Limits...
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.

Show MeSH

Related in: MedlinePlus

Both ALK1 and ALK5 target gene expression is down-regulated by blunted ALK1 signaling. (a–c) Id1 (a), Id3 (b), and PAI-1 (c) mRNA expression in tumors from RIP1-Tag2; Alk1+/− mice compared with that of WT littermates at 12 wk of age, as well as RIP1-Tag2 mice treated with control Fc (Ctrl Fc) protein or RAP-041 between 10 and 12 wk of age. The values for each gene represent mean and SD of at least three independent experiments with three to seven tumors per experimental condition. (d) Expression of PAI-1 mRNA in MS1 endothelial cells treated with TGF-β, BMP9, RAP-041, SB431542, and combinations thereof. Error bars show the mean ± SD. (e) Western blot analysis of PAI-1 protein levels and phosphorylated Smad2 (pSmad2) levels in lysates from MS1 cells subjected to single or combined stimulation of TGF-β and BMP9. Relative expression levels were calculated by densitometric quantification of PAI-1 or pSmad2 relative to the reference protein calnexin. All analyses were independently performed at least three times.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812548&req=5

fig7: Both ALK1 and ALK5 target gene expression is down-regulated by blunted ALK1 signaling. (a–c) Id1 (a), Id3 (b), and PAI-1 (c) mRNA expression in tumors from RIP1-Tag2; Alk1+/− mice compared with that of WT littermates at 12 wk of age, as well as RIP1-Tag2 mice treated with control Fc (Ctrl Fc) protein or RAP-041 between 10 and 12 wk of age. The values for each gene represent mean and SD of at least three independent experiments with three to seven tumors per experimental condition. (d) Expression of PAI-1 mRNA in MS1 endothelial cells treated with TGF-β, BMP9, RAP-041, SB431542, and combinations thereof. Error bars show the mean ± SD. (e) Western blot analysis of PAI-1 protein levels and phosphorylated Smad2 (pSmad2) levels in lysates from MS1 cells subjected to single or combined stimulation of TGF-β and BMP9. Relative expression levels were calculated by densitometric quantification of PAI-1 or pSmad2 relative to the reference protein calnexin. All analyses were independently performed at least three times.

Mentions: To elucidate the molecular mechanism behind the observed angiogenic effects of TGF-β and BMP9 signaling, we examined the expression of known target genes with pro-angiogenic properties for ALK1 (inhibitor of differentiation [Id] 1, 2, and 3) and ALK5 (plasminogen activator inhibitor [PAI] 1, PDGF-B, and fibronectin; Goumans et al., 2002; David et al., 2007). All target genes examined were found to be dramatically up-regulated in RIP1-Tag2 angiogenic islets and/or tumors, compared with normal islets (Fig. S6, a–d; and not depicted), which is consistent with our observation that TGF-β and BMP9 were similarly induced during tumor progression. Expression analysis using mRNA from isolated endothelial cells from RIP1-Tag2 tumors revealed that Id1 was exclusively expressed by endothelial cells, whereas Id3 exhibited some expression also by other cell types (Fig. S6, e and f). Similarly, the prototypical ALK5 target genes PAI-1 and PDGF-B were predominantly expressed by endothelial cells, as ascertained by immunostaining (Fig. S6, g and h). Notably, either diminution of ALK1 gene dosage or treatment with RAP-041 gave rise to a lower expression level of Id1 and Id3 in tumors (Fig. 7, a and b). Surprisingly, tumors from RIP1-Tag2; Alk1+/− mice or from RIP1-Tag2 mice treated with RAP-041 exhibited reduced expression of PAI-1, indicating that ALK1 signaling affects also ALK5 target gene expression in vivo (Fig. 7 c).


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)

Both ALK1 and ALK5 target gene expression is down-regulated by blunted ALK1 signaling. (a–c) Id1 (a), Id3 (b), and PAI-1 (c) mRNA expression in tumors from RIP1-Tag2; Alk1+/− mice compared with that of WT littermates at 12 wk of age, as well as RIP1-Tag2 mice treated with control Fc (Ctrl Fc) protein or RAP-041 between 10 and 12 wk of age. The values for each gene represent mean and SD of at least three independent experiments with three to seven tumors per experimental condition. (d) Expression of PAI-1 mRNA in MS1 endothelial cells treated with TGF-β, BMP9, RAP-041, SB431542, and combinations thereof. Error bars show the mean ± SD. (e) Western blot analysis of PAI-1 protein levels and phosphorylated Smad2 (pSmad2) levels in lysates from MS1 cells subjected to single or combined stimulation of TGF-β and BMP9. Relative expression levels were calculated by densitometric quantification of PAI-1 or pSmad2 relative to the reference protein calnexin. All analyses were independently performed at least three times.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812548&req=5

fig7: Both ALK1 and ALK5 target gene expression is down-regulated by blunted ALK1 signaling. (a–c) Id1 (a), Id3 (b), and PAI-1 (c) mRNA expression in tumors from RIP1-Tag2; Alk1+/− mice compared with that of WT littermates at 12 wk of age, as well as RIP1-Tag2 mice treated with control Fc (Ctrl Fc) protein or RAP-041 between 10 and 12 wk of age. The values for each gene represent mean and SD of at least three independent experiments with three to seven tumors per experimental condition. (d) Expression of PAI-1 mRNA in MS1 endothelial cells treated with TGF-β, BMP9, RAP-041, SB431542, and combinations thereof. Error bars show the mean ± SD. (e) Western blot analysis of PAI-1 protein levels and phosphorylated Smad2 (pSmad2) levels in lysates from MS1 cells subjected to single or combined stimulation of TGF-β and BMP9. Relative expression levels were calculated by densitometric quantification of PAI-1 or pSmad2 relative to the reference protein calnexin. All analyses were independently performed at least three times.
Mentions: To elucidate the molecular mechanism behind the observed angiogenic effects of TGF-β and BMP9 signaling, we examined the expression of known target genes with pro-angiogenic properties for ALK1 (inhibitor of differentiation [Id] 1, 2, and 3) and ALK5 (plasminogen activator inhibitor [PAI] 1, PDGF-B, and fibronectin; Goumans et al., 2002; David et al., 2007). All target genes examined were found to be dramatically up-regulated in RIP1-Tag2 angiogenic islets and/or tumors, compared with normal islets (Fig. S6, a–d; and not depicted), which is consistent with our observation that TGF-β and BMP9 were similarly induced during tumor progression. Expression analysis using mRNA from isolated endothelial cells from RIP1-Tag2 tumors revealed that Id1 was exclusively expressed by endothelial cells, whereas Id3 exhibited some expression also by other cell types (Fig. S6, e and f). Similarly, the prototypical ALK5 target genes PAI-1 and PDGF-B were predominantly expressed by endothelial cells, as ascertained by immunostaining (Fig. S6, g and h). Notably, either diminution of ALK1 gene dosage or treatment with RAP-041 gave rise to a lower expression level of Id1 and Id3 in tumors (Fig. 7, a and b). Surprisingly, tumors from RIP1-Tag2; Alk1+/− mice or from RIP1-Tag2 mice treated with RAP-041 exhibited reduced expression of PAI-1, indicating that ALK1 signaling affects also ALK5 target gene expression in vivo (Fig. 7 c).

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