hes1-protein--human and Bone-Neoplasms

Controlling metastasis is the key to improving outcomes for osteosarcoma patients; yet our knowledge of the mechanisms regulating the metastatic process is incomplete. Clearly Fas and Ezrin are important, but other genes must play a role in promoting tumor spread. Early developmental pathways are often recapitulated in malignant tissues, and these genes are likely to be important in regulating the primitive behaviors of tumor cells, including invasion and metastasis. The Notch pathway is a highly conserved regulatory signaling network involved in many developmental processes and several cancers, at times serving as an oncogene and at others, behaving as a tumor suppressor. In normal limb development, Notch signaling maintains the apical ectodermal ridge in the developing limb bud and regulated size of bone and muscles. Here, we examine the role of Notch signaling in promoting metastasis of osteosarcoma, and the underlying regulatory processes that control Notch pathway expression and activity in the disease. We have shown that, compared to normal human osteoblasts and non-metastatic osteosarcoma cell lines, osteosarcoma cell lines with the ability to metastasize have higher levels of Notch 1, Notch 2, the Notch ligand DLL1 and the Notch-induced gene Hes1. When invasive osteosarcoma cells are treated with small molecule inhibitors of gamma-secretase, which blocks Notch activation, invasiveness is abrogated. Direct retroviral expression has shown that Hes1 expression was necessary for osteosarcoma invasiveness and accounted for the observations. In a novel orthotopic murine xenograft model of osteosarcoma pulmonary metastasis, blockade of Hes1 expression and Notch signaling eliminated spread of disease from the tibial primary tumor. In a sample of archival human osteosarcoma tumor specimens, expression of Hes1 mRNA was inversely correlated with survival (n=16 samples, p=0.04). Expression of the microRNA 34 cluster, which is known to downregulate DLL1, Notch 1 and Notch 2, was inversely correlated with invasiveness in a small panel of osteosarcoma tumors, suggesting that this family of microRNAs may be responsible for regulating Notch expression in at least some tumors. Further, exposure to valproic acid at therapeutic concentrations induced expression of Notch genes and caused a 250-fold increase in invasiveness for non-invasive cell lines, but had no discernible effect on those lines that expressed high levels of Notch without valproic acid treatment, sugg

Topics: Basic Helix-Loop-Helix Transcription Factors; Bone Development; Bone Neoplasms; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Neoplasm Invasiveness; Osteosarcoma; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Valproic Acid

This study aimed to investigate the role of long non-coding RNA MEG3 (lncRNA MEG3) in osteosarcoma (OS) and further explore the underlying molecular mechanism.. The expression profiles of MEG3 in OS cell lines and normal osteoblast cell line were detected by qRT-PCR. MEG3 was over-expressed in OS cell line by using LV-MEG3. MTT and colony-formation assays were applied for cell proliferation analysis. Cell migration assay was applied to investigate the cell migration ability. In addition, the expression levels of cell growth and metastasis related factors (Notch1, Hes1, TGF-β, N-cadheren and E-cadheren) were determined to illustrate the mechanisms.. We found that compared with normal osteoblast hFOB1.19 cell line, MEG3 was significantly down-regulated in MG63 and U2OS cell lines, particularly in MG-63 cells. MEG3 was significantly up-regulated in MG63 cells by LV-MEG3. Cell proliferation and migration ability were obviously repressed by MEG3 over-expression. In addition, MEG3 over-expression markedly inhibited Notch1, Hes1,TGF-β and N-cadheren expression, and the expression level of E-cadheren was improved.. These results indicated that MEG3 could prevent cell growth and metastasis of OS by repressing Notch and TGF-β signaling pathway, thus providing a potential therapeutic target for OS treatment (Tab. 1, Fig. 4, Ref. 30).

Topics: Antigens, CD; Bone Neoplasms; Cadherins; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Osteoblasts; Osteosarcoma; Receptor, Notch1; RNA, Long Noncoding; Transcription Factor HES-1; Transforming Growth Factor beta; Tumor Stem Cell Assay; Up-Regulation

Osteosarcomas are the most common primary malignant tumors of bone, showing complex chromosomal rearrangements with multiple gains and losses. A frequent deletion within the chromosomal region 3q13.31 has been identified by us and others, and is mainly reported to be present in osteosarcomas. The purpose of the study was to further characterize the frequency and the extent of the deletion in an extended panel of osteosarcoma samples, and the expression level of the affected genes within the region. We have identified LSAMP as the target gene for the deletion, and have studied the functional implications of LSAMP-reexpression.. LSAMP copy number, expression level and protein level were investigated by quantitative PCR and western blotting in an osteosarcoma panel. The expression of LSAMP was restored in an osteosarcoma cell line, and differences in proliferation rate, tumor formation, gene expression, migration rate, differentiation capabilities, cell cycle distribution and apoptosis were investigated by metabolic dyes, tumor formation in vivo, gene expression profiling, time-lapse photography, differentiation techniques and flow cytometry, respectively.. We found reduced copy number of LSAMP in 45/76 osteosarcoma samples, reduced expression level in 25/42 samples and protein expression in 9/42 samples. By restoring the expression of LSAMP in a cell line with a homozygous deletion of the gene, the proliferation rate in vitro was significantly reduced and tumor growth in vivo was significantly delayed. In response to reexpression of LSAMP, mRNA expression profiling revealed consistent upregulation of the genes hairy and enhancer of split 1 (HES1), cancer/testis antigen 2 (CTAG2) and kruppel-like factor 10 (KLF10).. The high frequency and the specificity of the deletion indicate that it is important for the development of osteosarcomas. The deletion targets the tumor suppressor LSAMP, and based on the functional evidence, the tumor suppressor function of LSAMP is most likely exerted by reducing the proliferation rate of the tumor cells, possibly by indirectly upregulating one or more of the genes HES1, CTAG2 or KLF10. To our knowledge, this study describes novel functions of LSAMP, a first step to understanding the functional role of this specific deletion in osteosarcomas.

Topics: Antigens, Neoplasm; Antigens, Surface; Basic Helix-Loop-Helix Transcription Factors; Bone Neoplasms; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Cell Proliferation; Chromosome Mapping; Chromosomes, Human, Pair 3; Early Growth Response Transcription Factors; Female; Gene Deletion; Gene Dosage; Gene Expression Regulation, Neoplastic; Genetic Complementation Test; GPI-Linked Proteins; Homeodomain Proteins; Homozygote; Humans; Kruppel-Like Transcription Factors; Male; Mutation Rate; Osteosarcoma; RNA, Messenger; Signal Transduction; Survival Analysis; Transcription Factor HES-1

Doxorubicin plays a major role in the treatment of osteosarcoma disorders. The Notch signaling pathway exerts various biological functions, including cell proliferation, differentiation, and apoptosis. In the present study, we investigated the effects of different doses of doxorubicin on proliferation and apoptosis of osteosarcoma cells with or without Notch signaling. Results found that cellular viability was downregulated while caspase 3 activity and expression were promoted in osteosarcoma cells following treatment with various doses of doxorubicin for 24, 48, and 72 h, and the effects showed a dose- and time-dependent manner. Furthermore, it was found that various doses of doxorubicin activated the Notch signaling pathway, shown by the elevated expression of Notch target genes NOTCH1, HEY1, HES1, AND HES5. It was further proved that, after small interfering RNA (siRNA)-mediated knockdown of Notch, the effects of doxorubicin on the viability and apoptosis of osteosarcoma cells were significantly reduced. It was indicated that doxorubicin treatment reduced the proliferation and promoted the apoptosis of osteosarcoma cells, and this effect was mediated by the Notch signaling pathway.

Topics: Antibiotics, Antineoplastic; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Bone Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Osteosarcoma; Receptor, Notch1; Receptors, Notch; Repressor Proteins; RNA Interference; Signal Transduction; Time Factors; Transcription Factor HES-1; Transfection; Up-Regulation

Notch can act as an oncogene or as a tumour suppressor and thus can either promote or inhibit tumour cell growth. To establish Notch status in Ewing's sarcoma family of tumours (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We found that although Notch receptors were highly expressed, Notch did not appear to be active, as evidenced by the absence of Notch receptors in cell nuclei. In contrast, we show that Notch receptors known to be active in colon adenocarcinoma, hepatocarcinoma, and pancreatic carcinoma stain cell nuclei in these tumours. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of ESFT, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation and reduced their clonogenic potential in soft agar. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and support a role for Notch in ESFT tumour suppression, at least partly through the Notch effector HEY1.

Topics: Basic Helix-Loop-Helix Transcription Factors; Bone Neoplasms; Cell Nucleus; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Neoplasm Proteins; Neoplastic Stem Cells; Receptors, Notch; Sarcoma, Ewing; Signal Transduction; Transcription Factor HES-1; Tumor Cells, Cultured

The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Bone Neoplasms; Carrier Proteins; Chromatin Immunoprecipitation; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Homeodomain Proteins; Humans; Immunoprecipitation; Luciferases; Neoplasm Invasiveness; Osteosarcoma; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription Factor HES-1; Transfection; Ubiquitination

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Bone Neoplasms; Calcium; Carbamates; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Core Binding Factor Alpha 1 Subunit; Dipeptides; DNA; Enzyme Activation; Homeodomain Proteins; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Metastasis; Neoplasm Proteins; Osteoblasts; Prostatic Neoplasms; Protein Binding; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Ribosomal, 18S; Signal Transduction; Time Factors; Transcription Factor HES-1; Transcription Factors