Publications

@article{nokey_88,

title = {Nanopore sequencing-derived methylation biomarker prediction for methylation-specific PCR in patients with head and neck squamous cell carcinoma},

author = {Daria Meyer and Anne Hennig and Anna-Bawany Hums and Orlando Guntinas-Lichius and Martina Schmitz and Manja Marz },

doi = {10.1186/s13148-025-01960-7},

year  = {2025},

date = {2025-09-13},

journal = {Clinical Epigenetics},

volume = {17},

abstract = {DNA methylation of CpG islands is altered in cancer cells. Hypermethylation of single CpG islands in the promoter regions of tumor-suppressor genes occurs already in the early stages of cancer. These methylation changes are cancer-type specific and therefore can serve as early cancer biomarker. Identifying good and reliable biomarkers is crucial for the development of diagnostic tests and their application in clinical practice and remains the most significant challenge to date.},

keywords = {cancer, nanopore, nucleic acid modifications},

pubstate = {published},

tppubtype = {article}

}

@article{nokey_73,

title = {diffONT: predicting methylation-specific PCR biomarkers based on nanopore sequencing data for clinical application},

author = {Daria Meyer and Emanuel Barth and Laura Wiehle and Manja Marz},

doi = {10.1101/2025.02.17.638597},

year  = {2025},

date = {2025-02-20},

urldate = {2025-02-20},

journal = {bioRxiv},

abstract = {DNA methylation is known to act as biomarker applicable for clinical diagnostics, especially in cancer detection. Methylation-specific PCR (MSP) is a widely used approach to screen patient samples fast and efficiently for differential methylation. During MSP, methylated regions are selectively amplified with specific primers. With nanopore sequencing, knowledge about DNA methylation is generated during direct DNA sequencing, without any need for pretreatment of the DNA. Multiple methods, mainly developed for whole-genome bisulfite sequencing (WGBS) data, exist to predict differentially methylated regions (DMRs) in the genome. However, the predicted DMRs are often very large, and not sufficiently discriminating to generate meaningful results in MSP creating a gap between theoretical cancer marker research and practical application, as no tool currently provides methylation difference predictions tailored for PCR-based diagnostics. Here we present diffONT, which predicts differentially methylated primer regions, directly suitable for MSP primer design and thus allowing a direct translation into practical approaches. diffONT takes into account (i) the specific length of primer and amplicon regions, (ii) the fact that one condition should be unmethylated, and (iii) a minimal required amount of differentially methylated cytosines within the primer regions. Based on two nanopore sequencing data sets we compared the results of diffONT to metilene, DSS and pycoMeth. We show that the regions predicted by diffONT are more specific towards hypermethylated regions and more usable for MSP. diffONT accelerates the design of methylation-specific diagnostic assays, bridging the gap between theoretical research and clinical application.Competing Interest Statement. The authors have declared no competing interest.},

keywords = {cancer, DNA / genomics, nanopore, nucleic acid modifications, software},

pubstate = {published},

tppubtype = {article}

}

@article{nokey_42,

title = {PAPAS Suppresses Breast Carcinogenesis by Promoting Differentiation of Mammary Epithelial Cells},

author = {Sijia Ren and Feng Bai and Clara Stanko and Muriel Ritsch and Tino Schenk and Emanuel Barth and Xin-Hai Pei and Holger Bierhoff

},

doi = {10.2139/ssrn.4436847},

year  = {2024},

date = {2024-01-23},

urldate = {2023-05-23},

journal = {Cell Reports},

abstract = {Extensive remodeling of the female mammary epithelium during development and pregnancy has been linked to cancer susceptibility. The faithful response of mammary epithelial cells (MECs) to hormone signaling is key to avoid breast cancer development. Here we show that lactogenic differentiation of murine MECs requires epigenetic silencing of genes encoding ribosomal RNA (rRNA) by the antisense transcript PAPAS. Accordingly, knockdown of PAPAS derepresses rRNA genes, attenuates the response to lactogenic hormones, and induces malignant transformation. Restoring PAPAS levels in breast cancer cells reduces tumorigenicity and, as revealed by transcriptomics, immune evasion potential. Mechanistically, we show that PAPAS transcription depends on R-loop formation at the 3’ end of rRNA genes, which is repressed by RNase H1 and replication protein A (RPA) overexpression in breast cancer cells. Depletion of PAPAS, and upregulation of RNase H1 and RPA in human breast cancer underpin the clinical relevance of our findings.},

keywords = {cancer, ncRNAs, RNA structure},

pubstate = {published},

tppubtype = {article}

}

@article{nokey_49,

title = {Decoding cell-type contributions to the cfRNA transcriptomic landscape of liver cancer},

author = {Aram Safrastyan and Christian {Höner Zu Siederdissen} and Damian Wollny},

doi = {10.1186/s40246-023-00537-w},

year  = {2023},

date = {2023-10-05},

journal = {Hum Genomics},

volume = {17},

issue = {1},

pages = {90},

abstract = {Background

Liquid biopsy, particularly cell-free RNA (cfRNA), has emerged as a promising non-invasive diagnostic tool for various diseases, including cancer, due to its accessibility and the wealth of information it provides. A key area of interest is the composition and cellular origin of cfRNA in the blood and the alterations in the cfRNA transcriptomic landscape during carcinogenesis. Investigating these changes can offer insights into the manifestations of tissue alterations in the blood, potentially leading to more effective diagnostic strategies. However, the consistency of these findings across different studies and their clinical utility remains to be fully elucidated, highlighting the need for further research in this area.



Results

In this study, we analyzed over 350 blood samples from four distinct studies, investigating the cell type contributions to the cfRNA transcriptomic landscape in liver cancer. We found that an increase in hepatocyte proportions in the blood is a consistent feature across most studies and can be effectively utilized for classifying cancer and healthy samples. Moreover, our analysis revealed that in addition to hepatocytes, liver endothelial cell signatures are also prominent in the observed changes. By comparing the classification performance of cellular proportions to established markers, we demonstrated that cellular proportions could distinguish cancer from healthy samples as effectively as existing markers and can even enhance classification when used in combination with these markers.



Conclusions

Our comprehensive analysis of liver cell-type composition changes in blood revealed robust effects that help classify cancer from healthy samples. This is especially noteworthy, considering the heterogeneous nature of datasets and the etiological distinctions of samples. Furthermore, the observed differences in results across studies underscore the importance of integrative and comparative approaches in the future research to determine the consistency and robustness of findings. This study contributes to the understanding of cfRNA composition in liver cancer and highlights the potential of cellular deconvolution in liquid biopsy.},

keywords = {cancer, ncRNAs},

pubstate = {published},

tppubtype = {article}

}

@article{nokey_48,

title = {Detection of reproducible liver cancer specific ligand-receptor signaling in blood},

author = {Aram Safrastyan and Damian Wollny},

doi = {10.1101/2023.09.25.559274},

year  = {2023},

date = {2023-09-25},

urldate = {2023-09-25},

journal = {bioRxiv},

abstract = {Cell-cell communication mediated by ligand-receptor interactions (LRI) is critical to coordinating diverse biological processes in homeostasis and disease. Lately, our understanding of these processes has greatly expanded through the inference of cellular communication, utilizing RNA extracted from bulk tissue or individual cells. Considering the challenge of obtaining tissue biopsies for these approaches, we considered the potential of studying cell-free RNA obtained from blood. To test the feasibility of this approach, we used the BulkSignalR algorithm across 295 cell-free RNA samples and compared the LRI profiles across multiple cancer types and healthy donors. Interestingly, we detected specific and reproducible LRIs particularly in the blood of liver cancer patients compared to healthy donors. We found an increase in the magnitude of hepatocyte interactions, notably hepatocyte autocrine interactions in liver cancer patients. Additionally, a robust panel of 30 liver cancer-specific LRIs presents a bridge linking liver cancer pathogenesis to discernible blood markers. In summary, our approach shows the plausibility of detecting liver LRIs in blood and builds upon the biological understanding of cell-free transcriptomes.},

keywords = {cancer, ncRNAs},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Network analysis of hepatocellular carcinoma liquid biopsies augmented by single-cell sequencing data},

author = {Aram Safrastyan and Damian Wollny},

doi = {10.3389/fgene.2022.921195},

year  = {2022},

date = {2022-08-25},

journal = {Front Genet},

volume = {13},

pages = {921195},

abstract = {Liquid biopsy, the analysis of body fluids, represents a promising approach for disease diagnosis and prognosis with minimal intervention. Sequencing cell-free RNA derived from liquid biopsies has been very promising for the diagnosis of several diseases. Cancer research, in particular, has emerged as a prominent candidate since early diagnosis has been shown to be a critical determinant of disease prognosis. Although high-throughput analysis of liquid biopsies has uncovered many differentially expressed genes in the context of cancer, the functional connection between these genes is not investigated in depth. An important approach to remedy this issue is the construction of gene networks which describes the correlation patterns between different genes, thereby allowing to infer their functional organization. In this study, we aimed at characterizing extracellular transcriptome gene networks of hepatocellular carcinoma patients compared to healthy controls. Our analysis revealed a number of genes previously associated with hepatocellular carcinoma and uncovered their association network in the blood. Our study thus demonstrates the feasibility of performing gene co-expression network analysis from cell-free RNA data and its utility in studying hepatocellular carcinoma. Furthermore, we augmented cell-free RNA network analysis with single-cell RNA sequencing data which enables the contextualization of the identified network modules with cell-type specific transcriptomes from the liver.},

keywords = {cancer, single-cell sequencing},

pubstate = {published},

tppubtype = {article}

}

@article{Sofer2022,

title = {A genome-wide CRISPR activation screen reveals Hexokinase 1 as a critical factor in promoting resistance to multi-kinase inhibitors in hepatocellular carcinoma cells},

author = {Summer Sofer and Kevin Lamkiewicz and Shir Armoza Eilat and Shirly Partouche and Manja Marz and Neta Moskovits and Salomon M Stemmer and Amir Shlomai and Ella H Sklan},

doi = {10.1096/fj.202101507RR},

year  = {2022},

date = {2022-02-11},

urldate = {2022-02-11},

journal = {FASEB J },

volume = {36},

issue = {3},

pages = {e22191},

abstract = {Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.},

keywords = {cancer, DNA / genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Heinze:21,

title = {RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis},

author = {Karolin Heinze and Martin Hölzer and Martin Ungelenk and Melanie Gerth and Jürgen Thomale and Regine Heller and Claire R. Morden and Kirk J. McManus and Alexander S. Mosig and Matthias Dürst and Ingo B. Runnebaum and Norman Häfner},

doi = {10.3390/cancers13030476},

year  = {2021},

date = {2021-01-26},

urldate = {2021-01-26},

journal = {Cancers},

volume = {13},

number = {3},

pages = {476},

publisher = {MDPI AG},

abstract = {The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis. },

keywords = {cancer, differential expression analysis, proteins, RNA / transcriptomics},

pubstate = {published},

tppubtype = {article}

}

@article{Kotolloshi:21,

title = {SLC35F2, a Transporter Sporadically Mutated in the Untranslated Region, Promotes Growth, Migration, and Invasion of Bladder Cancer Cells},

author = {Roland Kotolloshi and Martin Hölzer and Mieczyslaw Gajda and Marc-Oliver Grimm and Daniel Steinbach},

doi = {10.3390/cells10010080},

year  = {2021},

date = {2021-01-06},

urldate = {2021-01-01},

journal = {Cells},

volume = {10},

number = {1},

pages = {80},

publisher = {MDPI AG},

abstract = {Bladder cancer is a very heterogeneous disease and the molecular mechanisms of carcinogenesis and progression are insufficiently investigated. From the DNA sequencing analysis of matched non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) samples from eight patients, we identified the tumour-associated gene SLC35F2 to be mutated in the 5′ and 3′ untranslated region (UTR). One mutation in 3′UTR increased the luciferase activity reporter, suggesting its influence on the protein expression of SLC35F2. The mRNA level of SLC35F2 was increased in MIBC compared with NMIBC. Furthermore, in immunohistochemical staining, we observed a strong intensity of SLC35F2 in single tumour cells and in the border cells of solid tumour areas with an atypical accumulation around the nucleus, especially in the MIBC. This suggests that SLC35F2 might be highly expressed in aggressive and invasive tumour cells. Moreover, knockdown of SLC35F2 repressed the growth of bladder cancer cells in the monolayer and spheroid model and suppressed migration and invasion of bladder cancer cells. In conclusion, we suggest that SLC35F2 is involved in bladder cancer progression and might provide a new therapeutic approach, for example, by the anti-cancer drug YM155, a cargo of the SLC35F2 transporter.},

keywords = {cancer, DNA / genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Gerresheim:20,

title = {Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication},

author = {Gesche K. Gerresheim and Carolin S. Hess and Lyudmila A. Shalamova and Markus Fricke and Manja Marz and Dmitri E. Andreev and Ivan N. Shatsky and Michael Niepmann},

doi = {10.3390/ijms21186955},

year  = {2020},

date = {2020-09-22},

urldate = {2020-09-22},

journal = {Int J Mol Sci},

volume = {21},

number = {18},

pages = {6955},

publisher = {MDPI AG},

abstract = {Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3′-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.

},

keywords = {cancer, liver, ncRNAs, RNA structure, viruses},

pubstate = {published},

tppubtype = {article}

}

@article{Gerresheim:19,

title = {Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling},

author = {Gesche Gerresheim and Jochen Bathke and Audrey Michel and Dmitri E. Andreev and Lyudmila Shalamova and Oliver Rossbach and Pan Hu and Dieter Glebe and Markus Fricke and Manja Marz and Alexander Goesmann and Stephen Kiniry and Pavel Baranov and Ivan Shatsky and Michael Niepmann},

doi = {10.3390/ijms20061321},

year  = {2019},

date = {2019-03-15},

urldate = {2019-03-15},

journal = {Int J Mol Sci},

volume = {20},

number = {6},

pages = {1321},

publisher = {MDPI AG},

abstract = {Background: Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known. Methods: We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days. Results: Established viral replication does not cause global changes in host gene expression—only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (PPP1R15A/GADD34, DDIT3/CHOP, and TRIB3) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes. Conclusion: After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming (“Warburg effect”) even in the hepatocellular carcinoma cells used here. },

keywords = {cancer, differential expression analysis, liver, RNA structure, virus host interaction, viruses},

pubstate = {published},

tppubtype = {article}

}

@article{Steinbach:18,

title = {Analysis of molecular mechanism of progression of non-muscle-invasive bladder cancer (NMIBC) by genome-wide exome and UTR mutation analysis},

author = {D Steinbach and Martin Hölzer and Manja Marz and M Gajda and F-C Von Rundstedt and M-O Grimm},

year  = {2018},

date = {2018-01-01},

urldate = {2018-01-01},

journal = {Eur Urol Suppl},

volume = {17},

number = {2},

pages = {e1523},

publisher = {Elsevier},

keywords = {cancer},

pubstate = {published},

tppubtype = {article}

}