Direct Regulation of Alternative Splicing by SMAD3 through PCBP1 Is Essential to the Tumor-Promoting Role of TGF-β

Abstract

Summary In advanced stages of cancers, TGF-β promotes tumor progression in conjunction with inputs from receptor tyrosine kinase pathways. However, mechanisms that underpin the signaling cooperation and convert TGF-β from a potent growth inhibitor to a tumor promoter are not fully understood. We report here that TGF-β directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated T179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. We show that TGF-β and EGF respectively induce SMAD3 and PCBP1 to colocalize in SC35 positive nuclear speckles, and the two proteins interact in the variable exon region of CD44 pre-mRNA to inhibit spliceosome assembly in favor of expressing the mesenchymal isoform CD44s over the epithelial isoform CD44E. We further show that the SMAD3-mediated alternative splicing is essential to the tumor-promoting role of TGF-β and has a global influence on protein products of genes instrumental to epithelial to mesenchymal transition and metastasis.

Redirecting RNA splicing by SMAD3 turns TGF-β into a tumor promoter

“…6 We also found that a nonphosphorytable T179V mutant of SMAD3 while still retaining its transcriptional activity was nevertheless inept in restoring the mesenchymal gene expression, TGF-b-mediated invasion, and formation of lung metastasis to SMAD3-depleted cells. These experimental clues suggested that phosphorylation at T179 could contribute to the contextual cue of cancer progression and prompted us to seek nontranscriptional mechanism of SMAD3 in mediating TGF-b-induced mesenchymal gene expression and cell invasion and metastasis.…”

“…In searching for proteins that confer regulation of the key TGF-b pathway transcription effector SMAD3 via phosphorylation of threonine 179 (T179) in the linker region, we identified an RNA-binding protein poly(RC) binding protein 1 (PCBP1, also known as hnRNP E1), and serendipitously discovered that by partnering with PCBP1, SMAD3 is brought onto the pre-mRNA of a cancer stem cell marker gene CD44 to regulate its alternative splicing. 6 In addition to CD44, our global RNA-seq study revealed a plethora of cancers genes whose splicing patterns are altered by the SMAD3-PCBP1 interaction in favor of tumor progression. These findings let us to propose that regulation of alternative splicing by the concerted action of receptor-activated SMAD3 and PCBP1 is a key mechanism that propels TGF-b to a tumor promoter (Fig.…”

“…Against this backdrop, PCBP1 was identified as a SMAD3-binding partner. 6 The notable features of this new interaction include phosphorylation-induced binding and colocalization of both proteins in the nuclear antigen serine and arginine-rich splicing factor 2 (SRSF2, also known as SC35) positive speckles that are intimately involved in RNA splicing. Modeling on a heavily spliced CD44 gene, we demonstrated that SMAD3 indeed participates directly in the splicing of CD44 mRNA isoforms that are preferentially expressed in association with EMT.…”

Phosphorylation status at Smad3 linker region modulates transforming growth factor‐β‐induced epithelial‐mesenchymal transition and cancer progression

“…Alternative splicing induced by Smad3 linker phosphorylation is now extended to occur in a large number of genes required for TGF‐β‐induced EMT and metastasis. Upon binding of either Pin1, Nedd4L, Smurf2, or PCBP1 to phosphorylated linker sites of Smad3, phosphorylation at the T179 residue has been determined to play a pivotal role in eliciting biological activities …”

“…Upon binding of either Pin1, Nedd4L, Smurf2, or PCBP1 to phosphorylated linker sites of Smad3, phosphorylation at the T179 residue has been determined to play a pivotal role in eliciting biological activities. 35…”