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

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

Ectodomain shedding of TßRIII is required for TßRIII-mediated suppression of TGF-ß signaling and breast cancer migration and invasion.

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

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

The majority of breast cancers originate from the highly polarized luminal epithelial cells lining the breast ducts. However, cell polarity is often lost during breast cancer progression. The type III transforming growth factor-ß cell surface receptor (TßRIII) functions as a suppressor of breast cancer progression and also regulates the process of epithelial-to-mesenchymal transition (EMT), a consequence of which is the loss of cell polarity. Many cell surface proteins exhibit polarized expression, being targeted specifically to the apical or basolateral domains. Here we demonstrate that TßRIII is basolaterally localized in polarized breast epithelial cells and that disruption of the basolateral targeting of TßRIII through a single amino acid mutation of proline 826 in the cytosolic domain results in global loss of cell polarity through enhanced EMT. In addition, the mistargeting of TßRIII results in enhanced proliferation, migration, and invasion in vitro and enhanced tumor formation and invasion in an in vivo mouse model of breast carcinoma. These results suggest that proper localization of TßRIII is critical for maintenance of epithelial cell polarity and phenotype and expand the mechanisms by which TßRIII prevents breast cancer initiation and progression.

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

Endoglin (CD105) is an endothelial-specific transforming growth factor ß (TGF-ß) coreceptor essential for angiogenesis and vascular homeostasis. Although endoglin dysfunction contributes to numerous vascular conditions, the mechanism of endoglin action remains poorly understood. Here we report a novel mechanism in which endoglin and Gα-interacting protein C-terminus-interacting protein (GIPC)-mediated trafficking of phosphatidylinositol 3-kinase (PI3K) regulates endothelial signaling and function. We demonstrate that endoglin interacts with the PI3K subunits p110α and p85 via GIPC to recruit and activate PI3K and Akt at the cell membrane. Opposing ligand-induced effects are observed in which TGF-ß1 attenuates, whereas bone morphogenetic protein-9 enhances, endoglin/GIPC-mediated membrane scaffolding of PI3K and Akt to alter endothelial capillary tube stability in vitro. Moreover, we employ the first transgenic zebrafish model for endoglin to demonstrate that GIPC is a critical component of endoglin function during developmental angiogenesis in vivo. These studies define a novel non-Smad function for endoglin and GIPC in regulating endothelial cell function during angiogenesis.

Type III TGF-ß receptor enhances colon cancer cell migration and anchorage-independent growth.

The type III TGF-ß receptor (TßRIII or betagylcan) is a TGF-ß superfamily coreceptor with emerging roles in regulating TGF-ß superfamily signaling and cancer progression. Alterations in TGF-ß superfamily signaling are common in colon cancer; however, the role of TßRIII has not been examined. Although TßRIII expression is frequently lost at the message and protein level in human cancers and suppresses cancer progression in these contexts, here we demonstrate that, in colon cancer, TßRIII messenger RNA expression is not significantly altered and TßRIII expression is more frequently increased at the protein level, suggesting a distinct role for TßRIII in colon cancer. Increasing TßRIII expression in colon cancer model systems enhanced ligand-mediated phosphorylation of p38 and the Smad proteins, while switching TGF-ß and BMP-2 from inhibitors to stimulators of colon cancer cell proliferation, inhibiting ligand-induced p21 and p27 expression. In addition, increasing TßRIII expression increased ligand-stimulated anchorage-independent growth, a resistance to ligand- and chemotherapy-induced apoptosis, cell migration and modestly increased tumorigenicity in vivo. In a reciprocal manner, silencing endogenous TßRIII expression decreased colon cancer cell migration. These data support a model whereby TßRIII mediates TGF-ß superfamily ligand-induced colon cancer progression and support a context-dependent role for TßRIII in regulating cancer progression.

Roles for the type III TGF-beta 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.

Altered senescence, apoptosis, and DNA damage response in a mutant p53 model of accelerated aging.

The tumor suppressors p16(INK4a) and p53 have been implicated as contributors to age-associated stem cell decline. Key functions of p53 are the induction of cell cycle arrest, senescence, or apoptosis in response to DNA damage. Here, we examine senescence, apoptosis, and DNA damage responses in a mouse accelerated aging model that exhibits increased p53 activity, the p53(+/m) mouse. Aged tissues of p53(+/m) mice display higher percentages of senescent cells (as determined by senescence-associated beta-galactosidase staining and p16(INK4a) and p21 accumulation) compared to aged tissues from p53(+/+) mice. Surprisingly, despite having enhanced p53 activity, p53(+/m) lymphoid tissues exhibit reduced apoptotic activity in response to ionizing radiation compared to p53(+/+) tissues. Ionizing radiation treatment of p53(+/m) tissues also induces higher and prolonged levels of senescence markers p16(INK4a) and p21, suggesting that in p53(+/m) tissues the p53 stress response is enhanced and is shifted away from apoptosis toward senescence. One potential mechanism for accelerated aging in the p53(+/m) mouse is a failure to remove damaged or dysfunctional cells (including stem and progenitor cells) through apoptosis. The increased accumulation of dysfunctional and senescent cells may contribute to reduced tissue regeneration, tissue atrophy, and some of the accelerated aging phenotypes in p53(+/m) mice.

Altered mammary gland development in the p53+/m mouse, a model of accelerated aging.

The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.