Mitogens play an integral role in resistance, proliferation, and survival of human tumors. Unraveling the intricate signal transduction pathways that regulate tumorigenesis is a critical step in identifying new therapeutic targets for disruption to impede the progression of cancer. It is not clear as to what extent tumor derived mitogens such as IGF-1 and EGF-1 signal to activate the oncogenic activity of the murine double minute protein (Mdm2). Mdm2 plays a role in tumorigenesis in part through the disruption of the tumor suppressor protein p53. It has been assumed that Mdm2 nuclear localization was a passive process requiring only the nuclear localization sequence (NLS). We show that IGF-1 activated PI3-K/Akt signaling induces the phosphorylation and nuclear translocation of Mdm2, which disrupts p53 activity and facilitates p53 degradation. The p53-Mdm2 complex is exported from the nucleus for both proteins to be degraded. Thus, it seems likely that there is a second signal transduction pathway responsible for mediating Mdm2 nuclear export. We hypothesize that Mdm2 nuclear export is regulated by post-translational modifications mediated by the mitogen activated protein kinase pathway, and culminates in the inactivation of p53.
The specific aims i n this proposal are designed to ascertain how signal transduction pathways directly regulate Mdm2 and its activity to block p53 function. [Aim 1] To elucidate the signal transduction pathway that mediates nuclear export of Mdm2 by post-translational modifications. We will use selective pharmacological inhibitors to signaling kinases, and dominant negative kinases to block nuclear export of Mdm2. We will determine post-translational modifications to Mdm2 by mass spectrometry in response to growth factor simulation and pharmacological blockade of growth factor stimulated signaling pathways. The identification of phosphorylation sites required for export will be alter to a non-phosphorylatable amino acids to confirm the requirement of these sites to promote Mdm2 nuclear export. [Aim 2] This specific aim is to characterize the effect of Mdm2 nuclear export on p53 activity and stability in response to growth factors and DNA damage. Preventing Mdm2 nuclear export should have a dramatic effect on p53 activity. We will examine p53 transcriptional activity when Mdm2 is unable to exit the nucleus. We will also determine if p53 ubiquitation mediated by Mdm2 occurs in the nucleus or cytoplasm and in what compartment the p53-Mdm2 complex is degraded. Biologically, we will test if Mdm2 rendered in the nucleus can attenuate p53 dependent apoptosis in response to DNA damage. Considering that nuclear Mdm2 is prevalent in human cancer, upon completion of this proposal we will understand the contribution of signal transduction pathways in promoting Mdm2 nuclear export and potentially how to regulate export of nuclear Mdm2 to increase p53 dependent apoptosis. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA109262-01A1
Application #
6919409
Study Section
Tumor Cell Biology Study Section (TCB)
Program Officer
Blair, Donald G
Project Start
2005-05-11
Project End
2010-04-30
Budget Start
2005-05-11
Budget End
2006-04-30
Support Year
1
Fiscal Year
2005
Total Cost
$241,740
Indirect Cost
Name
Case Western Reserve University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Cheng, Ying-Hua; Streicher, Drew A; Waning, David L et al. (2015) Signaling pathways involved in megakaryocyte-mediated proliferation of osteoblast lineage cells. J Cell Physiol 230:578-86
Wang, Haiyan; Cai, Shanbao; Ernstberger, Aaron et al. (2013) Temozolomide-mediated DNA methylation in human myeloid precursor cells: differential involvement of intrinsic and extrinsic apoptotic pathways. Clin Cancer Res 19:2699-709
Cheng, Ying-Hua; Hooker, R Adam; Nguyen, Khanh et al. (2013) Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation. J Bone Miner Res 28:1434-45
Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua et al. (2012) Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts. J Biol Chem 287:17257-68
Waning, David L; Lehman, Jason A; Batuello, Christopher N et al. (2011) c-Abl phosphorylation of Mdm2 facilitates Mdm2-Mdmx complex formation. J Biol Chem 286:216-22
Lehman, Jason A; Waning, David L; Batuello, Christopher N et al. (2011) Induction of apoptotic genes by a p73-phosphatase and tensin homolog (p73-PTEN) protein complex in response to genotoxic stress. J Biol Chem 286:36631-40
Waning, David L; Lehman, Jason A; Batuello, Christopher N et al. (2010) Controlling the Mdm2-Mdmx-p53 Circuit. Pharmaceuticals (Basel) 3:1576-1593
Cipriano, Rocky; Patton, John T; Mayo, Lindsey D et al. (2010) Inactivation of p53 signaling by p73 or PTEN ablation results in a transformed phenotype that remains susceptible to Nutlin-3 mediated apoptosis. Cell Cycle 9:1373-9
Araki, Shinako; Eitel, Jacob A; Batuello, Christopher N et al. (2010) TGF-beta1-induced expression of human Mdm2 correlates with late-stage metastatic breast cancer. J Clin Invest 120:290-302
Eitel, Jacob A; Bijangi-Vishehsaraei, Khadijeh; Saadatzadeh, M Reza et al. (2009) PTEN and p53 are required for hypoxia induced expression of maspin in glioblastoma cells. Cell Cycle 8:896-901

Showing the most recent 10 out of 14 publications