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Activation of the transforming growth factor β (TGFβ) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFβ induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFβ-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFβ signaling, expression of TGFβ-target genes, and TGFβ-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFβ signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFβ1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFβ1 cytokine secretion. In addition, TGFβ regulated let-7a-2-3p, miR-125b-5p, and miR-125b-1-3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFβ/SMAD-mediated responses (let-7a-2-3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFβ induces MIR100HG, which then enhances TGFβ1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFβ signaling via TGFβ1 autoinduction and secretion in carcinomas.

Activator protein (AP)-1 transcription factors are essential elements of the pro-oncogenic functions of transforming growth factor-β (TGFβ)-SMAD signaling. Here we show that in multiple HER2+ and/or EGFR+ breast cancer cell lines these AP-1-dependent tumorigenic properties of TGFβ critically rely on epidermal growth factor receptor (EGFR) activation and expression of the ΔN isoform of transcriptional regulator p63. EGFR and ΔNp63 enabled and/or potentiated the activation of a subset of TGFβ-inducible invasion/migration-associated genes, e.g., ITGA2, LAMB3, and WNT7A/B, and enhanced the recruitment of SMAD2/3 to these genes. The TGFβ- and EGF-induced binding of SMAD2/3 and JUNB to these gene loci was accompanied by p63-SMAD2/3 and p63-JUNB complex formation. p63 and EGFR were also found to strongly potentiate TGFβ induction of AP-1 proteins and, in particular, FOS family members. Ectopic overexpression of FOS could counteract the decrease in TGFβ-induced gene activation after p63 depletion. p63 is also involved in the transcriptional regulation of heparin binding (HB)-EGF and EGFR genes, thereby establishing a self-amplification loop that facilitates and empowers the pro-invasive functions of TGFβ. These cooperative pro-oncogenic functions of EGFR, AP-1, p63, and TGFβ were efficiently inhibited by clinically relevant chemical inhibitors. Our findings may, therefore, be of importance for therapy of patients with breast cancers with an activated EGFR-RAS-RAF pathway.

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

Glioblastoma multiforme (GBM) is an aggressive brain malignancy characterized by high heterogeneity and invasiveness. It is increasingly accepted that the refractory feature of GBM to current therapies stems from the existence of few tumorigenic cells that sustain tumor growth and spreading, the so-called glioma-initiating cells (GICs). Previous studies showed that cytokines of the bone morphogenetic protein (BMP) family induce differentiation of the GICs, and thus act as tumor suppressors. Molecular pathways that explain this behavior of BMP cytokines remain largely elusive. Here, we show that BMP signaling induces Smad-dependent expression of the transcriptional regulator Snail in a rapid and sustained manner. Consistent with its already established promigratory function in other cell types, we report that Snail silencing decreases GBM cell migration. Consequently, overexpression of Snail increases GBM invasiveness in a mouse xenograft model. Surprisingly, we found that Snail depletes the GBM capacity to form gliomaspheres in vitro and to grow tumors in vivo, both of which are important features shared by GICs. Thus Snail, acting downstream of BMP signaling, dissociates the invasive capacity of GBM cells from their tumorigenic potential.

Epithelial-mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor β (TGFβ) is a well-studied inducer of EMT. We demonstrate that TGFβ potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGFβ receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGFβ-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGFβ-induced EMT. Interestingly, HAS2 suppression completely abolished the TGFβ-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGFβ-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGFβ-induced EMT.

Deregulation of the transforming growth factor β (TGFβ) signal transduction cascade is functionally linked to cancer. In early phases, TGFβ acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFβ responses, but relatively little is known about the Smad-containing complexes that are important for epithelial-mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFβ signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFβ-induced invasion and found that various mesenchymal and invasion-associated TGFβ-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFβ signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFβ stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFβ-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFβ-induced invasion program.

A dual role of transforming growth factor β (TGF-β), to both suppress and promote tumor progression and metastasis, has been well established, but its molecular basis has remained elusive. In this review, we focus on Smad proteins, which are central mediators of the signal transduction of TGF-β family members. We describe current knowledge of cell-type-specific binding patterns of Smad proteins and mechanisms of transcriptional regulation, obtained from recent studies on genome-wide binding sites of Smad molecules. We also discuss potential application of the genome-wide analyses for cancer research, which will allow clarification of the complex mechanisms occurring during cancer progression, and the identification of potential biomarkers for future cancer diagnosis, prognosis and therapy.

High-grade gliomas, including glioblastomas, are malignant brain tumors for which improved treatment is urgently needed. Genetic studies have demonstrated the existence of biologically distinct subsets. Preliminary studies have indicated that platelet-derived growth factor (PDGF) receptor signaling contributes to the growth of some of these tumors. In this study, human high-grade glioma primary cultures were analysed for sensitivity to treatment with the PDGF receptor inhibitor imatinib/Glivec/Gleevec/STI571. Six out of 15 cultures displayed more than 40% growth inhibition after imatinib treatment, whereas seven cultures showed less than 20% growth inhibition. In the sensitive cultures, apoptosis contributed to growth inhibition. Platelet-derived growth factor receptor status correlated with imatinib sensitivity. Supervised analyses of gene expression profiles and real-time PCR analyses identified expression of the chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) as a predictor of imatinib sensitivity. Exogenous addition of CXCL12 to imatinib-insensitive cultures conferred some imatinib sensitivity. Finally, coregulation of CXCL12 and PDGF alpha-receptor was observed in glioblastoma biopsies. We have thus defined the characteristics of a novel imatinib-sensitive subset of glioma cultures, and provided evidence for a functional relationship between imatinib sensitivity and chemokine signaling. These findings will assist in the design and evaluation of clinical trials exploring therapeutic effects of imatinib on malignant brain tumors.

Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor-beta (TGFbeta) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGFbeta signaling pathway, forms a complex with BRCA1 in vitro and in vivo. The interaction is mediated by the MH1 domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGFbeta1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGFbeta1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.

Smad3 is an essential component in the intracellular signaling of transforming growth factor-beta (TGFbeta), which is a potent inhibitor of tumor cell proliferation. BRCA2 is a tumor suppressor involved in early onset of breast, ovarian and prostate cancer. Both Smad3 and BRCA2 possess transcription activation domains. Here, we show that Smad3 and BRCA2 interact functionally and physically. We found that BRCA2 forms a complex with Smad3 in vitro and in vivo, and that both MH1 and MH2 domains of Smad3 contribute to the interaction. TGFbeta1 stimulates interaction of endogenous Smad3 and BRCA2 in non-transfected cells. BRCA2 co-activates Smad3-dependent transcriptional activation of luciferase reporter and expression of plasminogen activator inhibitor-1 (PAI-1). Smad3 increases the transcriptional activity of BRCA2 fused to the DNA-binding domain (DBD) of Gal4, and reciprocally, BRCA2 co-activates DBD-Gal4-Smad3. Thus, our results show that BRCA2 and Smad3 form a complex and synergize in regulation of transcription.

We have previously shown that the binding site for GTPase activating protein of Ras (RasGAP) in the PDGF beta-receptor, Tyr771, is phosphorylated to a much lower extent in the heterodimeric configuration of PDGF alpha- and beta-receptors, compared to the PDGF beta-receptor homodimer. The decreased recruitment of the RasGAP to the receptor leads to prolonged activation of the Ras/MAP kinase pathway, which could explain the increase in mitogenicity seen upon induction of heterodimers. The molecular mechanism underlying these differences was investigated. We could show that the loss of phosphorylation of Tyr771 was dependent on presence of intact binding sites for the protein tyrosine phosphatase SHP-2 on the PDGF beta-receptor. Thus, in PDGF receptor mutants in which binding of SHP-2 was lost, a higher degree of phosphorylation of Tyr771 was seen, while other phosphorylation sites in the receptor remained virtually unaffected. Thus, SHP-2 appears to play an important role in modulating phosphorylation of Y771, thereby controlling RasGAP recruitment and Ras/MAP kinase signaling in the heterodimeric configuration of the PDGF receptors.

Smads, the intracellular effectors of transforming growth factor-beta (TGF-beta) family members, are somatically mutated at high frequency in particular types of human cancers. Certain of these mutations affect the Smad amino-terminal domain, which, in the case of Smad3 and Smad4, binds DNA. We investigated the functional consequences of four missense mutations in the Smad4 amino-terminal domain found in human tumors. The mutant proteins were found to have impaired abilities to bind DNA although they were fully capable of forming complexes with Smad3. All four Smad4 mutants showed decreased protein stability compared to wild-type Smad4. Two of the Smad4 mutants (G65V and P130S) were translocated to the nucleus and were capable of transactivating a Smad-dependent promoter in a ligand-dependent manner. In contrast, the L43S and R100T mutants were not translocated efficiently to the nucleus and consequently resulted in severely defective transcriptional responses to TGF-beta. Moreover, we demonstrate here the critical importance of two basic residues in the beta-hairpin loop of Smad3 or Smad4 for DNA binding, consistent with predictions from the Smad3 crystal structure. In addition, our results reveal that in the TGF-beta-induced heteromeric signaling complex, loss of DNA binding of Smad4 can be compensated by Smad3, however, both Smad3 and Smad4 are needed for efficient DNA binding and signaling. In conclusion, mutations in the amino-terminal domain of Smad4, that are found in cancer, show loss of multiple functional properties which may contribute to tumorigenesis.

The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGF beta R, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF beta-receptor (PDGF beta R). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGF beta R and also indicated that the TEL moiety activates the tyrosine kinase of the PDGF beta R through the formation of TEL-PDGF beta R oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGF beta R, suggesting a strategy for antagonizing the signaling of TEL-PDGF beta R, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGF beta R and ligand-stimulated PDGF beta R revealed that only TEL-PDGF beta R expression conferred IL-3-independent growth, suggesting differences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL-PDGF beta R was identified as autophosphorylation sites in TEL-PDGF beta R.

Activation of the beta-receptor for platelet-derived growth factor (PDGF) by its ligand leads to autophosphorylation on a number of tyrosine residues. Here we show that Tyr763 in the kinase insert region is a novel autophosphorylation site, which after phosphorylation binds the protein tyrosine phosphatase SHP-2. SHP-2 has also previously been shown to bind to phosphorylated Tyr1009 in the PDGF beta-receptor. Porcine aortic endothelial (PAE) cells transfected with a PDGF beta-receptor in which Tyr763 and Tyr1009 were mutated to phenylalanine residues failed to associate with SHP-2 after ligand stimulation. Moreover, PDGF-BB-induced Ras GTP-loading and Erk2 activation were severely compromised in the receptor mutant. Whereas the mitogenic response to PDGF-BB remained at the same level as in cells expressing wild-type PDGF beta-receptor, chemotaxis induced by PDGF-BB was significantly decreased in the case of the Y763F/Y1009F mutant cells, suggesting an important role for SHP-2 in chemotactic signaling.

The different platelet-derived growth factor (PDGF) isoforms cause activation of their alpha and beta protein tyrosine kinase receptors through dimerization. Homodimerization as well as heterodimerization of receptors occur. It has been shown previously that the heterodimeric receptor complex mediates a stronger mitogenic response than either of the homodimeric complexes. In this report, we show that in cells expressing both PDGF alpha- and beta-receptors, stimulation with PDGF-AB, which leads to preferential heterodimer formation, leads to a very low degree of phosphorylation of Tyr771 in the beta-receptor. In contrast, Tyr771 is phosphorylated in a homodimeric complex of beta-receptors. Phosphorylated Tyr771 is a binding site for RasGAP; an analogous site is not present in the alpha-receptor, which lacks the ability to associate with RasGAP. The lowered phosphorylation of Tyr771 in the heterodimeric receptor complex correlates with lowered association with RasGAP, as well as with a more efficient activation of Ras and MAP kinase, which is consistent with the increased mitogenicity elicited by PDGF-AB, compared to PDGF-AA or PDGF-BB.

Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) alpha-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, affinity purification using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the purified proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF alpha-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was specifically inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF alpha-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type alpha-receptor. In contrast, the Y762F mutant PDGF alpha-receptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF alpha-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF alpha-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF beta-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the beta-receptor, which is localized at an analogous position to Tyr-762 in the alpha-receptor, binds RasGAP. RasGAP is not bound to the alpha-receptor. Thus, this region in the kinase inserts of the two receptors may be important for the divergency in signaling from the two PDGF receptors.

Signal transducers and activators of transcription (Stats) are known to transduce signals from the cell surface to the nucleus in cytokine receptor signaling. We examined the capacity of platelet-derived growth factor (PDGF) receptor to interact with and activate Stat molecules. Activation of the PDGF beta-receptor led to tyrosine phosphorylation of Stat1, Stat3 and Stat5, which was accompanied by specific DNA-binding activities. These events were only weakly stimulated by the activated PDGF alpha-receptor. In cells expressing PDGF beta-receptors mutated at Tyr579, Tyr581 or Tyr775, tyrosine phosphorylation as well as DNA-binding activity of Stat5 was reduced. Immobilized peptides containing phosphorylated Tyr579, Tyr581 or Tyr775 bound Stat5, suggesting direct binding of Stat5 to these tyrosine residues of the PDGF beta-receptor. Members of the Janus kinase family were also shown to interact with the PDGF beta-receptor, and to a lesser extent with the alpha-receptor, but their importance for PDGF-induced Stat activation remains to be determined.

Activation of the platelet-derived growth factor (PDGF) beta-receptor results in motility responses in the forms of membrane ruffling and chemotaxis. Porcine aortic endothelial cells expressing the PDGF beta-receptor or a chimeric fibroblast growth factor (FGF) receptor, in which the endogenous kinase insert was replaced with the corresponding region from the PDGF beta-receptor, migrated efficiently towards a concentration gradient of PDGF-BB and bFGF, respectively, and exhibited both pronounced edge ruffling and circular membrane ruffling in response to ligand-stimulation. The wildtype FGF receptor-1 showed weak or no response in these assays. Further analyses were conducted on mutant receptors, in which tyrosine residues that can serve as autophosphorylation sites and thereby mediate interactions with specific signal transduction molecules, were changed to phenylalanine residues. Each one of the analysed mutants were mitogenically active, however, a mutant in which Tyr740 and Tyr751 were replaced failed to mediate ruffling and chemotaxis. These two residues are implicated in the binding of phosphatidylinositol 3' kinase. The notion that this enzyme is involved in PDGF beta-receptor-induced cell motility is furthermore supported by the finding that another mutant, in which Met743 and Met754 were replaced, and which failed to interact with phosphatidylinositol 3' kinase, was also unable to mediate motility responses.

Human cDNA clones encoding four novel putative transmembrane protein serine/threonine kinases, denoted activin receptor-like kinase (ALK) -1, -2, -3 and -4, were obtained using a polymerase chain reaction (PCR)-based strategy. The PCR primers were designed based upon the sequence similarity between the activin receptor type II and Daf-1. The cDNA clones for ALK-1, -2 and -3 encode complete proteins of 503, 509 and 532 amino acids respectively. The ALK-4 cDNA is incomplete and the predicted protein of 383 amino acids has a truncated extracellular domain. The ALKs share similar domain structures, comprising predicted signal sequences at the N-terminals, followed by hydrophilic cysteine-rich ligand-binding domains, single hydrophobic transmembrane regions and C-terminal intracellular portions that consist almost entirely of putative serine/threonine kinase domains. The ALKs have approximately 40% sequence identity to activin receptors type II and IIB, transforming growth factor-beta (TGF-beta) type II receptor and Daf-1 in the kinase domains. However, the sequence identities are higher (60-79%) between ALK-1, -2, -3 and -4, suggesting that they form a subfamily among the putative receptor serine/threonine kinases. The extracellular domains of ALKs show only little sequence identity to other putative receptor serine/threonine kinases, but the cysteine residues are conserved. Their structural properties suggest that ALK-1 to -4 are receptors that may bind ligands that are members of the TGF-beta superfamily. The expression of mRNA in human tissues varied for the different ALKs; ALK-2 and ALK-4 showed ubiquitous tissue expression patterns, whereas the distribution of ALK-1 and ALK-3 varied strongly between different tissues with more restricted expression patterns. These results suggest that each ALK may have different in vivo functions.