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Pivotal Role of the Chromatin Protein Nupr1 in Kras-Induced Senescence and Transformation.

Grasso D, Bintz J, Lomberk G, Molejon MI, Loncle C, Garcia MN, Lopez MB, Urrutia R, Iovanna JL - Sci Rep (2015)

Bottom Line: In the current study, we report that Nupr1 acts as a gene modifier of the effect of Kras(G12D)-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation.This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1(-/-) mice the expression of Kras(G12D) induces senescence instead of transformation.Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as Kras(G12D), during carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.

ABSTRACT
Nupr1 is a chromatin protein, which cooperates with Kras(G12D) to induce PanIN formation and pancreatic cancer development in mice, though the molecular mechanisms underlying this effect remain to be fully characterized. In the current study, we report that Nupr1 acts as a gene modifier of the effect of Kras(G12D)-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation. Congruently, 5-aza-2'-deoxycytydine, a general inhibitor of DNA methylation, reverses the Kras(G12D)-induced PanIN development by promoting senescence. This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1(-/-) mice the expression of Kras(G12D) induces senescence instead of transformation. Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as Kras(G12D), during carcinogenesis. The biomedical relevance of these findings lies in that they support the rational for developing similar therapeutic interventions in human aimed at controlling either the initiation or progression of cancer.

No MeSH data available.


Related in: MedlinePlus

Nupr1 depletion reduces Dnmt1 expression.(A) RT-qPCR showing decrease of Dnmt1 transcript in KrasG12D expressing mouse pancreas Nupr1–/– (n = 3) compared to Nupr1+/+ (n = 3). (B) By RT-qPCR, little modification in Dnmt3a and Dnmt3b but important Dnmt1 decrease is observed in siNupr1-treated MiaPaCa2 cells. (C) MiaPaCa2 cells were transfected with siNupr1 or siDNMT1 and expression of DNMT1 was measured by western blotting (D) MiaPaCa2 cells were transfected with pCDNA3 vectors containing a Flag-tagged DNMT1, an irrelevant Cytochrome C or an empty vector (Empty). ChIP was performed using an anti-Flag antibody or a non-relevant IgG. (Top) Occupancy of Nupr1 on Dnmt1 promoter, (bottom) DNA input (10%). (E) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with siControl or siNupr1, and pSV40-RL as an internal control. After 48 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. (F) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with increasing amounts of pNupr1-Flag construct, and pSV40-RL as an internal control. After 24 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. Means ± SD; *p < 0.05, **p < 0.01***p < 0.001.
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f1: Nupr1 depletion reduces Dnmt1 expression.(A) RT-qPCR showing decrease of Dnmt1 transcript in KrasG12D expressing mouse pancreas Nupr1–/– (n = 3) compared to Nupr1+/+ (n = 3). (B) By RT-qPCR, little modification in Dnmt3a and Dnmt3b but important Dnmt1 decrease is observed in siNupr1-treated MiaPaCa2 cells. (C) MiaPaCa2 cells were transfected with siNupr1 or siDNMT1 and expression of DNMT1 was measured by western blotting (D) MiaPaCa2 cells were transfected with pCDNA3 vectors containing a Flag-tagged DNMT1, an irrelevant Cytochrome C or an empty vector (Empty). ChIP was performed using an anti-Flag antibody or a non-relevant IgG. (Top) Occupancy of Nupr1 on Dnmt1 promoter, (bottom) DNA input (10%). (E) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with siControl or siNupr1, and pSV40-RL as an internal control. After 48 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. (F) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with increasing amounts of pNupr1-Flag construct, and pSV40-RL as an internal control. After 24 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. Means ± SD; *p < 0.05, **p < 0.01***p < 0.001.

Mentions: We evaluated whether key epigenetic regulators were differentially regulated in the pancreas of KrasG12D; Nupr1+/+vs. KrasG12D; Nupr1–/– mice at 5 week after birth, a stage preceding the appearance of PanINs, by generating and analyzing genome-wide expression profiles (accession number GSE45232), which lead to the observation that DNMT is downregulated under this conditions. Indeed, using quantitative RT-PCR, we found that Nupr1 inactivation decreased Dnmt1 expression in the pancreas tissue by 2.1 ± 0.3 folds (p < 0.01) (Fig. 1A). Complementary knockdown experiments using 2 specific Nupr1 siRNAs to transfect pancreatic cancer cells (MiaPaCa2) decreased Dnmt1 mRNA levels (3.8 fold ± 0.7; p < 0.01), without affecting either Dnmt3a or Dnmt3b expression (Fig. 1B). The, downregulation of Dnmt1 at the protein level was confirmed using western blot analyses (Fig. 1C). ChIP assays demonstrated that Nupr1 binds to the Dnmt1 promoter, revealing a direct effect for this protein in the transcriptional regulation of this methylase (Fig. 1D). Luciferase-based gene reporter assay in MiaPaCa2 cells transfected with siNupr1 or siControl demonstrated that the activation of the Dnmt1 promoter is dependent on the expression of this protein (Fig. 1E) since the reporter activity decrease from 1.0 ± 0.2 to 0.2 ± 0.02; (p < 0.001) when Nupr1 was knockdown. Conversely, overexpression of Nupr1 increased Dnmt1 promoter activity in a dose-dependent manner as showed in Fig. 1F. The increase in the reporter activity was of 1.8 ± 0.3, 4.3 ± 0.9 and 5.1 ± 0.3 folds with 100, 200 and 400 ng of pNupr1-Flag construct. Similar results were obtained using another pancreatic cancer cells, Panc1 (supplemental Figure 1). Together, these results suggest that expression of Nurp1 can impact on DNA methylation. To test the validity of this idea, we performed a genome-wide MeDIP-based 5-methyl-cytosine analysis on DNA samples derived from MiaPaCa2 pancreatic cancer cells treated with either siNupr1 or siControl. Methylated DNA was first enriched using 5-methyl cytosine antibody-based capture method, labeled with C3 and C5 and hybridized to NimbleGen Human DNA Methylation 3 × 720 K CpG Island Plus RefSeq Promoter Arrays. Methylation data was generated using C3 vs. C5 subtractive image processing with peak calling as previously described24. The results of these experiments, shown in both supplemental Fig. 2 and supplemental Table 1, demonstrate that Nupr1 downregulation clearly modifies the DNA methylation status in pancreatic cancer cells. Depletion of Nupr1 resulted in 877 newly methylated genes and demethylation of 2026 genes (supplemental Figure 2 and supplemental Table 1). To validate the functional consequences of this epigenetic change, we used RT-qPCR to measure the changes in expression of a subset of mRNA encoded by the hypomethylated genes. The results showed in supplemental Fig. 3 demonstrate that 8 out of 10 methylated genes become downregulated. Ontological analysis revealed a modification pattern of genes which functions are primarily related to histone modification and chromatin organization, signaling, cell proliferation, cell adhesion and motility. Regarding cell signaling cascades, pathway enrichment analyses show that Nupr1 depletion induces methylation of genes related to the JAK-STAT signaling and the EGF pathway, while causing demethylation of genes related to Wnt pathway and Fork head family. These results demonstrate, that consistent with its effects on Dnmt1 expression, the genetic inactivation of Nupr1 gives rise to changes in genome-wide methylation events that affect various gene expression networks.


Pivotal Role of the Chromatin Protein Nupr1 in Kras-Induced Senescence and Transformation.

Grasso D, Bintz J, Lomberk G, Molejon MI, Loncle C, Garcia MN, Lopez MB, Urrutia R, Iovanna JL - Sci Rep (2015)

Nupr1 depletion reduces Dnmt1 expression.(A) RT-qPCR showing decrease of Dnmt1 transcript in KrasG12D expressing mouse pancreas Nupr1–/– (n = 3) compared to Nupr1+/+ (n = 3). (B) By RT-qPCR, little modification in Dnmt3a and Dnmt3b but important Dnmt1 decrease is observed in siNupr1-treated MiaPaCa2 cells. (C) MiaPaCa2 cells were transfected with siNupr1 or siDNMT1 and expression of DNMT1 was measured by western blotting (D) MiaPaCa2 cells were transfected with pCDNA3 vectors containing a Flag-tagged DNMT1, an irrelevant Cytochrome C or an empty vector (Empty). ChIP was performed using an anti-Flag antibody or a non-relevant IgG. (Top) Occupancy of Nupr1 on Dnmt1 promoter, (bottom) DNA input (10%). (E) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with siControl or siNupr1, and pSV40-RL as an internal control. After 48 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. (F) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with increasing amounts of pNupr1-Flag construct, and pSV40-RL as an internal control. After 24 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. Means ± SD; *p < 0.05, **p < 0.01***p < 0.001.
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Related In: Results  -  Collection

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f1: Nupr1 depletion reduces Dnmt1 expression.(A) RT-qPCR showing decrease of Dnmt1 transcript in KrasG12D expressing mouse pancreas Nupr1–/– (n = 3) compared to Nupr1+/+ (n = 3). (B) By RT-qPCR, little modification in Dnmt3a and Dnmt3b but important Dnmt1 decrease is observed in siNupr1-treated MiaPaCa2 cells. (C) MiaPaCa2 cells were transfected with siNupr1 or siDNMT1 and expression of DNMT1 was measured by western blotting (D) MiaPaCa2 cells were transfected with pCDNA3 vectors containing a Flag-tagged DNMT1, an irrelevant Cytochrome C or an empty vector (Empty). ChIP was performed using an anti-Flag antibody or a non-relevant IgG. (Top) Occupancy of Nupr1 on Dnmt1 promoter, (bottom) DNA input (10%). (E) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with siControl or siNupr1, and pSV40-RL as an internal control. After 48 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. (F) Cells were transfected with combinations of a Dnmt1 promoter-Luc vector with increasing amounts of pNupr1-Flag construct, and pSV40-RL as an internal control. After 24 h, luciferase activity was determined and expressed as the ratio of specific luciferase activity/internal standard. Means ± SD; *p < 0.05, **p < 0.01***p < 0.001.
Mentions: We evaluated whether key epigenetic regulators were differentially regulated in the pancreas of KrasG12D; Nupr1+/+vs. KrasG12D; Nupr1–/– mice at 5 week after birth, a stage preceding the appearance of PanINs, by generating and analyzing genome-wide expression profiles (accession number GSE45232), which lead to the observation that DNMT is downregulated under this conditions. Indeed, using quantitative RT-PCR, we found that Nupr1 inactivation decreased Dnmt1 expression in the pancreas tissue by 2.1 ± 0.3 folds (p < 0.01) (Fig. 1A). Complementary knockdown experiments using 2 specific Nupr1 siRNAs to transfect pancreatic cancer cells (MiaPaCa2) decreased Dnmt1 mRNA levels (3.8 fold ± 0.7; p < 0.01), without affecting either Dnmt3a or Dnmt3b expression (Fig. 1B). The, downregulation of Dnmt1 at the protein level was confirmed using western blot analyses (Fig. 1C). ChIP assays demonstrated that Nupr1 binds to the Dnmt1 promoter, revealing a direct effect for this protein in the transcriptional regulation of this methylase (Fig. 1D). Luciferase-based gene reporter assay in MiaPaCa2 cells transfected with siNupr1 or siControl demonstrated that the activation of the Dnmt1 promoter is dependent on the expression of this protein (Fig. 1E) since the reporter activity decrease from 1.0 ± 0.2 to 0.2 ± 0.02; (p < 0.001) when Nupr1 was knockdown. Conversely, overexpression of Nupr1 increased Dnmt1 promoter activity in a dose-dependent manner as showed in Fig. 1F. The increase in the reporter activity was of 1.8 ± 0.3, 4.3 ± 0.9 and 5.1 ± 0.3 folds with 100, 200 and 400 ng of pNupr1-Flag construct. Similar results were obtained using another pancreatic cancer cells, Panc1 (supplemental Figure 1). Together, these results suggest that expression of Nurp1 can impact on DNA methylation. To test the validity of this idea, we performed a genome-wide MeDIP-based 5-methyl-cytosine analysis on DNA samples derived from MiaPaCa2 pancreatic cancer cells treated with either siNupr1 or siControl. Methylated DNA was first enriched using 5-methyl cytosine antibody-based capture method, labeled with C3 and C5 and hybridized to NimbleGen Human DNA Methylation 3 × 720 K CpG Island Plus RefSeq Promoter Arrays. Methylation data was generated using C3 vs. C5 subtractive image processing with peak calling as previously described24. The results of these experiments, shown in both supplemental Fig. 2 and supplemental Table 1, demonstrate that Nupr1 downregulation clearly modifies the DNA methylation status in pancreatic cancer cells. Depletion of Nupr1 resulted in 877 newly methylated genes and demethylation of 2026 genes (supplemental Figure 2 and supplemental Table 1). To validate the functional consequences of this epigenetic change, we used RT-qPCR to measure the changes in expression of a subset of mRNA encoded by the hypomethylated genes. The results showed in supplemental Fig. 3 demonstrate that 8 out of 10 methylated genes become downregulated. Ontological analysis revealed a modification pattern of genes which functions are primarily related to histone modification and chromatin organization, signaling, cell proliferation, cell adhesion and motility. Regarding cell signaling cascades, pathway enrichment analyses show that Nupr1 depletion induces methylation of genes related to the JAK-STAT signaling and the EGF pathway, while causing demethylation of genes related to Wnt pathway and Fork head family. These results demonstrate, that consistent with its effects on Dnmt1 expression, the genetic inactivation of Nupr1 gives rise to changes in genome-wide methylation events that affect various gene expression networks.

Bottom Line: In the current study, we report that Nupr1 acts as a gene modifier of the effect of Kras(G12D)-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation.This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1(-/-) mice the expression of Kras(G12D) induces senescence instead of transformation.Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as Kras(G12D), during carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.

ABSTRACT
Nupr1 is a chromatin protein, which cooperates with Kras(G12D) to induce PanIN formation and pancreatic cancer development in mice, though the molecular mechanisms underlying this effect remain to be fully characterized. In the current study, we report that Nupr1 acts as a gene modifier of the effect of Kras(G12D)-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation. Congruently, 5-aza-2'-deoxycytydine, a general inhibitor of DNA methylation, reverses the Kras(G12D)-induced PanIN development by promoting senescence. This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1(-/-) mice the expression of Kras(G12D) induces senescence instead of transformation. Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as Kras(G12D), during carcinogenesis. The biomedical relevance of these findings lies in that they support the rational for developing similar therapeutic interventions in human aimed at controlling either the initiation or progression of cancer.

No MeSH data available.


Related in: MedlinePlus