ABSTRACT
Glioma is the most common adult primary brain tumor. Its most malignant form, glioblastoma multiforme (GBM), is almost invariably fatal, due in part to the intrinsic resistance of GBM to radiation- and chemotherapy-induced apoptosis. We analyzed B-cell leukemia-2 (Bcl-2) anti-apoptotic proteins in GBM and found myeloid cell leukemia-1 (Mcl-1) to be the highest expressed in the majority of malignant gliomas. Mcl-1 was functionally important, as neutralization of Mcl-1 induced apoptosis and increased chemotherapy-induced apoptosis. To determine how Mcl-1 was regulated in glioma, we analyzed the promoter and identified a novel functional single nucleotide polymorphism in an uncharacterized E26 transformation-specific (ETS) binding site. We identified the ETS transcription factor ELK4 as a critical regulator of Mcl-1 in glioma, since ELK4 downregulation was shown to reduce Mcl-1 and increase sensitivity to apoptosis. Importantly the presence of the single nucleotide polymorphism, which ablated ELK4 binding in gliomas, was associated with lower Mcl-1 levels and a greater dependence on Bcl-xL. Furthermore, in vivo, ELK4 downregulation reduced tumor formation in glioblastoma xenograft models. The critical role of ELK4 in Mcl-1 expression and protection from apoptosis in glioma defines ELK4 as a novel potential therapeutic target for GBM.
Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Glioblastoma/prevention & control , Proto-Oncogene Proteins c-bcl-2/metabolism , ets-Domain Protein Elk-4/metabolism , Adult , Animals , Base Sequence , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Brain Neoplasms/prevention & control , Cell Line, Tumor , Chromatin Immunoprecipitation , Down-Regulation , Electrophoretic Mobility Shift Assay , Glioblastoma/metabolism , Humans , Luciferases/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Sequence Data , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Grading , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Transfection , bcl-X Protein/genetics , bcl-X Protein/metabolism , ets-Domain Protein Elk-4/antagonists & inhibitors , ets-Domain Protein Elk-4/geneticsABSTRACT
Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.
