Phosphorylation status of TGF-beta receptor-regulated SMADs in the pathophysiology of triple negative breast cancers

2018 Fiscal Year Research-status Report

• Project/Area Number: 18K15252
• Research Institution: Tokyo Medical University
• Principal Investigator: 裴 恩真 東京医科大学, 医学部, 兼任助教 (40773388)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Keywords: Breast cancer / TGF-β / Smad / Phosphorylation / Chemoresistance

Outline of Annual Research Achievements

I have uncovered the phosphorylation state-specific roles of transforming growth factor-β (TGF-β)-regulated receptor Smads: Smad2 and Smad3 in chemoresistance of triple-positive breast cancer (TPBC) and epithelial-mesenchymal transition (EMT) of triple-negative breast cancer (TNBC) as summarized below in the first fiscal year, 2018.
<1> TGF-β induced apoptosis of MCF-7 TPBC cells via C-terminal phosphorylation of Smad3.
<2> IL-6 rendered MCF7 TPBC cells resistant to paclitaxel and tamoxifen via linker phosphorylation of Smad2 at serine 255 and 245 residues (S255 and S245).
<3> Linker-phosphorylated Smad2 at S255 interacted with Stat3 and Stat5 to distinctively regulate their opposing effects on apoptosis of MCF-7 TPBC cells.
<4> Linker-phosphorylated Smad3 induced EMT of MDA-MB-231 TNBC cells.
I am preparing a manuscript based on the achievements: <1>, <2> and <3> for submission.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Proposed research plan and methods <1> Phosphorylation status of R-SMADs and localization of SMADs in TNBC and TPBC human breast cancer cell lines (FY2018 1st half): I have completed this part using MCF-7 TPBC and MDA-MB 231 TNBC cells.
<2> Effects of phosphorylation status of R-SMADs on the survival, proliferation, apoptosis, EMT, cancer stem cell populations, and gene regulation (FY2018~FY2019 1st half): I have examined the effects of various linker and C-terminally-phosphorylated Smad2 and Smad3 on survival, proliferation, apoptosis, EMT of MCF-7 TPBC and MDA-MB-231 TNBC cells. I have found that IL-6-induced linker-phosphorylation of Smad2 at S255 and S245 rendered MCF7 TPBC cells resistant to paclitaxel and tamoxifen, whereas C-terminal phosphorylation of Smad3 induced apoptosis of MCF7 TPBC cells. By contrast, linker-phosphorylated Smad3 induced EMT of MDA-MB-231 TNBC cells.
<3> Signaling network of R-SMADs and kinase pathways(FY2018 2nd half~FY2019): I have discovered that the interaction of linker-phosphorylated Smad2 with Stat3 or Stat5 affects the chemosensitivity of MCF-7 TPBC cells more potently than the reported kinases.
<4> Phosphorylation status of R-SMADs and chemoresistance of TNBCs (FY2018 2nd half~FY2019): I have found the involvement of linker-phosphorylated Smad3 in EMT and chemoresistance of MDA-MB-231 TNBC cells.

Strategy for Future Research Activity

I will continue the research plan and methods <2>, <3>, <4>. I will especially focus on the novel finding on the interaction between linker-phosphorylated Smad and Stat as described above in <3>.
I will check the phosphorylation status of Smad2/3 in Asian breast cancer patient samples by human breast cancer tissue array (SuperBioChips, http://www.tissue-array.com/main.html) as proposed in <5> Screening of R-SMAD phosphorylation status in breast cancer tissue samples <FY2019>.

Causes of Carryover

Because the remaining amount for FY2018 was not enough to purchase the required reagents, I have decided to purchase those FY2019.