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.

Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation.

Age-related defects in stem cells can limit proper tissue maintenance and hence contribute to a shortened lifespan. Using highly purified hematopoietic stem cells from mice aged 2 to 21 mo, we demonstrate a deficit in function yet an increase in stem cell number with advancing age. Expression analysis of more than 14,000 genes identified 1,500 that were age-induced and 1,600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the up-regulated expression profile, while the down-regulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling. Many chromosomal regions showed coordinate loss of transcriptional regulation; an overall increase in transcriptional activity with age and inappropriate expression of genes normally regulated by epigenetic mechanisms was also observed. Hematopoietic stem cells from early-aging mice expressing a mutant p53 allele reveal that aging of stem cells can be uncoupled from aging at an organismal level. These studies show that hematopoietic stem cells are not protected from aging. Instead, loss of epigenetic regulation at the chromatin level may drive both functional attenuation of cells, as well as other manifestations of aging, including the increased propensity for neoplastic transformation.

Tumor suppressor dosage regulates stem cell dynamics during aging.

The ability of tissues to maintain homeostasis is dependent in part on the function of adult tissue stem cells, which have the capability to self-renew and differentiate into multiple lineages. It has been hypothesized that the ability of stem cells to maintain tissue homeostasis declines functionally with age and that this decline may account for many of the biological phenotypes associated with aging. Recently, tumor suppressors such as p53 have been implicated in both aging and the regulation of stem cell dynamics. Our recent findings suggest that p53 may impact hematopoietic stem cell (HSC) dynamics during mammalian aging. Utilizing mouse models of varying levels of p53 dosage, we have shown that alteration of p53 activity affects stem cell number, proliferation, and functionality with age. Several other recent studies have implicated other tumor suppressors in potential age-related regulation of HSC dynamics as well. These data support a model in which aging is caused in part by a decline in tissue stem cell regenerative function, regulated in part by tumor suppressors.

Analysis of cellular senescence in culture in vivo: the senescence-associated beta-galactosidase assay.

Replicative senescence, a process first described almost 40 years ago, entails irreversible growth arrest with sustained metabolic functions, and it is also associated with increased resistance to apoptotic signals. Interest in this process has increased greatly over the last 10 years, as it has been demonstrated that senescence can function as a potent tumor suppressor mechanism. Although mounting evidence suggests that the senescent phenotype is associated with an extraordinarily complex array of gene expression patterns and interactions with the microenvironment, there is only one widely accepted marker for distinguishing such cells in vitro and in vivo. This marker is the senescence-associated expression of a pH 6 beta-galactosidase (SA-beta-gal). Here, a method for analyzing SA-beta-gal expression in cultured cells and in human and animal tissues is described, and important parameters to consider when performing such assays are also highlighted.

Insights into aging obtained from p53 mutant mouse models.

Cancer suppression is an integral component of longevity in organisms with renewable tissues. A number of genes in the mammalian genome function in cancer prevention, and some of these have been directly implicated in longevity assurance. One such longevity assurance gene is the tumor suppressor p53, a transcription factor that is mutated or dysregulated in most human cancers. Early studies have linked p53 to the induction of cellular senescence, whereas recent reports implicate it as a potential regulator of organismal aging. We have shown by gene inactivation studies that loss of p53 function enhances tumor susceptibility and reduces longevity in the mouse. A recent serendipitously generated p53 mutant allele resulted in a hypermorphic version of p53 that displays increased cancer resistance, yet also mediates decreased longevity. The reduced longevity is accompanied by the accelerated onset of a variety of aging phenotypes. These include a 20% decrease in median life span, early osteoporosis, lordokyphosis, organ atrophy, delayed wound healing, and a reduced regenerative response after various stresses. Since the initial characterization of these mutant mice, we have attempted to elucidate the underlying molecular and cellular mechanisms that could be influencing the early aging phenotypes. Molecular studies of the p53 mutant allele product indicate that it induces an increase in p53 activity in both in vitro and in vivo contexts. The age-associated loss of organ cellularity and reduced tissue regenerative responses in the mutant mice are consistent with an accelerated loss of stem cell functional capacity. Our model is that enhanced growth inhibitory activity of p53 produces an earlier loss of the ability of stem cells to produce adequate numbers of progenitor and mature differentiated cells in each organ. Currently, we are performing stem cell functional assays from p53 mutant and wild-type mice to test this model. One challenge for the future will be to find ways to manipulate p53 function to provide increased cancer resistance, yet still enhance overall organismal longevity.