA. We show that OMVs from A. kielensis and from P. marina both harbour DNA larger than 20 or 30 kbp. Transmission electron microscopical inspection of OMVs of A. kielensis and P. marina showed two types of vesicles: bilayered OMVs with a diameter between 30 and 250 nm and double bilayered OMVs ranging between 80 and 200 nm. Bilayered OMVs are either characterized by the presence of a large electron-dense substance or are elctron translucent. Double bilayered OMVs contained an electron dense substance in the core region surrounded by the second bilayer. 30,094 bp of the genome from OMV of A. kielensis and 45,981 bp of that from P. marina were sequenced. The results indicated that all sequences were single copy and that all sequences, with one exception, were similar to prokaryotic sequences, inserted viral sequences were not detected.

Journal article

Melanie Kappelmann-Fenzl
A.-K. Bosserhoff
S. Kuphal

AP-1/c-Jun transcription factors: regulation and function in malignant melanoma.

In: European Journal of Cell Biology vol. 93 pg. 76-81

(2014)

DOI: 10.1016/j.ejcb.2013.10.003

show abstract

Malignant melanoma is an aggressive form of skin cancer with an increasing incidence worldwide. One way to address the pathology of the disease is through molecular research. In addition to the analysis of melanoma-relevant signaling pathways, the investigation of important transcription factors is a fundamental objective. The AP-1 transcription factor family is known to play an important role in melanoma progression and development. The AP-1 family member c-Jun is highly expressed and active in melanoma cells, and the mechanisms and signaling pathways regulating c-Jun protein are diverse. In addition to the common regulation and activation of c-Jun by mitogen-activated protein kinases (MAPKs), there are several other signaling pathways and interactions leading to c-Jun protein expression and thus AP-1 activation. In malignant melanoma, and many other cancer types, c-Jun has mainly oncogenic functions; however, other AP-1 proteins also have anti-oncogenic roles. Interestingly, several studies have revealed that a strong AP-1 activity in melanoma mainly depends on c-Jun. Recently, it has also been shown that the c-Jun protein is regulated and activated by several other mechanisms, including miRNAs and the cytoskeleton. In summary, there are a variety of mechanisms underlying the induction of c-Jun protein expression and activity leading to tumor progression and development, and this diverse regulatory machinery is due to the heterogeneity of different tumor types, particularly in malignant melanoma.

Journal article

P. Schummer
S. Kuphal
L. Vardimon
A.-K. Bosserhoff
Melanie Kappelmann-Fenzl

Specific c-Jun target genes in malignant melanoma.

In: Cancer Biology & Therapy vol. 17 pg. 486-497

(2016)

DOI: 10.1080/15384047.2016.1156264

show 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.

Journal article

Melanie Kappelmann-Fenzl
S. Kuphal
R. Krupar
D. Schadendorf
V. Umansky
L. Vardimon
C. Hellerbrand
A.-K. Bosserhoff

Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun's Nuclear Translocation and Activity.

In: Cancers vol. 11 pg. 1-12

(2019)

DOI: 10.3390/cancers11111806

show abstract

Microtubules are highly dynamic structures, which consist of α- and β-tubulin heterodimers. They are essential for a number of cellular processes, including intracellular trafficking and mitosis. Tubulin-binding chemotherapeutics are used to treat different types of tumors, including malignant melanoma. The transcription factor c-Jun is a central driver of melanoma development and progression. Here, we identify the microtubule network as a main regulator of c-Jun activity. Monomeric α-tubulin fosters c-Jun protein stability by protein-protein interaction. In addition, this complex formation is necessary for c-Jun's nuclear localization sequence binding to importin 13, and consequent nuclear import and activity of c-Jun. A reduction in monomeric α-tubulin levels by treatment with the chemotherapeutic paclitaxel resulted in a decline in the nuclear accumulation of c-Jun in melanoma cells in an experimental murine model and in patients' tissues. These findings add important knowledge to the mechanism of the action of microtubule-targeting drugs and indicate the newly discovered regulation of c-Jun by the microtubule cytoskeleton as a novel therapeutic target for melanoma and potentially also other types of cancer.

Journal article

L. Feuerer
S. Lamm
I. Henz
Melanie Kappelmann-Fenzl
S. Haferkamp
S. Meierjohann
C. Hellerbrand
S. Kuphal
A.-K. Bosserhoff

Role of MIA (melanoma inhibitory activity) in melanocyte senescence.

In: Pigment Cell & Melanoma Research pg. 777-791

(2019)

DOI: 10.1111/pcmr.12801

show abstract

The protein MIA is known to be expressed in melanoma and to support melanoma progression. Interestingly, previous studies also observed the expression of MIA in nevi. Concentrating on these findings, we revealed that MIA expression is correlated with a senescent state in melanocytes. Induction of replicative or oncogene‐induced senescence resulted in increased MIA expression in vitro. Notably, MIA‐knockdown in senescent melanocytes reduced the percentage of senescence‐associated beta‐Gal‐positive cells and enhanced proliferation. Using the melanoma mouse model Tg(Grm1), MIA‐deficient mice supported the impact of MIA on senescence by showing a significantly earlier tumor onset compared to controls. In melanocytes, MIA‐knockdown led to a downregulation of the cell cycle inhibitor p21 in vitro and in vivo. In contrast, after induction of hTERT in human melanoma cells, p21 regulation by MIA was lost. In summary, our data show for the first time that MIA is a regulator of cellular senescence in human and murine melanocytes.

Journal article

Melanie Kappelmann-Fenzl
C. Gebhardt
A. Matthies
S. Kuphal
M. Rehli
A.-K. Bosserhoff

C-Jun drives melanoma progression in PTEN wild type melanoma cells.

In: Cell Death & Disease vol. 10

(2019)

DOI: 10.1038/s41419-019-1821-9

show abstract

Due to the critical impact of active AP-1 transcription factors in melanoma, it is important to define their target genes and to identify and ultimately inhibit oncogenic signals. Here we mapped the genome-wide occupancy of the AP-1 family member c-Jun in different melanoma cells and correlated AP-1 binding with transcriptome data to detect genes in melanoma regulated by c-Jun. Our analysis shows that c-Jun supports the malignant phenotype by deregulating genes in cancer-relevant signaling pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. Moreover, we demonstrate that the importance of c-Jun depends on melanoma stage and mutation status of the tumor suppressor PTEN. Our study reveals that activation of c-Jun overrules the tumor suppressive effect of PTEN in early melanoma development. These findings help to understand the relevance of c-Jun within cancer pathways in different melanoma cell types, especially in relation to MAPK and PI3K pathways, which are commonly deregulated in melanomas. Consequently, targeting c-Jun in PTEN+ melanoma cells may represent a promising therapeutic strategy to inhibit survival of melanoma cells to prevent the development of a metastatic phenotype.

Lecture

Melanie Kappelmann-Fenzl
C. Gebhardt
A. Matthies
S. Kuphal
M. Rehli
A.-K. Bosserhoff

C-Jun drives melanoma progression in PTEN wild type melanoma cells.

In: German Conference on Bioinformatics 2019

Deutsches Krebsforschungszentrum Heidelberg

16.-19.09.2019 (2019)

Contribution

M. Heppt
A. Wessely
E. Hornig
C. Kammerbauer
S. Graf
R. Besch
L. French
S. Kuphal
Melanie Kappelmann-Fenzl
A.-K. Bosserhoff
C. Bosserhoff
C. Berking

Brn3a expression is epigenetically controlled by HDAC2 in melanocytes and melanoma.

In: Experimental Dermatology - 47th Annual Meeting of the Arbeitsgemeinschaft Dermatologische Forschung (ADF) in collaboration with the Deutsche Dermatologische Gesellschaft (DDG). pg. P.196

(2021)

Contribution

A.-K. Bosserhoff
Melanie Kappelmann-Fenzl

Next Generation Sequencing (NGS): What Can Be Sequenced?.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 1-15

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_1

Contribution

Melanie Kappelmann-Fenzl

NGS Data.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 79-104

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_7

Contribution

Melanie Kappelmann-Fenzl

Reference Genome.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 105-109

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_8

Journal article

M. Fuentes-Chandía
A. Vierling
Melanie Kappelmann-Fenzl
M. Monavari
G. Letort
L. Höne
B. Parma
S. Antara
Ö. Ertekin
R. Palmisano
M. Dong
K. Böpple
A. Boccaccini
P. Ceppi
A.-K. Bosserhoff
A. Leal-Egaña

3D Spheroids Versus 3D Tumor-Like Microcapsules: Confinement and Mechanical Stress May Lead to the Expression of Malignant Responses in Cancer Cells.

In: Advanced Biology pg. e2000349

07.05.2021 (2021)

DOI: 10.1002/adbi.202000349

show abstract

As 2D surfaces fail to resemble the tumoral milieu, current discussions are focused on which 3D cell culture strategy may better lead the cells to express in vitro most of the malignant hints described in vivo. In this study, this question is assessed by analyzing the full genetic profile of MCF7 cells cultured either as 3D spheroids-considered as "gold standard" for in vitro cancer research- or immobilized in 3D tumor-like microcapsules, by RNA-Seq and transcriptomic methods, allowing to discriminate at big-data scale, which in vitro strategy can better resemble most of the malignant features described in neoplastic diseases. The results clearly show that mechanical stress, rather than 3D morphology only, stimulates most of the biological processes involved in cancer pathogenicity, such as cytoskeletal organization, migration, and stemness. Furthermore, cells entrapped in hydrogel-based scaffolds are likely expressing other physiological hints described in malignancy, such as the upregulated expression of metalloproteinases or the resistance to anticancer drugs, among others. According to the knowledge, this study represents the first attempt to answer which 3D experimental system can better mimic the neoplastic architecture in vitro, emphasizing the relevance of confinement in cancer pathogenicity, which can be easily achieved by using hydrogel-based matrices.

Contribution

Melanie Kappelmann-Fenzl

Alignment.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 111-122

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_9

Journal article

N. Rachinger
Stefan Fischer
I. Böhme
L. Linck-Paulus
S. Kuphal
Melanie Kappelmann-Fenzl
A.-K. Bosserhoff

Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR.

In: International Journal of Molecular Sciences vol. 22 pg. 9349

(2021)

DOI: 10.3390/ijms22179349

show abstract

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

Contribution

M. Eisele
Melanie Kappelmann-Fenzl

NGS Technologies.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 47-58

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_4

Contribution

Melanie Kappelmann-Fenzl

Library Construction for NGS.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 39-45

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_3

Contribution

Melanie Kappelmann-Fenzl

Computer Setup.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 59-69

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_5

Contribution

Melanie Kappelmann-Fenzl

Design and Analysis of Epigenetics and ChIP-Sequencing Data.

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 177-192

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_12

Contribution

Melanie Kappelmann-Fenzl

Introduction to Command Line (Linux/Unix).

In: Next Generation Sequencing and Data Analysis. (Learning Materials in Biosciences) pg. 71-78

Eds.:
Melanie Kappelmann-Fenzl

Springer Nature Switzerland AG Cham, Switzerland

(2021)

DOI: 10.1007/978-3-030-62490-3_6

Journal article

Melanie Kappelmann-Fenzl
S. Schmidt
Stefan Fischer
R. Schmid
L. Lämmerhirt
L. Fischer
S. Schrüfer
I. Thievessen
D. Schubert
A. Matthies
Rainer Detsch
Aldo B.
A. Arkudas
A. Kengelbach-Weigand
A.-K. Bosserhoff

Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels.

In: Cancers vol. 13 pg. 4111

(2021)

DOI: 10.3390/cancers13164111

show abstract

Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.

Lecture

S. Ranganathan
D. Riewe
A. Feiner
S. Steiner
Melanie Kappelmann-Fenzl
S. Fischer

De novo assembly of Humulus lupulus transcriptome comparing different bioinformatic tools. Posterpräsentation.

In: German Conference on Bioinformatics (GCB) 2021

Online

06.-08.09.2021

Journal article

Ö. Ertekin
M. Monavari
R. Krüger
M. Fuentes-Chandía
B. Parma
G. Letort
P. Tripal
A. Boccaccini
A.-K. Bosserhoff
P. Ceppi
Melanie Kappelmann-Fenzl
A. Leal-Egaña

3D hydrogel-based microcapsules as an in vitro model to study tumorigenicity, cell migration and drug resistance.

In: Acta Biomaterialia

13.02.2022 (2022)

DOI: 10.1016/j.actbio.2022.02.010

show abstract

In this work, we analyzed the reliability of alginate-gelatin microcapsules as artificial tumor model. These tumor-like scaffolds are characterized by their composition and stiffness (∼25 kPa), and their capability to restrict -but not hinder- cell migration, proliferation and release from confinement. Hydrogel-based microcapsules were initially utilized to detect differences in mechano-sensitivity between MCF7 and MDA-MB-231 breast cancer cells, and the endothelial cell line EA.hy926. Additionally, we used RNA-seq and transcriptomic methods to determine how the culture strategy (i.e. 2D v/s 3D) may pre-set the expression of genes involved in multidrug resistance, being then validated by performing cytotoxicological tests and assays of cell morphology. Our results show that both breast cancer cells can generate elongated multicellular spheroids inside the microcapsules, prior being released (mimicking intravasation stages), a behavior which was not observed in endothelial cells. Further, we demonstrate that cells isolated from 3D scaffolds show resistance to cisplatin, a process which seems to be strongly influenced by mechanical stress, instead of hypoxia. We finally discuss the role played by aneuploidy in malignancy and resistance to anticancer drugs, based on the increased number of polynucleated cells found within these microcapsules. Overall, our outcomes demonstrate that alginate-gelatin microcapsules represent a simple, yet very accurate tumor-like model, enabling us to mimic the most relevant malignant hints described in vivo, suggesting that confinement and mechanical stress need to be considered when studying pathogenicity and drug resistance of cancer cells in vitro. STATEMENT OF SIGNIFICANCE: In this work, we analyzed the reliability of alginate-gelatin microcapsules as an artificial tumor model. These scaffolds are characterized by their composition, elastic properties, and their ability to restrict cell migration, proliferation, and release from confinement. Our results demonstrate four novel outcomes: (i) studying cell migration and proliferation in 3D enabled discrimination between malignant and non-pathogenic cells, (ii) studying the cell morphology of cancer aggregates entrapped in alginate-gelatin microcapsules enabled determination of malignancy degree in vitro, (iii) determination that confinement and mechanical stress, instead of hypoxia, are required to generate clones resistant to anticancer drugs (i.e. cisplatin), and (iv) evidence that resistance to anticancer drugs could be due to the presence of polynucleated cells localized inside polymer-based artificial tumors.

Journal article

L. Linck-Paulus
T. Meißgeier
K. Pieger
A. Horn
A. Matthies
Stefan Fischer
G. Meister
H. Sticht
Melanie Kappelmann-Fenzl
A. Bosserhoff

A previously unknown Argonaute 2 variant positively modulates the viability of melanoma cells.

In: Cellular and Molecular Life Sciences : CMLS vol. 79 pg. 475

09.08.2022 (2022)

DOI: 10.1007/s00018-022-04496-8

show abstract

In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.

Journal article

I. Stüfchen
R. Beckervordersandforth
Stefan Fischer
Melanie Kappelmann-Fenzl
A.-K. Bosserhoff
F. Beyer

Two novel CreERT2 transgenic mouse lines to study melanocytic cells in vivo.

In: Pigment Cell & Melanoma Research pg. 1-9

03.08.2022 (2022)

DOI: 10.1111/pcmr.13061

show abstract

The skin of adult mammals protects from radiation, physical and chemical insults. While melanocytes and melanocyte-producing stem cells contribute to proper skin function in healthy organisms, dysfunction of these cells can lead to the generation of malignant melanoma-the deadliest type of skin cancer. Addressing cells of the melanocyte lineage in vivo represents a prerequisite for the understanding of melanoma on cellular level and the development of preventive and treatment strategies. Here, the inducible Cre-loxP-system has emerged as a promising tool to specifically target, monitor, and modulate cells in adult mice. Re-analysis of existing sequencing data sets of melanocytic cells revealed that genes with a known function in neural cells, including neural stem cells (Aldh1L1 and Nestin), are also expressed in melanocytic cells. Therefore, in this study, we explored whether the promoter activity of Nestin and Aldh1L1 can serve to target cells of the melanocyte lineage using the inducible CreERT2 -loxP-system. Using an immunohistochemical approach and different time points of analysis, we were able to map the melanocytic fate of recombined stem cells in the adult hair follicle of Nestin-CreERT2 and Aldh1L1-CreERT2 transgenic mice. Thus, we here present two new mouse models and propose their use to study and putatively modulate adult melanocytic cells in vivo.

Journal article

L. Linck-Paulus
A. Horn
A. Matthies
Stefan Fischer
G. Meister
H. Sticht
Melanie Kappelmann-Fenzl
A. Bosserhoff

Eine neue Spleißvariante von Argonaut 2 beeinflusst microRNA-Zielgene und die Zellviabilität von Melanomzellen. ePoster zum 32. Deutschen Hautkrebskongress (ADO) in Hannover.

In: JDDG: Journal der Deutschen Dermatologischen Gesellschaft vol. 20 pg. 28-114

(2022)

DOI: 10.1111/ddg.14901

Journal article

M. Heppt
A. Wessely
E. Hornig
C. Kammerbauer
S. Graf
R. Besch
L. French
A. Matthies
S. Kuphal
Melanie Kappelmann-Fenzl
A.-K. Bosserhoff
C. Berking

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma.

In: International Journal of Molecular Sciences vol. 23 pg. 849

(2022)

DOI: 10.3390/ijms23020849

show abstract

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Contribution

S. Schmidt
Stefan Fischer
R. Schmid
A. Arkudas
A. Kengelbach-Weigand
Melanie Kappelmann-Fenzl
A. Bosserhoff

Molecular analysis of tumor dormancy in a 3D printed melanoma model.

In: ABSTRACTS - 19th International Congress of the Society for Melanoma Research. pg. 176

(2022)

show abstract

Late recurrence is an issue in many cancers including malignant melanoma. Studies suggest that these recurrences may be caused by undetected former sleeping tumor cells within the patient, which “awake” and form aggressive metastases. The mechanisms coordinating this so called “tumor dormancy”, as well as the awakening are barely understood. Still, the tumor microenvironment (TME) is supposed to play a critical role in the signaling processes involved. In this study, a biofabricated 3D dormancy model was developed and characterized in order to investigate the molecular mechanisms underlying tumor dormancy in consideration of the TME. For this purpose, melanoma cells were 3D-printed in hydro-gels, serving as surrogates for the extracellular matrix. Gels allowing proliferation (Matrigel) or not allowing proliferation (Cellink Bioink) were defined. After 2 days in culture, RNA of proliferating or dormant cells was isolated and differences in gene expression patterns were analyzed using RNA-sequencing. Selected data were functionally confirmed via reporter cell and promoter assays, or immunofluorescence. Evaluation of RNA-seq data confirmed decreased expression of gene sets associated with cell division, DNA and RNA organization in dormant cells in Cellink Bioink, going in line with reporter cell analyses revealing a G1 cell cycle arrest in this matrix. Interestingly, highly expressed genes in dormant cells are associated with morphogenesis, actin cytoskeleton, and with migration and adhesion, among others. Functional analyses in vitro confirm a migratory potential of dormant melanoma cells and suggest that the quiescent phenotype behaves according to the migratory phenotype of the phenotype-switching model proposed by Hoek & Goding. Based on the 3D dormancy model and the RNA-seq data set presented, relevant target genes can be defined and their influence on the dormant phenotype can be investigated.

Journal article

M. Schott
Melanie Kappelmann-Fenzl
Stefan Fischer
M. Fernandez-Barrena
A. Pineda-Lucena
M. Ávila
S. Kuphal
A.-K. Bosserhoff

Impact of CYLD on chromatin structure and histone methylation in malignant melanoma.

In: International Journal of Molecular Medicine vol. 49

16.03.2022 (2022)

DOI: 10.3892/ijmm.2022.5122

show abstract

The tumor suppressive role of CYLD lysine 63 deubiquitinase (CYLD) is known in melanoma. To the best of our knowledge, however, the precise mechanism underlying the tumor suppressive function of CYLD has yet to be clarified. In the present study, a novel melanoma mouse model was generated, which revealed accelerated tumor growth in Cyld‑knockout (Cyld‑/‑) compared with Cyld‑wild‑type (Cyld+/+) mice. To determine the underlying molecular mechanism, mutation analysis of primary tumor‑derived cell lines from Cyld+/+ and Cyld‑/‑ mice was performed using RNA sequencing data. Variant calling revealed no common mutations in Cyld‑/‑ compared with Cyld+/+ cells. Thus, the epigenetic processes influencing development and progression of melanoma were investigated. Initial analysis of expression pattern of known hypermethylated genes in melanoma (suppressor of cytokine signalling, methylthioadenosine phosphorylase, cadherin 1) in the presence or absence of 5'‑Aza‑deoxyctidine treatment revealed that CYLD does not play a key role in DNA methylation. Chromatin accessibility and histone H3 modification assay uncovered a role of CYLD in the formation of chromatin structure. Subsequent inhibitor experiments confirmed the effect of CYLD on H3K9me2 level associated with heterochromatin. Furthermore, enhanced H3K9 dimethylation in Cyld‑/‑ melanoma cells was associated with upregulation of euchromatic histone lysine methyltransferase 2 (EHMT2). Moreover, the specific inhibitor of EHMT2, CM272, resulted in decreased proliferation and relaxation of compact chromatin in Cyld‑deficient melanoma cells. These results reveal a novel role of CYLD in histone methylation and chromatin packaging.

Journal article

L. Lämmerhirt
Melanie Kappelmann-Fenzl
Stefan Fischer
M. Pommer
T. Zimmermann
V. Kluge
A. Matthies
S. Kuphal
A.-K. Bosserhoff

Knockdown of Lamin B1 and the Corresponding Lamin B Receptor Leads to Changes in Heterochromatin State and Senescence Induction in Malignant Melanoma.

In: Cells vol. 11 pg. 2154

(2022)

DOI: 10.3390/cells11142154

show abstract

Modifications in nuclear structures of cells are implicated in several diseases including cancer. They result in changes in nuclear activity, structural dynamics and cell signalling. However, the role of the nuclear lamina and related proteins in malignant melanoma is still unknown. Its molecular characterisation might lead to a deeper understanding and the development of new therapy approaches. In this study, we analysed the functional effects of dysregulated nuclear lamin B1 (LMNB1) and its nuclear receptor (LBR). According to their cellular localisation and function, we revealed that these genes are crucially involved in nuclear processes like chromatin organisation. RNA sequencing and differential gene expression analysis after knockdown of LMNB1 and LBR revealed their implication in important cellular processes driving ER stress leading to senescence and changes in chromatin state, which were also experimentally validated. We determined that melanoma cells need both molecules independently to prevent senescence. Hence, downregulation of both molecules in a BRAFV600E melanocytic senescence model as well as in etoposide-treated melanoma cells indicates both as potential senescence markers in melanoma. Our findings suggest that LMNB1 and LBR influence senescence and affect nuclear processes like chromatin condensation and thus are functionally relevant for melanoma progression.

Lecture

Melanie Kappelmann-Fenzl

Bioinformatische Analyse molekularer Mechanismen der AP-1 Transkriptionsaktivität und deren funktioneller Bedeutung im malignen Melanom.

In: Deggendorfer Wissenschaftliches Kolloquium

Technische Hochschule Deggendorf Technische Hochschule Deggendorf

23.06.2022 (2022)

Lecture

Stefan Fischer
S. Schmidt
Melanie Kappelmann-Fenzl
R. Schmid
L. Lämmerhirt
L. Fischer
S. Schrüfer
I. Thievessen
D. Schubert
A. Matthies
R. Detsch
A. Boccaccini
A. Arkudas
A. Kengelbach-Weigand
A. Bosserhoff

Bioinformatic analysis of different 2D and 3D culture conditions of an established melanoma cell model.

In: Fall Meeting of the German Society for Biochemistry and Molecular Biology

Heinrich-Heine-Universität Düsseldorf Düsseldorf

14.-16.09.2022 (2022)

Journal article

I. Stüfchen
F. Beyer
S. Staebler
Stefan Fischer
Melanie Kappelmann-Fenzl
R. Beckersvordersandforth
A. Bosserhoff

Sox9 regulates melanocytic fate decision of adult hair follicle stem cells.

In: iScience vol. 26 pg. 106919

19.05.2023 (2023)

DOI: 10.1016/j.isci.2023.106919

show abstract

The bulge of hair follicles harbors Nestin+ (neural crest like) stem cells, which exhibit the potential to generate various cell types including melanocytes. In this study, we aimed to determine the role of Sox9, an important regulator during neural crest development, in melanocytic differentiation of those adult Nestin+ cells. Immunohistochemical analysis after conditional Sox9 deletion in Nestin+ cells of adult mice revealed that Sox9 is crucial for melanocytic differentiation of these cells and that Sox9 acts as a fate determinant between melanocytic and glial fate. A deeper understanding of factors that regulate fate decision, proliferation and differentiation of these stem cells provides new aspects to melanoma research as melanoma cells share many similarities with neural crest cells. In summary, we here show the important role of Sox9 in melanocytic versus glial fate decision of Nestin+ stem cells in the skin of adult mice.

Lecture

Melanie Kappelmann-Fenzl

Mutationen & Co. KG.

In: 17. Deggendorfer Forum zur digitalen Datenanalyse 2023: "Wenn Maschinen lernen: was ist unsere Lektion?"

Deggendorf

11.05.2023 (2023)

show abstract

Anders und doch ähnlich: sind Fragen in der Betriebswirtschaft und des menschlichen Körpers vergleichbar? Mit etwas Fantasie durchaus denkbar und sind die Analysemethoden beider komplexen Systeme womöglich übertragbar? Wagt man den Blick in andere Fachbereiche, erkennt man oft Parallelen, die man nicht erwartet hätte. Die computergestützte Biologie hat sich in den letzten Jahrzehnten rasant entwickelt und wird es auch weiterhin tun, dabei ist die Analyse biologischer Sequenzen (z.B. unseres Erbguts) ein sehr wichtiger Teil der Bioinformatik geworden. Um biomedizinische Daten professionell handhaben und spezifische prognostische, prädiktive, diagnostische etc. Fragen beantworten zu können nutzt man Algorithmen, Skripte, Software-Tools, Datenbanken usw. Die Fragestellung ist primär die gleiche wie in der Wirtschaftsprüfung: Wo ist der Fehler? Gibt es eine Systematik? Hat der Fehler Allgemeingültigkeit oder ist er individuell? Mutationen in Genen oder Bilanzierungsfehler - same same, but different!

Journal article

L. Lämmerhirt
Melanie Kappelmann-Fenzl
Stefan Fischer
P. Meier
S. Staebler
S. Kuphal
A. Bosserhoff

Loss of miR-101-3p in melanoma stabilizes genomic integrity, leading to cell death prevention.

In: Cellular & Molecular Biology Letters vol. 29 pg. 29

02.03.2024 (2024)

DOI: 10.1186/s11658-024-00552-2

show abstract

Malignant melanoma remains the most lethal form of skin cancer, exhibiting poor prognosis after forming distant metastasis. Owing to their potential tumor-suppressive properties by regulating oncogenes and tumor suppressor genes, microRNAs are important player in melanoma development and progression. We defined the loss of miR-101-3p expression in melanoma cells compared with melanocytes and melanoblast-related cells as an early event in tumor development and aimed to understand the tumor suppressive role of miR-101-3p and its regulation of important cellular processes. Reexpression of miR-101-3p resulted in inhibition of proliferation, increase in DNA damage, and induction of apoptosis. We further determined the nuclear structure protein Lamin B1, which influences nuclear processes and heterochromatin structure, ATRX, CASP3, and PARP as an important direct target of miR-101-3p. RNA sequencing and differential gene expression analysis after miR-101-3p reexpression supported our findings and the importance of loss of mir-101-3p for melanoma progression. The validated functional effects are related to genomic instability, as recent studies suggest miRNAs plays a key role in mediating this cellular process. Therefore, we concluded that miR-101-3p reexpression increases the genomic instability, leading to irreversible DNA damage, which leads to apoptosis induction. Our findings suggest that the loss of miR-101-3p in melanoma serves as an early event in melanoma progression by influencing the genomic integrity to maintain the increased bioenergetic demand.

Journal article

S. Staebler
U. Rottensteiner-Brandl
Z. El Ahmad
Melanie Kappelmann-Fenzl
A. Arkudas
A. Kengelbach-Weigand
A. Bosserhoff
S. Schmidt

Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma.

In: Cell Death & Disease vol. 15 pg. 351

21.05.2024 (2024)

DOI: 10.1038/s41419-024-06733-3

show abstract

Malignant melanoma, the most aggressive form of skin cancer, is often incurable once metastatic dissemination of cancer cells to distant organs has occurred. We investigated the role of Transcription Factor Activating Enhancer-Binding Protein 2ε (AP2ε) in the progression of metastatic melanoma. Here, we observed that AP2ε is a potent activator of metastasis and newly revealed AP2ε to be an important player in melanoma plasticity. High levels of AP2ε lead to worsened prognosis of melanoma patients. Using a transgenic melanoma mouse model with a specific loss of AP2ε expression, we confirmed the impact of AP2ε to modulate the dynamic switch from a migratory to a proliferative phenotype. AP2ε deficient melanoma cells show a severely reduced migratory potential in vitro and reduced metastatic behavior in vivo. Consistently, we revealed increased activity of AP2ε in quiescent and migratory cells compared to heterogeneously proliferating cells in bioprinted 3D models. In conclusion, these findings disclose a yet-unknown role of AP2ε in maintaining plasticity and migration in malignant melanoma cells.

Journal article

V. Kluge
Melanie Kappelmann-Fenzl
Stefan Fischer
T. Zimmermann
M. Pommer
S. Kuphal
A.-K. Bosserhoff

Alternative Wnt-signaling axis leads to a break of oncogene-induced senescence.

In: Cell Death & Disease vol. 15 pg. 166

22.02.2024 (2024)

DOI: 10.1038/s41419-024-06550-8

show abstract

Oncogene-induced senescence (OIS) is an important process that suppresses tumor development, but the molecular mechanisms of OIS are still under investigation. It is known that BRAFV600E-mutated melanocytes can overcome OIS and develop melanoma, but the underlying mechanism is largely unknown. Using an established OIS model of primary melanocytes transduced with BRAFV600E, YAP activity was shown to be induced in OIS as well as in melanoma cells compared to that in normal epidermal melanocytes. This led to the assumption that YAP activation itself is not a factor involved in the disruption of OIS. However, its role and interaction partners potentially change. As Wnt molecules are known to be important in melanoma progression, these molecules were the focus of subsequent studies. Interestingly, activation of Wnt signaling using AMBMP resulted in a disruption of OIS in BRAFV600E-transduced melanocytes. Furthermore, depletion of Wnt6, Wnt10b or β-catenin expression in melanoma cells resulted in the induction of senescence. Given that melanoma cells do not exhibit canonical Wnt/β-catenin activity, alternative β-catenin signaling pathways may disrupt OIS. Here, we discovered that β-catenin is an interaction partner of YAP on DNA in melanoma cells. Furthermore, the β-catenin-YAP interaction changed the gene expression pattern from senescence-stabilizing genes to tumor-supportive genes. This switch is caused by transcriptional coactivation via the LEF1/TEAD interaction. The target genes with binding sites for LEF1 and TEAD are involved in rRNA processing and are associated with poor prognosis in melanoma patients. This study revealed that an alternative YAP-Wnt signaling axis is an essential molecular mechanism leading to OIS disruption in melanocytes.

Journal article

T. Meißgeier
Melanie Kappelmann-Fenzl
S. Staebler
A. Ahari
C. Mertes
J. Gagneur
L. Linck-Paulus
A. Bosserhoff

Splicing control by PHF5A is crucial for melanoma cell survival.

In: Cell Proliferation pg. e13741

30.08.2024 (2024)

DOI: 10.1111/cpr.13741

show abstract

Abnormalities in alternative splicing are a hallmark of cancer formation. In this study, we investigated the role of the splicing factor PHD finger protein 5A (PHF5A) in melanoma. Malignant melanoma is the deadliest form of skin cancer, and patients with a high PHF5A expression show poor overall survival. Our data revealed that an siRNA-mediated downregulation of PHF5A in different melanoma cell lines leads to massive splicing defects of different tumour-relevant genes. The loss of PHF5A results in an increased rate of apoptosis by triggering Fas- and unfolded protein response (UPR)-mediated apoptosis pathways in melanoma cells. These findings are tumour-specific because we did not observe this regulation in fibroblasts. Our study identifies a crucial role of PHF5A as driver for melanoma malignancy and the described underlying splicing network provides an interesting basis for the development of new therapeutic targets for this aggressive form of skin cancer.

Journal article

Y. Antonelli
R. Krüger
A. Buehler
M. Monavari
M. Fuentes-Chandía
F. Colombo
R. Palmisano
A.-K. Bosserhoff
Melanie Kappelmann-Fenzl
et al.

When Mechanical Stress Matters: Generation of Polyploid Giant Cancer Cells in Tumor‐Like Microcapsules.

In: Advanced Functional Materials pg. 1-14

(2024)

DOI: 10.1002/adfm.202311139

show abstract

Biofabrication techniques enable the performance of bioinspired three-dimensional (3D) matrices resembling primary tumors. To validate their reliability, embedded cells may express complex biophysical responses. Among others, the emergence of tumor heterogeneity and the generation of Polyploid Giant Cancer Cells (PGCC), as a result of the mechanical stress, are two of the most challenging hallmarks to resemble in vitro. Here, these phenomena are studied in cells cultured on two-dimensional (2D) flasks, in 3D spheroids, or immobilized within 3D polymer-based tumor-like microcapsules. These results show that cells cultured in 3D microcapsules exhibited an enhanced biomechanical heterogeneity, a higher number of PGCC, and an increased exertion of cell-matrix attachment forces with respect to the other two experimental conditions. Additionally, cells isolated from tumor-like microcapsules redistribute and align the cytoplasmatic protein Caveolin-1, and upregulate markers involved in cell proliferation (i.e., Ki67), metastasis (i.e., TGF-β1, TGF-β-R2), and epithelial to mesenchymal transition, to name a few. These hallmarks are barely described in the past as a result of the confinement and mechanical stress. Thus, in this work it is demonstrated that both the mechanical stress and confinement are required to stimulate cell polyploidy and biomechanical heterogeneity, which can be easily addressed by immobilizing breast cancer cells in tumor-like microcapsules.

Journal article

Melanie Kappelmann-Fenzl
A. Matthies
A. Bosserhoff

Regulatory relevance of the AP-1 associated co-factor TEAD in melanoma.

In: Pigment Cell & Melanoma Research (The 20th International Congress of the Society for Melanoma Research) vol. 37 pg. 90-226

(2024)

DOI: 10.1111/pcmr.13152

show abstract

The development and progression of malignant melanoma is characterized by dedifferentiation and molecular changes in melanocytes induced by various gene defects, deregulated signaling pathways and the deregulation of tumor-relevant transcription factors. Especially members of the AP-1 transcription factor family play an important role in malignant melanoma leading to changes in expression of specific target genes, which can result in critical functional alterations and thus in the development of a malignant cell type. However, the molecular mechanisms of AP-1-DNA-binding modalities, its regulation and effects on gene expression are still largely unexplored. Yet unpublished data led to new essential insights in the regulatory mechanisms of AP-1 factors in melanoma. Based on c-Jun- and Fra-1- ChIP-Seq experiments, we were able to identify common as well as specific c-Jun and Fra-1 DNA-binding sites in different melanoma cell lines. Within the bioinformatical analyses we focused on motifs near the AP-1 consensus sequence as potential co-regulatory factors. Therefore, the respective peak files were centered to the identified motifs and subsequently allow to detect surrounding co-factor motifs. Here, we observed an enrichment of TEAD binding motifs in the immediate vicinity of AP-1 motifs. Previous studies already showed increased AP-1, as well as TEAD activity in melanoma. Thus, we were further interested in the combinatorial effect of AP-1 and TEAD on transcriptional activity. Hence, we newly generated reporter gene constructs to further analyze the effect on AP-1 activity with a nearby TEAD co-factor binding site. The designed construct was cloned into a pGL3 promoter vector to investigate the AP-1/TEAD activity via luciferase reporter gene assays in various melanoma cell lines. Surprisingly, no increased activity could be detected. Consequently, we addressed the question of whether the predicted target genes of the identified AP-1/TEAD motifs were differentially expressed in melanoma cells compared to normal melanocytes. Here, we focus on differential expression analysis results of performed RNA-Seq experiments comparing different melanoma cell lines and NHEMs. Further in-depth studies of the identified melanoma-related, regulated genes will clarify whether the observed decrease in AP-1/TEAD activity is a possible molecular regulator of decreased target gene expression in melanoma cells.