Here we report that RNA interference against ATM inhibited p53 accumulation in cells expressing oncogenic STAT5 and cooperated with Rb inactivation to suppress STAT5A-induced senescence. Knocking down ATM was also effective to bypass E2F1-induced senescence and in combination with Rb inactivation, inhibited RasV12-induced senescence. Cells that senesced in response to ca-STAT5A or RasV12 accumulated DNA damage foci and activated ATM, ATR, Chk1, and Chk2, indicating that aberrant oncogene activation induces a DNA damage signaling response. Intriguingly, bypassing oncogene-induced senescence by inactivation of p53 and Rb did not eliminate the accumulation of oncogene-induced DNA damage foci (ODDI), suggesting a mechanism that may limit transformation in immortalized cells.
The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. Here, we show that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1a. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors.
Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAimediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.
Cellular senescence is a permanent cell cycle arrest that can be triggered by a variety of stresses including short telomeres and activated oncogenes. Promyelocytic leukemia protein (PML) is a central component of the senescence response, and is able to trigger the process when overexpressed in human diploid fibroblasts (HDFs). Senescence induced by PML in HDFs is characterized by a modest increase in p53 levels and activity, the accumulation of hypophosphorylated Rb and a reduced expression of E2F-dependent genes. To dissect the p53 and Rb family requirements for PML-induced senescence, we used the oncoproteins E6 and E7 from human papillomavirus type 16. We found that the coexpression of E6 and E7 inhibited the growth arrest and senescence induced by PML. In addition, these viral oncoproteins blocked the formation of PML bodies and excluded both p53 and Rb from PML bodies. Expression of dominantnegative p53 alone failed to block PML-induced senescence and expression of E6 only delayed the process. On the other hand, expression of E7 was sufficient to block PML-induced senescence, while an E7 mutant unable to bind Rb did not. Together, these data indicate that PMLinduced senescence engages the Rb tumor-suppressor pathway predominantly.
Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2.
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