Biology subjects

Meyer, T. F.

Publications and source records attributed to Meyer, T. F..

13 recordsLinked to original sources

bioRxiv2025

Infection and herbicide exposure implicate c-Abl kinase in α-Synuclein Ser129 phosphorylation

BackgroundParkinsons disease is a complex multifactorial neurodegenerative disorder characterized by -Synuclein aggregation in Lewy bodies, with phosphorylation at serine 129 (pSer129) being a critical pathological hallmark. However, the exact mechanisms by which environmental triggers lead to this disease phenotype remain poorly understood. In this study we compare the effects of an exemplary infection and a certain pesticide exposure on the generation of pSer129 -Synuclein, with a focus on the involvement of cellular kinases in this process. MethodsTwo distinct environmental stressors were applied to neuronal cells: the pesticide rotenone and the well-studied gastric bacterium Helicobacter pylori (H. pylori). Phosphorylation of Ser129 -Synuclein was assessed by immunofluorescent staining and Western blotting. Cells were treated with mechanistically distinct c-Abl inhibitors, and pSer129 -Synuclein was detected using Western blotting and activities of the upstream serine-threonine kinase were predicted by kinase profiling and Western blotting, analyzed by one-way ANOVA followed by Tukeys multiple comparisons test. Moreover, transcriptome analyses of treated cells were performed and ingenuity pathway analysis and Deseq2 were applied to unravel the affected neurodegenerative pathways. ResultsThe functional analysis of our RNA sequence data demonstrated that both H. pylori and rotenone induced oxidative stress and neuroinflammation by stimulating neurodegenerative pathways. Rotenone and H. pylori activated c-Abl, likely through the induced oxidative stress and promoted -Synuclein phosphorylation. The kinase inhibitors Ponatinib and Asciminib effectively prevented pSer129 -Synuclein accumulation and reversed associated gene expression changes induced by rotenone or H. pylori. Moreover, GSK3{beta} appeared to be involved in the induction of Ser129 phosphorylation via activated c-Abl. Furthermore, H. pyloris vacuolating cytotoxin appeared to play a crucial role in the phosphorylation of pSer129 -Synuclein by c-Abl. ConclusionsThese findings highlight the pivotal role of c-Abl in -Synucleopathies and provide insights into shared mechanisms between infection and pesticide exposure, offering potential therapeutic targets for Parkinsons disease and related pathologies involving -Synuclein modification.

neuroscience↗

bioRxiv2024

Inhibition of ADAM17 increases cytotoxic effect of cisplatin in cervical spheroids and organoids

BackgroundCervical cancer represents one of the main causes of female, cancer-related mortality worldwide. The majority of cancers are caused by human papillomaviruses such as HPV16 and HPV18. As chemotherapeutic resistance to first-line platinum treatment is still a predominant clinical challenge in advanced cervical cancer, novel treatment options including combinatorial therapies are urgently required to overcome chemotherapeutic resistance. Inhibition of A Disintegrin And Metalloproteinase (ADAM)-family members, heavily involved in tumour progression of a vast range of solid tumours, strongly improved response to chemotherapeutic treatment in other tumour entities including ovarian cancer. MethodsWe established two- and three-dimensional models derived from three traditional cervical cancer cell lines and ectocervical cancer-derived organoids. Following characterisation, these models were used to investigate their response to cisplatin treatment in the absence and presence of ADAM inhibitors using viability assays and automated live cell imaging. ResultsThe pivotal role of the metalloprotease ADAM17 driving chemotherapy resistance was detectable in all ectocervical cultures irrespective of the model system used, whereas ADAM10 inhibition was predominantly effective only in loosely aggregated spheroids. We showed prominent differences regarding treatment responses between 2D monolayers compared to 3D spheroid and 3D organoid model systems. Particularly, the organoid system, regarded as the closest representation of primary tumours, exhibited reliably the combinatorial effect of ADAM17 inhibition and cisplatin in all three individual donors. ConclusionsAs two- and three-dimensional models of the same cell lines differ in their responses to chemotherapy it is essential to validate treatment strategies in more advanced model systems representing the patient situation more realistically. Ectocervical organoids showed reliable results regarding treatment responses closely mimicking the primary tumours and could therefore serve as an important tool for personalized medicine in cervical cancer. These findings strengthen the role of ADAM17 as a potential novel target for combinatorial treatments to overcome chemoresistance in cervical cancer.

cancer biology↗

bioRxiv2023

Helicobacter pylori cancer associated CagA protein drives intestinal metaplastic transition in human gastric organoids

Gastric intestinal metaplasia (GIM) constitutes a pre-neoplastic stage in the development of stomach cancer. While strong evidence points to a role of infection with CagA positive Helicobacter pylori in the development of GIM, currently available experimental models have not provided mechanistic clues on this association. Here, we ectopically expressed the H. pylori CagA protein in human gastric organoids derived from normal, primary epithelial cells. Native CagA protein was produced and rapidly processed to yield a tyrosine-phosphorylated C-terminal fragment of [~]35 kDa. It led to an activation of the STAT3 pathway and aberrant elevation of CDX2 expression, a marker of intestinal type of cells, as well as other intestinal markers. Thus, CagA drives re-programming of gastric cells towards an intestinal-like phenotype, towards GIM. In summary, we describe a cooperative mechanism of CagA-induced STAT3 signaling and intestinal-like trans-differentiation, promoting a pre-neoplastic state. Our model provides mechanistic evidence for a direct role of CagA in driving premalignancy in gastric pathogenesis.

cell biology↗

bioRxiv2023

γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to {beta} T cells and natural killer (NK) cells, {gamma}{delta} T cells represent a cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of {gamma}{delta} T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded {gamma}{delta} T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to {gamma}{delta} T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential {gamma}{delta} T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for {gamma}{delta} T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of {gamma}{delta} T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.

immunology↗

bioRxiv2022

DNA methylation in human gastric epithelial cells allows cell type-related plasticity and defines regional identity

BackgroundEpigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail. ResultsHere, we describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach (i.e., antrum, corpus, fundus) together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1, and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. ConclusionsBy extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation already characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics as a starting point for further studies.

molecular biology↗

bioRxiv2021

Single Object Profiles Regression Analysis (SOPRA): A novel method for analyzing high content cell-based screens

MotivationHigh content screening (HCS) experiments generate complex data from multiple object features for each cell within a treated population. Usually these data are analyzed by using population-averaged values of the features of interest, increasing the amount of false positives and the need for intensive follow-up validation. Therefore, there is a strong need for novel approaches with reproducible hit prediction by identifying significantly altered cell populations. ResultsHere we describe SOPRA, a workflow for analyzing image-based HCS data based on regression analysis of non-averaged object features from cell populations, which can be run on hundreds of samples using different cell features. Following plate-wise normalization the values are counted within predetermined binning intervals, generating unique frequency distribution profiles (histograms) for each population, which are then normalized to control populations. Statistically significant differences are identified using a regression model approach. Significantly changed profiles can be used to generate a heatmap from which altered cell populations with similar phenotypes are identified, enabling detection of siRNAs and compounds with the same on-target profile, reducing the number of false positive hits. A screen for cell cycle progression was used to validate the workflow, which identified statistically significant changes induced by siRNA-mediated gene perturbations and chemical inhibitors of different cell cycle stages.

bioinformatics↗

bioRxiv2021

Spatial organisation and homeostasis of epithelial lineages at the gastroesophageal junction is regulated by the divergent Wnt mucosal microenvironment

The gastroesophageal junction (GEJ), where squamous and columnar epithelia meet, is a hotspot for Barretts metaplasia development, dysbiosis and carcinogenesis. However, the mechanisms regulating GEJ homeostasis remain unclear. Here, by employing organoids, bulk and single-cell transcriptomics, single-molecule RNA in situ hybridisations and lineage tracing, we identified the spatial organisation of the epithelial, stromal compartment and the regulators that maintain the normal GEJ homeostasis. During development, common KRT8 progenitors generate committed unilineage p63/KRT5-squamous and KRT8-columnar stem cells responsible for the regeneration of postnatal esophagus and gastric epithelium that meet at GEJ. A unique spatial distribution of Wnt regulators in the underlying stromal compartment of these stem cells creates diverging Wnt microenvironments at GEJ and supports their differential regeneration. Further, we show that these tissue-resident stem cells do not possess the plasticity to transdifferentiate to the other lineage with the altered Wnt signals. Our study provides invaluable insights into the fundamental process of GEJ homeostasis and is crucial for understanding disease development.

developmental biology↗

bioRxiv2021

Chlamydia coinfection inhibits HPV-induced safeguards of the cellular and genomic integrity in patient-derived ectocervical organoids

Cervical mucosa is continually confronted by coinfections with pathogenic microbes. In addition to human papillomavirus, coinfections with Chlamydia trachomatis have been associated with an increased risk of cervical cancer. However, the dynamics of coinfections, their impact on the epithelia, and their contribution to pathogenesis remain obscure. Using a novel human ectocervical squamous stratified epithelial organoids, we recapitulated the natural infections of the cervix by Chlamydia, HPV, and their coinfections. Towards this, we genetically manipulated the healthy organoids to mimic in vivo HPV persistence by introducing E6E7 oncogenes into the host genome. HPV persistent organoids show enhanced tissue regeneration, increased proliferation and differentiation of stem cells, and nuclear atypia resembling cervical intraepithelial neoplasia grade 1. We found that HPV interferes with normal Chlamydia development. Further, a unique transcriptional host response induced by Chlamydia and HPV leads to distinct reprogramming of host cell processes. Strikingly, in coinfections, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair (MMR). Distinct post-translational proteasomal-degradation and E2F-mediated transcriptional regulation delineate the inverse regulation of MMR during coinfections. Our study employing organoids demonstrates the jeopardy of multiple sequential infection processes and the unique cellular microenvironment they create, accelerating neoplastic progression.

cancer biology↗

bioRxiv2019

Colibactin DNA damage signature indicates causative role in colorectal cancer

Colibactin, a potent genotoxin of Escherichia coli, causes DNA double strand breaks (DSBs) in human cells. We investigated if colibactin creates a particular DNA damage signature in infected cells by identifying DSBs in colon cells after infection with pks+ E.coli. Interestingly, genomic contexts of DSBs were enriched for AT-rich penta-/hexameric sequence motifs, exhibiting a particularly narrow minor groove width and extremely negative electrostatic potential. This corresponded with the binding characteristics of colibactin to double-stranded DNA, as elucidated by docking and molecular dynamics simulations. A survey of somatic mutations at the colibactin target sites of several thousand cancer genomes revealed significant enrichment of the identified motifs in colorectal cancers. Our work provides direct evidence for a role of colibactin in the etiology of human cancer.\n\nOne sentence summaryWe identify a mutational signature of colibactin, which is significantly enriched in human colorectal cancers.

microbiology↗

bioRxiv2019

Preservation of stemness in high-grade serous ovarian cancer organoids requires low Wnt environment

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the parental tumor mutational profile and phenotype. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for generation of HGSOC organoids, while healthy FT organoids depend on BMP suppression by Noggin. Interestingly, FT organoids modified by stable shRNA knockdown (KD) of p53, PTEN, and Retinoblastoma (RB), also require a low Wnt environment for long-term growth, while FT organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotype of normal and KD organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

cancer biology↗

bioRxiv2018

Transition of Wnt signaling microenvironment delineates the squamo-columnar junction and emergence of squamous metaplasia of the cervix

The transition zones (TZ) between squamous and columnar epithelium constitute hotspots for the emergence of cancers. Carcinogenesis at these sites is often preceded by the development of metaplasia, where one epithelial type invades the neighboring one. It remains unclear how these niches are restrained at the boundary between the two epithelial types and what factors contribute to metaplasia. Here we show that the cervical squamo-columnar junction derives from two distinct stem cell lineages that meet at the TZ. In contrast to the prevailing notion, our analysis of cervical tissue showed that the TZ is devoid of any locally restricted, specialized stem cell population, which has been implicated as precursor of both cervical squamous cell carcinoma and adenocarcinoma. Instead, we reveal that these cancers originate from two separate stem cell lineages. We show that the switch in the underlying Wnt signaling milieu of the stroma is a key determinant of proliferation or quiescence of epithelial stem cell lineages at the TZ. Strikingly, while the columnar lineage of the endocervix is driven by Wnt signaling, the maintenance of squamous stratified epithelium of the ectocervix and emergence of squamous metaplasia requires inhibition of Wnt signaling via expression of Dickkopf2 (Dkk2) in the underlying stroma. Moreover, Notch signaling is required for squamous cell stratification. Thus, our results indicate that homeostasis at the TZ is not maintained by a transition from one epithelial type to another but rather results from alternative signals from the stromal compartment driving the differential proliferation of the respective cell lineages at the squamo-columnar junction.

cancer biology↗

bioRxiv2018

ADP heptose, a novel pathogen-associated molecular pattern associated with Helicobacter pylori type 4 secretion

The gastric pathogen Helicobacter pylori activates the NF-{kappa}B pathway in human epithelial cells via the -kinase 1 (Alpk1)-TIFA axis. We and others have previously shown that heptose 1,7-bisphosphate (HBP) acts as a pathogen-associated molecular pattern (PAMP). HBP is an intermediate of lipopolysaccharide (LPS) synthesis in H. pylori and other gram-negative bacteria. Deletion of the hldE (rfaE) gene encoding the enzyme responsible for HBP synthesis, as well as deletion of further upstream genes, causes loss of NF-{kappa}B stimulation by H. pylori, while deletion of the downstream phosphatase encoding gene gmhB does not. This has led to the conclusion that HBP is the PAMP responsible for NF-{kappa}B induction. Here, our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts that fail to explain NF-{kappa}B activation. Instead, we identified ADP heptose, a major downstream metabolite of HdlE, as the predominant PAMP in H. pylori lysates, exhibiting [~]100-fold stronger activity compared to HBP. It therefore appears that synthesis of ADP heptose from HBP in H. pylori occurs independently of GmhB. The data lead us to conclude that ADP heptose constitutes the key PAMP, secreted via the pathogens cagPAI encoded type 4 secretion (T4SS).

microbiology↗

bioRxiv2017

ALPK1 And TIFA Dependent Innate Immune Response Triggered By The Helicobacter Pylori Type IV Secretion System

Activation of transcription factor NF-{kappa}B is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1{beta}- and TNF-- dependent NF-{kappa}B regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-{beta}-D-manno-heptose 1,7-bisphosphate ({beta}HBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-{kappa}B activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-{kappa}B activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-{kappa}B activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogens T4SS.

molecular biology↗