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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

DNA hypomethylation triggers OIS in pancreatic cancer cells.(A) Arrest in cell cycle and accumulation of G1 cells is observed in 5-aza-dC and siDnmt1 treated cells. (B) Proliferation curve showing that either 5-aza-dC treatment or siRNA-mediated Dnmt1 silencing stops cell proliferation. The relative cell number at each time point on the growth curves represents the means value ± SD of triplicates normalized to the cell number at day 1. (C) SA-βGal activity staining in MiaPaCa2 cells. As with siNupr1 treatment (upper middle panel), 5-aza-dC (lower left panel) and siDnmt1 (lower middle panel) treatments induce cells enter in senescence. On the other hand, the constitutive overexpression of Dnmt1 in MiaPaCa2 cells significantly rescues the siNupr1-induced senescence (lower right panel and right plot). (D) quantification of SA-βGal staining. (E) Expression of DNMT1 in MiaPaCa2 transfected with empty and pCMV6-Dnmt1 plasmid (F) SA-βGal staining in pancreas from Nupr1+/+ KrasG12D-expressing animals treated with 5-aza-dC (n = 6) or vehicle (n = 6). Means ± SD; ns = no significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 20 μm.
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f3: DNA hypomethylation triggers OIS in pancreatic cancer cells.(A) Arrest in cell cycle and accumulation of G1 cells is observed in 5-aza-dC and siDnmt1 treated cells. (B) Proliferation curve showing that either 5-aza-dC treatment or siRNA-mediated Dnmt1 silencing stops cell proliferation. The relative cell number at each time point on the growth curves represents the means value ± SD of triplicates normalized to the cell number at day 1. (C) SA-βGal activity staining in MiaPaCa2 cells. As with siNupr1 treatment (upper middle panel), 5-aza-dC (lower left panel) and siDnmt1 (lower middle panel) treatments induce cells enter in senescence. On the other hand, the constitutive overexpression of Dnmt1 in MiaPaCa2 cells significantly rescues the siNupr1-induced senescence (lower right panel and right plot). (D) quantification of SA-βGal staining. (E) Expression of DNMT1 in MiaPaCa2 transfected with empty and pCMV6-Dnmt1 plasmid (F) SA-βGal staining in pancreas from Nupr1+/+ KrasG12D-expressing animals treated with 5-aza-dC (n = 6) or vehicle (n = 6). Means ± SD; ns = no significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 20 μm.

Mentions: We first treated MiaPaCa2 cells with 1 μM 5-aza-2′-deoxycytydine (5-aza-dC), a general inhibitor of DNA methylation. Figure 3 show that 5-aza-dC induced cell cycle arrest (mean 81 ± 11% of cells in G1 vs. 46 ± 8 in control cells; p < 0.01) as judged by the standard propidium iodide staining protocol followed by flow cytometry analysis (Fig. 3A); inhibits cell proliferation (mean 62 ± 9 folds increase in control cells vs. 8 ± 1 in 5-aza-dC treated cells after 8 days of growth; p < 0.001) (Fig. 3B); and become positive for SA-βGal staining (mean 42% ± 8 vs. 2% ± 1 of siControl; p < 0.001) (Fig. 3C,D). Altogether, these results demonstrate that drug-induced DNA demethylation triggers cell growth arrest and cellular senescence. Similarly, knock down of Dnmt1 using specific siRNAs induces senescence in these cells (SA-βGal mean 29% ± 7 vs. 2% ± 1; p < 0.001) (Fig. 3C,D). We also performed senescence assays in MiaPaCa2 cells that stably overexpresses Dnmt1 but had either decreased or wild-type levels of Nurp1. We find that the downregulation in Nurp1 levels induces senescence in a manner that can be antagonized by overexpressing Dnmt1 (37% ± 7 vs. 11% ± 3 of Dnmt1 overexpressing cells; p < 0.01) as shown in Fig. 3C,D. We then validate this hypothesis in vivo by showing that SA-βGal staining was readily detected in these pancreata from Nupr1+/+; KrasG12D treated with 5-aza-dC but not in untreated pancreas (Fig. 3F). Morphometric analysis reveals that mice treated with 5-aza-dC display a 13 ± 4% of their area stained with SA-βGal whereas in the vehicle-treated pancreas this staining occupied 2.5 ± 2% (p < 0.01; n = 6) of the total pancreas sections. Thus, combined, the data from pharmacological, RNAi knockdown, and overexpression experiments are congruent in revealing that Nupr1 maintains a genome-wide level and pattern of Dnmt1-mediated DNA methylation that is necessary to counteract the induction of KrasG12D-induced senescence. This result are consistent, but more importantly, extend previous studies showing that the knockout of Nurp1 inhibits KrasG12D-induced PanINs formation, which is known to require a bypass of oncogene-mediated senescence16.


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)

DNA hypomethylation triggers OIS in pancreatic cancer cells.(A) Arrest in cell cycle and accumulation of G1 cells is observed in 5-aza-dC and siDnmt1 treated cells. (B) Proliferation curve showing that either 5-aza-dC treatment or siRNA-mediated Dnmt1 silencing stops cell proliferation. The relative cell number at each time point on the growth curves represents the means value ± SD of triplicates normalized to the cell number at day 1. (C) SA-βGal activity staining in MiaPaCa2 cells. As with siNupr1 treatment (upper middle panel), 5-aza-dC (lower left panel) and siDnmt1 (lower middle panel) treatments induce cells enter in senescence. On the other hand, the constitutive overexpression of Dnmt1 in MiaPaCa2 cells significantly rescues the siNupr1-induced senescence (lower right panel and right plot). (D) quantification of SA-βGal staining. (E) Expression of DNMT1 in MiaPaCa2 transfected with empty and pCMV6-Dnmt1 plasmid (F) SA-βGal staining in pancreas from Nupr1+/+ KrasG12D-expressing animals treated with 5-aza-dC (n = 6) or vehicle (n = 6). Means ± SD; ns = no significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 20 μm.
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f3: DNA hypomethylation triggers OIS in pancreatic cancer cells.(A) Arrest in cell cycle and accumulation of G1 cells is observed in 5-aza-dC and siDnmt1 treated cells. (B) Proliferation curve showing that either 5-aza-dC treatment or siRNA-mediated Dnmt1 silencing stops cell proliferation. The relative cell number at each time point on the growth curves represents the means value ± SD of triplicates normalized to the cell number at day 1. (C) SA-βGal activity staining in MiaPaCa2 cells. As with siNupr1 treatment (upper middle panel), 5-aza-dC (lower left panel) and siDnmt1 (lower middle panel) treatments induce cells enter in senescence. On the other hand, the constitutive overexpression of Dnmt1 in MiaPaCa2 cells significantly rescues the siNupr1-induced senescence (lower right panel and right plot). (D) quantification of SA-βGal staining. (E) Expression of DNMT1 in MiaPaCa2 transfected with empty and pCMV6-Dnmt1 plasmid (F) SA-βGal staining in pancreas from Nupr1+/+ KrasG12D-expressing animals treated with 5-aza-dC (n = 6) or vehicle (n = 6). Means ± SD; ns = no significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 20 μm.
Mentions: We first treated MiaPaCa2 cells with 1 μM 5-aza-2′-deoxycytydine (5-aza-dC), a general inhibitor of DNA methylation. Figure 3 show that 5-aza-dC induced cell cycle arrest (mean 81 ± 11% of cells in G1 vs. 46 ± 8 in control cells; p < 0.01) as judged by the standard propidium iodide staining protocol followed by flow cytometry analysis (Fig. 3A); inhibits cell proliferation (mean 62 ± 9 folds increase in control cells vs. 8 ± 1 in 5-aza-dC treated cells after 8 days of growth; p < 0.001) (Fig. 3B); and become positive for SA-βGal staining (mean 42% ± 8 vs. 2% ± 1 of siControl; p < 0.001) (Fig. 3C,D). Altogether, these results demonstrate that drug-induced DNA demethylation triggers cell growth arrest and cellular senescence. Similarly, knock down of Dnmt1 using specific siRNAs induces senescence in these cells (SA-βGal mean 29% ± 7 vs. 2% ± 1; p < 0.001) (Fig. 3C,D). We also performed senescence assays in MiaPaCa2 cells that stably overexpresses Dnmt1 but had either decreased or wild-type levels of Nurp1. We find that the downregulation in Nurp1 levels induces senescence in a manner that can be antagonized by overexpressing Dnmt1 (37% ± 7 vs. 11% ± 3 of Dnmt1 overexpressing cells; p < 0.01) as shown in Fig. 3C,D. We then validate this hypothesis in vivo by showing that SA-βGal staining was readily detected in these pancreata from Nupr1+/+; KrasG12D treated with 5-aza-dC but not in untreated pancreas (Fig. 3F). Morphometric analysis reveals that mice treated with 5-aza-dC display a 13 ± 4% of their area stained with SA-βGal whereas in the vehicle-treated pancreas this staining occupied 2.5 ± 2% (p < 0.01; n = 6) of the total pancreas sections. Thus, combined, the data from pharmacological, RNAi knockdown, and overexpression experiments are congruent in revealing that Nupr1 maintains a genome-wide level and pattern of Dnmt1-mediated DNA methylation that is necessary to counteract the induction of KrasG12D-induced senescence. This result are consistent, but more importantly, extend previous studies showing that the knockout of Nurp1 inhibits KrasG12D-induced PanINs formation, which is known to require a bypass of oncogene-mediated senescence16.

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