Activated Akt pathway promotes genome instability through suppression of Mre11

Activating mutations in RAS are often found in different human cancers. The expression of an oncogene is called a driver mutation because it provides the bases for tumour initiation, but it still requires additional mutations to achieve tumour progression. 90% of invasive pancreatic ductal adenocarcinoma (PDAC) present activating KRAS mutation, in conjunction with inactivation of various tumour suppressor genes such as BRCA1, TP53, SMAD4 and CDKN2A. The most important effectors of RAS are PI3K and its downstream kinases. These function as mediators of RAS-induced cell survival and proliferation. Interestingly, concurrent mutations of RAS and PI3K/PTEN/Akt pathway have been described in the same human tumour types. Endometrial cancer, thyroid cancer and acute lymphoblastic leukemia have all been shown to harbor the simultaneous mutation of RAS gene and those encoding various members of the PI3K signalling pathway. Published data suggest that 25% of human colon cancers contain mutations in both K‐RAS and PI3K-associated genes. Moreover, 60% of human PDAC show PTEN loss, due to deletions, mutations or epigenetic silencing. Despite this prevalence, the molecular mechanism for the cooperation between RAS and PI3K pathway in tumourigenesis is poorly understood. A fundamental barrier for tumourigenesis is senescence. The activation of an oncogene such as RAS in a primary cell line drives cells into unscheduled DNA synthesis, resulting in a high frequency of stalled replication forks and DNA double strand breaks (DSBs). A DSB is one of the most deleterious lesions if unrepaired, and it is the primary trigger of oncogene-induced senescence (OIS). DSBs activate the ATM/ATR signalling pathway and senescence-associated cell cycle arrest. However, various oncogenes differ in their ability to induce senescence, for example activated Akt is a weak inducer of senescence compared to RAS. Previous work from our lab and others has suggested that the co-activation of these two oncogenes may serve to bypass certain aspects of the senescence program, but the precise mechanism by which this is achieved remains unclear. Surprisingly, detailed cell cycle analyses in this study demonstrate that the simultaneous activation of Akt in primary fibroblasts expressing oncogenic RAS reinforces RAS-induced senescence. This correlates with an increased accumulation of unrepaired damage, which is known to directly contribute to establishment of senescence. Interestingly, the expression of activated Akt in these cells correlates with reduced expression of MRN complex components, which in presence of RAS-induced damage impairs the activation of the checkpoint kinases. The inhibition of Mre11, the nuclease component of the MRN complex, in RAS expressing cells recapitulates the phenotype of RAS/Akt cells. Thus, Akt downregulates Mre11 to exacerbate RAS-induced DNA damage and induce a qualitatively stronger senescence. Multiple studies have previously reported the negative regulation of DDR by Akt, however a mechanism for this has not been described. Experiments on two colon cancer lines, HCT116 and DLD1, have revealed that inactivation of PTEN/activation of Akt suppresses DDR via a reduction in MRN complex expression and activity. In these cells, the components of the MRN complex display low protein stability and are rapidly degraded by an unknown mechanism. MRN complex is central in the DNA damage response to DSBs. Its suppression impairs the activation of the two checkpoint kinases Chk1 and Chk2, which mediates the G2/M arrest, and also impairs HR repair. The inhibition of these two events severely affects cell survival in presence of DSBs, and the surviving fraction present high levels of genome instability. The use of specific inhibitors targeting S6K1 activity rescues the levels of MRN complex in these cells, suggesting a role of this kinase in DDR suppression. Thus, the enhanced RAS-induced senescence in cells caused by Akt can be ascribed to the high levels of unrepaired damage due to the suppression of MRN complex. Despite compounding senescence, the simultaneous mutation of RAS and Akt allow the cells to acquire genome instability, which in vivo significantly contributes to bypassing senescence and promotes tumour progression

KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage

The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

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Additional file 1: of Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Six additional supplementary figures and legends. (PDF 10.1 MB

Additional file 3: Dataset 1. of Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Ranked list of the 500 genes containing the greatest H4K20me3 enrichment in RS cells. Dataset 2. Ranked list of the 500 genes containing the greatest H4K20me3 enrichment in OIS cells. (XLS 109 kb