ABSTRACT
Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.
Subject(s)
Gene Expression Regulation, Neoplastic , Mechanistic Target of Rapamycin Complex 2 , Medulloblastoma , Meningeal Neoplasms , Otx Transcription Factors , Animals , Female , Humans , Male , Mice , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Cerebellar Neoplasms/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , Medulloblastoma/genetics , Medulloblastoma/pathology , Medulloblastoma/metabolism , Meningeal Neoplasms/genetics , Meningeal Neoplasms/pathology , Meningeal Neoplasms/metabolism , Meningeal Neoplasms/secondary , Otx Transcription Factors/metabolism , Otx Transcription Factors/genetics , Signal TransductionABSTRACT
Glioblastoma is one of the most devastating neoplasms of the central nervous system. This study focused on the development of serum extracellular vesicle (EV)-based glioblastoma tumor marker panels that can be used in a clinic to diagnose glioblastomas and to monitor tumor burden, progression, and regression in response to treatment. RNA sequencing studies were performed using RNA isolated from serum EVs from both patients (n = 85) and control donors (n = 31). RNA sequencing results for preoperative glioblastoma EVs compared to control EVs revealed 569 differentially expressed genes (DEGs, 2XFC, FDR < 0.05). By using these DEGs, we developed serum-EV-based biomarker panels for the following glioblastomas: wild-type IDH1 (96% sensitivity/80% specificity), MGMT promoter methylation (91% sensitivity/73% specificity), p53 gene mutation (100% sensitivity/89% specificity), and TERT promoter mutation (89% sensitivity/100% specificity). This is the first study showing that serum-EV-based biomarker panels can be used to diagnose glioblastomas with a high sensitivity and specificity.
ABSTRACT
Studies on tumor-associated antigens in brain tumors are sparse. There is scope for enhancing our understanding of molecular pathology, in order to improve on existing forms, and discover new forms, of treatment, which could be particularly relevant to immuno-oncological strategies. To elucidate immunological differences, and to provide another level of biological information, we performed antibody profiling, based on a high-density protein array (containing 8173 human transcripts), using IgG isolated from the sera of n = 12 preoperative and n = 16 postoperative glioblastomas, n = 26 preoperative and n = 29 postoperative meningiomas, and n = 27 healthy, cancer-free controls. Differentially reactive antigens were compared to gene expression data from an alternate public GBM data set from OncoDB, and were analyzed using the Reactome pathway browser. Protein array analysis identified approximately 350-800 differentially reactive antigens, and revealed different antigen profiles in the glioblastomas and meningiomas, with approximately 20-30%-similar and 10-15%-similar antigens in preoperative and postoperative sera, respectively. Seroreactivity did not correlate with OncoDB-derived gene expression. Antigens in the preoperative glioblastoma sera were enriched for signaling pathways, such as signaling by Rho-GTPases, COPI-mediated anterograde transport and vesicle-mediated transport, while the infectious disease, SRP-dependent membrane targeting cotranslational proteins were enriched in the meningiomas. The pre-vs. postoperative seroreactivity in the glioblastomas was enriched for antigens, e.g., platelet degranulation and metabolism of lipid pathways; in the meningiomas, the antigens were enriched in infectious diseases, metabolism of amino acids and derivatives, and cell cycle. Antibody profiling in both tumor entities elucidated several hundred antigens and characteristic signaling pathways that may provide new insights into molecular pathology and may be of interest for the development of new treatment strategies.
Subject(s)
Glioblastoma , Meningeal Neoplasms , Meningioma , Humans , Meningioma/genetics , Antibodies , Antigens, Neoplasm , Meningeal Neoplasms/geneticsABSTRACT
Extracellular vesicles (EVs) may be used as a non-invasive screening platform to discover markers associated with early diagnosis, prognosis, and treatment response. Such an approach is invaluable for diseases such as glioblastoma, for which only a few non-invasive diagnostic or prognostic markers are available. We used mass spectrometry to analyze proteomics profiles of EVs derived from four glioblastoma cell lines and human primary astrocytes (HPAs) and found that SRPX is the only protein enriched in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Then, we evaluated the relationship between SRPX protein expression and tumor grade using immunohistochemical staining (IHC) and performed colony formation and viability assays to analyze the possible function of SRPX in glioblastoma. SRPX mRNA and protein expression were associated with tumor grade. Moreover, temozolomide (TMZ)-resistant tumor tissues showed highly positive SRPX staining, compared to all other tumor grades. Additionally, glioblastoma cells displayed enhanced SRPX gene expression when exposed to TMZ. Knockdown of SRPX gene expression via siRNA inhibited cell viability. Taken together, the results of this study suggest that SRPX can be used as a novel tumor marker for diagnostic and prognostic purposes and can also be a therapeutic target for glioblastomas.
ABSTRACT
Cancer metastasis is the major cause of death from cancer (Massague and Obenauf, 2016; Steeg, 2016). The extensive genetic heterogeneity and cellular plasticity of metastatic tumors set a prime barrier for the current cancer treatment protocols (Boumahdi and de Sauvage, 2020). In addition, acquired therapy resistance has become an insurmountable obstacle that abolishes the beneficial effects of numerous anti-cancer regimens (De Angelis et al., 2019; Boumahdi and de Sauvage, 2020). Here we report that deficiency of Ku leads to the exploitation of host cells in human cancer cell line models. We found that, upon conditional deletion of XRCC6 that codes for Ku70, HCT116 human colorectal cancer cells gain a parasitic lifestyle that is characterized by the continuous cycle of host cell exploitation. We also found that DAOY cells, a human medulloblastoma cell line, innately lack nuclear Ku70/Ku86 proteins and utilize the host-cell invasion/exit mechanism for maintenance of their survival, similarly to the Ku70 conditionally-null HCT116 cells. Our study demonstrates that a functional loss of Ku protein promotes an adaptive, opportunistic switch to a parasitic lifestyle in human cancer cells, providing evidence for a previously unknown mechanism of cell survival in response to severe genomic stress. We anticipate that our study will bring a new perspective for understanding the mechanisms of cancer cell evolution, leading to a shift in the current concepts of cancer therapy protocols directed to the prevention of cancer metastasis and therapy resistance.
ABSTRACT
Meningiomas are primary central nervous system (CNS) tumors that originate from the arachnoid cells of the meninges. Recurrence occurs in higher grade meningiomas and a small subset of Grade I meningiomas with benign histology. Currently, there are no established circulating tumor markers which can be used for diagnostic and prognostic purposes in a non-invasive way for meningiomas. Here, we aimed to identify potential biomarkers of meningioma in patient sera. For this purpose, we collected preoperative (n = 30) serum samples from the meningioma patients classified as Grade I (n = 23), Grade II (n = 4), or Grade III (n = 3). We used a high-throughput, multiplex immunoassay cancer panel comprising of 92 cancer-related protein biomarkers to explore the serum protein profiles of meningioma patients. We detected 14 differentially expressed proteins in the sera of the Grade I meningioma patients in comparison to the age- and gender-matched control subjects (n = 12). Compared to the control group, Grade I meningioma patients showed increased serum levels of amphiregulin (AREG), CCL24, CD69, prolactin, EGF, HB-EGF, caspase-3, and decreased levels of VEGFD, TGF-α, E-Selectin, BAFF, IL-12, CCL9, and GH. For validation studies, we utilized an independent set of meningioma tumor tissue samples (Grade I, n = 20; Grade II, n = 10; Grade III, n = 6), and found that the expressions of amphiregulin and Caspase3 are significantly increased in all grades of meningiomas either at the transcriptional or protein level, respectively. In contrast, the gene expression of VEGF-D was significantly lower in Grade I meningioma tissue samples. Taken together, our study identifies a meningioma-specific protein signature in blood circulation of meningioma patients and highlights the importance of equilibrium between tumor-promoting factors and anti-tumor immunity.
ABSTRACT
Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy.
Subject(s)
Extracellular Vesicles/metabolism , Melanoma, Experimental/pathology , Stress, Physiological , Animals , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Doxorubicin/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/pathology , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Extracellular Vesicles/drug effects , Extracellular Vesicles/ultrastructure , Male , Melanoma, Experimental/ultrastructure , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/metabolism , Proteome/metabolism , Silver/chemistry , Stress, Physiological/drug effects , Titanium/chemistry , Tumor Microenvironment/drug effectsABSTRACT
Gene therapy involves the introduction of genes (termed transgenes) into cells to compensate for a deficiency or to make a beneficial protein. Gene therapy can used as a form of cancer treatment. A particularly attractive paradigm in this regard involves the selective introduction of transgenes into cancer cells that converts inactive prodrugs into active chemotherapeutic agents, thereby triggering the death of cancer cells. Since prodrugs are inactive, they tend not to cause significant side-effects and are well-tolerated by patients relative to conventional chemotherapy. Several viral and nonviral vectors have been used as delivery tools for suicide gene therapy. Extracellular vesicles (EVs) are now recognized as a promising class of nonviral delivery vectors. Here, we describe a method in which a suicide fusion gene construct is loaded into EVs derived from a non-tumorigenic cell line. Delivery of these modified EVs to glioblastoma cell lines and spheroids decreases glioblastoma cell viability, induces apoptotic cell death, and inhibits tumor growth in vivo.
Subject(s)
Drug Carriers , Extracellular Vesicles , Genes, Transgenic, Suicide , Genetic Therapy/methods , Glioblastoma/therapy , Cell Line, Tumor , Cytosine Deaminase/metabolism , Fungal Proteins/metabolism , Glioblastoma/drug therapy , HEK293 Cells , Humans , Pentosyltransferases/metabolism , Prodrugs/metabolism , Prodrugs/therapeutic use , RNA, Messenger , Yeasts/enzymologyABSTRACT
Ape1 is the major apurinic/apyrimidinic (AP) endonuclease activity in mammalian cells, and a key factor in base-excision repair of DNA. High expression or aberrant subcellular distribution of Ape1 has been detected in many cancer types, correlated with drug response, tumor prognosis, or patient survival. Here we present evidence that Ape1 facilitates BRCA1-mediated homologous recombination repair (HR), while counteracting error-prone non-homologous end joining of DNA double-strand breaks. Furthermore, Ape1, coordinated with checkpoint kinase Chk2, regulates drug response of glioblastoma cells. Suppression of Ape1/Chk2 signaling in glioblastoma cells facilitates alternative means of damage site recruitment of HR proteins as part of a genomic defense system. Through targeting "HR-addicted" temozolomide-resistant glioblastoma cells via a chemical inhibitor of Rad51, we demonstrated that targeting HR is a promising strategy for glioblastoma therapy. Our study uncovers a critical role for Ape1 in DNA repair pathway choice, and provides a mechanistic understanding of DNA repair-supported chemoresistance in glioblastoma cells.
Subject(s)
DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Drug Tolerance , Glioblastoma/pathology , Metabolic Networks and Pathways , Ubiquitin-Protein Ligases/metabolism , Checkpoint Kinase 2/metabolism , Homologous Recombination , HumansABSTRACT
BACKGROUND: Overexpression of minichromosome maintenance (MCM) proteins 2, 3, and 7 is associated with migration and invasion in medulloblastoma (MB). However, expression profiling of all prereplication complex (pre-RC) has not been addressed in MBs. PROCEDURE: We performed mRNA expression profiling of a large set of pre-RC elements in cell lines and tumor tissues of MB. RNAi technology was employed for functional studies in MB cell lines. RESULTS: Our data showed that most of the pre-RC components are significantly overexpressed in MB. Among all pre-RC mRNAs, MCM10 showed the highest level of expression (â¼500- to 1,000-fold) in MB cell lines and tissues compared to the levels detected in cerebellum. In addition, RNAi silencing of MCM10 caused reduced cell proliferation and cell viability in MB cells. CONCLUSIONS: Taken together, our study reveals that the pre-RC is dysregulated in MB. In addition, MCM10, a member of this complex, is significantly overexpressed in MB and is required for tumor cell proliferation.
Subject(s)
Cerebellar Neoplasms/chemistry , Medulloblastoma/chemistry , Minichromosome Maintenance Proteins/physiology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cerebellar Neoplasms/pathology , Humans , Immunohistochemistry , Medulloblastoma/pathology , Minichromosome Maintenance Proteins/analysisABSTRACT
BACKGROUND: Head and neck cancers comprise the sixth most common cancer type worldwide. One of the most remarkable malignancies of the head and neck is the cancer of the nasopharynx, with a strong metastatic tendency already in the early stage. Besides the conventional pathways of metastasis formation, the information content of exosomes produced by the cancer cells may play a key role in metastatic transformation. The aim of this study was to investigate how stressors alter the characteristic of tumor derived exosomes. METHODS: In our experimental model, we compared the quantity and content of exosomes produced by a nasopharyngeal carcinoma cell line (5-8F) under conventional (chemotherapy) and alternative (Ag-TiO2 -catalyzed reactive oxygen species generation) cytostatic treatment. After isolation, exosomes were identified by atomic force microscopy and quantified with Nanosight NS500 device. MicroRNA content of them was analyzed using SOLiD 5500xl technology. The sequences were annotated in CLC Genomics Workbench version 5.5.1. RESULTS: Beyond the classic chemotherapeutic agent (doxorubicin), Ag-TiO2 in a photo-catalytic process also showed cytostatic activity. Tumor cell damage induced by the cytostatic treatments significantly altered the number of released exosomes and led to the predominance of tumor suppressors in the exosomal miRNA profile. CONCLUSIONS: Our results suggest that the intercellular communication between tumor cells and surrounding stroma cells can be altered by microenvironment which increased quantity of exosomes and diversity of miRNAs in this study. Imbalance of oncogenic and tumor suppressor miRNAs caused by cytostatic treatments may influence the antiproliferative and metastasis inhibitory effect of cytostatic agents.
Subject(s)
Exosomes/metabolism , Nasopharyngeal Neoplasms/metabolism , Antibiotics, Antineoplastic/pharmacology , Cell Communication , Cell Line, Tumor , Cytostatic Agents/pharmacology , Doxorubicin/pharmacology , Exosomes/drug effects , Humans , MicroRNAs/metabolism , Nasopharyngeal Neoplasms/pathology , Tumor MicroenvironmentABSTRACT
Extracellular vesicles (EVs) are different types of membrane-derived vesicles that originate from the endosomal pathway or the plasma membrane. These vesicles are used as "carriers" in intercellular communication, and are responsible for the transfer of biological cargo (lipids, proteins, RNA species, and DNA) between different cells. Despite the shortcomings in our knowledge of EV biology, attempts to employ EVs as natural delivery tools for therapeutic purposes have been partly successful in different settings. In this review, we highlight this unique potential of EVs, and discuss previous examples and future scenarios.
Subject(s)
Drug Delivery Systems , Extracellular Vesicles/metabolism , Neoplasms/drug therapy , HumansABSTRACT
The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for Ape1-dependent DNA repair activity, deeper molecular mechanisms of the fundamental role of Ape1 in cell survival have not been defined. Here, we report that Ape1 is an essential factor stabilizing telomeric DNA, and its deficiency is associated with telomere dysfunction and segregation defects in immortalized cells maintaining telomeres by either the alternative lengthening of telomeres pathway (U2OS) or telomerase expression (BJ-hTERT), or in normal human fibroblasts (IMR90). Through the expression of Ape1 derivatives with site-specific changes, we found that the DNA repair and N-terminal acetylation domains are required for the Ape1 function at telomeres. Ape1 associates with telomere proteins in U2OS cells, and Ape1 depletion causes dissociation of TRF2 protein from telomeres. Consistent with this effect, we also observed that Ape1 depletion caused telomere shortening in both BJ-hTERT and in HeLa cells. Thus, our study describes a unique and unpredicted role for Ape1 in telomere protection, providing a direct link between base excision DNA repair activities and telomere metabolism.
Subject(s)
DNA Repair/physiology , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Telomere Homeostasis/genetics , Blotting, Western , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Primers/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Fluorescent Antibody Technique , Humans , Immunoprecipitation , In Situ Hybridization, Fluorescence , Telomerase/metabolism , Telomere Homeostasis/physiologyABSTRACT
Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein-cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)-an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived "liposomes" to effectively deliver therapeutic mRNA/proteins to treatment of diseases.
Subject(s)
Cytosine Deaminase/genetics , Genetic Engineering , Neurilemmoma/pathology , RNA, Messenger/genetics , Animals , Cell Line , Humans , Injections, Intralesional , Mice , Neurilemmoma/genetics , Pentosyltransferases/genetics , Polymerase Chain ReactionABSTRACT
Herpes simplex virus type 1 (HSV-1)-based amplicon vectors conserve most properties of the parental virus: broad host range, the ability to transduce dividing and nondiving cells, and a large transgene capacity. This permits incorporation of genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell-specific expression, multiple transgene cassettes, or genetic elements from other viruses. Hybrid vectors use elements from HSV-1 that allow replication and packaging of large-vector DNA into highly infectious particles, and elements from other viruses that confer genetic stability to vector DNA in the transduced cell. For example, adeno-associated virus (AAV) has the unique ability to integrate its genome into a specific site on human chromosome 19. The viral rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. However, AAV-based vectors have a very small transgene capacity and do not conventionally contain the rep gene to support site-specific genomic integration. HSV/AAV hybrid vectors contain both HSV-1 replication and packaging functions and the AAV rep gene and a transgene cassette flanked by the AAV ITRs. This combines the large transgene capacity of HSV-1 with the capability of site-specific genomic transgene integration and long-term transgene expression of AAV. This protocol describes the preparation of HSV/AAV hybrid vectors using a replication-competent/conditional, packaging-defective HSV-1 genome cloned as a bacterial artificial chromosome (BAC) to provide helper functions for vector replication and packaging. The advantages and limitations of such vectors compared to standard HSV-1 amplicon vectors are also discussed.
Subject(s)
Dependovirus/physiology , Genetic Vectors , Herpesvirus 1, Human/physiology , Molecular Biology/methods , Virus Assembly , Animals , Chlorocebus aethiops , Dependovirus/genetics , Herpesvirus 1, Human/genetics , Host Specificity , Recombination, Genetic , Transduction, Genetic , Vero Cells , Virology/methodsABSTRACT
Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3'-untranslated region (3'UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3'UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs.
ABSTRACT
miRNAs have been recently implicated as drivers in several carcinogenic processes, where they can act either as oncogenes or as tumor suppressors. Schwannomas arise from Schwann cells, the myelinating cells of the peripheral nervous system. These benign tumors typically result from loss of the neurofibromatosis type 2 (NF2) tumor suppressor gene. We have recently carried out high-throughput miRNA expression profiling of human vestibular schwannomas using an array representing 407 known miRNAs in order to explore the role of miRNAs in the tumorigenesis of schwannomas. We found that miR-7 functions as a "tumor suppressor" by targeting proteins in three major oncogenic pathways - EGFR, Pak1, and Ack1. Interestingly, in this study, we also observed that several previously described potential tumor suppressor miRNAs that are down-regulated in malignant tumors were up-regulated in schwannomas. Here we discuss the possibility that "tumor suppressor" miRNAs may play a role in the transition stage(s) of cancer from benign to malignant forms.
Subject(s)
Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , MicroRNAs/genetics , Neurilemmoma/genetics , Neurofibromatosis 2/genetics , Animals , Cell Transformation, Neoplastic/genetics , Humans , MicroRNAs/biosynthesisABSTRACT
Micro RNAs (miRNA) negatively regulate protein-coding genes at the posttranscriptional level and are critical in tumorigenesis. Schwannomas develop from proliferation of dedifferentiated Schwann cells, which normally wrap nerve fibers to help support and insulate nerves. In this study, we carried out high-throughput miRNA expression profiling of human vestibular schwannomas by using an array representing 407 known miRNAs to explore the role of miRNAs in tumor growth. Twelve miRNAs were found to be significantly deregulated in tumor samples as compared with control nerve tissue, defining a schwannoma-typical signature. Among these miRNAs, we focused on miR-7, which was one of the most downregulated in these tumors and has several known oncogene targets, including mRNAs for epidermal growth factor receptor (EGFR) and p21-activated kinase 1 (Pak1). We found that overexpression of miR-7 inhibited schwannoma cell growth both in culture and in xenograft tumor models in vivo, which correlated with downregulation of these signaling pathways. Furthermore, we identified a novel direct target of miR-7, the mRNA for associated cdc42 kinase 1 (Ack1), with the expression levels of miR-7 and Ack1 being inversely correlated in human schwannoma samples. These results represent the first miRNA profiling of schwannomas and the first report of a tumor suppressor function for miR-7 in these tumors that is mediated by targeting the EGFR, Pak1, and Ack1 oncogenes. Our findings suggest miR-7 as a potential therapeutic molecule for schwannoma treatment, and they prompt clinical evaluation of drugs that can inhibit the EGFR, Pak1, and Ack1 signaling pathways to treat this tumor type.
Subject(s)
MicroRNAs/genetics , Neurilemmoma/genetics , Neurilemmoma/metabolism , Animals , Cell Growth Processes/genetics , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/biosynthesis , ErbB Receptors/metabolism , Female , Humans , Mice , Mice, Nude , MicroRNAs/biosynthesis , Neurilemmoma/pathology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Transfection , Transplantation, Heterologous , Up-Regulation , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/biosynthesis , p21-Activated Kinases/metabolismABSTRACT
Benign schwannomas are common tumors of the cranial and peripheral nerves, causing pain and loss of function. The development of effective therapy for these tumors has been hampered by the lack of relevant experimental in vivo models for convenient testing. Here, we describe a novel schwannoma model in which an immortalized human schwannoma cell line, HEI-193, established from an neurofibromatosis type 2 patient, has been stably transduced with fluorescent protein and luciferase reporters and implanted within the sciatic nerve of nude mice. These cells reliably formed a tumor within several weeks which had pathologic characteristics of schwannoma tumors. This model system will be useful for investigation of schwannoma biology and for preclinical assessment of therapeutic agents.
Subject(s)
Disease Models, Animal , Luciferases, Firefly , Luminescent Proteins , Neurilemmoma/pathology , Sciatic Nerve/pathology , Animals , Cell Line, Tumor , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Mice , Mice, Nude , Transduction, GeneticABSTRACT
Meningiomas are among the most frequent tumors of the brain and spinal cord accounting for 15-20% of all central nervous system tumors and frequently associated with neurofibromatosis type 2. In this study, we aimed to unravel molecular meningioma tumorigenesis and discover novel protein biomarkers for diagnostic and/or prognostic purposes and performed in-depth proteomic profiling of meningioma cells compared to human primary arachnoidal cells. We isolated proteins from meningioma cell line SF4433 and human primary arachnoidal cells and analyzed the protein profiles by Gel-nanoLC-MS/MS in conjunction with protein identification and quantification by shotgun nanoLC tandem mass spectrometry and spectral counting. Differential analysis of meningiomas revealed changes in the expression levels of 281 proteins (P < 0.01) associated with various biological functions such as DNA replication, recombination, cell cycle, and apoptosis. Among several interesting proteins, we focused on a subset of the highly significantly up-regulated proteins, the minichromosome maintenance (MCM) family. We performed subsequent validation studies by qRT-PCR in human meningioma tissue samples (WHO grade I, 14 samples; WHO grade II, 7 samples; and WHO grade III, 7 samples) compared to arachnoidal tissue controls (from fresh autopsies; 3 samples) and found that MCMs are highly and significantly up-regulated in human meningioma tumor samples compared to arachnoidal tissue controls. We found a significant increase in MCM2 (8 fold), MCM3 (5 fold), MCM4 (4 fold), MCM5 (4 fold), MCM6 (3 fold), and MCM7 (5 fold) expressions in meningiomas. This study suggests that MCM family proteins are up-regulated in meningiomas and can be used as diagnostic markers.