ABSTRACT
BACKGROUND: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood-brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets. METHODS: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis. RESULTS: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis. CONCLUSION: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease.
Subject(s)
ADAM Proteins/metabolism , Biomimetic Materials/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/secondary , Breast Neoplasms/drug therapy , Intracellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/metabolism , Peptides/pharmacology , ADAM Proteins/biosynthesis , ADAM Proteins/genetics , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Gene Expression Profiling , Humans , Molecular Targeted Therapy , Neoplasm Recurrence, Local/metabolism , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/geneticsABSTRACT
BACKGROUND: Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer. However, its efficacy is limited by intrinsic and acquired resistance. Here the potential of S100ß as a biomarker and inhibition of its signaling network as a therapeutic strategy in endocrine treated patients was investigated. METHODS: The expression of S100ß in tissue and serum was assessed by immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The S100ß signaling network was investigated in cell line models of endocrine resistance by western blot, PCR, immunoprecipitation, and chromatin-immunoprecipitation. Endocrine resistant xenografts and tumor explants from patients with resistant tumors were treated with endocrine therapy in the presence and absence of the p-Src kinase inhibitor, dasatinib. RESULTS: Tissue and serum levels of S100ß were found to predict poor disease-free survival in endocrine-treated patients (n = 509, HR 2.32, 95% CI is 1.58-3.40, p < 0.0001 and n = 187, HR 4.009, 95% CI is 1.66-9.68, p = 0.002, respectively). Moreover, elevated levels of serum S100ß detected during routine surveillance over the patient treatment period significantly associated with subsequent clinically confirmed disease recurrence (p = 0.019). In vivo studies demonstrated that endocrine treatment induced transcriptional regulation of S100ß which was successfully disrupted with tyrosine kinase inhibition. In endocrine resistant xenografts and tumor explants from patients with endocrine resistant breast cancer, combined endocrine and dasatinib treatment reduced tumor proliferation and down-regulated S100ß protein expression in comparison to endocrine treatment alone. CONCLUSIONS: S100ß has potential as a new surveillance tool for patients with ER-positive breast cancer to monitor ongoing response to endocrine therapy. Moreover, endocrine resistant breast cancer patients with elevated S100ß may benefit from combined endocrine and tyrosine-kinase inhibitor treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01840293 ). Registered on 23 April 2013. Retrospectively registered.
Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Biomarkers/blood , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm , S100 Calcium Binding Protein beta Subunit/blood , Adult , Aged , Aged, 80 and over , Animals , Biomarkers/metabolism , Breast Neoplasms/blood , Breast Neoplasms/pathology , Cell Line, Tumor , Disease-Free Survival , Drug Resistance, Neoplasm/drug effects , Enzyme-Linked Immunosorbent Assay , Female , Humans , MCF-7 Cells , Mice , Middle Aged , Neoplasm Recurrence, Local , S100 Calcium Binding Protein beta Subunit/genetics , Signal Transduction/drug effects , Tamoxifen/therapeutic use , Tissue Array Analysis , Xenograft Model Antitumor AssaysABSTRACT
Targeted therapeutic options and prognostic biomarkers for hormone receptor- or Her2 receptor-negative breast cancers are severely limited. The sigma-1 receptor, a stress-activated chaperone, is frequently dysregulated in disease. However, its significance in breast cancer (BCa) has not been adequately explored. Here, we report that the sigma-1 receptor gene (SIGMAR1) is elevated in BCa, particularly in the aggressive triple-negative (TNBC) subtype. By examining several patient datasets, we found that high expression at both the gene (SIGMAR1) and protein (Sig1R) levels associated with poor survival outcomes, specifically in ER-Her2- groups. Our data further show that high SIGMAR1 was predictive of shorter survival times in patients treated with adjuvant chemotherapy (ChT). Interestingly, in a separate cohort who received neoadjuvant taxane + anthracycline treatment, elevated SIGMAR1 associated with higher rates of pathologic complete response (pCR). Treatment with a Sig1R antagonist, 1-(4-iodophenyl)-3-(2-adamantyl)guanidine (IPAG), activated the unfolded protein response (UPR) in TNBC (high-Sig1R expressing) and ER + (low-Sig1R expressing) BCa cell lines. In tamoxifen-resistant LY2 cells, IPAG caused Sig1R to aggregate and co-localise with the stress marker BiP. These findings showcase the potential of Sig1R as a novel biomarker in TNBC as well as highlight its ligand-induced interference with the stress-coping mechanisms of BCa cells.
Subject(s)
Breast Neoplasms , Receptors, sigma , Triple Negative Breast Neoplasms , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Ligands , Receptors, sigma/genetics , Receptors, sigma/therapeutic use , Endoplasmic Reticulum Stress , Sigma-1 ReceptorABSTRACT
PURPOSE: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. EXPERIMENTAL DESIGN: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor-positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. RESULTS: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. CONCLUSIONS: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.
Subject(s)
Androstenedione/physiology , Aromatase Inhibitors/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/etiology , Receptors, Androgen/physiology , Androstenedione/blood , Breast Neoplasms/blood , Drug Resistance, Neoplasm , Female , Humans , Ligands , Signal Transduction , Tumor Cells, CulturedABSTRACT
The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.
Subject(s)
Cytidine Deaminase/genetics , Minor Histocompatibility Antigens/genetics , Neoplasms/genetics , Transcription Factor RelA/genetics , Tumor Suppressor Protein p53/genetics , Ataxia Telangiectasia Mutated Proteins/genetics , CRISPR-Cas Systems/genetics , Cisplatin/pharmacology , DNA Repair/drug effects , Etoposide/pharmacology , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Genetic Heterogeneity , HCT116 Cells , Humans , MCF-7 Cells , Mutation/genetics , NF-kappa B/genetics , Neoplasms/pathologyABSTRACT
Steroid regulated cancer cells use nuclear receptors and associated regulatory proteins to orchestrate transcriptional networks to drive disease progression. In primary breast cancer, the coactivator AIB1 promotes estrogen receptor (ER) transcriptional activity to enhance cell proliferation. The function of the coactivator in ER+ metastasis however is not established. Here we describe AIB1 as a survival factor, regulator of pro-metastatic transcriptional pathways and a promising actionable target. Genomic alterations and functional expression of AIB1 associated with reduced disease-free survival in patients and enhanced metastatic capacity in novel CDX and PDX ex-vivo models of ER+ metastatic disease. Comparative analysis of the AIB1 interactome with complementary RNAseq characterized AIB1 as a transcriptional repressor. Specifically, we report that AIB1 interacts with MTA2 to form a repressive complex, inhibiting CDH1 (encoding E-cadherin) to promote EMT and drive progression. We further report that pharmacological and genetic inhibition of AIB1 demonstrates significant anti-proliferative activity in patient-derived models establishing AIB1 as a viable strategy to target endocrine resistant metastasis. This work defines a novel role for AIB1 in the regulation of EMT through transcriptional repression in advanced cancer cells with a considerable implication for prognosis and therapeutic interventions.
Subject(s)
Breast Neoplasms/drug therapy , Cadherins/genetics , Histone Deacetylases/genetics , Nuclear Receptor Coactivator 3/genetics , Repressor Proteins/genetics , Antigens, CD/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation/genetics , Disease-Free Survival , Epithelial-Mesenchymal Transition/drug effects , Estrogen Receptor alpha/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Neoplasm Metastasis , Nuclear Receptor Coactivator 3/antagonists & inhibitors , Phenotype , Prognosis , Tamoxifen/pharmacologyABSTRACT
While conventional cancer treatments, such as surgery, radiotherapy and chemotherapy, have been combined for decades in an effort to treat cancer patients, the emergence of novel fields of cancer research have led to a renewed interest in combining conventional treatments with more innovative approaches. The realisation that cancer progression is not exclusively due to changes in the cancer epithelial cells, but also involves changes in the tumour microenvironment, has opened new avenues for combination treatments. Here we discuss the use of combination therapies presented at the 55th Irish Association for Cancer Research (IACR) Annual Conference, highlighting examples of novel therapeutic strategies which, combined with conventional therapies, may greatly enhance not only the overall outcome for patients, but also the quality of life for cancer survivors. Among the novel treatment strategies, immune metabolism, epigenetic therapies and physical exercise are presented. In addition, novel technologies in the field of precision medicine, which will be useful to discover new therapeutics and to stratify patients for combination treatments, are also discussed.
ABSTRACT
Conventional therapies for cancer such as chemotherapy and radiotherapy remain a mainstay in treatment, but in many cases a targeted approach is lacking, and patients can be vulnerable to drug resistance. In recent years, novel concepts have been emerging to improve the traditional therapeutic options in cancers with poor survival outcomes. New therapeutic strategies involving areas like energy metabolism and extracellular vesicles along with advances in immunotherapy and nanotechnology are driving the next generation of cancer treatments. The development of fields such as theranostics in nanomedicine is also opening new doors for targeted drug delivery and nano-imaging. Here we discuss the use of innovative technologies presented at the Irish Association for Cancer Research (IACR) Annual Meeting, highlighting examples of where new approaches may lead to promising new treatment options for a range of cancer types.
ABSTRACT
Steroid receptor coactivator 1 (SRC-1) interacts with nuclear receptors and other transcription factors (TFs) to initiate transcriptional networks and regulate downstream genes which enable the cancer cell to evade therapy and metastasise. Here we took a top-down discovery approach to map out the SRC-1 transcriptional network in endocrine resistant breast cancer. First, rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was employed to uncover new SRC-1 TF partners. Next, RNA sequencing (RNAseq) was undertaken to investigate SRC-1 TF target genes. Molecular and patient-derived xenograft studies confirmed STAT1 as a new SRC-1 TF partner, important in the regulation of a cadre of four SRC-1 transcription targets, NFIA, SMAD2, E2F7 and ASCL1. Extended network analysis identified a downstream 79 gene network, the clinical relevance of which was investigated in RNAseq studies from matched primary and local-recurrence tumours from endocrine resistant patients. We propose that SRC-1 can partner with STAT1 independently of the estrogen receptor to initiate a transcriptional cascade and control regulation of key endocrine resistant genes.
Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Gene Regulatory Networks , Nuclear Receptor Coactivator 1/physiology , Animals , Breast Neoplasms/pathology , Chromatin Assembly and Disassembly/drug effects , Chromatin Assembly and Disassembly/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks/drug effects , Humans , MCF-7 Cells , Mice , Mice, Inbred NOD , Mice, SCID , Microarray Analysis , Transcriptional Activation/genetics , Transcriptome/drug effects , Tumor Cells, CulturedABSTRACT
Purpose: Despite the clinical utility of endocrine therapies for estrogen receptor-positive (ER) breast cancer, up to 40% of patients eventually develop resistance, leading to disease progression. The molecular determinants that drive this adaptation to treatment remain poorly understood. Methylome aberrations drive cancer growth yet the functional role and mechanism of these epimutations in drug resistance are poorly elucidated.Experimental Design: Genome-wide multi-omics sequencing approach identified a differentially methylated hub of prodifferentiation genes in endocrine resistant breast cancer patients and cell models. Clinical relevance of the functionally validated methyl-targets was assessed in a cohort of endocrine-treated human breast cancers and patient-derived ex vivo metastatic tumors.Results: Enhanced global hypermethylation was observed in endocrine treatment resistant cells and patient metastasis relative to sensitive parent cells and matched primary breast tumor, respectively. Using paired methylation and transcriptional profiles, we found that SRC-1-dependent alterations in endocrine resistance lead to aberrant hypermethylation that resulted in reduced expression of a set of differentiation genes. Analysis of ER-positive endocrine-treated human breast tumors (n = 669) demonstrated that low expression of this prodifferentiation gene set significantly associated with poor clinical outcome (P = 0.00009). We demonstrate that the reactivation of these genes in vitro and ex vivo reverses the aggressive phenotype.Conclusions: Our work demonstrates that SRC-1-dependent epigenetic remodeling is a 'high level' regulator of the poorly differentiated state in ER-positive breast cancer. Collectively these data revealed an epigenetic reprograming pathway, whereby concerted differential DNA methylation is potentiated by SRC-1 in the endocrine resistant setting. Clin Cancer Res; 24(15); 3692-703. ©2018 AACR.
Subject(s)
Breast Neoplasms/drug therapy , Cell Differentiation/drug effects , Receptors, Estrogen/genetics , src-Family Kinases/genetics , Breast/drug effects , Breast/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , CRISPR-Cas Systems/genetics , Cell Proliferation/drug effects , DNA Methylation/genetics , Disease-Free Survival , Drug Resistance, Neoplasm/genetics , Epigenomics , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing , Heterografts , Humans , MCF-7 Cells , Microarray Analysis , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm MetastasisABSTRACT
The use of monoclonal antibodies (mAbs) and molecules derived from them has achieved considerable attention and success in recent years, establishing this mode of therapy as an important therapeutic strategy in many cancers, in particular hematological tumors. mAbs recognize cell surface antigens expressed on target cells and mediate their function through various mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or immune system modulation. The efficacy of mAb therapy can be improved when they are conjugated to a highly potent payloads, including cytotoxic drugs and radiolabeled isotopes. The Eph family of proteins has received considerable attention in recent years as therapeutic targets for treatment of both solid and hematological cancers. High expression of Eph receptors on cancer cells compared with low expression levels in normal adult tissues makes them an attractive candidate for cancer immunotherapy. In this review, we detail the modes of action of antibody-based therapies with a focus on the Eph family of proteins as potential targets for therapy in hematological malignancies.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Gene Expression Regulation, Neoplastic , Hematologic Neoplasms/drug therapy , Molecular Targeted Therapy , Neoplasm Proteins/antagonists & inhibitors , Receptors, Eph Family/antagonists & inhibitors , Antibodies, Monoclonal/biosynthesis , Antibody-Dependent Cell Cytotoxicity/drug effects , Antineoplastic Agents/therapeutic use , Complement Activation/drug effects , Cytotoxins/chemistry , Cytotoxins/therapeutic use , Hematologic Neoplasms/genetics , Hematologic Neoplasms/immunology , Hematologic Neoplasms/pathology , Humans , Immunoconjugates/chemistry , Immunoconjugates/therapeutic use , Immunomodulation/drug effects , Immunotherapy/methods , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Receptors, Eph Family/genetics , Receptors, Eph Family/immunologyABSTRACT
Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects.
Subject(s)
Biomarkers, Tumor/analysis , Brain Neoplasms/pathology , Glioblastoma/pathology , NFI Transcription Factors/metabolism , Animals , Cell Line, Tumor , Genes, Tumor Suppressor/physiology , Heterografts , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Tumor Suppressor Proteins/metabolismABSTRACT
Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.
Subject(s)
Antibodies, Monoclonal/pharmacology , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Leukemia/therapy , Radioimmunotherapy , Receptor, EphA2/physiology , Animals , Cell Differentiation , Female , Flow Cytometry , Gene Rearrangement , Hematopoietic Stem Cells/cytology , Leukemia/genetics , Leukemia/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid-Lymphoid Leukemia Protein/genetics , Real-Time Polymerase Chain Reaction , Receptor, EphA2/antagonists & inhibitors , Translocation, GeneticABSTRACT
Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.
Subject(s)
Brain Neoplasms/prevention & control , Glioblastoma/prevention & control , Mitogen-Activated Protein Kinases/metabolism , Neoplastic Stem Cells/pathology , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis , Blotting, Western , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Differentiation , Cell Proliferation , Flow Cytometry , Fluorescent Antibody Technique , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Immunoprecipitation , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Small Interfering/genetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor, EphA3 , Tumor Cells, CulturedABSTRACT
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.