2016 Fiscal Year Research-status Report
Hotspot Synonymous Cancer Mutations Part 1: Effect on Cap-independent Translation of New HRAS Isoform
| Project/Area Number |
16K21111
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Keywords | cancer / synonymous mutation / IRES / HRas / HRas mRNA / HRas isoform |
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| Outline of Annual Research Achievements |
Our purpose for the whole period of the grant was to understand why the synonymous mutation H27H in HRas is so common in cancer. In order to accomplish that we had laid out 5 tasks: 2 for the first year [1. Investigate the mechanisms that regulate the translation of H-Ras isoforms & 2. Define and characterize the RNA structures responsible for HRas isoforms’ expression] and 3 for the second year [3. Test the impact of HRas synonymous mutation on IRES function and isoforms expression; 4. Investigate the role of IRESs and SCM on carcinogenesis in cell and mouse models & 5. Bioinformatic & statistical analyses of synonymous mutations in HRas and other cancer-related genes]. We have now successfully accomplished task 1 and partially completed tasks 2 and 3, so we are progressing as planned.
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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
The progress has been smooth. Even though we have not concluded task 2 completely, we have instead advanced with task 3 already and obtained some good results on that front as well. Briefly, our progress so far is as follows: With task 1 we have identified a cellular stress that induces the new p14HRas isoform. We have tested this in 6 different cell lines so our data is very robust. We have then partially accomplished task 2 (we could identify the site of the HRas IRES and confirm its function and induction by stress using bicistronic reporter constructs) and task 3 (confirmed our theory that the very common synonymous mutation in cancer is changed IRES function and p14HRas expression). We had a little trouble with the sensitivity of the p14HRas to very small alterations in cell culture, but we could overcome that difficulty. We were also surprised to identify one extra WB band for most cell lines. We are still trying to identify its origin.
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| Strategy for Future Research Activity |
Our plan will not diverge from last year’s estimation. We will conclude task 2 by analyzing the secondary structures of the HRas IRES. For this we will perform DMS modifications followed by primer extension assays (toeprinting). We will performed the last experiments to conclude task 3 and then focus on tasks 4 and 5. In task 4 we will examine the roles of the synonymous mutation, IRES and the new isoform p14 in cell proliferation and transformation. If these results are encouraging, we would then like to test the tumorigenicity of this new isoform using xenografts in mice. Finally, with the aid of Research Collaborator JB Brown, we will search for new hotspot synonymous mutations that are likely to have an impact on cancer. We will promote our research in 2 international meetings this year, with a book in “Experimental Medicine” and by organizing a workshop at the MBSJ annual meeting. We published an article in EMBO Reports end of last year.
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| Causes of Carryover |
Due to the small change in the plan (since we already advanced with task 3 (almost completed) but did not yet conclude task 2), there was also a small change in the use of money. Task 3 is cheaper than task 2 because it uses mostly the same methods as in task 1, so we saved a little money that will now be required in the second year in order to conclude task 2.
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| Expenditure Plan for Carryover Budget |
The carryover money will be used in the second year in order to complete task 2 (this task has not yet been concluded because we focused on task 3 first). To conclude task 2 new products are necessary (such as DMS) as well as different methods (RNA work) and the use of different equipment (sequencer).
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