TMEPAI/PMEPA1 inhibits Wnt signaling by regulating β-catenin stability and nuclear accumulation in triple negative breast cancer cells

doi:10.1016/j.cellsig.2019.03.016

Cellular Signalling

Volume 59, July 2019, Pages 24-33

https://doi.org/10.1016/j.cellsig.2019.03.016 Get rights and content

Highlights

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TMEPAI/PMEPA1 suppresses β-catenin/TCF4 transcriptional activity on Wnt target genes.
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TMEPAI regulates β-catenin stability and nuclear accumulation.
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TMEPAI PY motifs are essential for Wnt signaling suppression.

Abstract

Transmembrane prostate androgen-induced protein (TMEPAI) is a type I transmembrane protein induced by several intracellular signaling pathways such as androgen, TGF-β, EGF, and Wnt signaling. It has been reported that TMEPAI functions to suppress TGF-β and androgen signaling but here, we report a novel function of TMEPAI in Wnt signaling suppression. First, we show that TMEPAI significantly inhibits TCF/LEF transcriptional activity stimulated by Wnt3A, LiCl, and β-catenin. Mechanistically, TMEPAI overexpression prevented β-catenin accumulation in the nucleus and TMEPAI knockout in triple negative breast cancer cell lines promoted β-catenin stability and nuclear accumulation together with increased mRNA levels of Wnt target genes AXIN2 and c-MYC. The presence of TGF-β type I receptor kinase inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells. These data suggest that TMEPAI suppresses Wnt signaling by interfering with β-catenin stability and nuclear translocation in a TGF-β signaling-independent manner.

Graphical abstract

Introduction

Transmembrane prostate androgen induced protein (TMEPAI), also known as prostate transmembrane protein, androgen induced 1 (PMEPA1), or solid tumor associated gene 1 (STAG1), was originally identified as a prostatic protein induced by testosterone or its derivatives [1,2]. It is a type 1b transmembrane protein consisting of a short extracellular domain, a single-pass transmembrane domain (TM), and intracellular domain, including two PPxY (PY) motifs and a Smad interaction motif (SIM) [3,4].

During tumor progression, cancer cells undergo a number of alterations in cellular signaling pathways which control cell proliferation, survival, motility, and metabolism. Many proteins in these signaling pathways are currently under investigation as cancer therapeutic targets [5,6]. TMEPAI is constitutively and highly expressed in many types of cancers including triple negative breast cancer (TNBC), an aggressive subtype of breast cancer compared with other types of breast cancers, and associated with poor prognoses in TNBC [7,8]. In consistent with these findings, we previously demonstrated that knockdown of TMEPAI in human lung cancer cells reduces tumorigenic activities (such as xenograft tumor formation and sphere formation) [9], TMEPAI is thought to be an oncogenic protein even though its role in tumorigenesis is still not fully understood.

TMEPAI itself is induced by TGF-β [4,7,8,10] and ERK signaling [2,11]. In addition, our previous study showed that TMEPAI is a downstream target of Wnt signaling in which the Wnt/β-catenin/TCF7L2 pathway preferentially activates TMEPAI/PMEPA1 gene transcription together with TGF-β signaling [12]. Since the Wnt signaling pathway controls cell proliferation and stem cell maintenance [13], we investigated the role of TMEPAI in Wnt signaling with respect to mechanistic interaction with Wnt downstream targets. Here, we demonstrate that TMEPAI inhibits Wnt signaling and subsequent AXIN2 and c-MYC transcription by regulating β-catenin protein stability and nuclear accumulation.

Section snippets

Cell culture

MDA-MB-231, Hs578T, human embryonic kidney (HEK) 293, and HEK-293 T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 10% fetal bovine serum (FBS) (Life Technologies) and penicillin-streptomycin solution (Wako). Hs578T cells and Hs578T cells stably expressing TMEPAI isoform A were cultured in the above medium supplemented with 10 μg/ml insulin and 8 μg/ml blasticidin hydrochloride. BT-549 cells were cultured in RPMI-1640 medium (Sigma) supplemented with 10%

TMEPAI inhibits Wnt signal-induced TOP-flash-luciferase reporter activity

Human TMEPAI transcription results in four isoforms (A, B, C and D) (Sup. Fig. 1) and is coordinated by both TGF-β and Wnt signaling [4,12]. First, we confirmed our previous finding of the inhibitory role of TMEPAI in TGF-β/Smad signaling [4]. As expected, overexpression of TMEPAI isoforms A, B, and D in HEK-293 cells inhibited the (CAGA)₁₂-luciferase reporter, which is specifically activated by the TGF-β-induced Smad3 and Smad4 complex (Sup. Fig. 2). We then tested the role of TMEPAI in Wnt

Discussion

TGF-β and Wnt signaling pathways are important for cellular function and activation of both pathways is required for many developmental and patterning events [28]. Multiple studies have described the signaling crosstalk between TGF-β and Wnt pathways by showing that Axin can associate with the TGF-β-regulated Smad2 and Smad3 [[22], [23], [24], [25], [26], [27]]. Mouse models have indicated the cooperation of TGF-β and Wnt signaling in cancer [28], although much remains to be understood on how

Conclusions

Collectively, our data support the inhibitory role of TMEPAI in Wnt signaling. TMEPAI regulates β-catenin stability, nuclear accumulation, and the resultant transcriptional activity that affects the expression of Wnt target genes AXIN2 and c-MYC.

Conflict of interest

The authors declare that there are no conflicts of interest.

Author contributions

Conception and design of the work: R.A., Y.W., M.K. Acquisition of data: R.A., M.A., M.U.P., J.H., F. A., Y.W. Analysis and interpretation of data: R.A., M.A., M.U.P., J.H., F. A., Y.W., M.K. Drafting or correcting the manuscript: R.A., Y.W., M.K. All authors read and approved of the final article.

Acknowledgements

This work was supported by Grant-in-Aid for Young Scientists (B) [JP25870093 (Y.W.), JP16K19100 (Y.W.)], for Scientific Research (B) [JP25293092 (M.K.)], for Scientific Research on Innovative Area [JP26116707 (M.K.)] and Research Grant from the Uehara Memorial Foundation (Y.W.). The authors thank George Church for the kind gifts of hCas9 expression vector (Addgene plasmid #41815) and gRNA_cloning vector (Addgene plasmid #41824). We also thank Dr. Bryan J. Mathis of the Medical English

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TMEPAI/PMEPA1 inhibits Wnt signaling by regulating β-catenin stability and nuclear accumulation in triple negative breast cancer cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Cell culture

TMEPAI inhibits Wnt signal-induced TOP-flash-luciferase reporter activity

Discussion

Conclusions

Conflict of interest

Author contributions

Acknowledgements

Geno.

Mol. Cell

J. Biol. Chem.

Biochem. Biophysical. Res. Com.

J. Biol. Chem.

Cell

Develop. Cell.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Cell. Sig.

J. Biol. Chem.

Cancer Cell

EGF- and cell-cycle-regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer

Mol. Carcinog.

PMEPA1, an androgen-regulated NEDD4-binding protein, exhibits cell growth inhibitory function and decreased expression during prostate cancer progression

Cancer Res.

Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers

PNAS.