ABSTRACT
The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.
Subject(s)
Forkhead Transcription Factors/metabolism , Muscle Proteins/antagonists & inhibitors , Paired Box Transcription Factors/metabolism , Polycomb Repressive Complex 2/physiology , Rhabdomyosarcoma, Alveolar/metabolism , SKP Cullin F-Box Protein Ligases/antagonists & inhibitors , Adolescent , Apoptosis , Cell Differentiation , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Cell Survival , Child , Enhancer of Zeste Homolog 2 Protein , Female , Forkhead Box Protein O1 , Gene Expression Regulation, Neoplastic , Gene Silencing , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Homeostasis , Humans , Male , Muscle Proteins/physiology , PAX3 Transcription Factor , SKP Cullin F-Box Protein Ligases/physiologyABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplastic disease. Gemcitabine, the currently used chemotherapeutic drug for PDAC, elicits only minor benefits, because of the development of escape pathways leading to chemoresistance. Herein, we aimed at investigating the involvement of the mitogen activating protein kinase interacting kinase (MNK)/eIF4E pathway in the acquired drug resistance of PDAC cells. Screening of a cohort of PDAC patients by immunohistochemistry showed that eIF4E phosphorylation correlated with disease grade, early onset of disease and worse prognosis. In PDAC cell lines, chemotherapeutic drugs induced MNK-dependent phosphorylation of eIF4E. Importantly, pharmacological inhibition of MNK activity synergistically enhanced the cytostatic effect of gemcitabine, by promoting apoptosis. RNA interference (RNAi) experiments indicated that MNK2 is mainly responsible for eIF4E phosphorylation and gemcitabine resistance in PDAC cells. Furthermore, we found that gemcitabine induced the expression of the oncogenic splicing factor SRSF1 and splicing of MNK2b, a splice variant that overrides upstream regulatory pathways and confers increased resistance to the drug. Silencing of SRSF1 by RNAi abolished this splicing event and recapitulated the effects of MNK pharmacological or genetic inhibition on eIF4E phosphorylation and apoptosis in gemcitabine-treated cells. Our results highlight a novel pro-survival pathway triggered by gemcitabine in PDAC cells, which leads to MNK2-dependent phosphorylation of eIF4E, suggesting that the MNK/eIF4E pathway represents an escape route utilized by PDAC cells to withstand chemotherapeutic treatments.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Carcinoma, Pancreatic Ductal/metabolism , Deoxycytidine/analogs & derivatives , Eukaryotic Initiation Factor-4E/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Pancreatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/mortality , Cell Line, Tumor , Cell Survival/drug effects , Deoxycytidine/pharmacology , Drug Resistance, Neoplasm , Drug Synergism , Enzyme Inhibitors/pharmacology , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Kaplan-Meier Estimate , Middle Aged , Nuclear Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/mortality , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA Splicing , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors , Signal Transduction , GemcitabineABSTRACT
A 27 years old man was admitted in our Institute for exertion dyspnea, palpitation and a single syncopal attack; five months before admission he was subjected to thoracotomy for pericardial and myocardial haemostatic suture because of a slash. Clinical, phonocardiographic and echocardiographic data suggested diagnosis of combined aortic stenosis and aortic regurgitation. An anterior myocardial necrosis was in ECG and VCG, Cardiac catheterization confirmed previous report. Coronary arteriography showed total occlusion of left anterior descending artery soon after the second diagonal branch. Right coronary was undamaged. Furthermore left ventricular angiocardiography showed a changed ventricular function. The Authors point out the iatrogenic cause of the coronary occlusion; it was due to the inclusion of left anterior descending artery in the surgical myocardial and pericardial haemostatic suture and/or post-traumatic fibrosis. It is necessary to watch over the patient as the changed ventricular function can develop in an aneurismatic area.
