Opposing USP19 splice variants in TGF-ß signaling and TGF-ß-induced epithelial-mesenchymal transition of breast cancer cells.

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-ß signaling by directly interacting with TGF-ß type I receptor (TßRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TßRI in the ER. By decreasing cell surface TßRI levels, USP19-ER inhibits TGF-ß/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-ß-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.

Role of soluble endoglin in BMP9 signaling.

Endoglin (ENG) is a coreceptor of the transforming growth factor-ß (TGFß) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor ß (TGF-ß) type I receptor, and endoglin, a TGF-ß 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 antiangiogenic 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 and TGF-ß 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/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.

Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and ß4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.

TGF-ß-Induced Endothelial to Mesenchymal Transition Is Determined by a Balance Between SNAIL and ID Factors.

Endothelial-to-mesenchymal transition (EndMT) plays an important role in embryonic development and disease progression. Yet, how different members of the transforming growth factor-ß (TGF-ß) family regulate EndMT is not well understood. In the current study, we report that TGF-ß2, but not bone morphogenetic protein (BMP)9, triggers EndMT in murine endothelial MS-1 and 2H11 cells. TGF-ß2 strongly upregulates the transcription factor SNAIL, and the depletion of Snail is sufficient to abrogate TGF-ß2-triggered mesenchymal-like cell morphology acquisition and EndMT-related molecular changes. Although SLUG is not regulated by TGF-ß2, knocking out Slug also partly inhibits TGF-ß2-induced EndMT in 2H11 cells. Interestingly, in addition to SNAIL and SLUG, BMP9 stimulates inhibitor of DNA binding (ID) proteins. The suppression of Id1, Id2, or Id3 expression facilitated BMP9 in inducing EndMT and, in contrast, ectopic expression of ID1, ID2, or ID3 abrogated TGF-ß2-mediated EndMT. Altogether, our results show that SNAIL is critical and indispensable for TGF-ß2-mediated EndMT. Although SLUG is also involved in the EndMT process, it plays less of a crucial role in it. In contrast, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. These data suggest that the control over endothelial vs. mesenchymal cell states is determined, at least in part, by a balance between the expression of SNAIL/SLUG and ID proteins.

Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors.

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

Studying TGF-ß Signaling and TGF-ß-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells.

Transforming growth factor-ß (TGF-ß) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-ß signaling can lead to breast cancer. TGF-ß elicits its effects on proliferation and differentiation via specific cell surface TGF-ß type I and type II receptors (i.e., TßRI and TßRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-ß-induced heteromeric complex formation, activated TßRI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-ß acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-ß signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-ß-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-ß-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-ß receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-ß-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-ß-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.

ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis.

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-ß (TGF-ß) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.

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

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.

ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes.

INTRODUCTION: TGF-beta is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF-beta action has been implicated in cartilage diseases such as osteoarthritis. TGF-beta signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF-beta receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF-beta signaling and responses in human chondrocytes. MATERIALS AND METHODS: ALK1 expression in human chondrocytes was examined by RT-PCR and Western blot. The ability of ALK1 to form complexes with other TGF-beta receptors was determined by affinity labeling/immunoprecipitation and by immunoprecipitation followed by Western blot. The effect of ALK1 on TGF-beta1-induced signaling and responses was determined by varying ALK1 expression levels and measuring transcriptional activity using promoter/luciferase assays, Smad1/5 and Smad3 phosphorylation, and expression of type II collagen, PAI-1, and fibronectin. RESULTS: Our results indicate that ALK1 is expressed in human chondrocytes and that it is a component of the TGF-beta receptor system, associating with ALK5, type II TGF-beta receptor, endoglin, and betaglycan. Furthermore, we show that both ALK1 and ALK5 are needed for TGF-beta-induced phosphorylation of intracellular mediators Smad1/5, whereas only ALK5 is essential for TGF-beta1-induced phosphorylation of Smad3. In addition, our results show that ALK1 inhibits, whereas ALK5 potentiates, TGF-beta-induced Smad3-driven transcriptional activity and the expression of PAI-1, fibronectin, and type II collagen in chondrocytes. CONCLUSIONS: Our results suggest that ALK1 and ALK5 display opposing functions in human chondrocytes, implicating an essential role for ALK1 in the regulation of TGF-beta signaling and function in these cells.

Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses.

Transforming growth factor-beta (TGF-beta) elicits cellular effects by activating specific Smad proteins that control the transcription of target genes. Whereas there is growing evidence that there are TGF-beta type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-beta signaling is not well understood. Therefore, we generated TGF-beta type I receptors (also termed ALK5s) with mutations in the L45 loop of the kinase domain, termed ALK5(D266A) and ALK5(3A). These mutants showed retained kinase activity but were unable to activate Smads. Characterization of their signaling properties revealed that the two L45 loop mutants did not mediate Smad-dependent transcriptional responses, TGF-beta-induced growth inhibition, and fibronectin and plasminogen activator-1 production in R4-2 mink lung epithelial cells lacking functional ALK5 protein. Mutation in the L45 loop region did not affect the binding of inhibitory Smads but did abrogate the weak binding of X-linked inhibitor of apoptosis protein and Disabled-2 to ALK5. This suggests that the L45 loop in the kinase domain is important for docking of other binding proteins. Interestingly, JNK MAP kinase activity was found to be activated by the ALK5(3A) mutant in various cell types. In addition, TGF-beta-induced inhibition of cyclin D1 expression and stimulation of PMEPA1 (androgen-regulated prostatic mRNA) expression were found to occur, albeit weakly, in an Smad-independent manner in normal murine mammary gland cells. However, the TGF-beta-induced epithelial to mesenchymal transdifferentiation was found to require an intact L45 loop and is likely to be dependent on the Smad pathways.

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction.

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.