RESUMO
Insulinoma-associated-1 (INSM1) is a key protein functioning as a transcriptional repressor in neuroendocrine differentiation and is activated by N-Myc in human neuroblastoma (NB). INSM1 modulates the phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-glycogen synthase kinase 3ß (GSK3ß) signaling pathway through a positive-feedback loop, resulting in N-Myc stabilization. Accordingly, INSM1 has emerged as a critical player closely associated with N-Myc in facilitating NB cell growth. Here, an INSM1 promoter-driven luciferase-based screen revealed that the compound 5'-iodotubercidin suppresses adenosine kinase (ADK), an energy pathway enzyme, and also INSM1 expression and NB tumor growth. Next, we sought to dissect how the ADK pathway contributes to NB tumor cell growth in the context of INSM1 expression. We also found that 5'-iodotubercidin inhibits INSM1 expression and induces an intra- and extracellular adenosine imbalance. The adenosine imbalance, which triggers adenosine receptor-3 signaling that decreases cAMP levels and AKT phosphorylation and enhances GSK3ß activity. We further observed that GSK3ß then phosphorylates ß-catenin and promotes the cytoplasmic proteasomal degradation pathway. 5'-Iodotubercidin treatment and INSM1 inhibition suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activity and the AKT signaling pathways required for NB cell proliferation. The 5'-iodotubercidin treatment also suppressed ß-catenin, lymphoid enhancer-binding factor 1 (LEF-1), cyclin D1, N-Myc, and INSM1 levels, ultimately leading to apoptosis via caspase-3 and p53 activation. The identification of the signaling pathways that control the proliferation of aggressive NB reported here suggests new options for combination treatments of NB patients.
Assuntos
Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Neuroblastoma/tratamento farmacológico , Proteínas Repressoras/biossíntese , Tubercidina/análogos & derivados , Apoptose/efeitos dos fármacos , Humanos , Células K562 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Neoplasias/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proto-Oncogene Mas , Proteínas Repressoras/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Tubercidina/farmacologiaRESUMO
BACKGROUND: Neurogenin3 (Ngn3) and neurogenic differentiation 1 (NeuroD1), two crucial transcriptional factors involved in human diabetes (OMIM: 601724) and islet development, have been previously found to directly target to the E-boxes of the insulinoma-associated 2 (Insm2) gene promoter, thereby activating the expression of Insm2 in insulin-secretion cells. However, little is known about the function of Insm2 in pancreatic islets and glucose metabolisms. METHODS: Homozygous Insm2-/- mice were generated by using the CRISPR-Cas9 method. Glucose-stimulated insulin secretion and islet morphology were analyzed by ELISA and immunostainings. Expression levels of Insm2-associated molecules were measured using quantitative RT-PCR and Western blots. RESULTS: Fasting blood glucose levels of Insm2-/- mice were higher than wild-type counterparts. Insm2-/- mice also showed reduction in glucose tolerance and insulin/C-peptide levels when compared to the wild-type mice. RT-PCR and Western blot analysis revealed that expression of Insm1 was significantly increased in Insm2-/- mice, suggesting a compensatory response of the homolog gene Insm1. Similarly, transcriptional levels of Ngn3 and NeuroD1 were also increased in Insm2-/- mice. Moreover, Insm2-/- female mice showed a significantly decreased reproductive capacity. CONCLUSIONS: Our findings suggest that Insm2 is important in glucose-stimulated insulin secretion and is involved in the development pathway of neuroendocrine tissues which are regulated by the transcription factors Ngn3, NeuroD1 and Insm1.
Assuntos
Deleção de Genes , Intolerância à Glucose/genética , Secreção de Insulina , Fatores de Transcrição/genética , Animais , Sequência de Bases , Feminino , Genótipo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos Knockout , Modelos Biológicos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismoRESUMO
INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity.
Assuntos
Células Epiteliais/metabolismo , Timo/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Linhagem Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Células Epiteliais/patologia , Humanos , Mutação , Estrutura Terciária de Proteína , Timo/patologia , Fatores de Transcrição/genética , Proteína AIRERESUMO
BACKGROUND: Insulinoma associated-1 (INSM1) gene is expressed exclusively in early embryonic neuroendocrine tissues, but has been found highly re-activated in most of the neuroendocrine tumors including small cell lung carcinoma. METHODS: In order to elucidate the functional effects of INSM1 in normal lung development, we used a conditional lung-specific INSM1 transgenic mouse model. Transgenic (Tet-on system) CMV-INSM1 responder mice were bred with the lung-specific, club cell secretory protein (CCSP) promoter-rtTA activator mice to produce bi-transgenic progeny carrying both alleles, CCSP-rtTA and Tet-on-INSM1. Mice were fed with doxycycline containing food at the initial mating day to the postnatal day 21. Lung samples were collected at embryonic day 17.5, newborn, and postnatal day 21 for analyses. RESULTS: Northern blot, RT-PCR, and immunohistochemical analyses revealed that doxycycline induced respiratory epithelium-specific INSM1 expression in bi-transgenic mice. Samples from postnatal day 21 mice revealed a larger lung size in the bi-transgenic mouse as compared to the single-transgenic or wild-type littermates. The histopathology results showed that the alveolar space in the bi-transgenic mice were 4 times larger than those in the single transgenic or wild-type littermates. In contrast, the size was not significantly different in the lungs collected at E17.5 or newborn among the bi-transgenic, single transgenic, or wild type mice. The respiratory epithelium with INSM1 ectopic expression suppressed cyclin D1 signal. Further in vitro studies revealed that the ectopic expression of INSM1 suppresses cyclin D1 expression and delays cell cycle progression. CONCLUSION: The current study suggests that CCSP promoter-driven INSM1 ectopic expression impairs normal lung development especially in postnatal alveologenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Expressão Ectópica do Gene/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Alvéolos Pulmonares/embriologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Fatores de Transcrição/genética , Animais , Northern Blotting , Western Blotting , Brônquios/citologia , Brônquios/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/metabolismo , Proteínas Repressoras , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Lung cancer is the leading cause of cancer-related deaths in the US. Recombinant vectors based on adeno-associated virus (AAV) and lentivirus are promising delivery tools for gene therapy due to low toxicity and long term expression. The efficiency of the gene delivery system is one of the most important factors directly related to the success of gene therapy. METHODS: We infected SCLC cell lines, SHP-77, DMS 53, NCI-H82, NCI-H69, NCI-H727, NCI-H1155, and NSCLC cell lines, NCI-H23, NCI-H661, and NCI-H460 with VSV-G pseudo-typed lentivirus or 5 AAV serotypes, AAV2/1, AAV2/2, AAV2/4, AAV2/5, and AAV2/8 expressing the CMV promoter mCherry or green fluorescent protein transgene (EGFP). The transduction efficiency was analyzed by fluorescent microscopy and flow cytometry. RESULTS: Of all the serotypes of AAV examined, AAV2/1 was the optimal serotype in most of the lung cancer cell lines except for NCI-H69 and NCI-H82. The highest transduction rate achieved with AAV2/1 was between 30-50% at MOI 100. Compared to all AAV serotypes, lentivirus had the highest transduction efficiency of over 50% at MOI 1. Even in NCI-H69 cells resistant to all AAV serotypes, lentivirus had a 10-40% transduction rate. To date, AAV2 is the most widely-used serotype to deliver a transgene. Our results showed the transduction efficiency of AAVs tested was AAV2/1 > AA2/5 = AAV2/2> > AAV2/4 and AAV2/8. CONCLUSIONS: This study demonstrated that VSV-G pseudotyped lentivirus and AAV2/1 can mediate expression of a transgene for lung cancer gene therapy. Overall, our results showed that lentivirus is the best candidate to deliver a transgene into lung cancer cells for treatment.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Lentivirus/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Glicoproteínas de Membrana/genética , Transdução Genética/métodos , Proteínas do Envelope Viral/genética , Linhagem Celular Tumoral , Dependovirus/fisiologia , Terapia Genética/instrumentação , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Lentivirus/metabolismo , Neoplasias Pulmonares/virologia , Glicoproteínas de Membrana/metabolismo , Transdução Genética/instrumentação , Proteínas do Envelope Viral/metabolismoRESUMO
Human neuroblastoma (NB) is the most common childhood extracranial tumor arising from the sympathetic nervous system. It is also a clinically heterogeneous disease that ranges from spontaneous regression to high-risk stage 4 disease. The cause of this disease remains elusive. However, the amplification of NMYC oncogene occurred in roughly 30% of NB patients, which strongly correlated with the advanced stage of disease subtype and the worse prognosis status. We discovered that N-Myc oncoprotein binds and activates INSM1, a zinc-finger transcription factor of neuroendocrine tumors. We also found that INSM1 modulates N-Myc stability mediated through PI3K/AKT/GSK3ß signaling pathway. Therefore, INSM1 emerges as a critical co-player with N-Myc in facilitating NB tumor cell growth and sustaining the advanced stage of malignancy. Using an INSM1-promoter driven luciferase screening-platform, we have recently identified fifteen small molecules that negatively regulate INSM1 expression. Interestingly, the identified small molecules can be divided into four large groups of compounds such as cell signaling inhibitor, DNA/RNA inhibitor, HDAC inhibitor, and cardiac glycoside. These findings support the presence of a unique mechanism associated with INSM1 and N-Myc interplay, which is critical in regulating NB tumor cell growth. We discuss the feasibility of identifying novel or repurposing small molecules targeting INSM1 as a potential treatment option for high-risk NB.
RESUMO
High-risk neuroblastoma (NB) is a heterogeneous and malignant childhood cancer that is frequently characterized by MYCN proto-oncogene amplification or elevated N-Myc protein (N-Myc) expression. An N-Myc downstream target gene, insulinoma associated-1 (INSM1) has emerged as a biomarker that plays a critical role in facilitating NB tumor cell growth and transformation. N-Myc activates endogenous INSM1 gene expression through binding to the E2-box of the INSM1 proximal promoter in NB. We identified a plant alkaloid, homoharringtonine (HHT), from a chemical library screening showing potent inhibition of INSM1 promoter activity. This positive-hit plant alkaloid exemplifies an effective screening approach for repurposed compound targeting INSM1 expression in NB cancer therapy. The elevated N-Myc and INSM1 expression in NB constitutes a positive-loop through INSM1 activation that promotes N-Myc stability. In the present study, the biological effects and anti-tumor properties of HHT against NB were examined. HHT either down regulates and/or interferes with the binding of N-Myc to the E2-box of the INSM1 promoter and the inhibition of PI3K/AKT-mediated N-Myc stability could lead to the NB cell apoptosis. HHT inhibition of NB cell proliferation is consistent with the INSM1 expression as higher level of INSM1 exhibits a more sensitive IC50 value. The combination treatment of HHT and A674563 provides a better option of increasing potency and reducing cellular cytotoxicity than HHT or A674563 treatment alone. Taken together, the suppression of the INSM1-associated signaling pathway axis promotes the inhibition of NB tumor cell growth. This study developed a feasible approach for repurposing an effective anti-NB drug.
Assuntos
Insulinoma , Neuroblastoma , Neoplasias Pancreáticas , Humanos , Criança , Mepesuccinato de Omacetaxina , Fosfatidilinositol 3-Quinases/metabolismo , Reposicionamento de Medicamentos , Linhagem Celular Tumoral , Neuroblastoma/genética , Neoplasias Pancreáticas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Repressoras/metabolismoRESUMO
In this study, the functional role of INSM1 is examined with an AR42J acinar cell model for trans-differentiation into insulin-positive cells. Islet transcription factors (ITFs: INSM1, Pdx-1, and NeuroD1) are over-expressed in AR42J cells using adenoviral vectors. Addition of Ad-INSM1 alone or the combination of three ITFs to the AR42J cells triggers cellular trans-differentiation. Ectopic expression of INSM1 directly induces insulin, Pax6, and Nkx6.1 expression, whereas Pdx-1 and NeuroD1 were slightly suppressed by INSM1. Addition of Pdx-1 and NeuroD1 with INSM1 further enhances endocrine trans-differentiation by increasing both the numbers and intensity of the insulin-positive cells with simultaneous activation of ITFs, Ngn3 and MafA. INSM1 expression alone partially inhibits dexamethasone-induced exocrine amylase expression. The combination of the three ITFs completely inhibits amylase expression and concomitantly induces greater acinar cell trans-differentiation into endocrine cells. Also, addition of the three ITFs promotes EGF and TGFß receptors expression. Stimulation by the three ITFs along with the EGF/TGFß growth factors strongly promotes insulin gene expression. The combination of the three ITFs and EGF/TGFß growth factors with the primary cultured pancreatic acini also facilitates exocrine to endocrine cell differentiation. Taken together, both the AR42J cell line and the primary cultured mouse acinar cells support INSM1 induced acini trans-differentiation model.
Assuntos
Células Acinares/metabolismo , Transdiferenciação Celular , Ilhotas Pancreáticas/metabolismo , Proteínas Repressoras/metabolismo , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Adenoviridae/genética , Amilases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Transdiferenciação Celular/efeitos dos fármacos , Cricetinae , Dexametasona/farmacologia , Receptores ErbB/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Vetores Genéticos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Repressoras/genética , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
CD1d-expressing cells present lipid Ag to CD1d-restricted NKT cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Overexpression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and downregulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific small interfering RNA increases CD1D transcripts in K562 and Jurkat cells but there are different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using small interfering RNA potentiates an acetylation state of histone H3/H4, supporting the notion that LEF-1 acts as a transcriptional repressor for the CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of the human CD1D gene.
Assuntos
Antígenos CD1d/genética , Regulação para Baixo/imunologia , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Regiões Promotoras Genéticas/imunologia , Proteínas Repressoras/genética , Antígenos CD1d/biossíntese , Antígenos CD1d/metabolismo , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Células K562 , Fator 1 de Ligação ao Facilitador Linfoide/antagonistas & inibidores , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Interferente Pequeno/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteínas Wnt/fisiologiaRESUMO
An aggressive form of neuroblastoma (NB), a malignant childhood cancer derived from granule neuron precursors and sympathoadrenal lineage, frequently comprises MYCN amplification/elevated N-Myc expression, which contributes to the development of neural crest-derived embryonal malignancy. N-Myc is an oncogenic driver in NB. Persistent N-Myc expression during the maturation of SA precursor cells can cause blockage of the apoptosis and induce abnormal proliferation, resulting in NB development. An insulinoma-associated-1 (INSM1) zinc-finger transcription factor has emerged as an NB biomarker that plays a critical role in facilitating tumor cell growth and transformation. INSM1 plays an essential role in sympathoadrenal cell differentiation. N-Myc activates endogenous INSM1 through an E2-box of the INSM1 proximal promoter, whereas INSM1 enhances N-Myc stability via RAC-α-serine/threonine protein kinase (AKT) phosphorylation in NB. The ectopic expression of INSM1 stimulates NB tumor growth in contrast to the knockdown of INSM1 that inhibits NB cell proliferation. The clinical pathological result and bioinformatics analysis show that INSM1 is a strong diagnostic and a prognostic biomarker for the evaluation of NB progression. The INSM1/N-Myc expression shows high clinical relevance in NB. Therefore, targeting the INSM1/N-Myc-associated signaling axis should be a feasible approach to identifying new drugs for the suppression of NB tumor growth.
RESUMO
Zinc-finger transcription factors are DNA-binding proteins that are implicated in many diverse biological functions. INSM1 (formerly IA-1) contains five zinc-finger motifs and functions as a transcription factor. INSM1 protein structure is highly conserved in homologues of different species. It is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals. INSM1 represents an important player in early embryonic neurogenesis. In pancreatic endocrine cell differentiation, Ngn3 first activates INSM1 and subsequently NeuroD/beta2. Conversely, INSM1 exerts a feedback mechanism to suppress NeuroD/beta2 and its own gene expression. INSM1 gene ablation in the mouse results in the impairment of pancreatic endocrine cell maturation. Further, deletion of INSM1 severely impairs catecholamine biosynthesis and secretion from the adrenal gland that results in early embryonic lethality. Genetically, INSM1 acts as a downstream factor of Mash 1 and Phox2b in the differentiation of the sympatho-adrenal lineage. In the developing neocortex, mouse embryos lacking INSM1 expression contain half the number of basal progenitors and show a reduction in cortical plate radial thickness. Cell signaling studies reveal that INSM1 contributes to the induction of cell cycle arrest/exit necessary to facilitate cellular differentiation. INSM1 is highly expressed in tumors of neuroendocrine origin. Hence, its promoter could serve as a tumor-specific promoter that drives a specific targeted cancer gene therapy for the treatment of neuroendocrine tumors. Taken together, all of these features of INSM1 strongly support its role as an important regulator during neuroendocrine differentiation.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Células Neuroendócrinas/citologia , Proteínas Repressoras/fisiologia , Carcinoma Neuroendócrino/química , Carcinoma Neuroendócrino/patologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Humanos , Pâncreas/citologia , Proteínas Repressoras/análise , Proteínas Repressoras/genética , Fatores de Transcrição/fisiologiaRESUMO
Aggressive form of neuroblastoma (NB) is a malignant childhood cancer derived from granule neuron precursors and sympatho-adrenal lineage with N-MYC amplification. An insulinoma associated-1 (INSM1) transcription factor has emerged as a NB biomarker that plays critical role in facilitating tumor cell growth and transformation. N-myc activates INSM1 in NB was discovered. In order to elucidate the signaling pathways associated with INSM1 expression and NB tumor cell growth, we developed an INSM1 promoter-driven luciferase assay for new drug discovery. Promoter-driven luciferase assay demonstrated high Z' factor (>0.7). Performance measures using signal-to-noise ratio, signal window, and Z' factor confirmed this assay is a robust screening method. A panel of FDA-approved oncology drugs set (147 compounds) was subjected to the INSM1 promoter-driven luciferase assay. The positive-hit compounds were validated for effects on cell viability and INSM1 expression. Screening a FDA-approved oncology drugs set revealed multiple inhibitors against various signaling pathways via suppression of INSM1 promoter activity. The positive-hit compounds consist of 9 signaling pathway inhibitors negatively regulates INSM1 expression and cell viability in NB. These inhibitors target DNA/RNA/protein synthesis, tubulin assembly, and histone deacetylase signaling pathways. The outcome of this assay indicates that the newly identified pathways could be critical for NB growth and transformation. This technology and the positive-hits of multiple pathways represent a promising option of identifying re-purposed FDA-approved drugs valuable for NB treatment in the context of a NB-specific marker, INSM1.
Assuntos
Antineoplásicos/farmacologia , Reposicionamento de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neuroblastoma/tratamento farmacológico , Proteínas Repressoras/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
Insulinoma-associated-1 (IA-1 or INSM1) encodes a zinc-finger transcription factor, which was isolated from a human insulinoma subtraction library, with specific expression patterns, predominantly in developing neuroendocrine tissues and tumors. INSM1 is key in early pancreatic endocrine, sympatho-adrenal lineage, and pan-neurogenic precursor development. Insm1 gene ablation results in impairment of pancreatic ß cells, catecholamine biosynthesis, and basal progenitor development during mammalian neocortex maturation. Recently, INSM1 has emerged as a superior, sensitive, and specific biomarker for neuroendocrine tumors. INSM1 regulates downstream target genes and exhibits extranuclear activities associated with multiple signaling pathways, including Sonic Hedgehog, PI3K/AKT, MEK/ERK1/2, ADK, p53, Wnt, histone acetylation, LSD1, cyclin D1, Ascl1, and N-myc. Novel strategies targeting INSM1-associated signaling pathways facilitate the suppression of neuroendocrine tumor growth. In addition, INSM1 promoter-driven reporter assay and/or suicide gene therapy are promising effective therapeutic approaches for targeted specific neuroendocrine tumor therapy. In this review, the current knowledge of the biological role of INSM1 as a neuroendocrine tumor biomarker is summarized, and novel strategies targeting multiple signaling pathways in the context of INSM1 expression in neuroendocrine tumors are further explored. IMPLICATIONS: Neuroendocrine transcription factor (INSM1) may serve as a neuroendocrine biomarker for the development of novel cancer therapeutics against neuroendocrine tumors.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Terapia de Alvo Molecular , Tumores Neuroendócrinos/diagnóstico , Proteínas Repressoras/metabolismo , Biomarcadores Tumorais/genética , Humanos , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/metabolismo , Proteínas Repressoras/genética , Transdução de SinaisRESUMO
Neuroendocrine (NE) lung tumors account for 20% of total lung cancer cases and represent a subset of aggressive tumors with metastatic potential. High-risk NE lung cancer patients display disseminated disease, N-myc expression/amplification, and poorly differentiated tumors. In this study, we investigate the molecular mechanisms underlying a zinc-finger transcription factor, INSM1 in NE lung cancer. Our study revealed that INSM1 crosstalk with the Shh-PI3K/AKT-N-myc/Ascl1-MEK/ERK1/2 transcriptional network in NE lung cancer. The INSM1 expression pattern and functional data demonstrated that INSM1 is not only critical for NE differentiation, but also served as a NE tumor-specific marker in small cell lung carcinoma (SCLC). The Shh signaling pathway activates INSM1 expression through N-myc and Ascl1 in aggressive SCLC. The E2-box in the INSM1 promoter is the direct target recognized by N-myc and Ascl1 transcription factors. N-myc or Ascl1 activates endogenous INSM1 expression in lung cancer cells. INSM1 functions as a key player in NE lung cancer via Shh signaling that crosstalk with PI3K/AKT and MEK/ERK1/2 pathway to enhance N-myc stability in NE lung cancer. We investigate the negative effects of Shh inhibitor and knockdown of INSM1 in NE lung cancer cells. The combination of different Shh signaling pathway inhibitors targeting INSM1 and N-myc inhibits lung cancer cell growth and could be used as a new treatment option for SCLC.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Hedgehog/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Tumores Neuroendócrinos/metabolismo , Proteínas Repressoras/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Transdução de SinaisRESUMO
Pax4, a paired-box transcription factor, is a key regulator of pancreatic islet cell growth and differentiation. Here, we report for the first time that the Pax4 protein can permeate into various cell types including pancreatic islets. The paired domain of Pax4 serves as a novel protein transduction domain (PTD). The Pax4 protein can transduce in a dose- and time-dependent manner. The cellular uptake of Pax4 PTD can be completely blocked by heparin, whereas cytochalasin D and amiloride were partially effective in blocking the Pax4 protein entry. Transduced intact Pax4 protein functions similarly to the endogenous Pax4. It inhibits the Pax6 mediated transactivation and protects Min6 cells against TNFalpha-induced apoptosis. These data suggest that Pax4 protein transduction could be a safe and valuable strategy for protecting islet cell growth in culture from apoptosis and promoting islet cell differentiation.
Assuntos
Permeabilidade da Membrana Celular , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/farmacologia , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/farmacologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
INSM1 is a downstream target gene of neurogenin 3 (ngn3). A promoter construct containing the -426/+40bp region transiently co-transfected into NIH-3T3 cells with a ngn3 expression plasmid resulted in a 12-fold increase in promoter activity. The ngn3/E47 heterodimer selectively binds and activates the E-box3 of the INSM1 promoter. The endogenous ngn3 and CREB-binding protein (CBP) co-activator occupy the INSM1 promoter, resulting in hyper-acetylation of histone H3/H4 chromatin in a human neuroblastoma cell line, IMR-32. Additionally, adenoviral ngn3 can induce endogenous INSM-1 expression in pancreatic ductal carcinoma-1 cells through the recruitment of CBP to the INSM1 promoter and increase the acetylation of the INSM1 promoter region.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Acetilação , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/genética , Linhagem Celular , Corticosterona , Primers do DNA/genética , Regulação da Expressão Gênica , Histonas/química , Humanos , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
INSM1/IA-1 (insulinoma-associated 1) is a developmentally regulated zinc-finger transcription factor, exclusively expressed in the foetal pancreas and nervous system, and in tumours of neuroendocrine origin. We have identified an INSM1 binding site in the neuroD/beta2 promoter and demonstrated transcriptional repressor activity of INSM1 by transient transfection assay. A chromatin immunoprecipitation assay confirmed that in vivo INSM1 is situated on the promoter region of the neuroD/beta2 gene. In an attempt to elucidate the molecular mechanism of transcriptional repression by the INSM1 gene, cyclin D1 was identified as an interacting protein by using a 45-day-old human foetal brain cDNA library and a yeast two-hybrid screen. The physical association between INSM1 and cyclin D1 was confirmed by in vitro and in vivo pull-down assay. Cyclin D1 co-operates with INSM1 and suppresses neuroD/beta2 promoter activity. Co-immunoprecipitation of INSM1, cyclin D1 and HDACs (histone deacetylases) in mammalian cells revealed that INSM1 interacts with HDAC-1 and -3 and that this interaction is mediated through cyclin D1. Overexpression of cyclin D1 and HDAC-3 significantly enhanced the transcriptional repression activity of INSM1 on the neuroD/beta2 promoter. A further chromatin immunoprecipitation assay confirmed that HDAC-3 occupies this same region of the neuroD/beta2 promoter, by forming a transcription complex with INSM1. Thus we conclude that INSM1 recruits cyclin D1 and HDACs, which confer transcriptional repressor activity.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Repressoras/fisiologia , Adenoviridae/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Neoplasias do Tronco Encefálico , Imunoprecipitação da Cromatina , Ciclina D1/fisiologia , Histona Desacetilase 1 , Histona Desacetilase 2 , Histona Desacetilases/metabolismo , Humanos , Rim/citologia , Meduloblastoma , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the majority of the repressor activity. Using a selected and amplified random oligonucleotide binding assay and bacterially expressed GST-IA-1DBD fusion protein (257-510 a.a.), we identified the consensus IA-1 binding sequence, TG/TC/TC/TT/AGGGGG/TCG/A. Further experiments showed that zinc-fingers 2 and 3 of IA-1 are sufficient to demonstrate transcriptional activity using an IA-1 consensus site containing a reporter construct. A database search with the consensus IA-1 binding sequence revealed target sites in a number of pancreas- and brain-specific genes consistent with its restricted expression pattern. The most significant matches were for the 5'-flanking regions of IA-1 and NeuroD/beta2 genes. Co-transfection of cells with either the full-length IA-1 or hEgr-1AD/IA-1DBD construct and IA-1 or NeuroD/beta2 promoter/CAT construct modulated CAT activity. These findings suggest that the IA-1 protein may be auto-regulated and play a role in pancreas and neuronal development, specifically in the regulation of the NeuroD/beta2 gene.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Encéfalo/metabolismo , Diferenciação Celular , Sequência Consenso , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas do Olho , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Pâncreas/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Alinhamento de Sequência , Transativadores/genética , Transcrição Gênica , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
BACKGROUND: Insulinomas are the most common type of neuroendocrine (NE) pancreatic islet tumors. Patients with insulinomas may develop complications associated with hyperinsulinemia. To increase the treatment options for insulinoma patients, we have tested a conditionally replicating adenovirus that has been engineered in such a way that it can specifically express therapeutic genes in NE tumors. METHODS: We used a promoter-specific adenoviral vector delivery system that is regulated by an INSM1 (insulinoma-associated-1) promoter, which is silent in normal adult tissues but active in developing NE cells and tumors. Through a series of modifications, using an insulator (HS4) and neuron-restrictive silencer elements (NRSEs), an oncolytic adenoviral vector was generated that retains tumor specificity and drives the expression of a mutated adenovirus E1A gene (Δ24E1A) and the herpes simplex virus thymidine kinase (HSV-tk) gene. The efficacy of this vector was tested in insulinoma-derived MIN, RIN, ßTC-1 and pancreatic (Panc-1) cells using in vitro cell survival and in vivo tumor growth assays. RESULTS: Using in vitro insulinoma-derived cell lines and an in vivo subcutaneous mouse tumor model we found that the INSM1 promoter-driven viruses were able to replicate specifically in INSM1-positive cells. INSM1-specific HSV-tk expression in combination with ganciclovir treatment resulted in dose-dependent tumor cell killing, leaving INSM1-negative cells unharmed. When we combined the INSM1-promoter driven HSV-tk with Δ24E1A and INSM1p-HSV-tk (K5) viruses, we found that the co-infected insulinoma-derived cells expressed higher levels of HSV-tk and exhibited more efficient tumor suppression than cells infected with INSM1p-HSV-tk virus alone. CONCLUSIONS: INSM1 promoter-driven conditionally replicating adenoviruses may serve as a new tool for the treatment of insulinoma and may provide clinicians with additional options to combat this disease.
Assuntos
Terapia Genética/métodos , Insulinoma , Terapia Viral Oncolítica/métodos , Neoplasias Pancreáticas , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Adenoviridae , Animais , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Ganciclovir/farmacologia , Vetores Genéticos , Humanos , Camundongos , Timidina Quinase/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Following the publication of this article, an interested reader drew to our attention an anomaly associated with the presentation of Figs. 5 and 8; essentially, the ß-actin control bands in Fig. 8 appeared to be a mirror image of the same control bands shown in Fig. 5A. After having reexamined the data, Fig. 5 was correctly presented as shown in the paper, although the ß-actin blot in Fig. 8 had been inadvertently misplaced as a mirror image of the control data from Fig. 5. Two similar experiments were performed at that time, yielding similar results, and the error with the control data did not affect the second of the experiments. Therefore, we present a corrected version of Fig. 8 containing the alternative data. The findings and conclusions of this paper are still supported by our experimental data, and were not affected by this error. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused. [the original article was published in the International Journal of Oncology 42: 1113-1119, 2013; DOI: 10.3892/ijo.2013.1789].