Abstract
The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients.
Highlights
Transforming growth factor beta (TGF-beta) comprises a group of multifunctional polypeptides that regulate different cellular processes, including cell proliferation, apoptosis, differentiation, migration, tumorigenecity and metastasis, through binding to TGF-beta receptors
To further confirm the suppressive effect of TGF-beta on vascular endothelial growth factor A (VEGFA) expression, a dominant negative TGF-beta RII (DNRII) was introduced into FET cells that express TGF-a (FETa) cells to abrogate endogenous TGF-beta signaling, which was shown by reduced Smad2 and Smad3 phosphorylation
Since TGFbeta acts as a metastasis suppressor in colon cancer cells [12], we examined whether suppression of VEGFA expression by TGFbeta contributes to metastasis suppression function of TGF-beta
Summary
Transforming growth factor beta (TGF-beta) comprises a group of multifunctional polypeptides that regulate different cellular processes, including cell proliferation, apoptosis, differentiation, migration, tumorigenecity and metastasis, through binding to TGF-beta receptors. Many studies indicate that TGF-beta signaling acts as either a tumor promoter or a tumor suppressor. The tumor promoter function of TGF-beta has been associated with its ability to induce an epithelial to mesenchymal transition (EMT), which confers resistance to the apoptotic effects of TGFbeta [1,2,3]. We and others have demonstrated experimentally that TGF-beta mediates tumor suppressor activity in a variety of cancers including colon cancer, and that loss of TGFbeta signaling leads to acquisition and progression of malignancy [4,5,6,7,8,9,10,11]. Our recent studies demonstrate that TGF-beta signaling suppresses metastasis in a subset of colon cancer cells in an orthotopic model in vivo [12]. Identifying the mechanism(s) by which TGF-beta elicits its metastasis suppressor function is crucial for our understanding of tumor progression and for developing effective therapeutic strategies
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