Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor and endoglin, a TGF-β co-receptor, play an essential role in... Show moreGenetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for anti-angiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti hALK1-antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting, but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF-VEGFR and the BMP9-ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously is an attractive strategy to overcome resistance to anti-angiogenesis therapy. Show less
Naber, H.P.H.; Wiercinska, E.; Pardali, E.; Laar, T. van; Nirmala, E.; Sundqvist, A.; ... ; Dijke, P. ten 2012
BACKGROUND The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological... Show moreBACKGROUND The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells. METHODS In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay. RESULTS BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion. CONCLUSION Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3). Show less
Naber, H.P.H.; Wiercinska, E.; Pardali, E.; Laar, T. van; Nirmala, E.; Sundqvist, A.; ... ; Dijke, P. ten 2012
Transforming growth factor-beta (TGF-beta) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later... Show moreTransforming growth factor-beta (TGF-beta) 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-beta induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-beta-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-beta-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. Basal invasion was strongly inhibited by the TGF-beta receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-beta or TGF-beta-like activity. TGF-beta-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-beta-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-beta 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-beta/Smad- and MMP2- and MMP9-dependent breast cancer invasion. Show less
Olieslagers, S.; Pardali, E.; Tchaikovski, V.; Dijke, P. ten; Waltenberger, J. 2011
Aims Monocytes contribute to arteriogenesis by infiltration to sites of collateral growth and subsequent production and release of growth factors. Transforming growth factor beta 1 (TGF-beta 1)... Show moreAims Monocytes contribute to arteriogenesis by infiltration to sites of collateral growth and subsequent production and release of growth factors. Transforming growth factor beta 1 (TGF-beta 1) mediates monocyte motility and stimulates arteriogenesis. TGF-beta 1 signalling mechanisms mediating monocyte motility are unknown so far. Moreover, the influence of cardiovascular risk factor diabetes on TGF-beta 1-induced monocyte migration remains to be elucidated. Methods and results Stimulation of primary human monocytes with TGF-beta 1 endorsed phosphorylation of v-Akt murine thymoma viral oncogene analogues protein (AKT), p38, and extracellular signal-related kinase 1/2 (ERK1/2), besides the activation of the SMA/MAD homologues protein (SMAD) pathway. Inhibition of the TGF-btype 1 receptor, alias activin receptor-like kinase 5 (ALK5), hindered monocyte chemotaxis towards TGF-beta 1 and TGF-beta 1-activated downstream signalling cascades. Individual genetic knock-downs for receptor-regulated SMAD2 and SMAD3 did not affect monocyte migration to TGF-beta 1. Inhibition of phosphoinositide 3 kinase (PI3K) activity, but not AKT, diminished both basal and TGF-beta 1-mediated monocyte motility. TGF-beta 1-induced monocyte chemotaxis did not rely on ERK1/2, but rather on p38. Remarkably, TGF-beta 1 was able to stimulate chemotaxis of diabetic monocytes. Conclusion The current study provides novel insights into the molecular mechanisms of TGF-beta 1-induced monocyte migration, requiring ALK5 kinase activity and signalling via PI3K and p38. TGF-beta 1-driven monocyte motogenicity is fully functional in diabetic conditions, which is in sharp contrast to the impaired chemotactic responses to certain other arteriogenic cytokines. Therefore, TGF-beta 1 may be a promising candidate for endogenously and exogenously stimulating collateral growth in diabetic patients. Show less
Pardali, E.; Schaft, D.W.J. van der; Wiercinska, E.; Gorter, A.; Hogendoorn, P.C.W.; Griffioen, A.W.; Dijke, P. ten 2011
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... Show moreTumor 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 tubelike structures, a phenotype that has been linked to aggressive behavior and poor prognosis. We demonstrate here that the transforming growth factor (TGF)-beta 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-beta 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. Oncogene (2011) 30, 334-345; doi:10.1038/onc.2010.418; published online 20 September 2010 Show less
Members of the transforming growth factor-beta (TGF-beta) family play pivotal roles in development and disease. These cytokines elicit their pleiotropic effects on cells, including endothelial and... Show moreMembers of the transforming growth factor-beta (TGF-beta) family play pivotal roles in development and disease. These cytokines elicit their pleiotropic effects on cells, including endothelial and mural cells, through specific type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. This review highlights recent progress in our understanding of TGF-beta signaling in vascular development and angiogenesis and of how perturbed TGF-beta signaling might contribute to vascular pathologies, tumor angiogenesis and tumor progression. Recent research has provided exciting insights into the role of the TGF-beta type I receptor (ALK1) in tumor angiogenesis and the curative effects of thalidomide on vascular malformations in hereditary hemorrhagic telangiectasia (HHT). These advances provide opportunities for the development of new therapies for diseases with vascular abnormalities. Show less
Baan, B.; Pardali, E.; Dijke, P. ten; Dam, H. van 2010
Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1)... Show moreGenetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material. Molecular & Cellular Proteomics 9:1982-1990, 2010. Show less
Wiercinska, E.; Naber, H.P.H.; Pardali, E.; Pluijm, G. van der; Dam, H. van; Dijke, P. ten 2010
Transforming growth factor-beta (TGF-beta) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later... Show moreTransforming growth factor-beta (TGF-beta) 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-beta induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-beta-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-beta-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. Basal invasion was strongly inhibited by the TGF-beta receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-beta or TGF-beta-like activity. TGF-beta-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-beta-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-beta 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-beta/Smad- and MMP2- and MMP9-dependent breast cancer invasion. Show less
