Laboratory for signal transduction mechanisms of TGF-ß

TGF-β signal transduction

Transforming growth factor-β (TGF-β) is a secreted homodimeric protein that regulates numerous responses, such as proliferation, differentiation, migration and apoptosis. TGF-β is the prototypic member of a family of 33 structurally related pleiotropic cytokines. Members of the TGF-β superfamily have crucial roles in development and tissue homeostasis. We are interested in unraveling mechanisms by which transforming growth factor-β (TGF-β) family members elicit their multifunctional cellular effects and how perturbation in their signal transduction pathways contribute to human diseases. Previous research from us and other laboratories have now firmly established the intracellular signaling cascade of TGF-β via serine/threonine kinase receptors and SMAD transcriptional effectors. However, how this pathway is (mis)regulated in cancer and other diseases, remains not well understood. The present focus within our group is on 1) the identification of novel critical regulators of TGF-β family signaling pathways using functional genetic screens, 2) how TGF-β specificity and bioavailability is regulated via known (co-)receptors and ligand binding proteins and 3) interplay with other signaling pathways. In particular, we study the involvement of TGF-β family members in the metastasis of cancer cells, tumor angiogenesis and high/low bone mass diseases. We use 3-dimensional cell culture systems that allow for a functional analysis of homo- and heterotypic cell-cell interactions in structures that resemble those found in vivo. Moreover, mouse models are used that mimic diseases with misregulated TGF-β signaling. Our long term aim is to translate our findings towards the development of novel treatment modalities.

2011/2012 highlights:

Cell-type specific regulation of myostatin signaling

TGF-β family member myostatin is an important regulator of myoblast, adipocyte, and fibroblast growth and differentiation, but the signaling mechanisms remain to be established. We therefore determined the contribution of myostatin type I receptors activin receptor-like kinase-4 (ALK4) and -5 (ALK5) and different co-receptors in C2C12 myoblasts, C3H10T1/2 mesenchymal stem cells, and 3T3-L1 fibroblasts, as well as in primary myoblast and fibroblasts. We find that myostatin utilizes ALK4 in myoblasts, whereas it has a preference for ALK5 in nonmyogenic cells. Notably, our results show that coreceptor Cripto is expressed in myoblasts but not in the nonmyogenic cells and that it regulates myostatin activity. More specifically, myostatin requires Cripto in myoblasts, whereas Cripto represses activin activity and TGF-β signaling is Cripto independent. Furthermore, Cripto down-regulation enhances myoblast differentiation, showing its importance in myostatin signaling. Together, our results identify a molecular mechanism that explains the cell-type specific aspects of signaling by myostatin and other TGF-β family members The TGF-b/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system.

Lack of primary cilia primes shear-induced endothelial-to-mesenchymal transition

Primary cilia are cellular protrusions that serve as mechanosensors for fluid flow. In endothelial cells (ECs), they function by transducing local blood flow information into functional responses, such as nitric oxide production and initiation of gene expression. Cilia are present on ECs in areas of low or disturbed flow and absent in areas of high flow. In the embryonic heart, high-flow regime applies to the endocardial cushion area, and the absence of cilia here coincides with the process of endothelial-to-mesenchymal transition (EndoMT). We investigated the role of the primary cilium in defining the responses of ECs to fluid shear stress and in EndoMT. Nonciliated mouse embryonic ECs with a mutation in Tg737/Ift88 were used to compare the response to fluid shear stress to that of ciliated ECs. In vitro, nonciliated ECs undergo shear-induced EndoMT, which is accompanied by downregulation of Klf4. This Tgfβ/Alk5-dependent transformation is prevented by blocking Tgfβ signaling, overexpression of Klf4, or rescue of the primary cilium. In the hearts of Tg737(orpk/orpk) embryos, Tgfβ/Alk5 signaling was activated in areas in which ECs would normally be ciliated but now lack cilia because of the mutation. In these areas, ECs show increased Smad2 phosphorylation and expression of α-smooth muscle actin. This study demonstrates the central role of primary cilia in rendering ECs prone to shear-induced activation of Tgfβ/Alk5 signaling and EndoMT and thereby provides a functional link between primary cilia and flow-related endothelial performance.

Critical role of endoglin in tumor cell plasticity of Ewing sarcoma and melanoma

Tumor cell plasticity enables certain types of highly malignant tumor cells to dedifferentiate and engage a plastic multipotent embryonic-like phenotype, which enables them to 'adapt' during tumor progression and escape conventional therapeutic strategies. This plastic phenotype of aggressive cancer cells enables them to express endothelial cell-specific markers and form tube-like structures, a phenotype that has been linked to aggressive behavior and poor prognosis. We demonstrate here that the transforming growth factor (TGF)-β co-receptor endoglin, an endothelial cell marker, is expressed by tumor cells and its expression correlates with tumor cell plasticity in two types of human cancer, Ewing sarcoma and melanoma. Moreover, endoglin expression was significantly associated with worse survival of Ewing sarcoma patients. Endoglin knockdown in tumor cells interferes with tumor cell plasticity and reduces invasiveness and anchorage-independent growth in vitro. Ewing sarcoma and melanoma cells with reduced endoglin levels showed reduced tumor growth in vivo. Mechanistically, we provide evidence that endoglin, while interfering with TGF-β signaling, is required for efficient bone morphogenetic protein, integrin, focal adhesion kinase and phosphoinositide-3-kinase signaling in order to maintain tumor cell plasticity. The present study delineates an important role of endoglin in tumor cell plasticity and progression of aggressive tumors.

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.