2014 Fiscal Year Research-status Report
Role of Smad7 in myeloid leukemogenesis
| Project/Area Number |
26830086
|
|---|
| Research Institution | Japanese Foundation for Cancer Research |
|---|
Principal Investigator |
アーノ クズィーネ 公益財団法人がん研究会, その他部局等, 研究員 (70725621)
|
|---|
| Project Period (FY) |
2014-04-01 – 2016-03-31
|
| Keywords | Leukemia / TGF-beta / Smad7 |
|---|
| Outline of Annual Research Achievements |
First, in order to clarify the effect of TGF-beta on leukemic cells, we performed in vitro experiments by incubating leukemic cells with different concentrations of TGF-beta followed by proliferation rate assay. We found that TGF-beta inhibits cell proliferation of leukemic cells at all doses tested. However, when leukemic cells were co-incubated with stromal cells (OP9 cell line), TGF-beta instead promoted cell proliferation of leukemic cells.
In vivo inhibition of TGF-beta also showed discrepant results. When TGF-beta inhibitor was injected every 2-3 days into Hoxa9-Meis1 inoculated mice, the onset of leukemia occured earlier, suggesting that TGF-beta inhibits leukemic cells in vivo. However, when TGF-beta inhibitor was injected every day, leukemia occured at later time, suggesting that TGF-beta is actually required for the onset of leukemia. It therefore seems that TGF-beta can either promote or inhibit the onset of leukemia depending on the in vivo concentration of the cytokine.
In order to elucidate whether TGF-beta acts directly on leukemic or indirectly, we created leukemic cells that stably overexpress Smad7 (a TGF-beta signaling inhibitor). Mice inoculated with Hoxa9-Smad7 overexpressing cells did not show any sign of leukemia, suggesting that Smad7 overexpression does not promote the onset of leukemia in vivo.
However, in vitro experiments showed that phosphorylation of Smad2 was identical between Hoxa9-Meis1 and Hoxa9-Smad7 overexpressing cells. This result suggests that Smad7 does not inhibit TGF-beta signaling in leukemic cells.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Following TGF-beta stimulation in leukemic cells, Smad7 overexpression did not inhibit Smad2 phosphorylation. We initially thought that this unexpected result was specific to leukemic cells. However, when Smad7 was overexpressed in other cell lines, such as HeLa cells, U2OS cells and NIH3T3 cells, either transiently or stably overexpressed, Smad7 could not inhibit phosphorylation of Smad2 in all of the cell lines tested. Moreover, Smad7 did not interfere with Smad2 translocation into the nucleus as viewed by immunofluorescence studies.
We therefore sequenced the Smad7 transgene but found no mutation. Therefore, for an unidentified reason, Smad7 does not inhibit TGF-beta signaling in our hands, although it is clearly overexpressed as seen by western blot.
Instead of overexpressing Smad7, we attempted a different strategy by knocking down Smad7 with shRNA in leukemic cells. However, we were unable to obtain Smad7 knocked down cells. Similarly, TGF-beta Receptor I knock down also did not work.
|
| Strategy for Future Research Activity |
We are planning to shed clarity onto the precise role of TGF-beta in myeloid leukemia onset. Indeed, it seems that TGF-beta has both positive and negative impact(s) on leukemia onset depending on the cell type it is acting on.
For instance, while TGF-beta is likely to be a repressive factor when acting directly on leukemic cells (as a cell cycle inhibitor), it seems however to be a promoting factor when acting on bone marrow stromal cells. We will therefore create leukemic cells using bone marrow from mice deficient in TGF-beta Receptor I or II, and inoculate these cells into wild type recipient mice to assess the onset of leukemia. In parallele, the usage of Smad7 overexpression in leukemic cells will also be reinvestigated using a new plasmid-inserted cDNA.
In addition, other cell types not necessarily restricted to the bone marrow environment are also sensitive to TGF-beta stimulation, such as regulatory T cells (Tregs) for differentiation and T cells for inhibition. We found that Tregs depletion delayed the onset of leukemia, demonstrating that Tregs activity promotes the onset of leukemia.
Tregs are therefore required for the rapid onset of leukemia, likely by inhibiting T cells activity through TGF-beta secretion. Moreover, we found that Tregs proportion increases in the spleen of mice developing leukemia.
Regarding its activity on the T cell lineage, TGF-beta seems therefore to be a promoting agent for the onset of leukemia since it promotes Tregs differentiation and inhibits T cells activity. This hypothesis will be investigated using Foxp3-/- and RAG2-/- mice.
|
| Causes of Carryover |
Since the response of AML cells to the TGFb stimulus in the presence of Smad7 was unexpected, I changed the direction of the current research to get insight into the role of Meis1 in the immune system. Therefore, new experiments are being prepared. In the next fiscal year , I need immune compromised mice and antibodies. That is why I incurred amount to be used next fiscal year.
|
| Expenditure Plan for Carryover Budget |
The amount will be used to purchase immune compromised mice such as NOD-SCID, and antibodies to T-cell surface markers which will be used for FACS analyses.
|
Research Products
(1 results)
