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.

Small Cell Lung Cancer (SCLC) is a specific subtype of lung cancer presenting as highly metastatic disease with extremely poor prognosis. Despite responding initially well to chemo- or radiotherapy, SCLC almost invariably relapses and develops resistance to chemotherapy. This is suspected to be related to tumor cell subpopulations with different characteristics resembling stem cells. Epithelial-Mesenchymal Transition (EMT) is known to play a key role in metastatic processes and in developing drug resistance. This is also true for NSCLC, but there is very little information on EMT processes in SCLC so far. SCLC, in contrast to NSCLC cell lines, grow mainly in floating cell clusters and a minor part as adherent cells. We compared these morphologically different subpopulations of SCLC cell lines for EMT and epigenetic features, detecting significant differences in the adherent subpopulations with high levels of mesenchymal markers such as Vimentin and Fibronectin and very low levels of epithelial markers like E-cadherin and Zona Occludens 1. In addition, expression of EMT-related transcription factors such as Snail/Snai1, Slug/Snai2, and Zeb1, DNA methylation patterns of the EMT hallmark genes, functional responses like migration, invasion, matrix metalloproteases secretion, and resistance to chemotherapeutic drug treatment all differed significantly between the sublines. This phenotypic variability might reflect tumor cell heterogeneity and EMT during metastasis in vivo, accompanied by the development of refractory disease in relapse. We propose that epigenetic regulation plays a key role during phenotypical and functional changes in tumor cells and might therefore provide new treatment options for SCLC patients.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

Circulating tumor cells (CTCs) are accessible by liquid biopsies via an easy blood draw. They represent not only the primary tumor site, but also potential metastatic lesions, and could thus be an attractive supplement for cancer diagnostics. However, the analysis of rare CTCs in billions of normal blood cells is still technically challenging and novel specific CTC markers are needed. The formation of metastasis is a complex process supported by numerous molecular alterations, and thus novel CTC markers might be found by focusing on this process. One example of this is specific changes in the cancer cell glycocalyx, which is a network on the cell surface composed of carbohydrate structures. Proteoglycans are important glycocalyx components and consist of a protein core and covalently attached long glycosaminoglycan chains. A few CTC assays have already utilized proteoglycans for both enrichment and analysis of CTCs. Nonetheless, the biological function of proteoglycans on clinical CTCs has not been studied in detail so far. Therefore, the present review describes proteoglycan functions during the metastatic cascade to highlight their importance to CTCs. We also outline current approaches for CTC assays based on targeting proteoglycans by their protein cores or their glycosaminoglycan chains. Lastly, we briefly discuss important technical aspects, which should be considered for studying proteoglycans.

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).

Esophageal cancers are a challenging upper gastrointestinal tract tumor entity for interdisciplinary oncology. For the two main histotypes, namely esophageal squamous cell carcinomas and Barrett's adenocarcinomas, several genetic aberrations have been shown to contribute to carcinogenesis and progression as well as to represent potential novel targets for therapeutic intervention. This is paralleled by growing insight into epigenetic alterations of esophageal cancers. Studies involving the analyses of human tissue specimens predominantly describe altered patterns of miRNA expression, DNA methylation patterns, and histone marks levels. This review provides a critical update on this increasing knowledge of epigenetic alteration in esophageal cancers by specifically focusing on the translational aspects of epigenetic analyses from human tissue specimens.

In cancer biology, the architectural concept "form follows function" is reflected by cell morphology, migration, and epithelial-mesenchymal transition protein pattern. In vivo, features of epithelial-mesenchymal transition have been associated with tumor budding, which correlates significantly with patient outcome. Hereby, the majority of tumor buds are not truly detached but still connected to a major tumor mass. For detailed insights into the different tumor bud types and the process of tumor budding, we quantified tumor cells according to histomorphological and immunohistological epithelial-mesenchymal transition characteristics. Three-dimensional reconstruction from adenocarcinomas (pancreatic, colorectal, lung, and ductal breast cancers) was performed as published. Tumor cell morphology and epithelial-mesenchymal transition characteristics (represented by zinc finger E-box-binding homeobox 1 and E-Cadherin) were analyzed qualitatively and quantitatively in a three-dimensional context. Tumor buds were classified into main tumor mass, connected tumor bud, and isolated tumor bud. Cell morphology and epithelial-mesenchymal transition marker expression were assessed for each tumor cell. Epithelial-mesenchymal transition characteristics between isolated tumor bud and connected tumor bud demonstrated no significant differences or trends. Tumor cell count correlated significantly with epithelial-mesenchymal transition and histomorphological characteristics. Regression curve analysis revealed initially a loss of membranous E-Cadherin, followed by expression of cytoplasmic E-Cadherin and subsequent expression of nuclear zinc finger E-box-binding homeobox 1. Morphologic changes followed later in this sequence. Our data demonstrate that connected and isolated tumor buds are equal concerning immunohistochemical epithelial-mesenchymal transition characteristics and histomorphology. Our data also give an insight in the process of tumor budding. While there is a notion that the epithelial-mesenchymal transition zinc finger E-box-binding homeobox 1-E-Cadherin cascade is initiated by zinc finger E-box-binding homeobox 1, our results are contrary and outline other possible pathways influencing the regulation of E-Cadherin.

Supporting Information available: This material is available free of charge via the internet. It includes further enzyme inhibition data (MS), enzyme kinetic analyses, detailed EPR spectroscopic analyses, NMR binding experiments of control compounds, docking structures, as well as further analyses of the cellular effects of deferasirox (cell proliferation, western blots, immunostaining). Additional experimental methods are presented. AUTHOR INFORMATION