P. ten Dijke

Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis

Transforming growth factor (TGF)-β can suppress and promote breast cancer progression. How TGF-β elicits these dichotomous functions and which roles the principle intracellular effector proteins Smad2 and Smad3 have therein, is unclear. Here, we investigated the specific functions of Smad2 and Smad3 in TGF-β-induced responses in breast cancer cells in vitro and in a mouse model for breast cancer metastasis. We stably knocked down Smad2 or Smad3 expression in MDA-MB-231 breast cancer cells. The TGF-β-induced Smad3-mediated transcriptional response was mitigated and enhanced by Smad3 and Smad2 knockdown, respectively. This response was also seen for TGF-β-induced vascular endothelial growth factor (VEGF) expression. TGF-β induction of key target genes involved in bone metastasis, were found to be dependent on Smad3 but not Smad2. Strikingly, whereas knockdown of Smad3 in MDA-MB-231 resulted in prolonged latency and delayed growth of bone metastasis, Smad2 knockdown resulted in a more aggressive phenotype compared with control MDA-MB-231 cells. Consistent with differential effects of Smad knockdown on TGF-β-induced VEGF expression, these opposing effects of Smad2 versus Smad3 could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo. Thus, Smad2 and Smad3 differentially affect breast cancer bone metastasis formation in vivo.

Interaction with colon cancer cells hyperactivates TGF-β signaling in cancer-associated fibroblasts

The interaction between epithelial cancer cells and cancer-associated fibroblasts (CAFs) has a major role in cancer progression and eventually in metastasis. In colorectal cancer (CRC), CAFs are present in high abundance, but their origin and functional interaction with epithelial tumor cells has not been elucidated. In this study we observed strong activation of the transforming growth factor-β (TGF-β)/Smad signaling pathway in CRC CAFs, accompanied by decreased signaling in epithelial tumor cells. We evaluated the TGF-β1 response and the expression of target genes including matrix metalloproteinases (MMPs) and plasminogen activator inhibitor (PAI)-1 of various epithelial CRC cell lines and primary CAFs in vitro. TGF-β1 stimulation caused high upregulation of MMPs, PAI-1 and TGF-β1 itself. Next we showed that incubation of CAFs with conditioned medium (CM) from epithelial cancer cells led to hyperactivation of the TGF-β signaling pathway, enhanced expression of target genes like PAI-1, and the expression of α-smooth muscle actin (α-SMA). We propose that the interaction of tumor cells with resident fibroblasts results in hyperactivated TGF-β1 signaling and subsequent transdifferentiation of the fibroblasts into α-SMA-positive CAFs. In turn this leads to cumulative production of TGF-β and proteinases within the tumor microenvironment, creating a cancer-promoting feedback loop.

ELAC2, a putative prostate cancer susceptibility gene product, potentiates TGF-β/Smad-induced growth arrest of prostate cells

Transforming growth factor-β (TGF-β) elicits a potent growth inhibitory effect on many normal cells by binding to specific serine/threonine kinase receptors and activating specific Smad proteins, which regulate the expression of cell cycle genes, including the p21 cyclin-dependent kinase (CDK) inhibitor gene. Interestingly, cancer cells are often insensitive to the anti-mitogenic effects of TGF-β for which the molecular mechanisms are not well understood. In this study, we found that the candidate prostate cancer susceptibility gene ELAC2 potentiates TGF-β/Smad-induced transcriptional responses. ELAC2 associates with activated Smad2; the C-terminal MH2 domain of Smad2 interacts with the N-terminal region of ELAC2. Small interfering siRNA-mediated knock-down of ELAC2 in prostate cells suppressed TGF-β-induced growth arrest. Moreover, ELAC2 was shown to specifically associate with the nuclear Smad2 partner, FAST-1 and to potentiate the interaction of activated Smad2 with transcription factor Sp1. Furthermore, activation of the p21 CDK inhibitor promoter by TGF-β is potentiated by ELAC2. Taken together our data indicate an important transcriptional scaffold function for ELAC2 in TGF-β/Smad signaling mediated growth arrest.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC

Background:Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the β-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the β-catenin paradox.Methods:We analysed the expression patterns of SMAD4, p53 and β-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity.Results:Eighty-four percent of CRCs with high nuclear β-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner.Conclusions:The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

PO-185 Towards dynamic targeting of tgf-β in metastatic melanoma using intravital microscopy

Introduction Melanoma patients diagnosed with liver metastasis have a poor prognosis. TGF-β inhibitors give mixed results in clinical trials with metastatic melanoma patients. To more specifically target TGF-β in liver metastasis melanoma patients, it is necessary to unravel the spatio-temporal function of the TGF-β pathway in hepatic colonisation of melanoma cells. TGF-β is a multifunctional cytokine that signals via TGF-β receptors and downstream SMAD effector proteins. SMAD proteins regulate transcription by binding, among others, to CAGA elements in the DNA. It can exert both pro-and anti-tumorigenic functions, depending on cellular context. It acts on tumour cells, as well as the tumour micro-environment and the immune system. In metastatic melanoma cells, TGF-β can stimulate invasion and metastasis. To unravel its exact role during the different processes of metastasis, we will investigate the spatio-temporal patterns of TGF-β signalling during metastatic colonisation using intravital microscopy (IVM). Material and methods Spatio-temporal patterns of TGF-β signalling will be studied in vitro and in vivo. For our in vivo studies, we are using an experimentally induced liver metastasis model, injecting highly aggressive B16F10 melanoma cells in immune competent C57BL/6 mice. By injecting these tumour cells in the mesenteric vein, cells will be transported directly to the liver, the first capillary network the cells will encounter. An abdominal imaging window will be placed after cell injection to visualise the different steps of metastasis in real-time using IVM. We developed a rapid CAGA12-GFP-based transcriptional reporter, which expresses a fluorophore upon TGF-β receptor activation and SMAD binding. Upon the expression of this reporter in B16F10 cells, activation of the TGF-β pathway can be monitored over time. By genetic manipulation of B16F10 cells to express dominant negative or constitutively active TGF-β receptors, the role of TGF-β can be assessed for the different steps of metastasis. Results and discussions We confirmed earlier reports showing that B16F10 cells show a transcriptional CAGA response upon TGF-β3 stimulation. Using our liver metastasis model, the injected B16F10 cells are able to perform the steps of metastasis and form liver metastasis within a short time frame. The use of the rapid TGF-β reporter during intravital imaging will show the involvement of the TGF-β pathway in the different phases of metastasis. Conclusion B16F10 cells have a functional TGF-β pathway and are able to colonise the liver.

132 Non-synonymous SMAD6 mutations impaired inhibition of bmp signalling in patients with congenital cardiovascular malformation

Introduction Congenital cardiovascular malformation (CVM) exhibits familial predisposition but the specific genetic factors involved are unknown. Bone morphogenetic proteins (BMPs) regulate many processes during development, including cardiac development. Five genes of the BMP signalling were surveyed for novel variants predisposing to CVM risk. One of the genes, SMAD6, functions as an inhibitory SMAD which preferentially inhibits BMP signalling. The SMAD6 knockout mouse is characterised by cardiac valve and outflow tract defects, including aortic ossification. We hypothesised that rare functional variation in SMAD6 could predispose to congenital cardiovascular malformation (CVM). Methods The coding regions of BMP2, BMP4, BMPR1A, BMPR2 and SMAD6 were sequenced in 90 unrelated Caucasian cases of CVM. The MH2 domain of SMAD6 were further sequenced in additional 346 CVM patients. Functional effects of the wild-type and variant SMAD6 proteins were expressed in C2C12 cells and their capacity to inhibit ALK3 activated expression of a BMP-responsive reporter, or to inhibit osteogenic differentiation (using an alkaline phosphatase assay) was assessed. Results We identified two novel non-synonymous variants, P415L and C484F, that were not present in 1000 ethnically-matched controls. P415L was identified in a patient with congenital aortic stenosis and C484F was identified in a patient with coarctation and calcification of the aorta. Both mutations are in evolutionarily conserved amino acid residues and are predicted to be damaging by in silico analysis. This was confirmed in functional assays as both SMAD6 variants failed to inhibit BMP signalling compared with wild-type SMAD6. The P415L mutant appeared to be hypomorphic whereas C484F appeared to be a null allele in the luciferase assay. The C484F mutant had a significantly (p<0.05) lower capacity to inhibit alkaline phosphatase generation in response to BMP signalling. Conclusions This is the first time that functional mutations in SMAD6 have been described in patients with CVM, specifically those with calcific aortic malformations. Our data suggest that inadequate inhibition of BMP signalling pathway due to genetic variation in SMAD6 may be an important factor in CVM.