Homo sapiens
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PROVIDER: PRJNA93301 | ENA |
REPOSITORIES: ENA
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Project description:The diterpene ester PEP005 is a novel anticancer agent that activates PKC and cures subcutaneous murine melanoma by topical application. We now describe the in vitro cytostatic effects of PEP005 and the diterpene ester TPA, observed in 20% of human melanoma cell lines. Primary cultures of normal human neonatal fibroblasts were uniformly resistant to growth arrest, indicating a potential for tumor selectivity. Sensitive cells were induced to senesce and exhibited a G1 and G2/M arrest. There was sustained expression of p21WAF1/CIP1, irreversible dephosphorylation of the retinoblastoma gene product (Rb) and transcriptional silencing of E2F-responsive genes in sensitive cell lines. Activation of MEK1/2 by PKC was required for diterpene ester-induced senescence. Expression profiling revealed that the MAPK inhibitor HREV107 was expressed at a higher transcript level in resistant compared to sensitive cell lines. We propose that activation of PKC over-stimulates the Ras/Raf/MEK/ERK pathway, resulting in sustained induction of p21WAF1/CIP1, dephosphorylation of Rb and transcriptional silencing of E2F-responsive genes required for DNA synthesis and mitosis. Keywords: expression profile following treatment
2006-01-20 | GSE3484 | GEO
Project description:We previously identified the induction of growth arrest with phenotypic characteristics of senescence in melanoma cell lines sensitive to diterpene esters, indicating a therapeutic potential. Here we compared the cytostatic effects of two diterpene esters namely TPA (12-O-tetradecanoylphorbol-13-acetate) and PEP008 (20-O-acetyl-ingenol-3-angelate) in sensitive and resistant cell lines derived from melanoma, breast cancer and colon cancer. We showed the diterpene esters to induce senescence-like growth arrest in the sensitive cells at 100-1000 ng/ml. Use of the pan-PKC inhibitor bisindolylmaleimide-l demonstrated that activation of PKC was required for growth arrest. Full genome expression profiling revealed that pivotal genes involved in DNA synthesis and cell cycle control were down-regulated by treatment in all three sensitive solid tumor models. At the protein level, prolonged down-regulation of E2F-1 and proliferating cell nuclear antigen (PCNA), sustained expression of p21WAF1/CIP1 and dephosphorylation of retinoblastoma (Rb) occurred in the sensitive cells. Although activation of extracellular signal-related kinase (ERK) 1/2 by the diterpene esters occurred in both sensitive and resistant cell lines, the HRASLS3 type II tumor suppressor, which appears to have a role in MAPK pathway suppression, was constitutively elevated in the resistant cell lines compared to their sensitive counterparts. Together, these results demonstrate the ability of the PKC activating drugs TPA and PEP008 to induce growth arrest with characteristics of senescence in solid tumor cell lines derived from a variety of tissue types through a similar mechanism. PKC-activating diterpene esters may therefore have therapeutic potential in a range of solid tumors. Experiment Overall Design: We analyzed the transcriptional profiles of the diterpene ester sensitive cell lines MCF7, COLO-205 and SK-MEL-5 following treatment with PEP008 using full genome expression profiling (Affymetrix, U133 Plus 2.0). Cells were treated for 24 h and 24 h plus 72 h recovery with 1000 ng/ml of the drug, before harvesting RNA for analysis. From the cell growth assays, all three cell lines demonstrated permanent growth arrest with diterpene ester treatments at the 1000 ng/ml dose. Mock controls were treated with solvent alone for 24 h. SK-MEL-5 cells were also treated with 1000 ng/ml TPA for 24 h.
2008-06-16 | E-GEOD-8742 | biostudies-arrayexpress
Project description:The diterpene ester PEP005 is a novel anticancer agent that activates PKC and cures subcutaneous murine melanoma by topical application. We now describe the in vitro cytostatic effects of PEP005 and the diterpene ester TPA, observed in 20% of human melanoma cell lines. Primary cultures of normal human neonatal fibroblasts were uniformly resistant to growth arrest, indicating a potential for tumor selectivity. Sensitive cells were induced to senesce and exhibited a G1 and G2/M arrest. There was sustained expression of p21WAF1/CIP1, irreversible dephosphorylation of the retinoblastoma gene product (Rb) and transcriptional silencing of E2F-responsive genes in sensitive cell lines. Activation of MEK1/2 by PKC was required for diterpene ester-induced senescence. Expression profiling revealed that the MAPK inhibitor HREV107 was expressed at a higher transcript level in resistant compared to sensitive cell lines. We propose that activation of PKC over-stimulates the Ras/Raf/MEK/ERK pathway, resulting in sustained induction of p21WAF1/CIP1, dephosphorylation of Rb and transcriptional silencing of E2F-responsive genes required for DNA synthesis and mitosis. To investigate the molecular changes associated with the senescent phenotype, we examined the unique transcriptional changes occurring in sensitive melanoma cell lines treated with TPA or PEP005. Initially a time-course cDNA microarray analysis was conducted on one sensitive and one resistant cell line treated with 1 µg/ml of TPA for 6, 24 h and 24 h recovery following 24 h treatment, to determine the earliest time point at which the most significant changes in transcription occurred. The results provided support for conducting array experiments with 24 h treatment, in which three sensitive and four resistant melanoma cell lines were treated with 1 µg/ml of either diterpene ester. Our primary objective was to identify those genes which were uniquely up or down-regulated in sensitive or resistant cell lines in response to treatment, which could reflect the phenotypic outcome. Through applying a series of stringent selective criteria (see Material and Methods) we found that the most significant changes occurred in the transcriptional repression of genes required for DNA synthesis and mitosis in cell lines sensitive to treatment (Table 1). To confirm that these changes were reflected at the protein level, western blot analysis was conducted on three sensitive and three resistant cell lines following 6 and 24 h treatment with TPA or PEP005. We also included a 24 h recovery time point to determine the irreversibility of the change.
2010-07-01 | E-GEOD-3484 | biostudies-arrayexpress
Project description:We previously identified the induction of growth arrest with phenotypic characteristics of senescence in melanoma cell lines sensitive to diterpene esters, indicating a therapeutic potential. Here we compared the cytostatic effects of two diterpene esters namely TPA (12-O-tetradecanoylphorbol-13-acetate) and PEP008 (20-O-acetyl-ingenol-3-angelate) in sensitive and resistant cell lines derived from melanoma, breast cancer and colon cancer. We showed the diterpene esters to induce senescence-like growth arrest in the sensitive cells at 100-1000 ng/ml. Use of the pan-PKC inhibitor bisindolylmaleimide-l demonstrated that activation of PKC was required for growth arrest. Full genome expression profiling revealed that pivotal genes involved in DNA synthesis and cell cycle control were down-regulated by treatment in all three sensitive solid tumor models. At the protein level, prolonged down-regulation of E2F-1 and proliferating cell nuclear antigen (PCNA), sustained expression of p21WAF1/CIP1 and dephosphorylation of retinoblastoma (Rb) occurred in the sensitive cells. Although activation of extracellular signal-related kinase (ERK) 1/2 by the diterpene esters occurred in both sensitive and resistant cell lines, the HRASLS3 type II tumor suppressor, which appears to have a role in MAPK pathway suppression, was constitutively elevated in the resistant cell lines compared to their sensitive counterparts. Together, these results demonstrate the ability of the PKC activating drugs TPA and PEP008 to induce growth arrest with characteristics of senescence in solid tumor cell lines derived from a variety of tissue types through a similar mechanism. PKC-activating diterpene esters may therefore have therapeutic potential in a range of solid tumors. Keywords: time course
2007-10-10 | GSE8742 | GEO
Project description:Activation of oncogene signaling in primary cells generally results in cellular senescence. This process is not only considered to be tumor-protective, but also irreversible. However, if senescence induction can be circumvented, e.g. by additional genetic or epigenetic changes, tumor progression occurs. An in-vivo example for oncogene-induced senescence are human nevus cells, most of which bear oncogenic mutations in RAS/RAF/MAPK pathway genes. Here, we show that expression of the human melanoma oncogene N-RAS61K in cultured pigment cells initially induces OIS characterized by a highly multinucleated phenotype. Surprisingly, after prolonged periods of oncogene expression, mononucleated cells emerge from the multinucleated cells. They are highly proliferative, anoikis-resistent and induce fast growing and metastatic melanoma upon transplantation into nude mice. During long-term oncogene expression and the corresponding development of anoikis iresistance, expression of melanocyte-specific genes is lost. Our data demonstrate that the induction of oncogene-induced senescence is not just a failsafe escape mechanism, but also provides a source for highly aggressive tumor cells. total samples analysed are 4
2014-01-20 | E-GEOD-27010 | biostudies-arrayexpress
Project description:Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma. MEL-ST cells expressing either empty vector or mutant oncogenic RAS genes (HRAS v12, KRAS v12, NRAS Q61K) were used to isolate total RNA. The RNA was then used to perform gene expression analyses using the Illumina HumanHT-12 V4.0 Expression BeadChip array.
2016-09-08 | E-GEOD-62827 | biostudies-arrayexpress
Project description:Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma. YUGASP cells stably expressing a non-silencing shRNA or two individual shRNAs against IFI6 were used to prepare the total RNA, which was then used to analyze for gene expression using Illumina expression array.
2016-09-08 | E-GEOD-69933 | biostudies-arrayexpress
Project description:Activation of oncogenes often leads to induction of the DNA damage responses and onset of the cell senescence. Given that DNA damage can also trigger production of type I interferons (IFN) that contribute to senescence development, we sought to determine the role of IFN in the oncogene-induced senescence. Our data in mouse model demonstrate that inactivation of IFN signaling is sufficient for inducing melanomas in melanocytes harboring mutant Braf. Restoration of IFN signaling in IFN-deficient melanoma cells induces cell senescence and suppresses melanoma progression. In addition, data in human patients that received high dose IFN therapy and in mouse transplanted tumor models strongly suggest the importance of the non-cell-autonomous IFN signaling. Suppression of IFN signaling mediated by the downregulation of IFN receptor IFNAR1 invariably occurs during development of mouse melanoma. Mice harboring the IFNAR1 mutant, which is relatively resistant to downregulation, delay melanoma development, suppress the metastatic disease, and better respond to treatment with BRAF or PD1 inhibitors. These results suggest that IFN signaling is an important tumor suppressive pathway that inhibits melanoma development and progression. Accordingly, the inhibition of IFN pathway via IFNAR1 downregulation plays a key role in melanoma pathogenesis. Conversely, these data also argue for targeting IFNAR1 downregulation to prevent the metastatic disease and improve the efficacy of molecularly targeted and immune-targeted therapies. Two genotypes of mice were examined at 2 to 3 times after tamoxifen adminstration, with 2 replicates for each condition, yielding 8 samples in total.
2016-01-14 | E-GEOD-76842 | biostudies-arrayexpress
Project description:The tumor suppressive miR-29 family of microRNAs is encoded by two clusters, miR-29b1~a and miR-29b2~c, and is regulated by several oncogenic and tumor suppressive stimuli. Here we investigated whether oncogenic MAPK hyperactivation regulates miR-29 abundance and how this signaling axis impacts melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1~a and pri-miR-29b2~c, respectively. Expression analyses revealed that while pri-miR-29a~b1 remains elevated, pri-miR-29b2~c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we showed that inactivation of miR-29 in vivo accelerates the development of frank melanomas and decreases overall survival. We identified MAFG as a relevant miR-29 target that has oncogenic potential in melanocytes and is required for growth of melanoma cells. Our findings suggest that MAPK-driven miR-29 induction constitutes a tumor suppressive barrier by targeting MAFG, which is overcome by attenuation of miR-29b2~c expression.
2023-04-10 | GSE148552 | GEO
Project description:Activation of oncogene signaling in primary cells generally results in cellular senescence. This process is not only considered to be tumor-protective, but also irreversible. However, if senescence induction can be circumvented, e.g. by additional genetic or epigenetic changes, tumor progression occurs. An in-vivo example for oncogene-induced senescence are human nevus cells, most of which bear oncogenic mutations in RAS/RAF/MAPK pathway genes. Here, we show that expression of the human melanoma oncogene N-RAS61K in cultured pigment cells initially induces OIS characterized by a highly multinucleated phenotype. Surprisingly, after prolonged periods of oncogene expression, mononucleated cells emerge from the multinucleated cells. They are highly proliferative, anoikis-resistent and induce fast growing and metastatic melanoma upon transplantation into nude mice. During long-term oncogene expression and the corresponding development of anoikis iresistance, expression of melanocyte-specific genes is lost. Our data demonstrate that the induction of oncogene-induced senescence is not just a failsafe escape mechanism, but also provides a source for highly aggressive tumor cells.
2014-01-20 | GSE27010 | GEO