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Supplementary Figures 1-4 from Long-term Epigenetic Therapy with Oral Zebularine Has Minimal Side Effects and Prevents Intestinal Tumors in Mice

Recent successes in the application of epigenetic drugs for the treatment of myelodysplastic syndrome have raised questions on the safety of long-term administration of DNA methylation inhibitors. We treated preweaned cancer prone ApcMin/+ (Min) mice continuously with the DNA methylation inhibitor zebularine in their drinking water to determine the effects of the drug on normal mouse development as well as cancer prevention. Zebularine caused a tissue-specific reduction in DNA methylation at B1 short interspersed nucleotide elements in the small and large intestines of female Min mice but not in other organs examined after chronic oral treatment. No significant difference in the average weights of mice was observed during the treatment. In addition, analysis of global gene expression of colonic epithelial cells from the females indicated that only 3% to 6% of the genes were affected in their expression. We did not detect toxicity and abnormalities from the histopathologic analysis of liver and intestinal tissues. Lastly, we tested whether prevention of tumorigenesis can be achieved with chronic oral administration of zebularine in Min mice. The average number of polyps in Min females decreased from 58 to 1, whereas the average polyp number remained unaffected in Min males possibly due to differential activity of aldehyde oxidase. Taken together, our results show for the first time that long-term oral administration of zebularine causes a gender-specific abrogation of intestinal tumors while causing a tissue-specific DNA demethylation. Importantly, prolonged treatment of mice with epigenetic drugs resulted in only minor developmental and histologic changes.

Perspective from Long-term Epigenetic Therapy with Oral Zebularine Has Minimal Side Effects and Prevents Intestinal Tumors in Mice

Supplementary Table 1 from Inhibition of Histone Deacetylation Does Not Block Resilencing of p16 after 5-Aza-2′-Deoxycytidine Treatment

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR only–treated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy. [Cancer Res 2007;67(1):346–53]

The identification of molecular determinants of prostate cancer development and progression represents a major clinical and biological undertaking. We integrated transcriptomic data of primary localized and advanced prostate cancer from two cancer databases. Transcriptomic analysis showed upregulation of genes encoding for cell surface proteins in metastatic tumors, which were associated with cell-matrix components and chemokine signaling. Integration of machine learning and weighted correlation network modules identified the EZH2-TROAP axis as the main trajectory from initial tumor development to lethal metastatic disease. In addition to known biomarkers, we identified several targets includingHROBandGEN1that are implicated in DNA damage repair pathways or prognostic markers includingSBK1,DLX2andE2F2, whose biological role has not yet been elucidated for prostate cancer. Our results demonstrate the usability of complex bioinformatics approaches to identify biological drivers of prostate cancer progression and their suitability for clinical applications as prognostic biomarkers.

Prostate cancer (PCa) lethality is driven by its progression to a metastatic castration-resistant state, yet the signaling mechanisms underlying metastatic spread remain unknown. Here we show that STAT3 converges with the LKB1/mTORC1 and CREB to control metastatic disease in PCa mouse models. Unexpectedly, STAT3 was found to be upregulated in diabetic PCa patients undergoing metformin therapy with a concomitant reduction in mTORC1 expression. In preclinical mouse models of PCa, genetic ablation or activation of STAT3 had opposing effects on LKB1/AMPK/mTORC1- dependent tumorigenesis. Using genetic and pharmacological approaches, we identified LKB1 as a direct STAT3 target while repressing CREB. Furthermore, PCa patients with high CREB expression had inferior clinical outcome with significantly increased risk of disease and metastatic recurrence. We observe that castration state lowers STAT3 abundance and increases AR and CREB levels, leading to castration-resistant PCa (CRPC). Our findings revealed that STAT3 controls mTORC1 and CREB in metastatic disease, suggesting CREB as a promising target for lethal CRPC.

Although extensive research on brown adipose tissue (BAT) has stimulated optimism in the battle against obesity and diabetes, BAT physiology and organ crosstalk are not fully understood. Besides BAT, melanin‐concentrating hormone (MCH) and its receptor (MCHR1) play an important role in energy homeostasis. Because of the link between hypothalamic MCH neurons and sympathetic BAT activation via β‐adrenoceptors, we investigated the expression and physiological role of the MCHR1 in BAT. MCHR1 was detected in rodent and human BAT with RT‐qPCR and western blot analyses. In vivo imaging in rats used the glucose analog [18F]FDG and the MCHR1‐tracer [11C]SNAP‐7941. We found that the β3‐adrenoceptor (ADRB3) agonist CL316,243 increased [11C]SNAP‐7941 uptake in BAT. Additionally, a pharmacological concentration of SNAP‐7941—a low‐affinity ADRB3 ligand—stimulated [18F]FDG uptake, reflecting BAT activation. In cultured human adipocytes, CL316,243 induced MCHR1 expression, further supporting a direct interaction between MCHR1 and ADRB3. These findings characterized MCHR1 expression in rodent and human BAT for the first time, including in vitro and in vivo data demonstrating a link between MCHR1 and the β3‐adrenergic system. The presence of MCHR1 in BAT emphasizes the role of BAT in energy homeostasis and may help uncover treatment approaches for obesity., Our findings here describe the expression of melanin‐concentrating hormone receptor 1 (MCHR1) in rodent and human brown adipose tissue (BAT). In vitro and in vivo data demonstrate a link between MCHR1 and the β3‐adrenergic system. The presence of MCHR1 in BAT emphasizes its role in energy homeostasis and may open new possibilities for treatment of obesity.

Anaplastic large cell lymphoma (ALCL) is a mature T cell neoplasm that often expresses the CD4(+) T cell surface marker. It usually harbors the t(2;5) (p23;q35) translocation, leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. We demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their immortalization and malignant transformation. The tumor cells displayed morphological and immunophenotypical characteristics of primary patient–derived anaplastic large cell lymphomas. Cell growth, proliferation, and survival were strictly dependent on NPM-ALK activity and include activation of the key factors STAT3 and DNMT1 and expression of CD30 (the hallmark of anaplastic large-cell lymphoma). Implantation of NPM-ALK–transformed CD4(+) T lymphocytes into immunodeficient mice resulted in the formation of tumors indistinguishable from patients’ anaplastic large cell lymphomas. Integration of “Omic” data revealed that NPM-ALK–transformed CD4(+) T lymphocytes and primary NPM-ALK(+) ALCL biopsies share similarities with early T cell precursors. Of note, these NPM-ALK(+) lymphoma cells overexpress stem cell regulators (OCT4, SOX2, and NANOG) and HIF2A, which is known to affect hematopoietic precursor differentiation and NPM-ALK(+) cell growth. Altogether, for the first time our findings suggest that NPM-ALK could restore progenitor-like features in mature CD30(+) peripheral CD4(+) T cells, in keeping with a thymic progenitor-like pattern.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3′‐triiodo‐l‐thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ‐Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage‐specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3‐ and androgen‐mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth., What's new? Thyroid hormones may play a role in the progression of prostate cancer (PCa). In this study, the authors found that PCa cells had decreased expression of the thyroid‐hormone‐binding protein μ‐Crystallin (CRYM). Lower CRYM was also associated with poor prognosis in men with PCa. In addition, CRYM inhibited thyroid‐hormone and androgen signaling, as well as 18F‐fluoromethylcholine uptake by PCa cells. These results suggest that CRYM may act as a novel antagonist against factors that fuel PCa progression, and therefore thyroid signalling may offer a new therapeutic target in slowing aggressive prostate cancer.

Patient-derived organoid (PDO) cancer models are generated from epithelial tumor cells. Although they reflect the molecular tumor characteristics, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we present a colorectal cancer (CRC) organoid model that incorporates epithelial cells and stromal fibroblasts from the same patient. Molecular characterization of primary cancer associated fibroblasts (CAFs) and matched normal fibroblasts (NF) revealed proteomic, secretome and gene expression differences in pathways associated with tumor related fibroblast function. Further, CAFs retained higher motility compared to NFs in vitro. Importantly, both CAFs and NFs supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. PDOs grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared to mono-cultures, and closely resembled the in vivo tumor morphology. This was also confirmed by the calculation of cellular proportions of epithelial cell subtypes in organoid mono-versus co-cultures, which were inferred through bioinformatics deconvolution of bulk RNA sequencing data using published single cell RNA sequencing datasets from CRC tissues. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by majorly deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. For the fibroblasts, we observed enhanced expression of tumor induced marker genes and cytokines characteristic for myo- and immunogenic fibroblasts. This model will be vital as a physiological personalized tumor model to study disease mechanisms and therapy response in CRC.One Sentence SummaryPatient matched fibroblasts support tumor organoid growth in 3D co-culture and maintain intratumoral cellular heterogeneity and histo-morphology.

Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide. Here, we established human patient-derived colorectal cancer organoid cultures (PDOs) to decipher the patient-specific DNA methylation profile and drug sensitivity towards 5-aza-2’-deoxycytidine, a DNA methyltransferase inhibitor. Organoid lines (n=15) were generated from adjacent normal mucosa and tumor tissue located in different anatomical sites of the colon and rectum. The derived PDOs were characterized on a histopathological level and reproduced the grade and differentiation capacity of their parental tumors. Additional genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. However, the stability of the DNA methylation landscape of human cancer organoids remains largely unknown. In genome-wide methylation analysis of long-term organoid cultures (up to 6 months in culture), we observed surprisingly stable methylation signatures that recapitulates the patient’s profile whilst demonstrating the vast intertumoral heterogeneity among patients. Strikingly, we identified a tumor-specific methylation signature that consisted of 39 CpG sites, which were unmethylated in normal epithelial cells but highly (91-96%) methylated in all tumor cells analyzed. This signature allows for the estimation of the percentage of tumor content in resected tissues and might represent potent biomarkers for early CRC diagnostics. Notably, it has been demonstrated that PDOs have the potential to predict clinical outcome and response to chemo- and radiation therapy in patients. Herein, drug screening with 5-aza-2’-deoxycytidine revealed heterogeneous responses and a clustering into drug sensitive, median and resistant organoid lines. ATAC- and RNA-seq analyses identified pathways rendering drug sensitivity and resistance. Our findings highlight the utility of PDOs as an advanced model system to study the role of the epigenome, especially DNA methylation, and its impact on tumor burden and vulnerability towards epigenetic modifiers. Citation Format: Loan Tran, Raheleh Sheibani Tezerji, Carlos Uziel Perez Malla, Anna Malzer, Ajna Logo, Katarina Misura, Theresia Mair, Thomas Dillinger, Madeleine Kuroll, Velina Atanasova, Julijan Kabiljo, Helga Schachner, Katharina Wöran, Judith Stift, Helmut Dolznig, Walter Pulverer, Michael Bergmann, Gerda Egger. Epigenetic vulnerabilities in patient-derived colorectal cancer organoids. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A011.

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis‐specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1‐deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy., Deletion of maintenance methyltransferase DNMT1 in keratinocytes causes formation of micronuclei, whose rupture and cytosolic DNA release activates inflammatory signalling via cGAS/STING.

Background: Bacillus Calmette-Gu&eacute, rin (BCG) immunotherapy, the standard adjuvant intravesical therapy for some intermediate and most high-risk non-muscle invasive bladder cancers (NMIBCs), suffers from a heterogenous response rate. Molecular markers to help guide responses are scarce and currently not used in the clinical setting. Methods: To identify novel biomarkers and pathways involved in response to BCG immunotherapy, we performed a genome-wide DNA methylation analysis of NMIBCs before BCG therapy. Genome-wide DNA methylation profiles of DNA isolated from tumors of 26 BCG responders and 27 failures were obtained using the Infinium MethylationEPIC BeadChip. Results: Distinct DNA methylation patterns were found by genome-wide analysis in the two groups. Differentially methylated CpG sites were predominantly located in gene promoters and gene bodies associated with bacterial invasion of epithelial cells, chemokine signaling, endocytosis, and focal adhesion. In total, 40 genomic regions with a significant difference in methylation between responders and failures were detected. The differential methylation state of six of these regions, localized in the promoters of the genes GPR158, KLF8, C12orf42, WDR44, FLT1, and CHST11, were internally validated by bisulfite-sequencing. GPR158 promoter hypermethylation was the best predictor of BCG failure with an AUC of 0.809 (p-value &lt, 0.001). Conclusions: Tumors from BCG responders and BCG failures harbor distinct DNA methylation profiles. Differentially methylated DNA regions were detected in genes related to pathways involved in bacterial invasion of cells or focal adhesion. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts.

NPM-ALK–driven lymphoma in mice is dependent on MYC signaling and epigenomic reprogramming via DNA methyltransferases, resulting in heterogenous DNA methylation signatures reminiscent of human lymphoma., Malignant transformation depends on genetic and epigenetic events that result in a burst of deregulated gene expression and chromatin changes. To dissect the sequence of events in this process, we used a T-cell–specific lymphoma model based on the human oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) translocation. We find that transformation of T cells shifts thymic cell populations to an undifferentiated immunophenotype, which occurs only after a period of latency, accompanied by induction of the MYC-NOTCH1 axis and deregulation of key epigenetic enzymes. We discover aberrant DNA methylation patterns, overlapping with regulatory regions, plus a high degree of epigenetic heterogeneity between individual tumors. In addition, ALK-positive tumors show a loss of associated methylation patterns of neighboring CpG sites. Notably, deletion of the maintenance DNA methyltransferase DNMT1 completely abrogates lymphomagenesis in this model, despite oncogenic signaling through NPM-ALK, suggesting that faithful maintenance of tumor-specific methylation through DNMT1 is essential for sustained proliferation and tumorigenesis.

BackgroundThe monoclonal antibody (mAb) trastuzumab is part of the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer. Antibody-dependent cell-mediated phagocytosis (ADCP) and cytotoxicity (ADCC) are major mechanisms of action of the mAb trastuzumab. Histone deacetylase inhibitors (HDACi), such as valproic acid (VPA) or vorinostat (SAHA), exert several immunostimulatory properties, which contribute at least in part to their anticancer effect. However, the impact of HDACi-induced immunostimulatory effects on trastuzumab-mediated anti-tumor immune response is not well characterized.MethodsWe analyzed the ADCP and ADCC activity of peripheral blood mononuclear cells (PBMCs) from age and gender-matched healthy volunteers (n=5) against HDACi-treated HER2-overexpressing breast cancer cells (SKBR3), using a well-established in vitro three-color imaging flow cytometry and flow cytometry approach.ResultsVPA and SAHA enhanced trastuzumab-mediated ADCP and trastuzumab-independent cytotoxicity. Mechanistically, VPA upregulated the activating antibody-binding receptor Fc-gamma receptor (FcγR) IIA (CD32A) on monocytes (CD14+). Moreover, VPA and SAHA downregulated the anti-apoptotic protein myeloid leukemia cell differentiation 1 (MCL1) in breast cancer cells. Additionally, VPA and SAHA induced an immunogenic cell death, characterized by the exposure of calreticulin (CALR), as well as decreased the “do not eat me” signal CD47 on tumor cells.ConclusionsHDACi VPA and SAHA increase trastuzumab-mediated phagocytosis and trastuzumab-independent cytotoxicity. The immunomodulatory activities of those HDACi support a rationale combined treatment approach with mAb for cancer treatment.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co‐expression network of transcriptomics data in addition to laser‐microdissected proteomics from human and murine prostate FFPE samples. We show up‐regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up‐regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis., Transcriptomic and proteomic analyses in prostate cancer (PCa) reveal high TCA/OXPHOS activity and low PDK4 expression in low STAT3 tumors. PDK4 is a promising independent predictor of biochemical recurrence in PCa.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3’-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRa/TRb) in target tissues. We show in this study that low CRYM expression levels in PCa patient samples are associated with early BCR and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in PET/MRI imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3 and androgen-mediated signalling. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.HighlightsThyroid and androgen hormone driven pathways in prostate cancer (PCa) are antagonized by μ- Crystallin (CRYM).[18F]fluoromethylcholine uptake and prognostic values in PCa correlate with CRYM protein levels.Reduced CRYM expression predicts early biochemical recurrence (BCR) in PCa patients.

// Ursula J. Lemberger 1, 2 , Claudia D. Fuchs 2 , Christian Schofer 3 , Andrea Bileck 4 , Christopher Gerner 4 , Tatjana Stojakovic 5 , Makoto M. Taketo 6 , Michael Trauner 2 , Gerda Egger 1, 7 and Christoph H. Osterreicher 8 1 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 2 Hans Popper Laboratory for Molecular Hepatology, Department of Internal Medicine, Medical University of Vienna, Vienna, Austria 3 Department of Cell and Developmental Biology, Medical University of Vienna, Vienna, Austria 4 Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria 5 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria 6 Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan 7 Ludwig Boltzmann Institute Applied Diagnostics, Vienna, Austria 8 Institute of Pharmacology, Medical University Vienna, Vienna, Austria Correspondence to: Ursula J. Lemberger, email: ursula.lemberger@meduniwien.ac.at , ursula.lemberger@gmx.at Keywords: Wnt signaling pathway; liver cancer; lipid metabolism; glucose metabolism; mitochondrial disorder Received: August 31, 2017 Accepted: January 25, 2018 Published: January 30, 2018 ABSTRACT Background: Wnt/β-catenin signaling plays a crucial role in embryogenesis, tissue homeostasis, metabolism and malignant transformation of different organs including the liver. Continuous β-catenin signaling due to somatic mutations in exon 3 of the Ctnnb1 gene is associated with different liver diseases including cancer and cholestasis. Results: Expression of a degradation resistant form of β-catenin in hepatocytes resulted in 100% mortality within 31 days after birth. Ctnnb1 CA hep mice were characterized by reduced body weight, significantly enlarged livers with hepatocellular fat accumulation around central veins and increased hepatic triglyceride content. Proteomics analysis using whole liver tissue revealed significant deregulation of proteins involved in fat, glucose and mitochondrial energy metabolism, which was also reflected in morphological anomalies of hepatocellular mitochondria. Key enzymes involved in transport and synthesis of fatty acids and cholesterol were significantly deregulated in livers of Ctnnb1 CA hep mice. Furthermore, carbohydrate metabolism was substantially disturbed in mutant mice. Conclusion: Continuous β-catenin signaling in hepatocytes results in premature death due to severe disturbances of liver associated metabolic pathways and mitochondrial dysfunction. Methods: To investigate the influence of permanent β-catenin signaling on liver biology we analyzed mice with hepatocyte specific expression of a dominant stable form of β-catenin ( Ctnnb1 CA hep ) and their WT littermates by serum biochemistry, histology, electron microscopy, mRNA profiling and proteomic analysis of the liver.

// Ursula J. Lemberger 1, 2, 3 , Claudia D. Fuchs 3 , Matthias Karer 1 , Stefanie Haas 1 , Tatjana Stojakovic 4 , Christian Schofer 5 , Hanns-Ulrich Marschall 6 , Fritz Wrba 2 , Makoto M. Taketo 7 , Gerda Egger 2 , Michael Trauner 3 , Christoph H. Osterreicher 1 1 Institute of Pharmacology, Medical University of Vienna, Vienna, Austria 2 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 3 Hans Popper Laboratory for Molecular Hepatology, Department of Internal Medicine, Medical University of Vienna, Vienna, Austria 4 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria 5 Department of Cell and Developmental Biology, Medical University of Vienna, Vienna, Austria 6 Department of Molecular and Clinical Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 7 Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan Correspondence to: Christoph H. Osterreicher, email: coesterr@gmail.com Keywords: β-catenin, bile acids, cholestasis, biliary fibrosis, liver cancer Received: April 03, 2016 Accepted: September 26, 2016 Published: November 23, 2016 ABSTRACT Background: The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous β-catenin signaling in hepatocytes remain elusive. Results: Livers of Ctnnb1 CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1 CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1 CA hep mice. Expression of compensatory bile acid transporters including Abcb1 , Abcb4 , Abcc2 and Abcc4 were significantly increased in Ctnnb1 CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1 CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated β-catenin. Materials and Methods: Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of β-catenin ( Ctnnb1 CA hep mice). Ctnnb1 CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. Conclusions: Expression of a dominant stable form of β-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis.

Prostate cancer (PCa) has a broad spectrum of clinical behaviour, hence biomarkers are urgently needed for risk stratification. We previously described the protective effect of STAT3 in a prostate cancer mouse model. By utilizing a gene co-expression network in addition to laser microdissected proteomics from human and murine prostate FFPE samples, we describe STAT3-induced downregulation of the TCA cycle/OXPHOS in PCa on transcriptomic and proteomic level. We identify pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging and PSA level. Furthermore, PDK4 expression is causally linked to type 2 diabetes mellitus, which is known to have a protective effect on PCa. We conclude that this effect is related to PDK4 expression and that PDK4 loss could serve as a biomarker for PCa with dismal prognosis.

Nuclear factor ‘κ‐light‐chain‐enhancer’ of activated B cells (NF‐κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR‐derived signals, thereby maintaining their suppressive features. However, the role of NF‐κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF‐κB proteins p50, p65, and c‐Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF‐κB signaling in FOXP3‐transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF‐κB signaling. Additionally, repression of residual NF‐κB signaling in Treg further enhances interleukin‐10 (IL‐10) production. Blockade of NF‐κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF‐κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg‐specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF‐κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy., Human regulatory T cells (Treg) differentially integrate extracellular signals (e.g., mTOR, MAP‐kinase, and JAK/STAT signaling) compared to effector T cells. In this work, Ziegler et al. for the first time show that Treg also attenuate TCR‐triggered canonical NF‐κB signaling. This unique feature can also be exploited for the generation of inducible Treg. Combination of mTOR and NF‐κB blockade leads to the induction of Treg with increased suppressive capacity and improved functional stability.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) β-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM., Author summary In tissues, cellular differentiation is normally associated with cell cycle arrest. However, in some cases differentiating cells continue their cyclic activity in the absence of cell division. This event, referred to as endoreduplication, leads to polyploidy, which means an increased number of chromosome sets per cell and has been demonstrated in rodent placental trophoblast giant cells (TGCs). However, it is unknown whether human placental trophoblasts also endoreduplicate their genome. To study this, we focused on extravillous trophoblasts (EVTs), a specific trophoblastic subtype that invades the uterus during pregnancy in order to control blood supply and nutrient transfer to the growing embryo. We show that initiation of EVT differentiation is characterized by induced endoreduplication leading to genomic tetraploidization. Different to TGCs, EVTs duplicate their genome in an even manner. Upon invasion into the uterus, EVTs stop their endoreduplicative cycle and undergo cellular senescence. We further show that EVTs of hyperplastic complete hydatidiform mole (CHM) placentas, a genetic pregnancy disorder, are characterized by exacerbated endoreduplication, a greater DNA content and reduced signs for senescence. In summary, we propose senescence as a ploidy limiting factor during placental development and describe its suppression in hyperploid CHM-EVTs.

Metastases account for most cancer-related deaths, yet the mechanisms underlying metastatic spread remain poorly understood. Recent evidence demonstrates that senescent cells, while initially restricting tumorigenesis, can induce tumor progression. Here, we identify the metalloproteinase inhibitor TIMP1 as a molecular switch that determines the effects of senescence in prostate cancer. Senescence driven either by PTEN deficiency or chemotherapy limits the progression of prostate cancer in mice. TIMP1 deletion allows senescence to promote metastasis, and elimination of senescent cells with a senolytic BCL-2 inhibitor impairs metastasis. Mechanistically, TIMP1 loss reprograms the senescence-associated secretory phenotype (SASP) of senescent tumor cells through activation of matrix metalloproteinases (MMPs). Loss of PTEN and TIMP1 in prostate cancer is frequent and correlates with resistance to docetaxel and worst clinical outcomes in patients treated in an adjuvant setting. Altogether, these findings provide insights into the dual roles of tumor-associated senescence and can potentially impact the treatment of prostate cancer.

Introduction Through conserved signalling pathways, Anaplastic Lymphoma Kinase (ALK) is well-described in driving haematological malignancies including Anaplastic Large Cell Lymphoma (ALCL) and Diffuse Large B-Cell Lymphoma (DLBCL) and as such presents itself as an amenable therapeutic target. Hence, directed therapeutics (ALK tyrosine kinase inhibitors; TKI) are being used in the treatment of ALK-driven cancers. Unfortunately, findings in the clinic and clinical research studies have taught us that resistance to ALK inhibitors can develop through the activation of ALK signalling bypass tracks. As such there is a need for the development of novel front-line, dual-combination, as well as second-line therapies. Methods A large-scale epigenetic targeted drug library consisting of approximately 300 FDA-approved drugs and novel agents was applied to a number of cell lines representing ALK-driven haematological malignancies: ALCL cell lines (DEL, JB-6, KARPAS-299, SU-DHL-1, SUP-M2) and the DLBCL cell line LM-1. Drugs which caused a >75% decrease in cell viability were classified as 'candidate drugs' and studied further. Results Several of the validated drugs have previously been used/trialled in the clinic for the treatment of various cancers, e.g. aurora kinase (XL-228), topoisomerase (Mitoxantrone HCl) and HDAC (Romidepsin) inhibitors - these functioned as internal controls for the drug screens. However, an assortment of novel drugs was also identified that have not previously been described in the context of the treatment of ALK-driven haematological malignancies; including the FLT3 inhibitor KW2449 which caused a >75% decrease in viability in all the tested cell lines and as such may serve as a novel front line therapy. In addition, a novel DNA methyltransferase (DNMT) inhibitor was identified which is efficacious and resulted in a >90% decrease in viability in all cell lines treated across both disease entities. Furthermore, we investigated the combinatorial potential of the identified DNMT inhibitor with ALK TKIs such as Brigatinib and observed the inhibitor acting synergistically (as per Bliss-Independence calculations) resulting in a further decrease in cell viability. Several cell lines that are resistant to ALK TKIs were also assessed for their sensitivity to the DNMT inhibitor and were shown to be susceptible to this drug, as demonstrated by a significant decrease in cell viability. Figure 1: (A) Viability following drug screen in a representative cell line. Those drugs which led to a >75% change in viability were taken forward for validation, as shown in (B). (C) Candidate drugs identified for each of the cell lines tested, grouped according to their molecular target. Conclusion In conclusion, an epigenetic drug library has been employed to identify novel therapeutic agents for the treatment of ALK-driven haematological malignancies including ALCL and DLBCL. Data reveal a potent inhibitor of DNA methylation as a candidate drug that suppresses the growth of ALK-driven malignancies both alone and in combination with ALK TKIs. Significantly, this identified drug also inhibits the growth of cell lines resistant to directed therapeutics such as ALK TKIs suggesting it has potential clinical use. Disclosures No relevant conflicts of interest to declare.

The recurrence rate of non-muscle-invasive bladder cancer (NMIBC) is up to 60% within the first year of therapy. Accurate risk stratification is necessary for patient counselling, follow-up scheduling and individualized therapeutic decision making. Current prognostic models rely on clinicopathologic features, but their discrimination remains limited when in external cohorts. Despite intense efforts regarding the value of biomarkers in prognosticating outcomes in NMIBC, clinical utility remains suboptimal. It is clear that a single biomarker is not enough for the prediction of disease recurrence. Therefore, panels of non-redundant biomarkers have been created and integrated in clinical prognostic model further research relying on high throughput technologies is required. Areas covered: We performed a systematic research of the English-language literature on tissue biomarkers for prediction of NMIBC outcomes up to December 2017. Expert commentary: Despite the essential milestones achieved in our knowledge and understanding of the molecular biology underlying NMIBC, no biomarker has been implemented together with clinical feature in clinical practice. Integration of such biomarkers into predictive and prognostic model could, however, improve our accuracy, thereby paving the way for personalized medicine in the management of NMIBC.

Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. Results: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. Conclusions: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer.