Dataset Information

C/EBP?-1 promotes transformation and chemoresistance in Ewing sarcoma cells.

ABSTRACT: CEBPB copy number gain in Ewing sarcoma was previously shown to be associated with worse clinical outcome compared to tumors with normal CEBPB copy number, although the mechanism was not characterized. We employed gene knockdown and rescue assays to explore the consequences of altered CEBPB gene expression in Ewing sarcoma cell lines. Knockdown of EWS-FLI1 expression led to a decrease in expression of all three C/EBP? isoforms while re-expression of EWS-FLI1 rescued C/EBP? expression. Overexpression of C/EBP?-1, the largest of the three C/EBP? isoforms, led to a significant increase in colony formation when cells were grown in soft agar compared to empty vector transduced cells. In addition, depletion of C/EBP? decreased colony formation, and re-expression of either C/EBP?-1 or C/EBP?-2 rescued the phenotype. We identified the cancer stem cell marker ALDH1A1 as a target of C/EBP? in Ewing sarcoma. Furthermore, increased expression of C/EBP? led to resistance to chemotherapeutic agents. In summary, we have identified CEBPB as an oncogene in Ewing sarcoma. Overexpression of C/EBP?-1 increases transformation, upregulates expression of the cancer stem cell marker ALDH1A1, and leads to chemoresistance.

SUBMITTER: Gardiner JD

PROVIDER: S-EPMC5432234 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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C/EBPβ-1 promotes transformation and chemoresistance in Ewing sarcoma cells.

Gardiner Jamie D JD Abegglen Lisa M LM Huang Xiaomeng X Carter Bryce E BE Schackmann Elizabeth A EA Stucki Marcus M Paxton Christian N CN Lor Randall R R Amatruda James F JF Putnam Angelica R AR Kovar Heinrich H Lessnick Stephen L SL Schiffman Joshua D JD

Oncotarget 20170401 16

CEBPB copy number gain in Ewing sarcoma was previously shown to be associated with worse clinical outcome compared to tumors with normal CEBPB copy number, although the mechanism was not characterized. We employed gene knockdown and rescue assays to explore the consequences of altered CEBPB gene expression in Ewing sarcoma cell lines. Knockdown of EWS-FLI1 expression led to a decrease in expression of all three C/EBPβ isoforms while re-expression of EWS-FLI1 rescued C/EBPβ expression. Overexpres ...[more]

PMID: 28148901

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Project description:The Cancer associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. Evidence indicates that CaSm modulates mRNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer â including transformation, proliferation, chemoresistance, and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells in vitro. CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1, and Bcl-XL. CaSm-overexpressing Panc-1 cells were 2-fold more migratory and 4-fold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR, and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer. Tet-on driver and tet-on CaSm Panc-1 cells were grown with and without doxycycline (1 Î¼g/mL) for 4 weeks. Levels of CaSm expression were confirmed using RT-PCR (Supplementary Figure 2a). RNA isolation was performed using RNeasy Plus kit (Qiagen, Valencia, CA) following the manufacturerâs protocols. The Hollings Cancer Center Tissue Biorepository core facility confirmed RNA quality using the 18S/28S Ratio Integrity Number using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA); RNA with a RIN of 10 was used for further analysis (Supplementary Figure 2b). Complement DNA synthesis was performed using RT2 First Strand kit and RT2 SYBR Green qPCR master mix was used for the microarray master mix reagent according to the manufacturerâs manual (SABiosciences). The Cancer PathwayFinder PCR array (SABiosciences) was run on the Roche 480 Lightcycler (Roche, Indianapolis, IN) with 2 plates run for each tet-on driver + doxycycline, tet-on CaSm, and tet-on CaSm + doxycycline. The plates included controls for genomic DNA contamination, reverse transcription, and positive controls. Analysis was performed using SABioscienceâs Free PCR Array Data Analysis Software (ÎÎCt method) with B2M, HPRT1, and GAPDH as pooled housekeeping genes.

Dataset Information

C/EBP?-1 promotes transformation and chemoresistance in Ewing sarcoma cells.

Publications

C/EBPβ-1 promotes transformation and chemoresistance in Ewing sarcoma cells.

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