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Retinoblastoma loss modulates DNA damage response favoring tumor progression.

Seoane M, Iglesias P, Gonzalez T, Dominguez F, Fraga M, Aliste C, Forteza J, Costoya JA - PLoS ONE (2008)

Bottom Line: It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome.In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively.Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology Lab, Departamento de Fisioloxia, Facultade de Medicina, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

ABSTRACT
Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12). Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

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Rb loss enhances DNA damage response (DDR) induced by oncogenic Ras and reduces the p-p38MAPK levels by upregulation of Wip1.a, HRasV12 is able to induce DDR markers expression, such as p16INK4a, p21Cip1 and p19ARF, p53 and p53ser15 and γ-H2AXser139. Rb loss, in the presence of oncogenic Ras, increases this response. Immunoblot analysis was performed on astrocytes lysates prepared at day 6 after co-infection and selection with puromycin. b, Immunoblot analysis of p-p38MAPK and Wip1 levels was performed on cell lysates prepared at day 5 from co-infection and selection with puromycin. Densitometric analysis (in relative densitometric units) of Wip1 and p-p38MAPK protein levels. c, TMA analysis of Wip1 expression in glioma clinical samples. Representative pictures of samples are shown. The table shows median values. Wip1 immunoreactivity intensity was assigned according to the following scale: NA, non-assessable cases; −, less than 10% of neoplastic cells displayed immunoreactivity; +, 11–29% of neoplastic cells displayed immunoreactivity; ++, 30% or greater percentage of neoplastic cells displayed immunoreactivity. Phospho-histone H3 was used as a proliferation marker. Scale bars, 50 µm.
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pone-0003632-g004: Rb loss enhances DNA damage response (DDR) induced by oncogenic Ras and reduces the p-p38MAPK levels by upregulation of Wip1.a, HRasV12 is able to induce DDR markers expression, such as p16INK4a, p21Cip1 and p19ARF, p53 and p53ser15 and γ-H2AXser139. Rb loss, in the presence of oncogenic Ras, increases this response. Immunoblot analysis was performed on astrocytes lysates prepared at day 6 after co-infection and selection with puromycin. b, Immunoblot analysis of p-p38MAPK and Wip1 levels was performed on cell lysates prepared at day 5 from co-infection and selection with puromycin. Densitometric analysis (in relative densitometric units) of Wip1 and p-p38MAPK protein levels. c, TMA analysis of Wip1 expression in glioma clinical samples. Representative pictures of samples are shown. The table shows median values. Wip1 immunoreactivity intensity was assigned according to the following scale: NA, non-assessable cases; −, less than 10% of neoplastic cells displayed immunoreactivity; +, 11–29% of neoplastic cells displayed immunoreactivity; ++, 30% or greater percentage of neoplastic cells displayed immunoreactivity. Phospho-histone H3 was used as a proliferation marker. Scale bars, 50 µm.

Mentions: These observations suggest that the presence of DNA lesions may trigger DDR activation. Although the mechanisms that generate DNA damage put in place by different oncogenic events are unclear presently, Ras activation induces not only ROS-induced DNA damage but also re-replication, an event known to cause DDR activation [21]. DDR is likely to be related to premalignant neoplastic lesions in human [18], [39]–[41], and has been linked to increased expression levels of p16INK4A and p19ARF [18], which appear mutated in a vast proportion of GBMs [31]. Consequently, and considering that these cells were proliferating, we expected these DDR checkpoints to appear down-regulated. Surprisingly, we observed a rise of p16INK4A, p19ARF and p21CIP1 expression levels in HRasV12-expressing astrocytes despite of the lack of senescence induction. Accordingly, detection of p-p53Ser15 and phosphorylated histone γ-H2AX in HRasV12 expressing cells suggested DNA damage induced by the Ras-driven DNA hyper-replication (Figure 4A). Moreover, protein levels and their phosphorylation status were significantly higher in cRb−/−/RasV12 astrocytes than cRbloxP/loxP/RasV12 indicating that cRb−/−/RasV12 cells showed higher checkpoint activation (Figure 4A).


Retinoblastoma loss modulates DNA damage response favoring tumor progression.

Seoane M, Iglesias P, Gonzalez T, Dominguez F, Fraga M, Aliste C, Forteza J, Costoya JA - PLoS ONE (2008)

Rb loss enhances DNA damage response (DDR) induced by oncogenic Ras and reduces the p-p38MAPK levels by upregulation of Wip1.a, HRasV12 is able to induce DDR markers expression, such as p16INK4a, p21Cip1 and p19ARF, p53 and p53ser15 and γ-H2AXser139. Rb loss, in the presence of oncogenic Ras, increases this response. Immunoblot analysis was performed on astrocytes lysates prepared at day 6 after co-infection and selection with puromycin. b, Immunoblot analysis of p-p38MAPK and Wip1 levels was performed on cell lysates prepared at day 5 from co-infection and selection with puromycin. Densitometric analysis (in relative densitometric units) of Wip1 and p-p38MAPK protein levels. c, TMA analysis of Wip1 expression in glioma clinical samples. Representative pictures of samples are shown. The table shows median values. Wip1 immunoreactivity intensity was assigned according to the following scale: NA, non-assessable cases; −, less than 10% of neoplastic cells displayed immunoreactivity; +, 11–29% of neoplastic cells displayed immunoreactivity; ++, 30% or greater percentage of neoplastic cells displayed immunoreactivity. Phospho-histone H3 was used as a proliferation marker. Scale bars, 50 µm.
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pone-0003632-g004: Rb loss enhances DNA damage response (DDR) induced by oncogenic Ras and reduces the p-p38MAPK levels by upregulation of Wip1.a, HRasV12 is able to induce DDR markers expression, such as p16INK4a, p21Cip1 and p19ARF, p53 and p53ser15 and γ-H2AXser139. Rb loss, in the presence of oncogenic Ras, increases this response. Immunoblot analysis was performed on astrocytes lysates prepared at day 6 after co-infection and selection with puromycin. b, Immunoblot analysis of p-p38MAPK and Wip1 levels was performed on cell lysates prepared at day 5 from co-infection and selection with puromycin. Densitometric analysis (in relative densitometric units) of Wip1 and p-p38MAPK protein levels. c, TMA analysis of Wip1 expression in glioma clinical samples. Representative pictures of samples are shown. The table shows median values. Wip1 immunoreactivity intensity was assigned according to the following scale: NA, non-assessable cases; −, less than 10% of neoplastic cells displayed immunoreactivity; +, 11–29% of neoplastic cells displayed immunoreactivity; ++, 30% or greater percentage of neoplastic cells displayed immunoreactivity. Phospho-histone H3 was used as a proliferation marker. Scale bars, 50 µm.
Mentions: These observations suggest that the presence of DNA lesions may trigger DDR activation. Although the mechanisms that generate DNA damage put in place by different oncogenic events are unclear presently, Ras activation induces not only ROS-induced DNA damage but also re-replication, an event known to cause DDR activation [21]. DDR is likely to be related to premalignant neoplastic lesions in human [18], [39]–[41], and has been linked to increased expression levels of p16INK4A and p19ARF [18], which appear mutated in a vast proportion of GBMs [31]. Consequently, and considering that these cells were proliferating, we expected these DDR checkpoints to appear down-regulated. Surprisingly, we observed a rise of p16INK4A, p19ARF and p21CIP1 expression levels in HRasV12-expressing astrocytes despite of the lack of senescence induction. Accordingly, detection of p-p53Ser15 and phosphorylated histone γ-H2AX in HRasV12 expressing cells suggested DNA damage induced by the Ras-driven DNA hyper-replication (Figure 4A). Moreover, protein levels and their phosphorylation status were significantly higher in cRb−/−/RasV12 astrocytes than cRbloxP/loxP/RasV12 indicating that cRb−/−/RasV12 cells showed higher checkpoint activation (Figure 4A).

Bottom Line: It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome.In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively.Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology Lab, Departamento de Fisioloxia, Facultade de Medicina, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

ABSTRACT
Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12). Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus