The phosphatase MKP1 as a target to enhance replicative stress and apoptosis in tumor cells
by Veena Jagannathan
Date of Examination:2015-05-06
Date of issue:2016-04-29
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:PD Dr. Wilfried Kramer
Referee:Prof. Dr. Holger Reichardt
Persistent Address:
http://dx.doi.org/10.53846/goediss-5633
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Abstract
English
DNA replication is a tightly regulated elementary process that ensures the exact duplication and transfer of genetic information to the next generation. However, a wide range of exogenous and endogenous genotoxic insults often impair the progression of a replication fork and give rise to a phenomenon termed as replicative stress. In such a scenario, it is imperative for cells to maintain their DNA integrity to prevent genomic instability that may lead to tumorigenesis. This is achieved by instigating the DNA damage response (DDR), a highly organized, enzyme-based signaling cascade. While the contribution of kinases in this network has been very well studied, less is known about the role of their negative regulators, the phosphatases, in the same. Hence, our studies were aimed at investigating the function of a dual-specificity phosphatase, MKP1 (alias DUSP1), in both DNA replication and DDR. Identified as a MAP kinase phosphatase, MKP1 preferentially de-phosphorylates and inactivates p38MAPK and JNK, and protects the cell from stress-induced apoptosis. Our studies show that MKP1 inhibition accumulates phospho H2AX (γH2AX) and activates the DDR, even in the absence of any exogenous DNA damage. This effect was attributed to a previously unknown role of MKP1 in regulating DNA replication. Using the DNA fiber assay, we could demonstrate impaired replication fork progression and reduced origin firing upon MKP1 inhibition. Moreover, in the presence of an external replicative stress stimulus, gemcitabine, MKP1 inhibition was able to further down-regulate the speed of progressing forks. Additional investigations identified the activation of checkpoint kinases, MK2 and ATM, to be responsible for mediating these replication fork defects. Besides this, prolonged inhibition or transient depletion of MKP1 led to a massive induction of apoptosis, indicating a crucial function of this phosphatase in cellular survival. Cell death was accompanied by the degradation of an anti-apoptotic protein, Mcl-1, in the MKP1 deficient cells. Furthermore, for the first time, we detected a physical interaction between these proteins in an in-vitro co-immunoprecipitation assay. Taken together, our findings indicate that MKP1 is required for efficient DNA replication and cellular survival. Furthermore, this protective nature of MKP1 can be exploited by pharmacological antagonists in various cancers which over-express this phosphatase. Thus, we propose MKP1 as an attractive druggable target in cancer therapy, inhibition of which can enhance replicative stress and promote cell death.
Keywords: MKP1; MK2; ATM; replicative stress; DNA damage response; apoptosis; Mcl-1; dual specificity phosphatase