Activated Akt pathway promotes genome instability through suppression of Mre11

Q Science (General)

Piscitello, Desiree ; Adams, Peter

University:University of Glasgow

Department:Institute of Cancer Sciences

关键词: Akt, DNA damage, RAS, senescence.;   

Others  :  http://theses.gla.ac.uk/6507/1/2015PiscitelloPhD.pdf

来源: University of Glasgow

【 摘 要 】

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.