Dominik Jung

Background: Protein prenylation is crucial for the function of hundreds of proteins. Aberrant protein prenylation can be caused by the aberrant expression of prenyltransferases (PTases), which has been reported for multiple cancer entities. The reasons for aberrant PTase expression in cancer have not yet been investigated. Methods: We analyzed CpG methylation within promoter-associated CpG islands of the PTase genes FNTA, FNTB, PGGT1B, and RABGGTA via bisulfite conversion and pyrosequencing to assess its role in PTase expression and gain deeper insight into the regulation of protein prenylation in cancer. We used DNA from three benign controls (whole blood samples, peripheral blood mononuclear cells, and HEK293) and 19 human cancer cell lines from various origins to assess DNA methylation within PTase gene promoter-associated CpG islands. For a subset of these cell lines, we measured mRNA expression via qPCR and correlated it with DNA methylation. Results: Methylation across all PTase genes ranged from 1.9 ± 0.9% to 11.4 ± 4.0% (mean methylation ± standard deviation) in benign cells, and 2.3 ± 1.0% to 16.0 ± 5.4% in cancer cells. DNA methylation and mRNA expression of PGGT1B correlated inversely (PCC = −0.75; p = 0.005). Conclusions: We saw no general differences between benign and malignant cells, but observed significant differences between non-malignant controls and multiple individual cancer cell lines regarding the methylation of PTase genes. This was prominently seen in PGGT1B in Caki-1 cells, raising the possibility that DNA methylation is involved in the dysregulation of PTase expression in cancer.

Background: Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that causes progressive cystic lung destruction and is often associated with renal angiomyolipomas (AMLs). Given evidence of pleiotropy linking LAM risk to pulmonary traits, we investigated whether glucocorticoid receptor (GR) signalling might influence LAM biology and clinical features. Methods: We combined cell-based studies, GR inhibition/activation assays, gene expression and single-cell RNA sequencing analyses, and hormone profiling in retrospective and prospective LAM cohorts. Cellular experiments employed murine Tsc2 ^−/− embryonic fibroblasts and human TSC2 ^−/− AML cells. Circulating steroid levels were measured in women with LAM and healthy controls, and associations with clinical variables were evaluated. Results: In LAM/AML models, GR activation by glucocorticoids elicited transcriptional responses, whereas GR inhibition reduced clonogenic potential. GR stimulation was associated with CDKN1C upregulation through enhancer binding, and single-cell profiling suggested a shift towards slower proliferation and differentiation-prone states enriched for a LAM cell signature. Clinically, our analyses suggest that women with LAM may show altered circulating hormone profiles, including elevated adrenocorticotropic hormone (ACTH) and cortisol levels, together with reduced 17-hydroxyprogesterone, compared with controls. In a prospective cohort, ACTH levels were suggestively associated with advanced radiological disease stage. AML cells showed elevated expression of POMC , which encodes the precursor of ACTH, and POMC peptide was detected in LAM lung tissue. Conclusions: Our findings suggest that GR signalling may contribute to aspects of LAM cell behaviour and disease status. Further investigation of this pathway could clarify its role as a disease modifier and potential therapeutic target.

Background: Protein prenylation is crucial for the function of hundreds of proteins. Aberrant protein prenylation can be caused by aberrant expression of prenyltransferases (PTases), which has been reported for multiple cancer entities. The reasons for aberrant PTase expression in cancer have not yet been investigated. Methods: We analyzed CpG methylation within promoter-associated CpG islands of PTase genes FNTA, FNTB, PGGT1B and RABGGTA via bisulfite conversion and pyrosequencing to assess its role in PTase expression and gain deeper insight into the regulation of protein prenylation in cancer. We used DNA from three benign controls (whole blood samples, peripheral blood mononuclear cells, HEK293) and 19 human cancer cell lines from various origins to assess DNA methylation within PTase gene promoter-associated CpG islands. For a subset of these cell lines, we measured mRNA expression via qPCR and correlated it with DNA methylation. Results: Methylation across all PTase genes ranged from 1.9 % ± 0.9 to 11.4 % ± 4.0 (mean methylation ± standard deviation) in benign cells, and 2.3 % ± 1.0 to 16.0 % ± 5.4 in cancer cells. DNA methylation and mRNA expression of PGGT1B correlated inversely (PCC = −0.75; p = 0.005). Conclusions: We saw no general differences between benign and malignant cells, but observed significant differences between non-malignant controls and multiple individual cancer cell lines regarding methylation of PTase genes, most prominently of FNTA and PGGT1B in Caki-1 and Kelly cells, raising the possibility that DNA methylation is involved in the dysregulation of PTase expression in cancer.

In breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical Phase II–III trials. The objective of this study was to explore the clinical relevance of farnesyltransferase β-subunit (FNTB) single nucleotide promoter polymorphisms (FNTB-173 6G > 5G (rs3215788), -609 G > C (rs11623866) and -179 T > A (rs192403314)) in early breast cancer. FNTB genotyping was performed by pyrosequencing in 797 patients from a prospective multicentre observational PiA trial (NCT 01592825). In the total cohort, the FNTB-173 6G > 5G polymorphism was an independent predictor of RFI (HR = 0.568; 95% CI = 0.339–0.949, p = 0.031), OS (HR = 0.629; 95% CI = 0.403–0.980, p = 0.040) and BCSS (HR = 0.433; 95% CI = 0.213–0.882; p = 0.021), whereas the FNTB-609 G > C polymorphism was an independent predictor of RFI (HR = 0.453; 95% CI = 0.226–0.910, p = 0.026) and BCSS (HR = 0.227; 95% CI = 0.075–0.687, p = 0.009). Subtype analysis revealed the independent prognostic relevance of FNTB promoter polymorphisms, particularly in TNBC but not in luminal or HER2-positive intrinsic subtypes. Finally, we used electrophoretic mobility shift assays (EMSAs) to confirm in vitro that the polymorphism FNTB-173 6G > 5G resulted in the differential binding of nuclear proteins from five different breast cancer cell lines. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms (i) are independent prognostic biomarkers, particularly in patients with early TNBC, and (ii) could modulate FNTB’s transcriptional activity.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

HMG-CoA-Reductase inhibitors (HMGRIs) are currently the most widely used group of drugs in patients with coronary artery disease (CAD) and are given preemptively to patients with high levels of cholesterol, including those with diabetes mellitus (DM). However, intake of HMGRIs also increases the progression of coronary artery calcification (CAC) and the risk of developing DM. This study aimed to investigate whether HMGRI intake interacts with the diabetes-associated genetic risk score (GRS) to affect CAC progression using data from the population-based Heinz Nixdorf Recall (HNR) study. CAC was measured in 3157 participants using electron-beam computed tomography twice, at baseline (CAC_b) and 5 years later (CAC_5y). CAC progression was classified as slow, expected, or rapid based on predicted values. Weighted DM GRS was constructed using 100 diabetes mellitus–associated single nucleotide polymorphisms (SNPs). We used log-linear regression to evaluate the interaction of HMGRI intake with diabetes-associated GRS and individual SNPs on CAC progression (rapid vs. expected/slow), adjusting for age, sex, and log(CAC_b + 1). The prevalence of rapid CAC progression in the HNR study was 19.6%. We did not observe any association of the weighted diabetes mellitus GRS with the rapid progression of CAC (relative risk (RR) [95% confidence interval (95% CI)]: 1.01 [0.94; 1.10]). Furthermore, no indication of an interaction between GRS and HMGRI intake was observed (1.08 [0.83; 1.41]). Our analyses showed no indication that the impact of HMGRIs on CAC progression is significantly more severe in patients with a high genetic risk of developing DM than in those with a low GRS.

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gα_s-coupled human adenosine receptors, A_2A (A_2AAR) and A_2B (A_2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A_2AAR and A_2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.