WIKI4, a Novel Inhibitor of Tankyrase and Wnt/?-Catenin Signaling

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

GDF5 Regulates TGF?-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGF? Peptides

Background

TGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß.

Methods

We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis.

Results

TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis.

Conclusions

TGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.     

A Dynein Motor Attachment Complex Regulates TGF?/Smad3 Signaling

Our previous results have demonstrated that km23-2 has functions in TGFß signaling that are distinct from those for km23-1. In the current report, we demonstrate that blockade of km23-2 decreased TGFß activation of the human Smad7 promoter Smad7-Luc, an endogenous Smad3-target promoter. Luminescence-based mammalian interaction mapping (LUMIER) analyses showed that TGFß stimulated the interaction of km23-2 preferentially with Smad3, relative to that with Smad2. Size exclusion chromatography experiments revealed that km23-2 and Smad3 were recruited into the same complex after TGFß treatment. Moreover, in the presence of TGFß, but not in the absence, km23-2 was present in early endosomes with the TGFß receptors (TßRs) and Smad3. Collectively, our data indicate that km23-2 is a critical signaling intermediate in a Smad3-dependent TGFß signaling pathway. We also provide evidence of the novel finding that TGFß stimulates the rapid recruitment of the km23-2 dimer to the dynein intermediate chain (DIC) of the dynein complex, whereas a kinase-deficient form of TßRII prevented this interaction. Finally, we demonstrate for the first time that TGFß stimulated not only assembly of the dynein motor attachment complex, but also triggered the tethering of the km23-2-Smad3 cargo to the other dynein components. Thus, our data demonstrate a novel function for km23-2 as a motor receptor to recruit Smad3 to the dynein complex for intracellular transport, thereby mediating Smad3-dependent TGFß signaling.     

Nodal·Gdf1 Heterodimers with Bound Prodomains Enable Serum-independent Nodal Signaling and Endoderm Differentiation

The TGFβ family member Nodal is central to control pluripotent stem cell fate, but its use as a stem cell differentiation factor is limited by low specific activity. During development, Nodal depends on growth and differentiation factor (Gdf)-1 and on the shared co-receptor Cryptic to specify visceral left-right axis asymmetry. We therefore asked whether the functionality of Nodal can be augmented by Gdf1. Because Nodal and Gdf1 coimmunoprecipitate each other, they were predicted to form heterodimers, possibly to facilitate diffusion or to increase the affinity for signaling receptors. Here, we report that Gdf1 suppresses an unexpected dependence of Nodal on serum proteins and that it is critically required for non-autonomous signaling in cells expressing Cryptic. Nodal, Gdf1, and their cleaved propeptides copurified as a heterodimeric low molecular weight complex that stimulated Activin receptor (Acvr) signaling far more potently than Nodal alone. Although heterodimerization with Gdf1 did not increase binding of Nodal to Fc fusions of co-receptors or Acvr extracellular domains, it was essential for soluble Acvr2 to inhibit Nodal signaling. This implies that Gdf1 potentiates Nodal activity by stabilizing a low molecular weight fraction that is susceptible to neutralization by soluble Acvr2. Finally, in differentiating human ES cells, endodermal markers were more efficiently induced by Nodal·Gdf1 than by Nodal, suggesting that Nodal·Gdf1 is an attractive new reagent to direct stem cell differentiation.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

Tumor Necrosis Factor-Receptor–associated Factor-4 Is a Positive Regulator of Transforming Growth Factor-β Signaling That Affects Neural Crest Formation

The transforming growth factor (TGF)-β superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFβ signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFβ. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor–associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.     

PEAK1 Acts as a Molecular Switch to Regulate Context-Dependent TGFβ Responses in Breast Cancer

Transforming Growth Factor β (TGFβ) has dual functions as both a tumor suppressor and a promoter of cancer progression within the tumor microenvironment, but the molecular mechanisms by which TGFβ signaling switches between these outcomes and the contexts in which this switch occurs remain to be fully elucidated. We previously identified PEAK1 as a new non-receptor tyrosine kinase that associates with the cytoskeleton, and facilitates signaling of HER2/Src complexes. We also showed PEAK1 functions downstream of KRas to promote tumor growth, metastasis and therapy resistance using preclinical in vivo models of human tumor progression. In the current study, we analyzed PEAK1 expression in human breast cancer samples and found PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse. At the cellular level, we also observed that PEAK1 expression was highest in mesenchymal breast cancer cells, correlated with migration potential and increased in response to TGFβ-induced epithelial-mesenchymal transition (EMT). Thus, we sought to evaluate the role of PEAK1 in the switching of TGFβ from a tumor suppressing to tumor promoting factor. Notably, we discovered that high PEAK1 expression causes TGFβ to lose its anti-proliferative effects, and potentiates TGFβ-induced proliferation, EMT, cell migration and tumor metastasis in a fibronectin-dependent fashion. In the presence of fibronectin, PEAK1 caused a switching of TGFβ signaling from its canonical Smad2/3 pathway to non-canonical Src and MAPK signaling. This report is the first to provide evidence that PEAK1 mediates signaling cross talk between TGFβ receptors and integrin/Src/MAPK pathways and that PEAK1 is an important molecular regulator of TGFβ-induced tumor progression and metastasis in breast cancer. Finally, PEAK1 overexpression/upregulation cooperates with TGFβ to reduce breast cancer sensitivity to Src kinase inhibition. These findings provide a rational basis to develop therapeutic agents to target PEAK1 expression/function or upstream/downstream pathways to abrogate breast cancer progression.     

Nodal expression in triple-negative breast cancer: Cellular effects of its inhibition following doxorubicin treatment

Triple-negative breast cancer (TNBC) represents an aggressive cancer subtype characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The independence of TNBC from these growth promoting factors eliminates the efficacy of therapies which specifically target them, and limits TNBC patients to traditional systemic neo/adjuvant chemotherapy. To better understand the growth advantage of TNBC – in the absence of ER, PR and HER2, we focused on the embryonic morphogen Nodal (associated with the cancer stem cell phenotype), which is re-expressed in aggressive breast cancers. Most notably, our previous data demonstrated that inhibition of Nodal signaling in breast cancer cells reduces their tumorigenic capacity. Furthermore, inhibiting Nodal in other cancers has resulted in improved effects of chemotherapy, although the mechanisms for this remain unknown. Thus, we hypothesized that targeting Nodal in TNBC cells in combination with conventional chemotherapy may improve efficacy and represent a potential new strategy. Our preliminary data demonstrate that Nodal is highly expressed in TNBC when compared to invasive hormone receptor positive samples. Treatment of Nodal expressing TNBC cell lines with a neutralizing anti-Nodal antibody reduces the viability of cells that had previously survived treatment with the anthracycline doxorubicin. We show that inhibiting Nodal may alter response mechanisms employed by cancer cells undergoing DNA damage. These data suggest that development of therapies which target Nodal in TNBC may lead to additional treatment options in conjunction with chemotherapy regimens – by altering signaling pathways critical to cellular survival.     

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.     

T Cell-Specific Overexpression of TGF?1 Fails to Influence Atherosclerosis in ApoE-Deficient Mice

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of TGFß1 in atherogenesis by using the murine CD2-TGFß1 transgenic strain which represents a well characterized model of T-cell specific TGFß1 overexpression. The CD2-TGFß1 transgenic mice were crossed to ApoE knock-out mice and quantity and quality of atherosclerosis regarding number of macrophages, smooth muscle cells, CD3 positive T-cells and collagen was analyzed in CD2-TGFß1 ApoE double mutants as well as non-transgenic ApoE controls on both normal and atherogenic diet of a duration of 8, 16 or 24 weeks, respectively. In all experimental groups investigated, we failed to detect any influence of TGFß1 overexpression on disease. Total number of CD3-positive T-lymphocytes was not significantly different in atherosclerotic lesions of CD2-TGFß1 ApoE^−/− females and isogenic ApoE^−/− controls, even after 24 weeks on the atherogenic diet. The synopsis of these data and our previous study on TGFß1 overexpressing macrophages suggests that potential effects of TGFß1 on atherosclerosis are most probably mediated by macrophages rather than T-cells.     

Cerberus-like is a secreted BMP and nodal antagonist not essential for mouse development

Mouse cerberus-like (cer-l) is a member of the Cerberus/Dan family of secreted factors. As other members of this family of proteins, Cer-l functions in the extracellular space, inhibiting signaling molecules. Here we show that the neural-inducing and mesoderm-inhibiting activities of Cer-l result from specific binding to BMP and Nodal molecules, respectively. These properties resemble the ones from the related factor Xenopus Cerberus. However, Xenopus Cerberus in addition to BMP4 and Nodal also binds to and inhibits Wnt proteins. We show that Cer-l does not directly inhibit Wnt signals. A null allele of the mouse Cer-l gene was generated by targeted inactivation in ES cells. Homozygous embryos show no anterior patterning defects, are born alive, and are fertile. Since mouse Cer-l and Xenopus Cerberus differ in biochemical activities, we propose the existence of additional members of this family of inhibitors, which may compensate for the loss of cer-l.     

Amniotic Membrane Modifies the Genetic Program Induced by TGF?, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds

Background

Post-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM) induced a robust epithelialisation in deep traumatic wounds.

Methods and Findings

To better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21) and CDK2B (p15). The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border.

Conclusions

The effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.