Riezki Amalia

Tsukuba Repository

The roles of TMEPAI in Wnt signaling
著者 Riezki Amalia
発行年 2017
その他のタイトル TMEPAIによるWntシグナル抑制機構の解明
学位授与大学 筑波大学 (University of Tsukuba)
学位授与年度 2017
報告番号 12102甲第8388号
URL http://hdl.handle.net/2241/00150020
  
氏     名 Riezki Amalia 
学 位 の 種 類       EA 博士（医学） 
A学 位 記 番 号       EA 博甲第  8388  号 
A学位授与年月       EA 平成 29年 9月 25日 
A学位授与の要件       EA 学位規則第４条第１項該当 
A審 査 研 究 科       EA 人間総合科学研究科 
A学位論文題目       EA The roles of TMEPAI in Wnt signaling       
              (TMEPAIによるWntシグナル抑制機構の解明) 
A主 査       EA     筑波大学教授 薬学博士    熊谷 嘉人     
副 査       EA   筑波大学教授 博士（医学） 藤本 学  
A副 査       EA   筑波大学准教授 医学博士     内田 和彦   
A副 査       EA   筑波大学准教授 博士（薬学） 大林 典彦 
 
 
論文の内容の要旨 
Abstract of thesis 
 
【背景 Background】 
TMEPAI (Transmembrane prostate androgen-induced protein) is involved in intracellular signaling pathways such 
as androgen, TGF-β, PI3K/ERK, and Wnt signaling. In the present study, the applicant elucidated TMEPAI’s function 
in Wnt signaling. TOP-Flash reporter luciferase assays showed that TMEPAI suppressed Wnt signaling induced by 
treatment of Wnt3A, and LiCl, and β-catenin overexpression. Moreover, the applicant found that TMEPAI inhibits 
Wnt signaling by binding to β-catenin and preventing β-catenin nuclear translocation, the mRNA levels of Wnt-target 
genes, AXIN2 and C-MYC, were increased in TMEPAI knockout breast cancer cell lines. Furthermore, SB431542, a 
TGF-β signaling inhibitor, did not change AXIN2 level in TEMEPAI knockout cells. Overall, the applicant suggests 
that TMEPAI might directly interfere Wnt signaling. 
 
【目的 Purpose】 
Expression of Transmembrane prostate androgen-induced protein (TMEPAI) is induced by androgen, TGF-β, 
PI3K/ ERK, and Wnt signaling. Structurally, TMEPAI consists of a short extracellular domain, a transmembrane 
domain, and a Smad interaction motif (SIM) between two PPxY (PY) motifs; however, protein structure has not been 
precisely clarified. In this thesis, the applicant investigated predicted protein structure of TMEPAI and found a novel 
motif, Shisa-like motif, in TMEPAI. Since Shisa protein has been known as a suppressor of Wnt signaling, the 
applicant examined the role and molecular mechanism of of TMEPAI in Wnt signaling pathway.  
 
【対象と方法 Materials and methods】 
Computational prediction methods for TMEPAI protein structure were performed by using I-TASSER and 
RaptorX software and I-TASSER and Phyre2. To understand the mechanism, the applicant used TMEPAI knockout 
breast cancer cell line and TMEPAI knockdown breast cancer cell lines established using CRISPR/ Cas9 system and 
small interference RNA (siRNA), respectively. Sphere formation assays were done to examine oncogenic activities 
of TMEPAI in breast and colon cancer cells.  
 
【結果 Results】 
The luciferase activity showed that TMEPAI could suppress not only Wnt3A ligand-induced Wnt signaling but 
also LiCl-induced, and β-catenin overexpression-induced Wnt signaling. In addition, the applicant found that PY 
motifs of TMEPAI are involved in the β-catenin-dependent Wnt signaling. Therefore, the applicant examined the 
molecular mechanism and found that TMEPAI binds to β-catenin and prevent β-catenin nuclear translocation. In 
contrast, nuclear accumulation of β-catenin and mRNA expression of Wnt target genes, AXIN2 and C-MYC, were 
enhanced in TMEPAI knockout cancer cell lines, MDAMB-231 and Hs578T cells. In addition, the applicant 
examined the involvement of TGF-β signaling in TMEPAI-mediated suppression of Wnt signaling and found that 
SB431542 inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells and TMEPAI 
SIM mutant could suppress Wnt signaling as well as TMEPAI wild type. 
 
【考察 Discussion】 
The applicant demonstrated that TMEPAI could interact with β-catenin and this interaction might be important for 
inhibition of β-catenin nuclear translocation and its transcriptional activity. TGF-β type I inhibitor did not affect the 
enhancement of Wnt target genes in TMEPAI knockout cells, suggesting that TMEPAI directly suppressed Wnt 
signaling in TGF-β-independent manner. Since both TGF-β and Wnt signaling might be involved in cell proliferation 
and stem cell maintenance, TMEPAI could play important roles in tumorigenesis by regulating both signaling. 
 
【結果 Conclusion】 
TMEPAI could inhibit Wnt/ β-catenin signaling via its PY motifs and prevent β-catenin nuclear translocation and 
gene transcription.  
 
審査の結果の要旨 
 Abstract of assessment result 
 
【批評 General Comments】 
The doctoral thesis of Ms. Riezki Amalia elucidated that TMEPAI directly interfered Wnt signaling. The applicant 
also suggested a novel motif in the TMEPAI structure, Shisa-like motif and found that SB431542 inhibitor did not 
affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells and TMEPAI SIM mutant could suppress 
Wnt signaling as well as TMEPAI wild type. Although further study is required to investigate the role in TMEPAI 
functions, these original findings constitute valuable contribution to this field of research. 
 
【最終試験の結果 Assessment】 
The final examination committee conducted a meeting as a final examination on June 15, 2017. The applicant 
provided an overview of dissertation, addressed questions and comments raised during Q&A session. All of the 
committee members reached a final decision that the applicant has passed the final examination.  
 
【結果 Conclusion】 
Therefore, the final examination committee approved that the applicant is qualified to be awarded Doctor of 
Philosophy in Medical Sciences. 


TMEPAIによるWntシグナル抑制機構の解明

この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201

Amalia Riezki

The roles of TMEPAI in Wnt signaling
著者 Riezki Amalia
内容記述 この博士論文は内容の要約のみの公開（または一部
非公開）になっています
year 2017
その他のタイトル TMEPAIによるWntシグナル抑制機構の解明
学位授与大学 筑波大学 (University of Tsukuba)
学位授与年度 2017
報告番号 12102甲第8388号
URL http://hdl.handle.net/2241/00150033
      
論  文  概  要 
    
    
    
    
    
    
  ○ 論 文 題 目 
               The roles of TMEPAI in Wnt signaling       
               (TMEPAIによるWntシグナル抑制機構の解明)                                       
              
  論文題目が外国語の場合には，（   ）書きで日本語訳を記入すること。   
  
  
    
    
  ○ 指 導 教 員   
            
    
   人間総合科学研究科 Biomedical Sciences専攻   
                    Prof. Mitsuyasu Kato教授   
  
  
  
    
      
（所  属）   筑波大学大学院人間総合科学研究科 Biomedical Sciences 専攻   
  
      
（氏  名）                                            Riezki Amalia  
              
    
    
    
    
    
      
 
 
 
 
目的：  
[Purpose] 
Transmembrane prostate androgen-induced protein (TMEPAI) is induced by androgen, TGF-β, 
PI3K/ ERK, and Wnt signaling. It is highly expressed in many types of cancer and associated 
with poor prognosis. Structurally, TMEPAI consists of a short extracellular domain, a 
transmembrane domain, and a Smad interaction motif (SIM) between two PPxY (PY) motifs. 
However, no solved protein structure has been reported. In this thesis, I performed evaluation of 
the predicted protein structure of TMEPAI. By structural analysis, I found a novel motif, Shisa-
like motif, in TMEPAI. Shisa protein has been known as a suppressor of Wnt signaling. Hence, I 
further examined the role of TMEPAI in Wnt signaling pathway and its molecular mechanism. 
 
対象と方法：  
(Material and method) 
I carried out computational prediction methods for TMEPAI protein structure. I used I-TASSER 
and RaptorX software to evaluate the secondary structure of human TMEPAI. Furthermore, I 
used I-TASSER and Phyre2 to establish three-dimensional (3D) prediction structure of TMEPAI 
protein. I carried out molecular biological methods such as polymerase chain reaction (PCR), 
quantitative PCR (qPCR), western blotting, immunoprecipitation (IP), and nuclear fractionation 
to examine the expression, interactions, and localization of the proteins. I used luciferase-
reporter assays to examine the transcriptional activity of intracellular signaling pathways. To 
understand the mechanism, I used TMEPAI knockout breast cancer cell line and TMEPAI 
knockdown breast cancer cell line that are established using CRISPR/ Cas9 system and small 
interference RNA (siRNA), respectively. I also performed sphere formation assays to examine 
oncogenic activities of TMEPAI in breast and colon cancer cells.  
 
結果：  
(Results)  
The luciferase activity result showed that TMEPAI could suppress not only Wnt3A ligand-
induced Wnt signaling but also LiCl-induced, and β-catenin overexpression-induced Wnt 
signaling. Targeted the interaction between TMEPAI and β-catenin, I found that TMEPAI binds 
to β-catenin and overexpression of TMEPAI prevents β-catenin nuclear translocation. TMEPAI 
knockout in breast cancer cell lines, MDA-MB-231 and Hs578T cells, promotes β-catenin 
nuclear accumulation and increases the mRNA expression of Wnt target genes, AXIN2 and C-
MYC. Additionally, I examined the involvement of TGF- signaling in TMEPAI-mediated 
suppression of Wnt signaling and I found that SB431542 inhibitor did not affect the enhanced 
mRNA expression of AXIN2 in TMEPAI knockout cells and TMEPAI SIM mutant could 
suppress Wnt signaling as well as TMEPAI wild type. 
 
考察：  
[Disscussion]  
I demonstrated that TMEPAI could interact with -catenin and this interaction may be important 
for inhibition of -catenin nuclear translocation and its transcriptional activity. Previous reports 
suggested that Smad3 and β-catenin interaction attenuated β-catenin degradation, facilitates β-
catenin nuclear translocation, and transcriptional activity. Furthermore, TMEPAI is also known 
as a negative regulator of TGF-β signaling by binding to Smad3 and inhibiting its 
phosphorylation. However, the addition of TGF- type I inhibitor did not affect the enhancement 
of Wnt target genes in TMEPAI knockout cells. These data indicated that TMEPAI directly 
suppressed Wnt signaling in a TGF--independent manner. Both TGF-β and Wnt signaling are 
involved in cell proliferation and stem cell maintenance and by regulating both signaling 
pathways, TMEPAI could play important roles in tumorigenesis. 
  
結論：  
[Conclusion]  
TMEPAI could inhibit Wnt/ β-catenin signaling via its PY motifs and prevent β-catenin nuclear 
translocation and gene transcription. Furthermore, TMEPAI knockout in breast cancer cell line 
increased the mRNA level of Wnt target genes, AXIN2 and C-MYC. I suggested a novel motif in 
the TMEPAI structure, Shisa-like motif, although I need to further investigate the role of it in 
TMEPAI functions.  


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TMEPAIによるWntシグナル抑制機構の解明

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