Catherine E. Gatza

Roles for the type III TGF-β receptor in human cancer

Transforming growth factor beta (TGF-beta) superfamily ligands have important roles in regulating cellular homeostasis, embryonic development, differentiation, proliferation, immune surveillance, angiogenesis, motility, and apoptosis in a cell type and context specific manner. TGF-beta superfamily signaling pathways also have diverse roles in human cancer, functioning to either suppress or promote cancer progression. The TGF-beta superfamily co-receptor, the type III TGF-beta receptor (TbetaRIII, also known as betaglycan) mediates TGF-beta superfamily ligand dependent as well as ligand independent signaling to both Smad and non-Smad signaling pathways. Loss of TbetaRIII expression during cancer progression and direct effects of TbetaRIII on regulating cell migration, invasion, proliferation, and angiogenesis support a role for TbetaRIII as a suppressor of cancer progression and/or as a metastasis suppressor. Defining the physiological function and mechanism of TbetaRIII action and alterations in TbetaRIII function during cancer progression should enable more effective targeting of TbetaRIII and TbetaRIII mediated functions for the diagnosis and treatment of human cancer.

Endoglin regulates PI3-kinase/Akt trafficking and signaling to alter endothelial capillary stability during angiogenesis

Endoglin interacts with PI3K via GIPC to recruit and activate PI3K/Akt at the cell membrane. TGF-β1 attenuates, whereas BMP-9 enhances, endoglin/GIPC-mediated membrane scaffolding of PI3K/Akt to alter endothelial capillary tube stability in vitro, and GIPC mediates endoglin function during developmental angiogenesis in vivo.

Ectodomain shedding of TβRIII is required for TβRIII-mediated suppression of TGF-β signaling and breast cancer migration and invasion

The type III transforming growth factor β (TGF-β) receptor (TβRIII), also known as betaglycan, is the most abundantly expressed TGF-β receptor. TβRIII suppresses breast cancer progression by inhibiting migration, invasion, metastasis, and angiogenesis. TβRIII binds TGF-β ligands, with membrane-bound TβRIII presenting ligand to enhance TGF-β signaling. However, TβRIII can also undergo ectodomain shedding, releasing soluble TβRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TβRIII on TGF-β signaling and breast cancer biology, we defined TβRIII mutants with impaired (ΔShed-TβRIII) or enhanced ectodomain shedding (SS-TβRIII). Inhibiting ectodomain shedding of TβRIII increased TGF-β responsiveness and abrogated TβRIIIs ability to inhibit breast cancer cell migration and invasion. Conversely, expressing SS-TβRIII, which increased soluble TβRIII production, decreased TGF-β signaling and increased TβRIII-mediated inhibition of breast cancer cell migration and invasion. Of importance, SS-TβRIII-mediated increases in soluble TβRIII production also reduced breast cancer metastasis in vivo. Taken together, these studies suggest that the ratio of soluble TβRIII to membrane-bound TβRIII is an important determinant for regulation of TβRIII- and TGF-β-mediated signaling and biology.

TβRIII/β-arrestin2 regulates integrin α5β1 trafficking, function, and localization in epithelial cells

The type III TGF-β receptor (TβRIII) is a ubiquitous co-receptor for TGF-β superfamily ligands with roles in suppressing cancer progression, in part through suppressing cell motility. Here we demonstrate that TβRIII promotes epithelial cell adhesion to fibronectin in a β-arrestin2 dependent and TGF-β/BMP independent manner by complexing with active integrin α5β1, and mediating β-arrestin2-dependent α5β1 internalization and trafficking to nascent focal adhesions. TβRIII-mediated integrin α5β1 trafficking regulates cell adhesion and fibronectin fibrillogenesis in epithelial cells, as well as α5 localization in breast cancer patients. We further demonstrate that increased TβRIII expression correlates with increased α5 localization at sites of cell-cell adhesion in breast cancer patients, while higher TβRIII expression is a strong predictor of overall survival in breast cancer patients. These data support a novel, clinically relevant role for TβRIII in regulating integrin α5 localization, reveal a novel crosstalk mechanism between the integrin and TGF-β superfamily signaling pathways and identify β-arrestin2 as a regulator of α5β1 trafficking.

Role of TGF-β receptor III localization in polarity and breast cancer progression.

TβRIII is basolaterally localized in polarized breast epithelial cells. The disruption of TβRIII targeting by mutation of proline 826 results in global loss of cell polarity through enhanced EMT. The mistargeting of TβRIII results in enhanced proliferation, migration, and invasion in vitro and enhanced tumor formation and invasion in vivo.

The balance of cell surface and soluble type III TGF-β receptor regulates BMP signaling in normal and cancerous mammary epithelial cells.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily that are over-expressed in breast cancer, with context dependent effects on breast cancer pathogenesis. The type III TGF-β receptor (TβRIII) mediates BMP signaling. While TβRIII expression is lost during breast cancer progression, the role of TβRIII in regulating BMP signaling in normal mammary epithelium and breast cancer cells has not been examined. Restoring TβRIII expression in a 4T1 murine syngeneic model of breast cancer suppressed Smad1/5/8 phosphorylation and inhibited the expression of the BMP transcriptional targets, Id1 and Smad6, in vivo. Similarly, restoring TβRIII expression in human breast cancer cell lines or treatment with sTβRIII inhibited BMP-induced Smad1/5/8 phosphorylation and BMP-stimulated migration and invasion. In normal mammary epithelial cells, shRNA-mediated silencing of TβRIII, TβRIII over-expression, or treatment with sTβRIII inhibited BMP-mediated phosphorylation of Smad1/5/8 and BMP induced migration. Inhibition of TβRIII shedding through treatment with TAPI-2 or expression of a non-shedding TβRIII mutant rescued TβRIII mediated inhibition of BMP induced Smad1/5/8 phosphorylation and BMP induced migration and/or invasion in both in normal mammary epithelial cells and breast cancer cells. Conversely, expression of a TβRIII mutant, which exhibited increased shedding, significantly reduced BMP-mediated Smad1/5/8 phosphorylation, migration, and invasion. These data demonstrate that TβRIII regulates BMP-mediated signaling and biological effects, primarily through the ligand sequestration effects of sTβRIII in normal and cancerous mammary epithelial cells and suggest that the ratio of membrane bound versus sTβRIII plays an important role in mediating these effects.

Abstract 3972: The role of the TGF-β type III receptor in colon carcinogenesis

The loss of the TGF-β type III receptor (TβRIII), a TGF-β superfamily co-receptor, correlates with disease progression in multiple cancer types, suggesting that TβRIII plays a role in regulating tumor progression. TGF-β superfamily signaling is frequently dysregulated in colon cancer but the specific role of TβRIII in colon carcinogenesis has not been examined. In contrast to other cancer types, TβRIII mRNA expression is not altered in colon cancer and there is not a significant change in TβRIII protein expression in tumors compared to normal colon tissue. HT29 colon cancer cells overexpressing TβRIII (HT29-RIII) exhibit an increase in proliferation in response to TGF-β and BMP2 stimulation compared to HT29-Neo cells. This increase in proliferation is mediated through the down-regulation of p21. In a soft agar assay HT29-RIII cells demonstrate increased colony formation in response to ligand treatment. Additionally, HT29-RIII cells are resistant to anoikis and 5FU-induced apoptosis. Colon cancer cells expressing TβRIII also exhibit increased migration potential and this increase in migration can be abrogated by knockdown of TβRIII expression. Furthermore, cells over-expressing TβRIII show alterations in E-cadherin and actin organization. Taken together, these data suggest that TβRIII may have a pro-tumorigenic role in colon cancer tumorigenesis as it mediates ligand stimulated proliferation, resistance to apoptosis and increased migration. Further work is currently underway to more fully elucidate the role of TβRIII in colon cancer progression in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3972.

Abstract 3971: The type III tgf-β receptor mediates bmp signaling in normal and cancerous mammary epithelial cells

BMP signaling has been shown to enhance breast cancer invasion and bone metastasis, while inhibition of BMP signaling abrogates bone metastasis in an in vivo model. The type III TGF-β receptor (TβRIII) is known to mediate BMP signaling and can inhibit migration, invasion, and metastasis in breast cancer, however the role of TβRIII in regulating BMP signaling in breast cancer has not been examined. The MDA-MB-231, MCF7, and mouse 4T1 breast cancer cell lines, which express low levels of TβRIII, are responsive to BMP2 and 4 treatment as demonstrated by increased Smad1/5/8 phosphorylation, cell migration, and invasion. Restoring TβRIII expression in these cell lines or treatment of MDA-MB-231-Neo cells with recombinant soluble TβRIII (sTβRIII) or with conditioned media from MDA-MB-231-RIII cells inhibited BMP2 and BMP4 induced Smad1/5/8 phosphorylation (pSmad1/5/8) and BMP mediated cell migration. MDA-MB-231-RIII cells also exhibited enhanced growth arrest in response to BMP2 compared to MDA-MB-231-Neo cells, mediated through the up-regulation of p21. In reciprocal studies, shRNA-mediated knockdown of TβRIII expression in the normal mammary epithelial cell lines HMEC and MCF10A, abrogated BMP mediated induction of pSmad1/5/8, demonstrating that TβRIII mediates BMP signaling in normal mammary epithelial cells. Additionally, treatment with both recombinant sTβRIII and conditioned media from MDA-MB-231-RIII cells inhibits BMP mediated induction of pSmad1/5/8, suggesting that sTβRIII is able to inhibit BMP signaling in normal mammary epithelial cells by sequestration of ligand. BMP2 treatment induces cell migration in HMEC cells, which was attenuated by knockdown of TβRIII or treatment with sTβRIII. These data demonstrate that TβRIII mediates BMP signaling in normal mammary epithelial cells and breast cancer cell lines, regulating cell migration, invasion, and proliferation. The effects of sTβRIII suggest that the ratio of membrane bound versus soluble TβRIII plays an important role in mediating BMP signaling and cellular effects in normal and cancer mammary epithelial cells. Further work is underway to elucidate the mechanism by which TβRIII regulates BMP signaling in breast cancer in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3971.