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Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis.

Cunha SI, Pardali E, Thorikay M, Anderberg C, Hawinkels L, Goumans MJ, Seehra J, Heldin CH, ten Dijke P, Pietras K - J. Exp. Med. (2010)

Bottom Line: Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor.In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli.We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institutet, Stockholm SE-171 77, Sweden.

ABSTRACT
Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

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BMP9 and TGF-β signaling is increased during the tumor progression pathway of the RIP1-Tag2 model. (a–d) Quantitative RT-PCR analysis of the expression of ALK1 (a) and ALK5 (b) receptors and TGF-β (c) and BMP9 (d) ligands in pancreatic islets from progressive tumor stages in RIP1-Tag2 mice (materials pooled from >20 mice per tumor stage, analysis independently performed at least three times). Error bars show the mean ± SD. (e–h) Immunostaining for ALK1 (red; e), ALK5 (red; f), TGF-β (red; g), and BMP9 (red; h) of sections from the pancreas of RIP1-Tag2 mice. As a comparison, immunostaining for the endothelial cell marker podocalyxin (green) was performed in e and for CD31 (green) in f. The angiogenic islet lesional area is outlined by white dashes. Bars, 50 µm.
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fig1: BMP9 and TGF-β signaling is increased during the tumor progression pathway of the RIP1-Tag2 model. (a–d) Quantitative RT-PCR analysis of the expression of ALK1 (a) and ALK5 (b) receptors and TGF-β (c) and BMP9 (d) ligands in pancreatic islets from progressive tumor stages in RIP1-Tag2 mice (materials pooled from >20 mice per tumor stage, analysis independently performed at least three times). Error bars show the mean ± SD. (e–h) Immunostaining for ALK1 (red; e), ALK5 (red; f), TGF-β (red; g), and BMP9 (red; h) of sections from the pancreas of RIP1-Tag2 mice. As a comparison, immunostaining for the endothelial cell marker podocalyxin (green) was performed in e and for CD31 (green) in f. The angiogenic islet lesional area is outlined by white dashes. Bars, 50 µm.

Mentions: The RIP1-Tag2 mouse model of endocrine pancreatic tumorigenesis has been widely studied (Hanahan, 1985). Because its multistep progression through a synchronous angiogenesis-dependent pathway from normal pancreatic islets to hyperplastic islets, angiogenic islets, and, finally, into overt insulinomas and because of its proven predictive value for assessing possible targets for anti-angiogenic therapies (Bergers et al., 1999), it constitutes a suitable model for investigating the role of TGF-β signaling in tumor angiogenesis. First, we determined the expression pattern of key ligands and receptors in the TGF-β signaling pathways during RIP1-Tag2 tumorigenesis. As judged by quantitative RT-PCR analysis, the expression of ALK1 was significantly increased in angiogenic islets from RIP1-Tag2 mice, as compared with other stages of normal or malignant islets (Fig. 1 a). Consistent with the reported endothelial cell–restricted expression of ALK1 (Roelen et al., 1997), other vascular markers, including CD31 and vascular endothelial cadherin, demonstrated a similar pattern of expression (Fig. S1, a and b). In contrast, the expression of ALK5 was comparatively low and remained unchanged during the tumorigenesis pathway of RIP1-Tag2 mice (Fig. 1 b). Further analysis of the βTC3 cell line established from a RIP1-Tag2 tumor (Efrat et al., 1988) revealed that tumor cells express high levels of ALK5 but not of ALK1 (Fig. S1 c). Notably, the messenger RNA (mRNA) of both TGF-β and BMP9 were present at dramatically increasing levels during the course of malignant progression in RIP1-Tag2 mice and were expressed at 5.2- and 4.4-fold higher levels in tumors compared with normal islets, respectively (Fig. 1, c and d). Expression of the ALK1 ligand BMP10 was up-regulated in angiogenic islets, albeit to a lower degree, but decreased to baseline levels in tumors (Fig. S1 d). The βTC3 cell line expressed high levels of both TGF-β and BMP9 but negligible amounts of BMP10 (Fig. S1 e). Given the structural similarity between BMP9 and BMP10, it is likely that the two ligands share many properties, so our subsequent efforts were focused on BMP9.


Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis.

Cunha SI, Pardali E, Thorikay M, Anderberg C, Hawinkels L, Goumans MJ, Seehra J, Heldin CH, ten Dijke P, Pietras K - J. Exp. Med. (2010)

BMP9 and TGF-β signaling is increased during the tumor progression pathway of the RIP1-Tag2 model. (a–d) Quantitative RT-PCR analysis of the expression of ALK1 (a) and ALK5 (b) receptors and TGF-β (c) and BMP9 (d) ligands in pancreatic islets from progressive tumor stages in RIP1-Tag2 mice (materials pooled from >20 mice per tumor stage, analysis independently performed at least three times). Error bars show the mean ± SD. (e–h) Immunostaining for ALK1 (red; e), ALK5 (red; f), TGF-β (red; g), and BMP9 (red; h) of sections from the pancreas of RIP1-Tag2 mice. As a comparison, immunostaining for the endothelial cell marker podocalyxin (green) was performed in e and for CD31 (green) in f. The angiogenic islet lesional area is outlined by white dashes. Bars, 50 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2812548&req=5

fig1: BMP9 and TGF-β signaling is increased during the tumor progression pathway of the RIP1-Tag2 model. (a–d) Quantitative RT-PCR analysis of the expression of ALK1 (a) and ALK5 (b) receptors and TGF-β (c) and BMP9 (d) ligands in pancreatic islets from progressive tumor stages in RIP1-Tag2 mice (materials pooled from >20 mice per tumor stage, analysis independently performed at least three times). Error bars show the mean ± SD. (e–h) Immunostaining for ALK1 (red; e), ALK5 (red; f), TGF-β (red; g), and BMP9 (red; h) of sections from the pancreas of RIP1-Tag2 mice. As a comparison, immunostaining for the endothelial cell marker podocalyxin (green) was performed in e and for CD31 (green) in f. The angiogenic islet lesional area is outlined by white dashes. Bars, 50 µm.
Mentions: The RIP1-Tag2 mouse model of endocrine pancreatic tumorigenesis has been widely studied (Hanahan, 1985). Because its multistep progression through a synchronous angiogenesis-dependent pathway from normal pancreatic islets to hyperplastic islets, angiogenic islets, and, finally, into overt insulinomas and because of its proven predictive value for assessing possible targets for anti-angiogenic therapies (Bergers et al., 1999), it constitutes a suitable model for investigating the role of TGF-β signaling in tumor angiogenesis. First, we determined the expression pattern of key ligands and receptors in the TGF-β signaling pathways during RIP1-Tag2 tumorigenesis. As judged by quantitative RT-PCR analysis, the expression of ALK1 was significantly increased in angiogenic islets from RIP1-Tag2 mice, as compared with other stages of normal or malignant islets (Fig. 1 a). Consistent with the reported endothelial cell–restricted expression of ALK1 (Roelen et al., 1997), other vascular markers, including CD31 and vascular endothelial cadherin, demonstrated a similar pattern of expression (Fig. S1, a and b). In contrast, the expression of ALK5 was comparatively low and remained unchanged during the tumorigenesis pathway of RIP1-Tag2 mice (Fig. 1 b). Further analysis of the βTC3 cell line established from a RIP1-Tag2 tumor (Efrat et al., 1988) revealed that tumor cells express high levels of ALK5 but not of ALK1 (Fig. S1 c). Notably, the messenger RNA (mRNA) of both TGF-β and BMP9 were present at dramatically increasing levels during the course of malignant progression in RIP1-Tag2 mice and were expressed at 5.2- and 4.4-fold higher levels in tumors compared with normal islets, respectively (Fig. 1, c and d). Expression of the ALK1 ligand BMP10 was up-regulated in angiogenic islets, albeit to a lower degree, but decreased to baseline levels in tumors (Fig. S1 d). The βTC3 cell line expressed high levels of both TGF-β and BMP9 but negligible amounts of BMP10 (Fig. S1 e). Given the structural similarity between BMP9 and BMP10, it is likely that the two ligands share many properties, so our subsequent efforts were focused on BMP9.

Bottom Line: Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor.In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli.We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institutet, Stockholm SE-171 77, Sweden.

ABSTRACT
Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

Show MeSH
Related in: MedlinePlus