Melanie Boerries

S100A1 is released from ischemic cardiomyocytes and signals myocardial damage via Toll-like receptor 4.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll‐like receptor 4 (TLR4)‐dependent activation of MAP kinases and NF‐κB. CFs exposed to S100A1 assumed an immunomodulatory and anti‐fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule‐1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody‐mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post‐MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI‐related inflammation and repair warrants further investigation.

STAT3 expression, activity and functional consequences of STAT3 inhibition in esophageal squamous cell carcinomas and Barrett’s adenocarcinomas

Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC, n=49) and Barrett’s adenocarcinomas (BAC, n=61) revealed similar STAT3 expression in ESCCs and BACs (P=0.109), but preferentially activated P-STAT3 in ESCCs (P=0.013). In vitro, strong STAT3 activation was seen by epidermal growth factor (EGF) stimulation in OE21 (ESCC) cells, whereas OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 (P=0.0148) and OE33 (P=0.0243) cells. Importantly, STAT3 knockdown reduced cell migration of OE33 cells by 2.5-fold in two types of migration assays (P=0.073, P=0.015), but not in OE21 cells (P=0.1079, P=0.386). Investigation of transcriptome analysis of STAT3 knockdown revealed a reduced STAT3 level associated with significant downregulation of cell cycle genes in both OE21 (P<0.0001) and OE33 (P=0.01) cells. In contrast, genes promoting cell migration (CTHRC1) were markedly upregulated in OE21 cells, whereas a gene linked to tight-junction stabilization and restricted cell motility (SHROOM2) was downregulated in OE21 but upregulated in OE33 cells. This study shows frequent, but distinct, patterns of STAT3 expression and activation in ESCCs and BACs. STAT3 knockdown reduces cell proliferation in ESCC and BAC cells, inhibits migration of BAC cells and may support cell migration of ESCC cells. Thereby, novel STAT3-regulated genes involved in ESCC and BAC cell proliferation and cell migration were identified. Thus, STAT3 may be further exploited as a potential novel therapeutic target, however, by careful distinction between the two histotypes of esophageal cancers.

Selective inhibition of esophageal cancer cells by combination of HDAC inhibitors and Azacytidine

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitro and in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach.

B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells.

Protein abundance of AKT and ERK pathway components governs cell type-specific regulation of proliferation

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Understanding the mTOR signaling pathway via mathematical modeling

The mechanistic target of rapamycin (mTOR) is a central regulatory pathway that integrates a variety of environmental cues to control cellular growth and homeostasis by intricate molecular feedbacks. In spite of extensive knowledge about its components, the molecular understanding of how these function together in space and time remains poor and there is a need for Systems Biology approaches to perform systematic analyses. In this work, we review the recent progress how the combined efforts of mathematical models and quantitative experiments shed new light on our understanding of the mTOR signaling pathway. In particular, we discuss the modeling concepts applied in mTOR signaling, the role of multiple feedbacks and the crosstalk mechanisms of mTOR with other signaling pathways. We also discuss the contribution of principles from information and network theory that have been successfully applied in dissecting design principles of the mTOR signaling network. We finally propose to classify the mTOR models in terms of the time scale and network complexity, and outline the importance of the classification toward the development of highly comprehensive and predictive models. WIREs Syst Biol Med 2017, 9:e1379. doi: 10.1002/wsbm.1379

Global gene expression profiling analysis reveals reduction of stemness after B-RAF inhibition in colorectal cancer cell lines

Cancer cell differentiation is an important field of discussion in the light of cancer stem cells. In a recent study by Herr et al. (2015) “B-RAF inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells” we described how inhibition of mutant BRAF in colorectal cancer cell lines induces cell re-differentiation that is correlated with the loss of tumor growth in vitro and in vivo. We used Illumina HumanHT-12 v4 Expression BeadChip to characterize the gain of differentiation of PLX4720-treated 3D cultures of HT29 and Colo-205 cells. Here, we describe the experimental design and statistical analysis that were performed on the data set leading to the above hypothesis. The data are publicly available at the Gene Expression Omnibus (GEO) database under the accession number GSE50791.

Cilia‐localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy‐related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell‐autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.

Personalized Clinical Decision Making Through Implementation of a Molecular Tumor Board: A German Single-Center Experience

Purpose Dramatic advances in our understanding of the molecular pathophysiology of cancer, along with a rapidly expanding portfolio of molecular targeted drugs, have led to a paradigm shift toward personalized, biomarker-driven cancer treatment. Here, we report the 2-year experience of the Comprehensive Cancer Center Freiburg Molecular Tumor Board (MTB), one of the first interdisciplinary molecular tumor conferences established in Europe. The role of the MTB is to recommend personalized therapy for patients with cancer beyond standard-of-care treatment. Methods This retrospective case series includes 198 patients discussed from March 2015 through February 2017. The MTB guided individual molecular diagnostics, assessed evidence of actionability of molecular alterations, and provided therapy recommendations, including approved and off-label treatments as well as available matched clinical trials. Results The majority of patients had metastatic solid tumors (73.7%), mostly progressive (77.3%) after a mean of 2.0 lines of standard treatment. Diagnostic recommendations resulted in 867 molecular diagnostic tests for 172 patients (five per case), including exome analysis in 36 cases (18.2%). With a median turnaround time of 28 days, treatment recommendations were given to 104 patients (52.5%). These included single-agent targeted therapies (42.3%), checkpoint inhibitors (37.5%), and combination therapies (18.3%). Treatment recommendations were implemented in 33 of 104 patients (31.7%), of whom 19 (57.6%) showed stable disease or partial response, including 14 patients (7.1% of the entire population) receiving off-label treatments. Conclusion Personalized extended molecular-guided patient care is effective for a small but clinically meaningful proportion of patients in challenging clinical situations. Limited access to targeted drugs, lack of trials, and submission at late disease stage prevents broader applicability, whereas genome-wide analyses are not a strict requirement for predictive molecular testing.