Definition of Smad3 Phosphorylation Events That Affect Malignant and Metastatic Behaviors in Breast Cancer Cells

Abstract

Smad3, a major intracellular mediator of TGFb signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFb, the TGFb receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFb-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelialmesenchymal transition, and invasive activity. Moreover, all TGFb responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer. Cancer Res; 74(21);

Palbociclib enhances activin‐SMAD‐induced cytostasis in estrogen receptor‐positive breast cancer

“…It has been shown that among the MCF‐10A series of cell lines, the TGF‐β/activin‐SMAD pathway has cytostatic effects in the premalignant and low‐grade breast carcinoma line MCF10A (M‐II and M‐III), whereas it enhances metastasis in a high‐grade breast cancer line MCF10A M‐IV . Introduction of a SMAD3 mutant with phosphorylation‐resistant mutations in the linker was reported to inhibit primary tumor growth, but it significantly increased lung metastasis of MCF10A M‐IV . This was in contrast to the results reported for a SMAD3 mutant with a truncated C‐terminus (Smad3ΔC), which functions as dominant negatives .…”

Inflammatory cytokines prime adipose tissue mesenchymal stem cells to enhance malignancy of MCF‐7 breast cancer cells via transforming growth factor‐β1

“…TGF‐β in complex with its type I and type II receptors, leads to activation of Smad proteins, with an important role in the mammary development and tumorigenesis (,). We showed that CM from cytokines‐primed hASCs induced phosphorylation and nuclear translocation of Smad3 in MCF‐7 cells, thus supporting previous findings which showed that Smad3 phosphorylation can be induced by direct cell contacts/interactions between bone marrow MSCs and colorectal cancer cells .…”

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

“…To see the effect of Smad3 linker phosphorylation on TGF‐β responses and Smad3 transcriptional activity, a high‐grade human breast cancer cell line (MCF‐10CA1a.cl1, hereafter known as CA1a) in culture was first treated with a pan‐CDK inhibitor, flavopiridol. TGF‐β transcriptional activity was significantly upregulated by treatment with flavopiridol concomitant with reduction of Smad3 linker phosphorylation at T179, S204, and S208, suggesting that Smad3 linker phosphorylation negatively regulates Smad3 transcriptional activity as determined by (CAGA) 12 ‐luciferase ([CAGA] 12 ‐Luc) reporter assay (Figure B) . Likewise, inhibition of Smad3 linker phosphorylation by a specific phosphatase (SCP1) acting on the Smad2/3 linker region has resulted in upregulation of TGF‐β signaling …”

“…It was initially reported that Ras‐induced Smad2/3 linker phosphorylation inhibits TGF‐β‐induced responses by interrupting nuclear translocation of Smad2/3 and subsequent Smad‐dependent transcription . Direct evidence that Smad3 linker phosphorylation suppresses TGF‐β responses comes from studies using a Smad3 mutant at linker phosphorylation sites . We have adopted recombinant adenoviruses constitutively expressing wild‐type Smad3 (Ad‐Smad3) or its mutant at the linker region (Ad‐EPSM), the C‐terminal (Ad‐3SA) or both the linker and the C‐terminal (Ad‐EPSM/3SA), in which all serine and threonine phosphorylation sites are replaced with alanine (A) and valine (V), respectively (Figure A) .…”

“…Direct evidence that Smad3 linker phosphorylation suppresses TGF‐β responses comes from studies using a Smad3 mutant at linker phosphorylation sites . We have adopted recombinant adenoviruses constitutively expressing wild‐type Smad3 (Ad‐Smad3) or its mutant at the linker region (Ad‐EPSM), the C‐terminal (Ad‐3SA) or both the linker and the C‐terminal (Ad‐EPSM/3SA), in which all serine and threonine phosphorylation sites are replaced with alanine (A) and valine (V), respectively (Figure A) . When Ad‐EPSM was infected with the breast cancer cell line (CA1a), TGF‐β‐induced Smad3 transcriptional activity as evaluated by (CAGA) 12 ‐Luc reporter assay was greatly enhanced in Ad‐EPSM‐infected cells as compared to Ad‐Smad3‐infected counterparts.…”