2015 Fiscal Year Research-status Report
Identification of global epigenetic alterations indispensable for malignant transformation and mediated by the RB-ATM pathway
| Project/Area Number |
15K06834
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| Research Institution | Kanazawa University |
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Principal Investigator |
Shamma A.A.Awad 金沢大学, がん進展制御研究所, 助教 (50402839)
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| Project Period (FY) |
2015-04-01 – 2018-03-31
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| Keywords | RB / ATM / DNA damage / Protein ubiquitination / Stem cell reprogramming / Cancer |
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| Outline of Annual Research Achievements |
The retinoblastoma (RB) tumor suppressor gene is implicated in many facets of cancer. We previously reported novel functions of Rb in regulation of the DNA damage response and cellular senescence that restrict malignant transformation in mice models (Shamma et al. Cancer Cell 2009). In addition, we discovered novel functions of the Rb-ATM pathway in regulation of protein ubiquitination and DNA methylation (Shamma et al. Mol Cell Biol 2013).
In the current study we initially planned to characterize the epigenetic functions of the Rb-ATM pathway at whole-genome level. However, the high cost of the whole-genome bisulfide sequencing (WGBS) was a limitation. We rather focused on the Rb-ATM functions in protein ubiquitination and their significance in stem cell reprogramming and cancer progression.
We discovered that ATM function is necessary for ubiquitination and subsequent degradation of many cancer related proteins such as oncogenes including cyclin D1, cyclin E and c Myc as well as stem cell reprogramming proteins including Oct3/4, Sox2, KLF4 and Nanog. In addition, ATM physically binds to these proteins in complex with the E3 ligases UHRF1 and FBXW7. Furthermore, ATM restoration into RbATM DKO MEFs destabilized stem cell reprogramming proteins and abolished the features of stemness including sphere-formating ability and the three-germ layers teratoma formation. Importantly, these functions of ATM were reversed after inhibition of the proteasome, revealing an important role of the ATM functions in the ubiquitin-proteasome system (UPS).
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Our recent studies (Shamma et al. Mol Cell Biol 2013) provided us with insights about the functions of the Rb-ATM pathway in regulation of DNMT1 protein stability and the associated epigenetic alterations. We speculated that these functions of ATM might not be limited to DNMT1 but this 350KDa protein might exert a general role in the UPS regulation and ubiquitination of other important proteins. This hypothesis was the immediate backup of our research direction such in case we face difficulties of studying global epigenetic functions of the Rb-ATM pathway. Then due to the high cost of the WGBS we switched our experimental plan in the appropriate time and focused on studying the role of the Rb-ATM pathway in protein ubiquitination to link the UPS to stem cell reprogramming and cancer progression.
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| Strategy for Future Research Activity |
During the FY 2015, we made efforts and generated data showing that the Rb-ATM pathway links the UPS to stem cell reprogramming. More specifically, our data indicate that ATM might functions as adaptor protein binds to stem cell reprogramming proteins and bring them into the proximity of E3 ligases for ubiquitin delivery. These are original and novel findings that have implications in cancer therapy and regeneration medicine. However, we still have to validate our findings by performing the followings:
1. Validation of the E3-ATM-stemness proteins complex by using mass spectrometry and crystallography.
2. Examination of the contribution of the RbATM DKO stem-like cells into chimra mice.
3. Determination of the therapeutic benefits of reactivation of the RB-ATM pathway in human cancer.
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