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Dror Avni - One of the best experts on this subject based on the ideXlab platform.
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abstract a15 mir 377 targets e2f3 and alters the nf κb signaling pathway through MAP3K7 in malignant melanoma
Cancer Research, 2016Co-Authors: Liron Zehavi, Jasmine Jacobhirsch, Yechezkel Sidi, Raya Leibowitzamit, Adi Layani, Dror AvniAbstract:Background: The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We were the first to report that a large micro-RNA (miRNA) cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma, and have been studying this ‘tumor-suppressor miRNA cluster’ for several years now. MiR-377, one of the miRNAs located within this cluster, was studied in this work. Methods: qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection and mRNA expression was assessed using mRNA arrays. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques and protein expression was assessed by western blot. Results: miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 39UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7, a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is known to be involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-κB signaling pathway. Conclusion Our results suggest that miR-377 is an important negative regulator of E2F and of the MAP3K7/NF-κB signaling pathway in melanoma cells. The NF-κB signaling has been implicated in the acquisition of resistance to B-Raf inhibition. It is tempting to speculate that silencing of miR-377 in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways. Indeed we found in preliminary results that overexpression of miR-377 in melanoma cells resistance to PLX-4032 (Zelboraf), re-sensitized the cells to the drug. Citation Format: Liron Zehavi, Adi Layani, Jasmine Jacob-Hirsch, Yechezkel Sidi, Raya Leibowitz-Amit, Dror Avni. MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A15.
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mir 377 targets e2f3 and alters the nf kb signaling pathway through MAP3K7 in malignant melanoma
Molecular Cancer, 2015Co-Authors: Liron Zehavi, Hagit Schayek, Jasmine Jacobhirsch, Yechezkel Sidi, Raya Leibowitzamit, Dror AvniAbstract:The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We recently reported that a large miRNAs cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma. miR-377, one of the miRNAs located within this cluster, was studied here. qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection, mRNA expression was assessed using mRNA arrays and protein expression was assessed by Western blot analysis. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques. miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3′UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7 (known as TAK1), a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-kB signaling pathway. Our results suggest that miR-377 is an important negative regulator of E2F and MAP3K7/NF-kB signaling pathway in melanoma cells; it is tempting to speculate that its silencing in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways.
Liron Zehavi - One of the best experts on this subject based on the ideXlab platform.
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abstract a15 mir 377 targets e2f3 and alters the nf κb signaling pathway through MAP3K7 in malignant melanoma
Cancer Research, 2016Co-Authors: Liron Zehavi, Jasmine Jacobhirsch, Yechezkel Sidi, Raya Leibowitzamit, Adi Layani, Dror AvniAbstract:Background: The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We were the first to report that a large micro-RNA (miRNA) cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma, and have been studying this ‘tumor-suppressor miRNA cluster’ for several years now. MiR-377, one of the miRNAs located within this cluster, was studied in this work. Methods: qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection and mRNA expression was assessed using mRNA arrays. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques and protein expression was assessed by western blot. Results: miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 39UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7, a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is known to be involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-κB signaling pathway. Conclusion Our results suggest that miR-377 is an important negative regulator of E2F and of the MAP3K7/NF-κB signaling pathway in melanoma cells. The NF-κB signaling has been implicated in the acquisition of resistance to B-Raf inhibition. It is tempting to speculate that silencing of miR-377 in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways. Indeed we found in preliminary results that overexpression of miR-377 in melanoma cells resistance to PLX-4032 (Zelboraf), re-sensitized the cells to the drug. Citation Format: Liron Zehavi, Adi Layani, Jasmine Jacob-Hirsch, Yechezkel Sidi, Raya Leibowitz-Amit, Dror Avni. MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A15.
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mir 377 targets e2f3 and alters the nf kb signaling pathway through MAP3K7 in malignant melanoma
Molecular Cancer, 2015Co-Authors: Liron Zehavi, Hagit Schayek, Jasmine Jacobhirsch, Yechezkel Sidi, Raya Leibowitzamit, Dror AvniAbstract:The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We recently reported that a large miRNAs cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma. miR-377, one of the miRNAs located within this cluster, was studied here. qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection, mRNA expression was assessed using mRNA arrays and protein expression was assessed by Western blot analysis. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques. miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3′UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7 (known as TAK1), a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-kB signaling pathway. Our results suggest that miR-377 is an important negative regulator of E2F and MAP3K7/NF-kB signaling pathway in melanoma cells; it is tempting to speculate that its silencing in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways.
Ying Xia - One of the best experts on this subject based on the ideXlab platform.
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sphingosine 1 phosphate activates the map3k1 jnk pathway to promote epithelial movement and morphogenesis
bioRxiv, 2020Co-Authors: Jingjing Wang, Maureen Mongan, Jerold Chun, Ying XiaAbstract:MAP 3 kinase 1 (MAP3K1) plays an essential role in embryonic eyelid development. It regulates epithelial morphogenesis through the spatial-temporal activation of Jun N-terminal kinases (JNKs), resulting in forward progression of the embryonic eyelid epithelial cells to enable eyelid closure. The developmental signals that activate the MAP3K1-JNK pathway are still unknown, mainly due to the lack of suitable keratinocyte lines to elucidate the mechanisms of pathway regulation. To address this deficiency, we developed a straightforward method for long-term culture of mouse keratinocytes in feeder-free conditions using Ca2+-free media. Cells grown under these conditions displayed characteristic basal epithelial morphology and keratin 14 expression, but did not form tight- or adherens-junctions. Increased extracellular Ca2+ levels restored the formation of cell-cell junctions. Using keratinocyte lines derived from wild type and Map3k1-deficient mice, we found that sphingosine 1-phosphate (S1P) activated the JNK-c-JUN pathways in a manner dependent on MAP3K1 kinase activity and that this MAP3K1-mediated signaling led to epithelial cell migration. The in vivo roles of this pathway were examined through crossing of genetic mutant mice. Loss-of-function of the S1P receptor (S1pr) 2/3 became haploinsufficient only when combined with Map3k1 and Jnk1 mutations such that the compound mutants displayed eyelid closure defects, suggesting these gene products cooperated in eye morphogenesis. Results of this work establish the S1PR-MAP3K1-JNK pathway as a crucial signaling mechanism for epithelial cell movement and morphogenesis.
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repression of map3k1 expression and jnk activity by canonical wnt signaling
Developmental Biology, 2018Co-Authors: Qinghang Meng, Maureen Mongan, Jingjing Wang, Ying XiaAbstract:Morphogenesis is a complex and highly coordinated process orchestrated by temporal spatial activity of developmental pathways. How the different pathways interact to guide the developmental program remains an intriguing and open question. MAP3K1-JNK and Wnt are signaling pathways crucial for embryonic eyelid closure, an epithelial morphogenetic event conserved in mammals. Here we used a mouse model of eyelid development and genetic and biochemistry tools to investigate the relationships between the two pathways. We found that Wnt activation repressed MAP3K1 expression. Using Axin-LacZ reporter mice, spatial Wnt activity was detected in the leading edge of the developing eyelid. Conditional knockout of Wntless (Wls) in ocular surface ectoderm blocked eyelid formation, and significantly increased MAP3K1 expression in eyelid cells at the nasal canthus region. Conversely, knockout of Dkk2, encoding a canonical Wnt antagonist, resulted in an increase of Wnt activity in cells at the upper eyelid margin near the nasal canthus. Up-regulation of Wnt signaling in the Dkk2-knockout embryos corresponded to down-regulation of MAP3K1 expression. In vitro data showed that Wnt3a treatment decreased MAP3K1 promoter activity, whereas activation of Wnt by lithium chloride inhibited MAP3K1 expression, and attenuated MAP3K1-mediated JNK activity. Our data identify a unique signal crosstalk between Wnt signaling and the MAP3K1-JNK pathway in epithelial morphogenesis.
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map3k1 function is essential for cytoarchitecture of the mouse organ of corti and survival of auditory hair cells
Disease Models & Mechanisms, 2015Co-Authors: Rizwan Yousaf, Qinghang Meng, Ying Xia, Robert B Hufnagel, Chandrakala Puligilla, Zubair M Ahmed, Saima RiazuddinAbstract:MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its effect through various downstream effecter molecules, including JNK, ERK1/2 and p38. In humans, mutant alleles of MAP3K1 are associated with 46,XY sex reversal. Until recently, the only phenotype observed in Map3k1(tm1Yxia) mutant mice was open eyelids at birth. Here, we report that homozygous Map3k1(tm1Yxia) mice have early-onset profound hearing loss accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic junctions in the organ of Corti are grossly preserved, Map3k1(tm1Yxia) mutant mice have supernumerary functional outer hair cells (OHCs) and Deiters' cells. Loss of MAP3K1 function resulted in the downregulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and Fgf10 have a role in induction of the otic placode or in otic epithelium development in mice, and their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear morphogenesis genes. Collectively, our data highlight the crucial role of MAP3K1 in the development and function of the mouse inner ear and hearing.
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gene environment interactions target mitogen activated protein 3 kinase 1 map3k1 signaling in eyelid morphogenesis
Journal of Biological Chemistry, 2015Co-Authors: Maureen Mongan, Qinghang Meng, Alvaro Puga, Winston W Y Kao, Jingjing Wang, Ying XiaAbstract:Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure.
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Epithelial sheet movement requires the cooperation of c-Jun and MAP3K1.
Developmental biology, 2014Co-Authors: Qinghang Meng, Maureen Mongan, Winston W Y Kao, Jingjing Wang, Xiaofang Tang, Jinling Zhang, Ying XiaAbstract:Epithelial sheet movement is an essential morphogenetic process during mouse embryonic eyelid closure in which Mitogen-Activated Protein 3 Kinase 1 (MAP3K1) and c-Jun play a critical role. Here we show that MAP3K1 associates with the cytoskeleton, activates Jun N-terminal kinase (JNK) and actin polymerization, and promotes the eyelid inferior epithelial cell elongation and epithelium protrusion. Following epithelium protrusion, c-Jun begins to express and acts to promote ERK phosphorylation and migration of the protruding epithelial cells. Homozygous deletion of either gene causes defective eyelid closure, but non-allelic non-complementation does not occur between Map3k1 and c-Jun and the double heterozygotes have normal eyelid closure. Results from this study suggest that MAP3K1 and c-Jun signal through distinct temporal-spatial pathways and that productive epithelium movement for eyelid closure requires the consecutive action of MAP3K1-dependent cytoskeleton reorganization followed by c-Jun-mediated migration.
David Cordas Dos M Santos - One of the best experts on this subject based on the ideXlab platform.
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MAP3K7 is recurrently deleted in pediatric t lymphoblastic leukemia and affects cell proliferation independently of nf κb
BMC Cancer, 2018Co-Authors: David Cordas Dos M Santos, Juliane Eilers, Alfonso Vizcaino, Elena Orlova, Martin Zimmermann, Martin Stanulla, Martin Schrappe, Kathleen BornerAbstract:Deletions of 6q15–16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4. MAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. MAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α. This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts.
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MAP3K7 is recurrently deleted in pediatric t lymphoblastic leukemia and affects cell proliferation independently of nf κb
Blood, 2017Co-Authors: David Cordas Dos M Santos, Juliane Eilers, Alfonso Vizcaino, Elena Orlova, Martin Zimmermann, Martin Stanulla, Martin Schrappe, Kathleen BornerAbstract:Abstract Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which is crucial for innate immune signaling. It is functionally and prognostically relevant both in hematologic malignancies and solid tumors. However, its distinct biological functions and relevance for different tumor entities are cell-type specific and remain controversial. Here, we show that MAP3K7 is recurrently deleted in pediatric T-ALL cases and that a residual expression of MAP3K7 is indispensable for T-ALL cell lines. We determined the frequency of MAP3K7 deletions in a cohort of 327 pediatric T-ALL patients by multiplex ligation-dependent probe amplification (MLPA). In a subgroup, we additionally employed targeted sequencing to identify point mutations. Deletions of MAP3K7 were identified in approximately 10% and point mutations in approximately 1% of children with T-ALL. All deletions of 6q15 were heterozygous and the adjacent CASP8AP2 gene was co-deleted in 32 of 33 cases. Clinically, the MAP3K7/CASP8AP2 deletions were significantly associated with a mature T-ALL immunophenotype, but not with any other patient characteristics or with the cumulative incidence of relapse and overall survival. Furthermore, deletions of MAP3K7/CASP8AP2 were preferentially associated with SIL-TAL1 fusions. Within the subgroup of SIL-TAL1 positive patients, those with MAP3K7/CASP8AP2 deletion showed a trend towards a higher risk of relapse compared to SIL-TAL1 -positive MAP3K7/CASP8AP2 -negative patients (25% vs 8%, p=0.13). We next analyzed the effects of shRNA-mediated MAP3K7 depletion on cell proliferation, apoptosis and NF-κB activation in three T-ALL cell lines (CCRF-CEM, Jurkat, MOLT4) that do not carry MAP3K7 deletions. We aimed at phenocopying a MAP3K7 deletion by using an Adeno-associated viral vector-mediated transduction system to efficiently deliver anti-MAP3K7 shRNA together with a GFP reporter to these cell lines. Transduction efficiency was above 80% for the cell lines CCRF-CEM and Jurkat. Depletion efficiency was uniformly in the range of 75%. Treatment with anti-MAP3K7 shRNA in comparison to non-silencing shRNA significantly slowed down proliferation in all three T-ALL cell lines and resulted in an increase of apoptotic cells up to 8-fold as measured by Annexin V staining. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. Stimulation with TNF-α led to rapid and transient degradation of IκB and an accumulation of phospho-NF-κB p65 (Ser536), while other NF-κB proteins (p100, p105, p50) remained unaffected. Depletion of MAP3K7 neither changed IκB levels in unstimulated cells nor did it prevent the degradation of IκB after stimulation with TNF-α. Furthermore mRNA expression of none of the NF-κB target genes was consistently changed by more than 2-fold in any of the cell lines after MAP3K7 depletion. We conclude that in T-ALL cell lines, MAP3K7 is not required for the degradation of IκB and subsequent activation of NF-κB after stimulation with TNF-α. This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts. Disclosures Schrappe: Medac: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
Yechezkel Sidi - One of the best experts on this subject based on the ideXlab platform.
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abstract a15 mir 377 targets e2f3 and alters the nf κb signaling pathway through MAP3K7 in malignant melanoma
Cancer Research, 2016Co-Authors: Liron Zehavi, Jasmine Jacobhirsch, Yechezkel Sidi, Raya Leibowitzamit, Adi Layani, Dror AvniAbstract:Background: The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We were the first to report that a large micro-RNA (miRNA) cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma, and have been studying this ‘tumor-suppressor miRNA cluster’ for several years now. MiR-377, one of the miRNAs located within this cluster, was studied in this work. Methods: qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection and mRNA expression was assessed using mRNA arrays. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques and protein expression was assessed by western blot. Results: miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 39UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7, a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is known to be involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-κB signaling pathway. Conclusion Our results suggest that miR-377 is an important negative regulator of E2F and of the MAP3K7/NF-κB signaling pathway in melanoma cells. The NF-κB signaling has been implicated in the acquisition of resistance to B-Raf inhibition. It is tempting to speculate that silencing of miR-377 in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways. Indeed we found in preliminary results that overexpression of miR-377 in melanoma cells resistance to PLX-4032 (Zelboraf), re-sensitized the cells to the drug. Citation Format: Liron Zehavi, Adi Layani, Jasmine Jacob-Hirsch, Yechezkel Sidi, Raya Leibowitz-Amit, Dror Avni. MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A15.
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mir 377 targets e2f3 and alters the nf kb signaling pathway through MAP3K7 in malignant melanoma
Molecular Cancer, 2015Co-Authors: Liron Zehavi, Hagit Schayek, Jasmine Jacobhirsch, Yechezkel Sidi, Raya Leibowitzamit, Dror AvniAbstract:The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We recently reported that a large miRNAs cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma. miR-377, one of the miRNAs located within this cluster, was studied here. qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection, mRNA expression was assessed using mRNA arrays and protein expression was assessed by Western blot analysis. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques. miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3′UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7 (known as TAK1), a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-kB signaling pathway. Our results suggest that miR-377 is an important negative regulator of E2F and MAP3K7/NF-kB signaling pathway in melanoma cells; it is tempting to speculate that its silencing in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways.