Publications by authors named "Regine Schneider-Stock"

156 Publications

miR-138-5p induces aggressive traits by targeting Trp53 expression in murine melanoma cells, and correlates with poor prognosis of melanoma patients.

Neoplasia 2021 Aug 8;23(8):823-834. Epub 2021 Jul 8.

Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Electronic address:

Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.
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http://dx.doi.org/10.1016/j.neo.2021.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8274245PMC
August 2021

New Oleoyl Hybrids of Natural Antioxidants: Synthesis and In Vitro Evaluation as Inducers of Apoptosis in Colorectal Cancer Cells.

Antioxidants (Basel) 2020 Nov 3;9(11). Epub 2020 Nov 3.

Department of Pharmacy, Health and Nutritional Sciences, Department of Excellence 2018-2022, University of Calabria, Edificio Polifunzionale, 87036 Rende (CS), Italy.

Nowadays, the beneficial role of a healthy lifestyle, particularly emphasizing the quality of foods and cancer management, is accepted worldwide. Polyphenols and oleic acid play a key role in this context, but are still scarcely used as anti-cancer agents due to their bio-accessibility limits. Therefore, we aimed to synthesize a set of new oleoyl-hybrids of quercetin, morin, pinocembrin, and catechin to overcome the low bioavailability of polyphenols, throughout a bio-catalytic approach using pancreatic porcine lipase as a catalyst. The in vitro assays, using a wide panel of human cancer cell lines showed, mainly for two novel regioisomer oleoyl-hybrids of quercetin, a remarkable increase in apoptotic cell populations. We suggested that the DNA damage shown as ɣH2AX signals might be the major cause of apoptotic cell death. Finally, we demonstrated convincing data about two novel polyphenol-based hybrids displaying a highly selective anti-cancer cytotoxicity and being superior compared to their reference/parental compounds.
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http://dx.doi.org/10.3390/antiox9111077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692320PMC
November 2020

The CAM Assay as an Alternative In Vivo Model for Drug Testing.

Handb Exp Pharmacol 2021 ;265:303-323

Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy.

In the last decade, the chicken chorioallantoic membrane (CAM) assay has been re-discovered in cancer research to study the molecular mechanisms of anti-cancer drug effects. Literature about the CAM assay as an alternative in vivo cancer xenograft model according to the 3R principles has exploded in the last 3 years. Following a summary of the basic knowledge about the chicken embryo, we compare advantages and disadvantages with the classical mouse xenograft model, exemplify established and innovative imaging techniques that are used in the CAM model, and give examples of its successful utilization for studying major hallmarks of cancer such as angiogenesis, proliferation, invasion, and metastasis.
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http://dx.doi.org/10.1007/164_2020_375DOI Listing
March 2021

EMT transcription factor ZEB1 alters the epigenetic landscape of colorectal cancer cells.

Cell Death Dis 2020 02 24;11(2):147. Epub 2020 Feb 24.

Institute of Pathology, Universitätsklinikum Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Bavaria, Germany.

Epigenetic deregulation remarkably triggers mechanisms associated with tumor aggressiveness like epithelial-mesenchymal transition (EMT). Since EMT is a highly complex, but also reversible event, epigenetic processes such as DNA methylation or chromatin alterations must be involved in its regulation. It was recently described that loss of the cell cycle regulator p21 was associated with a gain in EMT characteristics and an upregulation of the master EMT transcription factor ZEB1. In this study, in silico analysis was performed in combination with different in vitro and in vivo techniques to identify and verify novel epigenetic targets of ZEB1, and to proof the direct transcriptional regulation of SETD1B by ZEB1. The chorioallantoic-membrane assay served as an in vivo model to analyze the ZEB1/SETD1B interaction. Bioinformatical analysis of CRC patient data was used to examine the ZEB1/SETD1B network under clinical conditions and the ZEB1/SETD1B network was modeled under physiological and pathological conditions. Thus, we identified a self-reinforcing loop for ZEB1 expression and found that the SETD1B associated active chromatin mark H3K4me3 was enriched at the ZEB1 promoter in EMT cells. Moreover, clinical evaluation of CRC patient data showed that the simultaneous high expression of ZEB1 and SETD1B was correlated with the worst prognosis. Here we report that the expression of chromatin modifiers is remarkably dysregulated in EMT cells. SETD1B was identified as a new ZEB1 target in vitro and in vivo. Our study demonstrates a novel example of an activator role of ZEB1 for the epigenetic landscape in colorectal tumor cells.
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http://dx.doi.org/10.1038/s41419-020-2340-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040187PMC
February 2020

The activating transcription factor 2: an influencer of cancer progression.

Mutagenesis 2019 12;34(5-6):375-389

Experimental Tumorpathology, Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany.

In contrast to the continuous increase in survival rates for many cancer entities, colorectal cancer (CRC) and pancreatic cancer are predicted to be ranked among the top 3 cancer-related deaths in the European Union by 2025. Especially, fighting metastasis still constitutes an obstacle to be overcome in CRC and pancreatic cancer. As described by Fearon and Vogelstein, the development of CRC is based on sequential mutations leading to the activation of proto-oncogenes and the inactivation of tumour suppressor genes. In pancreatic cancer, genetic alterations also attribute to tumour development and progression. Recent findings have identified new potentially important transcription factors in CRC, among those the activating transcription factor 2 (ATF2). ATF2 is a basic leucine zipper protein and is involved in physiological and developmental processes, as well as in tumorigenesis. The mutation burden of ATF2 in CRC and pancreatic cancer is rather negligible; however, previous studies in other tumours indicated that ATF2 expression level and subcellular localisation impact tumour progression and patient prognosis. In a tissue- and stimulus-dependent manner, ATF2 is activated by upstream kinases, dimerises and induces target gene expression. Dependent on its dimerisation partner, ATF2 homodimers or heterodimers bind to cAMP-response elements or activator protein 1 consensus motifs. Pioneering work has been performed in melanoma in which the dual role of ATF2 is best understood. Even though there is increasing interest in ATF2 recently, only little is known about its involvement in CRC and pancreatic cancer. In this review, we summarise the current understanding of the underestimated 'cancer gene chameleon' ATF2 in apoptosis, epithelial-to-mesenchymal transition and microRNA regulation and highlight its functions in CRC and pancreatic cancer. We further provide a novel ATF2 3D structure with key phosphorylation sites and an updated overview of all so-far available mouse models to study ATF2 in vivo.
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http://dx.doi.org/10.1093/mutage/gez041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923166PMC
December 2019

DAPK1 loss triggers tumor invasion in colorectal tumor cells.

Cell Death Dis 2019 11 26;10(12):895. Epub 2019 Nov 26.

Experimental Tumor Pathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Universitaetsstrasse 22, 91054, Erlangen, Germany.

Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.
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http://dx.doi.org/10.1038/s41419-019-2122-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879526PMC
November 2019

Epigenetic Regulation of p21 in Human Cancer.

Cancers (Basel) 2019 Sep 11;11(9). Epub 2019 Sep 11.

Experimental Tumor Pathology, Institute of Pathology, University Hospital, Friedrich-Alexander University Erlangen-Nuremberg, 91054 Erlangen, Germany.

p21 is a central regulator of cell cycle control and survival. While mutations are rare, it is commonly dysregulated in several human cancers due to epigenetic mechanisms influencing its transcriptional control. These mechanisms include promoter hypermethylation as well as additional pathways such as histone acetylation or methylation. The epigenetic regulators include writers, such as DNA methyltransferases (DNMTs); histone acetyltransferases (HATs) and histone lysine methyltransferases; erasers, such as histone deacetylases (HDACs); histone lysine demethylases [e.g., the Lysine Demethylase (KDM) family]; DNA hydroxylases; readers, such as the methyl-CpG-binding proteins (MBPs); and bromodomain-containing proteins, including the bromo- and extraterminal domain (BET) family. We further discuss the roles that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play in the epigenetic control of p21 expression and its function in human cancers.
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http://dx.doi.org/10.3390/cancers11091343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769618PMC
September 2019

Loss of enhancer of zeste homologue 2 (EZH2) at tumor invasion front is correlated with higher aggressiveness in colorectal cancer cells.

J Cancer Res Clin Oncol 2019 Sep 17;145(9):2227-2240. Epub 2019 Jul 17.

Experimental Tumorpathology, Institute of Pathology, Friedrich-Alexander-University of Erlangen-Nürnberg, Universitätsstr. 22, 91054, Erlangen, Germany.

Purpose: Enhancer of zeste homolog 2 (EZH2) is associated with epigenetic gene silencing and aggressiveness in many tumor types. However, the prognostic impact of high EZH2 expression is controversially discussed for colorectal cancer. For this reason, we immunohistochemically analyzed EZH2 expression in 105 specimens from colon cancer patients separately for tumor center and invasion front.

Methods: All sections from tissue microarrays were evaluated manually and digitally using Definiens Tissue Studio software (TSS). To mirror-image the EZH2 status at the tumor invasion front, we treated HCT116 colon cancer cells with the EZH2 inhibitor 3-Deazaneplanocin A (DZNep) and studied the growth of in ovo xenografts in the chorioallantoic membrane (CAM) assay.

Results: We showed a significant decrease in EZH2 expression and the repressive H3K27me3 code at the tumor invasion front as supported by the TSS-constructed heatmaps. Loss of EZH2 at tumor invasion front, but not in tumor center was correlated with unfavorable prognosis and more advanced tumor stages. The observed cell cycle arrest in vitro and in vivo was associated with higher tumor aggressiveness. Xenografts formed by DZNep-treated HCT116 cells showed loosely packed tumor masses, infiltrative growth into the CAM, and high vessel density.

Conclusion: The differences in EZH2 expression between tumor center and invasion front as well as different scoring and cutoff values can most likely explain controversial literature data concerning the prognostic value of EZH2. Epigenetic therapies using EZH2 inhibitors have to be carefully evaluated for each specific tumor type, since alterations in cell differentiation might lead to unfavorable results.
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http://dx.doi.org/10.1007/s00432-019-02977-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708512PMC
September 2019

Combination of 5-fluorouracil and thymoquinone targets stem cell gene signature in colorectal cancer cells.

Cell Death Dis 2019 05 16;10(6):379. Epub 2019 May 16.

University Hospital, Friedrich-Alexander-University Erlangen-Nuremberg, Experimental Tumorpathology, Institute of Pathology, Erlangen, Germany.

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here, we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both, Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells, but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly, two principle stem cell regulatory pathways WNT/ß-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore, both treatments strikingly eliminated CD133+ CSC population, accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear ß-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of ß-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU, both, Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together, combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells.
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http://dx.doi.org/10.1038/s41419-019-1611-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522523PMC
May 2019

Gene expression and promoter methylation of angiogenic and lymphangiogenic factors as prognostic markers in melanoma.

Mol Oncol 2019 06 25;13(6):1433-1449. Epub 2019 May 25.

Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.

The high mortality rate of melanoma is broadly associated with its metastatic potential. Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis. Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients. Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay. Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P-value = 0.044) and the lymphangiogenic receptor VEGFR-3 (P-value = 0.002) were independent prognostic factors of overall survival in melanoma patients. Enhanced reduced representation bisulfite sequencing-based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells. In patients, VEGFC (P-value = 0.031), ANGPT2 (P-value < 0.001), and SIX1 (P-value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival. Hence, our data suggest that these angio- and lymphangiogenesis factors are potential biomarkers of melanoma prognosis. Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.
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http://dx.doi.org/10.1002/1878-0261.12501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547615PMC
June 2019

DNA methylation and chromatin modifiers in colorectal cancer.

Mol Aspects Med 2019 10 30;69:73-92. Epub 2019 Apr 30.

Experimental Tumorpathology, Institute of Pathology, University Hospital of Friedrich-Alexander-University Erlangen-Nürnberg, Universitätsstrasse 22, 91054, Erlangen, Germany. Electronic address:

Colorectal carcinogenesis is a multistep process involving the accumulation of genetic alterations over time that ultimately leads to disease progression and metastasis. Binding of transcription factors to gene promoter regions alone cannot explain the complex regulation pattern of gene expression during this process. It is the chromatin structure that allows for a high grade of regulatory flexibility for gene expression. Posttranslational modifications on histone proteins such as acetylation, methylation, or phosphorylation determine the accessibility of transcription factors to DNA. DNA methylation, a chemical modification of DNA that modulates chromatin structure and gene transcription acts in concert with these chromatin conformation alterations. Another epigenetic mechanism regulating gene expression is represented by small non-coding RNAs. Only very recently epigenetic alterations have been included in molecular subtype classification of colorectal cancer (CRC). In this chapter, we will provide examples of the different epigenetic players, focus on their role for epithelial-mesenchymal transition and metastatic processes and discuss their prognostic value in CRC.
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http://dx.doi.org/10.1016/j.mam.2019.04.002DOI Listing
October 2019

Malignancies associated with GIST: a retrospective study with molecular analysis of KIT and PDGFRA.

Langenbecks Arch Surg 2019 Aug 15;404(5):605-613. Epub 2019 Mar 15.

Department of Pathology, FAU Erlangen-Nürnberg, Erlangen, Germany.

Purpose: Gastrointestinal stromal tumors (GISTs) are the most common soft tissue tumors of the GI tract. Studies have been published reporting additional neoplasms in GIST patients. This study aimed to evaluate possible associations of mutation type, morphology, and clinical aspects of GISTs.

Methods: All cases of GIST were identified from our pathology files. Coding exons of KIT and PDGFRA in GISTs with additional malignancies were sequenced.

Results: A total of 70 of 188 (37%) retrieved patients with confirmed diagnosis of GIST showed at least one additional malignant neoplasm. Fifty of these GISTs were located in the stomach (71%), 8 in the small intestine (11%), 5 in the colon/rectum (7%), and 7 cases (6.2%) were of undetermined sites of origin. The distribution of identified mutations was similar to that described in GISTs without secondary malignancies. A total of 37 of 57 cases (65%) showed mutations in the KIT gene exon 11, 3 (5%) cases in exon 9, and 1 (2%) case in exon 13. Nine tumors (16%) had mutations of the PDGFRA gene. KIT and PDGFRA wild-type status were found in seven cases (12%). Most of the secondary neoplasms were located within the GI tract (34%), in the urogenital system (24%), or the breast/female genital tract (20%).

Conclusion: This study confirms the high rate of additional malignant tumors in GIST patients. GIST features in these cases are very similar to those with sole GIST.
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http://dx.doi.org/10.1007/s00423-019-01773-2DOI Listing
August 2019

Carborane-Based Analogues of 5-Lipoxygenase Inhibitors Co-inhibit Heat Shock Protein 90 in HCT116 Cells.

ChemMedChem 2019 01 21;14(2):255-261. Epub 2018 Dec 21.

Faculty of Chemistry and Mineralogy, Institute of Inorganic Chemistry, Universität Leipzig, Johannisallee 29, 04103, Leipzig, Germany.

5-Lipoxygenase converts arachidonic acid into leukotrienes, which are involved in inflammation and angiogenesis. The introduction of carboranes can improve the pharmacokinetic behavior of metabolically less stable pharmaceutics. Herein we report the syntheses of several carborane-based inhibitors of the 5-lipoxygenase pathway. The isosteric replacement of phenyl rings by carboranes leads to improved cytotoxicity toward several melanoma and colon cancer cell lines. For the colon cancer cell line HCT116, the co-inhibition of heat shock protein 90 was observed.
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http://dx.doi.org/10.1002/cmdc.201800651DOI Listing
January 2019

Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2.

Cancers (Basel) 2018 Oct 9;10(10). Epub 2018 Oct 9.

Experimental Tumor Pathology, University Hospital of Friedrich-Alexander University Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany.

The oncogenic cytoplasmic p21 contributes to cancer aggressiveness and chemotherapeutic failure. However, the molecular mechanisms remain obscure. Here, we show for the first time that cytoplasmic p21 mediates 5-Fluorouracil (5FU) resistance by shuttling p-Chk2 out of the nucleus to protect the tumor cells from its pro-apoptotic functions. We observed that cytoplasmic p21 levels were up-regulated in 5FU-resistant colorectal cancer cells in vitro and the in vivo Chorioallantoic membrane (CAM) model. Kinase array analysis revealed that p-Chk2 is a key target of cytoplasmic p21. Importantly, cytoplasmic form of p21 mediated by p21 transfection diminished p-Chk2-mediated activation of E2F1 and apoptosis induction. Co-immunoprecipitation, immunofluorescence, and proximity ligation assay showed that p21 forms a complex with p-Chk2 under 5FU exposure. Using in silico computer modeling, we suggest that the p21/p-Chk2 interaction hindered the nuclear localization signal of p-Chk2, and therefore, the complex is exported out of the nucleus. These findings unravel a novel mechanism regarding an oncogenic role of p21 in regulation of resistance to 5FU-based chemotherapy. We suggest a possible value of cytoplasmic p21 as a prognosis marker and a therapeutic target in colorectal cancer patients.
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http://dx.doi.org/10.3390/cancers10100373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210175PMC
October 2018

Generation and characterization of hepatocellular carcinoma cell lines with enhanced cancer stem cell potential.

J Cell Mol Med 2018 12 2;22(12):6238-6248. Epub 2018 Oct 2.

Experimental Tumor Pathology, Institute of Pathology, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany.

Hepatocellular carcinoma (HCC) is one of the most common causes for cancer-related death worldwide with rapidly increasing incidence and mortality rates. As for other types of cancers, also in HCC cancer stem cells (CSCs) are thought to be responsible for tumour initiation, progression and therapy failure. However, as rare subpopulations of tumour tissue, CSCs are difficult to isolate, thus making the development of suitable and reliable model systems necessary. In our study, we generated HepG2 subclones with enriched CSC potential by application of the spheroid formation method and subsequent single-cell cloning. Analyses in several 2D and 3D cell culture systems as well as a panel of functional assays both in vitro and in vivo revealed that the generated subclones displayed characteristic and sustained features of tumour initiating cells as well as highly aggressive properties related to tumour progression and metastasis. These characteristics could clearly be correlated with the expression of CSC markers that might have prognostic value in the clinical HCC setting. Therefore, we conclude that our CSC enriched HepG2 clones certainly represent suitable model systems to study the role of CSCs during HCC initiation, progression and drug resistance.
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http://dx.doi.org/10.1111/jcmm.13911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237557PMC
December 2018

BLU-285-the breakthrough in treatment of patients with aggressive systemic mastocytosis and gastrointestinal stromal tumor.

Ann Transl Med 2018 Jun;6(11):232

Experimental Tumorpathology, Institute of Pathology, University Hospital of FAU Erlangen-Nürnberg, Erlangen, Germany.

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http://dx.doi.org/10.21037/atm.2018.05.21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036000PMC
June 2018

SIRT1 regulates Mxd1 during malignant melanoma progression.

Oncotarget 2017 Dec 3;8(70):114540-114553. Epub 2017 Oct 3.

Ontogeny and Epigenetics Laboratory, Pharmacology Department, Universidade Federal de São Paulo - UNIFESP, São Paulo, SP, Brazil.

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the expression. Sirt1 stable silencing increased mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. was identified as a new SIRT1 target gene. SIRT1 promoted silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.
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http://dx.doi.org/10.18632/oncotarget.21457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777712PMC
December 2017

Deep hypothermic circulatory arrest or tepid regional cerebral perfusion: impact on haemodynamics and myocardial integrity in a randomized experimental trial.

Interact Cardiovasc Thorac Surg 2018 04;26(4):667-672

Department of Pediatric Cardiac Surgery, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Objectives: Organ protective management during aortic arch surgery comprises deep hypothermic (18°C) circulatory arrest (DHCA), or moderate hypothermia (28°C/ 'tepid') with regional cerebral perfusion (TRCP). The aim of this experimental study was to evaluate the effect of distinct organ protective management on hemodynamic performance and myocardial integrity.

Methods: Ten male piglets were randomized to group DHCA (n = 5) or TRCP (n = 5) group and operated on cardiopulmonary bypass (CPB) with 60 min of aortic cross-clamping. Blood gas analysis was performed throughout the experiment. Haemodynamic assessment was performed using a thermodilution technique before and after CPB. Myocardial biopsies were taken 2 h after CPB and evaluated using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labelling assay and western blot analysis.

Results: At reperfusion, levels of central venous saturation were significantly higher (P = 0.016) and levels of lactate significantly lower (P = 0.029) in the DHCA group. After CPB, thermodilution measurements revealed higher stroke volume and lower peripheral resistance in the TRCP group (P = 0.012 and 0.037). At the end of the experiment, no significant differences regarding laboratory and haemodynamic parameters were evident. All specimens showed enrichment of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labelling-positive cells exclusively at the left ventricular subendocardium with no difference between groups and equal concentrations of cyclo-oxygenase-2.

Conclusions: TRCP is associated with decreased peripheral resistance and higher stroke volume immediately after CPB. However, this beneficial effect is contrasted by signs of lower body hypoperfusion, which is expressed by lower central venous saturations and higher lactate levels. Distinct strategies of organ protection did not seem to affect apoptotic/necrotic and inflammatory changes in the left ventricular myocardium.
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http://dx.doi.org/10.1093/icvts/ivx393DOI Listing
April 2018

Identification of miRNA-mRNA Modules in Colorectal Cancer Using Rough Hypercuboid Based Supervised Clustering.

Sci Rep 2017 02 21;7:42809. Epub 2017 Feb 21.

Laboratory of Systems Tumor Immunology, Department of Dermatology, Erlangen University Hospital and Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Differences in the expression profiles of miRNAs and mRNAs have been reported in colorectal cancer. Nevertheless, information on important miRNA-mRNA regulatory modules in colorectal cancer is still lacking. In this regard, this study presents an application of the RH-SAC algorithm on miRNA and mRNA expression data for identification of potential miRNA-mRNA modules. First, a set of miRNA rules was generated using the RH-SAC algorithm. The mRNA targets of the selected miRNAs were identified using the miRTarBase database. Next, the expression values of target mRNAs were used to generate mRNA rules using the RH-SAC. Then all miRNA-mRNA rules have been integrated for generating networks. The RH-SAC algorithm unlike other existing methods selects a group of co-expressed miRNAs and mRNAs that are also differentially expressed. In total 17 miRNAs and 141 mRNAs were selected. The enrichment analysis of selected mRNAs revealed that our method selected mRNAs that are significantly associated with colorectal cancer. We identified novel miRNA/mRNA interactions in colorectal cancer. Through experiment, we could confirm that one of our discovered miRNAs, hsa-miR-93-5p, was significantly up-regulated in 75.8% CRC in comparison to their corresponding non-tumor samples. It could have the potential to examine colorectal cancer subtype specific unique miRNA/mRNA interactions.
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http://dx.doi.org/10.1038/srep42809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318911PMC
February 2017

Synthesis of Novel Hybrids of Thymoquinone and Artemisinin with High Activity and Selectivity Against Colon Cancer.

ChemMedChem 2017 02 11;12(3):226-234. Epub 2017 Jan 11.

Organic Chemistry Chair I and Interdisciplinary Center for Molecular Materials (ICMM), Friedrich Alexander University of Erlangen-Nürnberg, Henkestr. 42, 91054, Erlangen, Germany.

Colorectal cancer causes 0.5 million deaths each year. To combat this type of cancer the development of new specific drug candidates is urgently needed. In the present work seven novel thymoquinone-artemisinin hybrids with different linkers were synthesized and tested for their in vitro anticancer activity against a panel of various tumor cell lines. The thymoquinone-artesunic acid hybrid 7 a, in which both subunits are connected via an ester bond, was found to be the most active compound and selectively decreased the viability of colorectal cancer cells with an IC value of 2.4 μm (HCT116) and 2.8 μm (HT29). Remarkably, hybrid 7 a was up to 20-fold more active than its parent compounds (thymoquinone and artesunic acid), while not affecting nonmalignant colon epithelial HCEC cells (IC >100 μm). Moreover, the activity of hybrid 7 a was superior to that of various 1:1 mixtures of thymoquinone and artesunic acid. Furthermore, hybrid 7 a was even more potent against both colon cancer cell lines than the clinically used drug 5-fluorouracil. These results are another excellent proof of the hybridization concept and confirm that the type and length of the linker play a crucial role for the biological activity of a hybrid drug. Besides an increase in reactive oxygen species (ROS), elevated levels of the DNA-damage marker γ-H2AX were observed. Both effects seem to be involved in the molecular mechanism of action for hybrid 7 a in colorectal cancer cells.
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http://dx.doi.org/10.1002/cmdc.201600594DOI Listing
February 2017

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Tissue Eng Part C Methods 2016 07;22(7):708-15

2 Department of Materials Science and Engineering, Institute of Biomaterials, Friedrich-Alexander University of Erlangen-Nuremberg , Germany .

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.
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http://dx.doi.org/10.1089/ten.TEC.2015.0452DOI Listing
July 2016

In vivo monitoring of the anti-angiogenic therapeutic effect of the pan-deacetylase inhibitor panobinostat by small animal PET in a mouse model of gastrointestinal cancers.

Nucl Med Biol 2016 Jan 20;43(1):27-34. Epub 2015 Oct 20.

Molecular Imaging and Radiochemistry, Department of Nuclear Medicine, Friedrich Alexander University (FAU), Erlangen, Germany. Electronic address:

Introduction: Deacetylase inhibitors have recently been established as a novel therapeutic approach to solid and hematologic cancers and have also been demonstrated to possess anti-angiogenic properties. Although these compounds show a good efficacy in vitro and in vivo, no data on monitoring and predicting treatment response are currently available. We therefore investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on gastrointestinal cancer models and the suitability of 2-[(18)F]FGlc-RGD as a specific agent for imaging integrin αvβ3 expression during tumor angiogenesis using small animal positron emission tomography (PET).

Methods: The effect of panobinostat on cell viability in vitro was assessed with a label-free impedance based real-time analysis. Nude mice bearing HT29 and HepG2 tumors were treated with daily i.p. injections of 10mg/kg panobinostat for 4 weeks. During this time, tumor size was determined with a calliper and mice were repeatedly subjected to PET imaging. Tumor tissues were analyzed immunohistochemically with a focus on proliferation and endothelial cell markers (Ki-67, Meca-32) and by Western blot applying specific markers of apoptosis.

Results: In vitro, panobinostat inhibited the proliferation of HepG2 and HT29 cells. Contrary to the situation in HepG2 tumors in vivo, where panobinostat treatment is known to reduce proliferation and vascularization resulting in a decreased tumor growth, HT29 tumors did not show any effect on these parameters. We demonstrated by Western blotting, that panobinostat induced apoptosis in HT29 tumors in vivo. Longitudinal PET imaging studies in HepG2 tumor-bearing mice using 2-[(18)F]FGlc-RGD demonstrated that the standard uptake value (SUVmax) in HepG2 tumors was significantly decreased by 39% at day 7 after treatment. The comparative PET study using HT29 tumor-bearing animals did not reveal any response of the tumors to panobinostat treatment.

Conclusions: Small-animal PET imaging using 2-[(18)F]FGlc-RGD was successfully applied to the non-invasive monitoring of the HepG2-tumor response to panobinostat in nude mice early after begin of treatment. Thus, PET imaging of angiogenesis using 2-[(18)F]FGlc-RGD could be a valuable tool to monitor panobinostat therapy in further preclinical studies.

Advances In Knowledge And Implications For Patient Care: When successfully translated to the clinical surrounding, PET imaging of angiogenesis could therefore facilitate therapy planning and monitoring of therapy success with panobinostat in hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.nucmedbio.2015.10.003DOI Listing
January 2016

HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo.

Oncotarget 2016 01;7(1):433-45

Sahlgrenska Cancer Center, Institute of Biomedicine, Department of Pathology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.
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http://dx.doi.org/10.18632/oncotarget.6336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808009PMC
January 2016

Metastatic Malignant Melanoma With Complete Loss of Differentiation Markers (Undifferentiated/Dedifferentiated Melanoma): Analysis of 14 Patients Emphasizing Phenotypic Plasticity and the Value of Molecular Testing as Surrogate Diagnostic Marker.

Am J Surg Pathol 2016 Feb;40(2):181-91

*Institute of Pathology, University of Erlangen-Nürnberg, Erlangen†Institute of Pathology, Technical University of München, Munich‡Institute of Pathology, Caritas-Krankenhaus, Bad Mergentheim gGmbH, Bad Mergentheim§Institute of Pathology, Klinikum Augsburg, Augsburg∥Institute of Pathology, Sana Klinikum Lichtenberg, Berlin¶Institute of Pathology#Department of Dermatology, University of Duisburg-Essen, Essen**Dermatopathologische Gemeinschaftspraxis, Friedrichshafen, Germany.

Metastatic malignant melanoma is notorious for its phenotypic diversity and loss of differentiation markers. We herein summarized our experience with 14 metastatic melanomas showing complete loss of immunohistochemical melanocytic markers (with or without heterologous differentiation). Patients included 11 men and 3 women aged 24 to 78 years (median, 67 y). Thirteen patients had histologically confirmed primary skin melanoma, and 1 had metastatic melanoma of unknown primary. Undifferentiated metastasis was diagnosed synchronous to primary tumor (n=1), following skin melanoma by 3 months to 9 years (n=11) and preceding it by 1 year (n=1). Sites of undifferentiated metastases were axillary (3), inguinal (1), or submandibular (1) lymph nodes, digestive tract (2), bone/soft tissue (2), lung/pleura (2), and disseminated (n=3). Histology of metastases mimicked undifferentiated pleomorphic or spindle cell sarcoma with variable myxoid and giant cell areas (n=10) and cytokeratin-positive undifferentiated small cell sarcoma (n=1). Three cases showed heterologous dedifferentiation: pleomorphic rhabdomyosarcoma (n=1), teratocarcinosarcoma-like with prominent rhabdomyoblasts (n=1), and adenocarcinoma-like with metaplastic bone (n=1). All cases were negative for S100, melanoma cocktail, HMB45, Melan A, and SOX10. Other markers showed following results: smooth muscle actin (1/14), p16 (1/14), TP53 (2/12), pancytokeratin (4/14), desmin (5/14), h-caldesmon (0/9), and MDM2/CDK4 (0/5). SMARCB1 was intact in 8/8 cases. Genotyping showed BRAF(V600E) mutation (5/14), NRAS mutation (5/14), and BRAF/NRAS wild-type (4/14). In conclusion, undifferentiated/dedifferentiated metastatic melanoma is likely underrecognized and frequently mistaken for undifferentiated sarcoma or other neoplasms. Diagnosis of undifferentiated sarcoma at sites where melanoma metastasis are frequent (eg, inguinal and axillary region) should be made with great caution and warrants exploration of the remote history. Genotyping is a helpful surrogate marker in classifying such difficult cases. In the light of available targeted therapies, recognition of undifferentiated/dedifferentiated metastatic melanoma is mandatory for appropriate treatment.
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http://dx.doi.org/10.1097/PAS.0000000000000527DOI Listing
February 2016

DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells.

Oncotarget 2015 Nov;6(34):36774-88

Experimental Tumor Pathology, Institute of Pathology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Erlangen, Germany.

Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPK's catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation.
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http://dx.doi.org/10.18632/oncotarget.4908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742210PMC
November 2015

Hsp90 inhibition by AUY922 as an effective treatment strategy against myxoid liposarcoma.

Cancer Lett 2015 Oct 28;367(2):147-56. Epub 2015 Jul 28.

Experimental Tumor Pathology, University Hospital Erlangen of Friedrich-Alexander University Erlangen-Nuremberg, Universitaetsstrasse 22, 91054 Erlangen, Germany; Institute of Pathology, University Hospital Erlangen of Friedrich-Alexander University Erlangen-Nuremberg, Krankenhausstr. 8-10, 91054 Erlangen, Germany. Electronic address:

Liposarcoma is one of the most common soft tissue sarcomas in adults. Recognized histological subtypes include well differentiated/dedifferentiated liposarcoma (WD/DDLS), myxoid liposarcoma (MLS) and pleomorphic liposarcoma. Currently, there are no proper subtype-specific treatments due to the genetic, histological and clinical heterogeneity of the liposarcoma subentities. In the past decade, the rising understanding of the various genetic and molecular aberrations in liposarcoma led to the development of novel alternative therapeutic strategies. One such therapy is the inhibition of the heat shock protein 90 (Hsp90) which is overexpressed in liposarcomas. In this study, we dissect the functional role of a novel potent Hsp90 inhibitor NVP-AUY922 (AUY922) in different cell lines of myxoid (MLS402, MLS1765) and undifferentiated (SW872) liposarcomas. We show that compared with 17-AAG treatment, lower concentrations of AUY922 achieve markedly cytotoxic effects on tumor cell viability. Combination treatment of AUY922 (20 nM) with Doxorubicin (300 nM) yielded a further reduction in cell viability in comparison to Doxorubicin alone. In vivo, we document an inhibition of tumor growth after AUY922 treatment. Further analyses revealed that Hsp90-inhibition induces apoptotic cell death and cell cycle arrest. In addition, we report striking perturbations of subtype-specific pattern in Raf/MEK/ERK and PI3K signaling after AUY922 application. In conclusion, our results provide evidence that Hsp90-inhibition by AUY922 may be a promising alternative therapeutic strategy for myxoid liposarcoma patients.
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http://dx.doi.org/10.1016/j.canlet.2015.07.025DOI Listing
October 2015
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