Genetic studies in mice and humans have shown that the transforming growth factor-β (TGF-β) type-I receptor activin receptor-like kinase 1 (ALK1) and its co-receptor endoglin play an important role in vascular development and angiogenesis. Here, we demonstrate that ALK1 is a signalling receptor for bone morphogenetic protein-9 (BMP-9) in endothelial cells (ECs). BMP-9 bound with high affinity to ALK1 and endoglin, and weakly to the type-I receptor ALK2 and to the BMP type-II receptor (BMPR-II) and activin type-II receptor (ActR-II) in transfected COS cells. Binding of BMP-9 to ALK2 was greatly facilitated when BMPR-II or ActR-II were co-expressed. Whereas BMP-9 predominantly bound to ALK1 and BMPR-II in ECs, it bound to ALK2 and BMPR-II in myoblasts. In addition, we observed binding of BMP-9 to ALK1 and endoglin in glioblastoma cells. BMP-9 activated Smad1 and/or Smad5, and induced ID1 protein and endoglin mRNA expression in ECs. Furthermore, BMP-9 was found to inhibit basic fibroblast growth factor (bFGF)-stimulated proliferation and migration of bovine aortic ECs (BAECs) and to block vascular endothelial growth factor (VEGF)-induced angiogenesis. Taken together, these results suggest that BMP-9 is a physiological ALK1 ligand that plays an important role in the regulation of angiogenesis.
Transforming growth factor B (TGF-B) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-B, but their function in TGF-B-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-B-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-B-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-B-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-B-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.
Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heterotopic ossification for which there is currently no treatment available. FOP has been linked recently to a heterozygous R206H mutation in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2). Expression of the mutant ALK2-R206H receptor (FOP-ALK2) results in increased phosphorylation of the downstream Smad1 effector proteins and elevated basal BMP-dependent transcriptional reporter activity, indicating that FOP-ALK2 is constitutively active. FOP-ALK2-induced transcriptional activity could be blocked by overexpressing either of the inhibitory Smads, Smad6 or -7, or by treatment with the pharmacological BMP type I receptor inhibitor dorsomorphin. However, in contrast to wild-type ALK2, FOP-ALK2 is not inhibited by the negative regulator FKBP12. Mesenchymal cells expressing the FOP-ALK2 receptor are more sensitive to undergoing BMP-induced osteoblast differentiation and mineralization. In vivo bone formation was assessed by loading human mesenchymal stem cells (hMSCs) expressing the ALK2-R206H receptor onto calcium phosphate scaffolds and implantation in nude mice. Compared with control cells FOP-ALK2-expressing cells induced increased bone formation. Taken together, the R206H mutation in ALK2 confers constitutive activity to the mutant receptor, sensitizes mesenchymal cells to BMP-induced osteoblast differentiation, and stimulates new bone formation. We have generated an animal model that can be used as a stepping stone for preclinical studies aimed at inhibiting the heterotopic ossification characteristic of FOP.
Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.
Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes, we characterized transforming growth factor β/BMP receptor and Smad expression in immortalized ovarian surface epithelial cells and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signaling in ovarian cancer. Using small interfering RNA, ligand trap, inhibitor, and ligand stimulation approaches, we show that BMP9 acts as a proliferative factor for immortalized ovarian surface epithelial cells and ovarian cancer cell lines, signaling predominantly through an ALK2/Smad1/Smad4 pathway rather than through ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signaling that is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that ∼25% of epithelial ovarian cancers express BMP9, whereas normal human ovarian surface epithelial specimens do not. Our data indicate that BMP9 signaling through ALK2 may be a novel therapeutic target in ovarian cancer. [Cancer Res 2009;69(24):9254-62]
Bone morphogenetic proteins (BMPs) are used clinically to induce new bone formation in spinal fusions and long bone nonunion fractures. However, large amounts of BMPs are needed to achieve these effects. BMPs were found to increase the expression of antagonists, which potentially limit their therapeutic efficacy. However, the relative susceptibility of osteoinductive BMPs to different antagonists is not well characterized. Here we show that BMP-6 is more resistant to noggin inhibition and more potent in promoting osteoblast differentiation in vitro and inducing bone regeneration in vivo when compared with its closely related BMP-7 paralog. Noggin was found to play a critical role as a negative feedback regulator of BMP-7 but not BMP-6-induced biological responses. Using BMP-6/7 chimeras, we identified lysine 60 as a key residue conferring noggin resistance within the BMP-6 protein. A remarkable correlation was found between the presence of a lysine at this position and noggin resistance among a panel of osteoinductive BMPs. Introduction of a lysine residue at the corresponding positions of BMP-2 and BMP-7 allowed for molecular engineering of recombinant BMPs with increased resistance to noggin antagonism. Bone morphogenetic proteins (BMPs)3 are dimeric secreted cytokines that were discovered based on their ability to induce ectopic bone and cartilage formation in vivo (1-4). BMPs belong to the transforming growth factor- superfamily, which also includes transforming growth factors- and activins. Over 15 distinct BMP family members have been identified that signal via specific BMP type I and type II serine/threonine kinase receptors (5). Three BMP type II receptors (BMPR-II (BMP type II receptor), ActR-II (activin type II receptor), and ActR-IIB) and four distinct BMP type I receptors (ALK1 (activin receptor-like kinase 1), ALK2, ALK3, and ALK6) have been described (5-8). Cell surface binding of BMPs to their receptors results in heteromeric complex formation, upon which the constitutively active type II receptor phosphorylates the type I receptor on specific serine and threonine residues in the juxtamembrane region. Different BMPs bind with different affinities and specificities to different BMPR complexes (6 -10). The activated BMP type I receptor initiates intracellular signaling by phosphorylating specific receptor-regulated Smad (R-Smad) proteins (Smad1, Smad5, and Smad8). Activated R-Smads form heteromeric complexes with Smad4, which translocate to the nucleus and regulate, in cooperation with transcriptional coactivators and co-repressors, the transcription of target genes (5). BMP signaling is controlled at different levels by both positive and negative regulators. At the extracellular level, BMP antagonists bind BMPs and interfere with their binding to BMP receptors. An important extracellular BMP antagonist of the osteogenic activity of BMPs is noggin. The crystal structure of the noggin-BMP-7 complex demonstrated that binding of noggin to BMPs resembles that of BMP receptors and thereby prevents the ...
Purpose: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies. Experimental Design: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations. Results: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFb signaling-induced metastasis by protecting TGFb type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression. Conclusions: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFb-induced breast cancer metastasis and may provide a potential target for TNBC treatment.
Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGFβ) are potent regulators of angiogenesis. How VEGF and TGFβ signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGFβ type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Sub-optimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGFβ type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins α5 and β3. Specific downregulation of α5-integrin expression or functional blocking of α5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an α5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of α5-integrin expression is a key determinant by which inhibitors of TGFβ type-I receptor kinase and VEGF synergistically promote angiogenesis.
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