Smad4-dependent Manner Research Articles

S100A2 induces epithelial–mesenchymal transition and metastasis in pancreatic cancer by coordinating transforming growth factor β signaling in SMAD4-dependent manner

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor and is associated with a poor prognosis. Treatment strategies for PDAC are largely ineffective primarily because of delay in its diagnosis and limited efficacy of systematic treatment. S100A2 is associated with the proliferation, migration, and differentiation of several tumors; however, its effects on PDAC and the associated molecular mechanisms remain to be explored. We studied the mechanisms underlying the effect of S100A2 on epithelial–mesenchymal transition (EMT) and metastasis in PDAC cells. We found that the level of S100A2 remarkably increased and was associated with poor PDAC prognosis. The overexpression of S100A2 in PANC-1 cells also induced EMT, in addition to increasing the invasion and migration of PDAC cells, whereas the knockdown of S100A2 markedly inhibited cell metastasis. Furthermore, S100A2 was found to enhance metastatic abilities in vivo. The overexpression of S100A2 increased SMAD4 expression, whereas the knockdown of S100A2 reduced SMAD4 expression. SMAD4 overexpression could effectively rescue the effects of S100A2 knockdown on EMT. S100A2 mechanistically activated the transforming growth factor (TGF)-β/Smad2/3 signaling pathway, upregulated SMAD4 expression, induced EMT, and increased PANC-1 cell metastasis. In conclusion, the S100A2/SMAD4 axis modulates EMT to accelerate PDAC development. Our results supplement and enrich the understanding of the pathogenesis underlying PDAC and provide a new theoretical basis and strategy targeting S100A2 for the diagnosis and treatment of PDAC.

TGF-β1 regulates the lncRNA transcriptome of ovarian granulosa cells in a transcription activity-dependent manner.

Transforming growth factor β1 (TGF-β1), an essential cytokine belongs to TGF-β superfamily, is crucial for female fertility. Increasing evidence show that long noncoding RNAs (lncRNAs) influence the state of granulosa cells (GCs). This study aimed to detect the effects of TGF-β1 on the lncRNA transcriptome, and investigate whether lncRNAs mediate the functions of TGF-β1 in GCs. RNA-seq and bioinformatics analyses were performed to identify and characterize the differentially expressed lncRNAs (DElncRNAs). The regulatory mechanism of TGF-β1 to lncRNA transcriptome was analyzed by chromatin immunoprecipitation. The effects of lncRNAs on the antiapoptotic and proproliferative functions of TGF-β1 were examined by morphological analysis, fluorescence-activated cell sorting, Cell Counting Kit-8, and Western blot. A total of 72 DElncRNAs highly sensitive to TGF-β1 were identified with the criteria of |log2 (fold chage)| ≥ 3 and false discovery rate < 0.05. Functional assessment showed that DElncRNAs were enriched in TGF-β, nuclear factor kappa B, p53, and Hippo pathways which are crucial for the normal state and function of GCs. Importantly, SMAD4 is essential for the regulation of TGF-β1 to lncRNA transcriptome. In vitro studies confirmed that TGF-β1 induced TEX14-IT1 transcription in a SMAD4-dependent manner, and TEX14-IT1 mediated the antiapoptotic and proproliferative effects of TGF-β1 in GCs. Our findings demonstrate that TGF-β1 alters lncRNA transcriptome in a SMAD4-dependent manner, and highlight that lncRNAs mediate the functions of TGF-β1 in GCs, which contribute to a better understanding of the epigenetic regulation of female fertility.

The SKI proto-oncogene restrains the resident CD103+CD8+ T cell response in viral clearance.

Acute viral infection causes illness and death. In addition, an infection often results in increased susceptibility to a secondary infection, but the mechanisms behind this susceptibility are poorly understood. Since its initial identification as a marker for resident memory CD8+ T cells in barrier tissues, the function and regulation of CD103 integrin (encoded by ITGAE gene) have been extensively investigated. Nonetheless, the function and regulation of the resident CD103+CD8+ T cell response to acute viral infection remain unclear. Although TGFβ signaling is essential for CD103 expression, the precise molecular mechanism behind this regulation is elusive. Here, we reveal a TGFβ-SKI-Smad4 pathway that critically and specifically directs resident CD103+CD8+ T cell generation for protective immunity against primary and secondary viral infection. We found that resident CD103+CD8+ T cells are abundant in both lymphoid and nonlymphoid tissues from uninfected mice. CD103 acts as a costimulation signal to produce an optimal antigenic CD8+ T cell response to acute viral infection. There is a reduction in resident CD103+CD8+ T cells following primary infection that results in increased susceptibility of the host to secondary infection. Intriguingly, CD103 expression inversely and specifically correlates with SKI proto-oncogene (SKI) expression but not R-Smad2/3 activation. Ectopic expression of SKI restricts CD103 expression in CD8+ T cells in vitro and in vivo to hamper viral clearance. Mechanistically, SKI is recruited to the Itgae loci to directly suppress CD103 transcription by regulating histone acetylation in a Smad4-dependent manner. Our study therefore reveals that resident CD103+CD8+ T cells dictate protective immunity during primary and secondary infection. Interfering with SKI function may amplify the resident CD103+CD8+ T cell response to promote protective immunity.

Tumor Microenvironment Remodeling Enables Bypass of Oncogenic KRAS Dependency in Pancreatic Cancer.

Oncogenic KRAS (KRAS*) is a key tumor maintenance gene in pancreatic ductal adenocarcinoma (PDAC), motivating pharmacologic targeting of KRAS* and its effectors. Here, we explored mechanisms involving the tumor microenvironment (TME) as a potential basis for resistance to targeting KRAS*. Using the inducible Kras G12D;Trp53 -/- PDAC mouse model, gain-of-function screens of epigenetic regulators identified HDAC5 as the top hit enabling KRAS* independent tumor growth. HDAC5-driven escaper tumors showed a prominent neutrophil-to-macrophage switch relative to KRAS*-driven tumors. Mechanistically, HDAC5 represses Socs3, a negative regulator of chemokine CCL2, resulting in increased CCL2, which recruits CCR2+ macrophages. Correspondingly, enforced Ccl2 promotes macrophage recruitment into the TME and enables tumor recurrence following KRAS* extinction. These tumor-associated macrophages in turn provide cancer cells with trophic support including TGFβ to enable KRAS* bypass in a SMAD4-dependent manner. Our work uncovers a KRAS* resistance mechanism involving immune cell remodeling of the PDAC TME. SIGNIFICANCE: Although KRAS* is required for PDAC tumor maintenance, tumors can recur following KRAS* extinction. The capacity of PDAC cancer cells to alter the TME myeloid cell composition to support KRAS*-independent tumor growth illuminates novel therapeutic targets that may enhance the effectiveness of therapies targeting KRAS* and its pathway components.See related commentary by Carr and Fernandez-Zapico, p. 910.This article is highlighted in the In This Issue feature, p. 890.

Eribulin rapidly inhibits TGF-\u03b2-induced Snail expression and can induce Slug expression in a Smad4-dependent manner

BackgroundEvidence shows that the anticancer effects of microtubule targeting agents are not due solely to their antimitotic activities but also their ability to impair microtubule-dependent oncogenic signalling.MethodsThe effects of microtubule targeting agents on regulators of TGF-β-induced epithelial-to-mesenchymal transition (EMT) were evaluated in breast cancer cell lines using high content imaging, gene and protein expression, siRNA-mediated knockdown and chromatin immunoprecipitation.ResultsMicrotubule targeting agents rapidly and differentially alter the expression of Snail and Slug, key EMT-promoting transcription factors in breast cancer. Eribulin, vinorelbine and in some cases, ixabepalone, but not paclitaxel, inhibited TGF-β-mediated Snail expression by impairing the microtubule-dependent nuclear localisation of Smad2/3. In contrast, eribulin and vinorelbine promoted a TGF-β-independent increase in Slug in cells with low Smad4. Mechanistically, microtubule depolymerisation induces c-Jun, which consequently increases Slug expression in cells with low Smad4.ConclusionThese results identify a mechanism by which eribulin-mediated microtubule disruption could reverse EMT in preclinical models and in patients. Furthermore, high Smad4 levels could serve as a biomarker of this response. This study highlights that microtubule targeting drugs can exert distinct effects on the expression of EMT-regulating transcription factors and that identifying differences among these drugs could lead to their more rational use.

BMP-4 enhances epithelial mesenchymal transition and cancer stem cell properties of breast cancer cells via Notch signaling

Bone morphogenetic protein (BMP) signaling and Notch signaling play important roles in tumorigenesis in various organs and tissues, including the breast. BMP-4 enhanced epithelial mesenchymal transition (EMT) and stem cell properties in both mammary epithelial cell line and breast carcinoma cell line. BMP-4 increased the expression of EMT biomarkers, such as fibronectin, laminin, N-cadherin, and Slug. BMP-4 also activated Notch signaling in these cells and increased the sphere forming efficiency of the non-transformed mammary epithelial cell line MCF-10A. In addition, BMP-4 upregulated the sphere forming efficiency, colony formation efficiency, and the expression of cancer stem cell markers, such as Nanog and CD44, in the breast carcinoma cell line MDA-MB-231. Inhibition of Notch signaling downregulated EMT and stem cell properties induced by BMP-4. Down-regulation of Smad4 using siRNA impaired the BMP-4-induced activation of Notch signaling, as well as the BMP-4-mediated EMT. These results suggest that EMT and stem cell properties are increased in mammary epithelial cells and breast cancer cells through the activation of Notch signaling in a Smad4-dependent manner in response to BMP-4.

Enhancement of gemcitabine cytotoxicity in pancreatic adenocarcinoma through controlled release of nitric oxide.

Gemcitabine (GEM) is the first-line treatment for pancreatic adenocarcinoma (PAC) yet chemoresistance is common. Nitric oxide (NO) is the predominant species responsible for the cytotoxic action of macrophages against cancer cells yet localized delivery is difficult given the short half-life. We sought to study the effect of locally delivered NO on GEM mediated PAC cytotoxicity and the potential role of SMAD4 in this effect. We hypothesized that NO would enhance the cytotoxicity of GEM in a SMAD4 dependent manner. NO-Silica nanoparticles (NO-Si) were synthesized via a co-condensation of tetraethoxysilane with aminoalkoxysilane under high-pressure nitrous oxide. NO release was measured using chemiluminescence. A SMAD4 negative PAC cell line (SMAD4-) was made using retroviral knockdown of Panc1 PAC cells. Panc1 and SMAD4- cells were treated with gemcitabine (100nm (hi) to 30μm (lo)), 30mg NOSi particles, or both (NOSihi or NOSilo) and cell viability assessed. NoSi reduced cell viability by 25.99% in Panc1 and 24.38% in SMAD4-. When combined with gemcitabine, further reductions were seen in a dose dependent manner for both cell lines. We have demonstrated the in-vitro dose dependent cytotoxic effects of NOSi. When combined with GEM there is a synergistic effect resulting in improved cytotoxicity seen in both Panc1 and SMAD4- PAC cells with a differential pattern of cell death seen at high concentrations of NO. These findings suggest not only that NO is useful chemosensitizing agent but that SMAD4- may play a role in its synergism with GEM.

SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4iΔEC). We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

TGF-β/SMAD4-Regulated LncRNA-LINP1 Inhibits Epithelial-Mesenchymal Transition in Lung Cancer.

Lung cancer is the leading cause of cancer related deaths worldwide. TGF-β-induced epithelial-mesenchymal transition (EMT) is a key cell-intrinsic identity for tumor cell migration, invasion, and stemness acquisition in cancer metastasis. Long noncoding RNAs (lncRNAs) have not been fully investigated for their involvement in regulating TGF-β-induced EMT and metastasis in lung cancer. Here, we demonstrated that the transcription of lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) was inhibited by TGF-β1 in a SMAD4-dependent manner. LINP1 suppressed EMT of lung cancer cells, thereby controlling cancer cell migration, invasion, and stemless. Moreover, LINP1 inhibited TGF-β-induced EMT and cell invasion in lung cancer cells. Our study reveals the role of LINP1 in the regulation of TGF-β-induced EMT in human lung cancer.

Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation.

Th17 cells are critically involved in host defense, inflammation, and autoimmunity1–5. TGF-β is instrumental in Th17 differentiation by cooperating with IL-66,7. Yet, the mechanism of how TGF-β enables Th17 differentiation remains elusive. Here we reveal that TGF-β licenses Th17 differentiation by releasing Ski-Smad4-complex suppressed RORγt expression. We found serendipitously that, unlike wild-type T cells, Smad4-deficient T cells differentiated into Th17 cells in the absence of TGF-β signaling in a RORγt-dependent manner. Ectopic Smad4 expression suppressed the RORγt expression and Th17 differentiation of Smad4-deficient T cells. Unexpectedly however, TGF-β neutralized Smad4 mediated suppression without affecting Smad4 binding to Rorc locus. Proteomic analysis revealed that Smad4 interacted with Ski, a transcriptional repressor degraded upon TGF-β stimulation. Ski controlled the histone acetylation/de-acetylation of Rorc locus and Th17 differentiation via Smad4 because ectopic Ski expression inhibited H3K9Ac of Rorc locus, Rorc expression and Th17 differentiation in a Smad4-dependent manner. Therefore, TGF-β-induced disruption of Ski releases Ski-Smad4 complex imposed suppression of RORγt to license Th17 differentiation. This study reveals a critical mechanism by which TGF-β controls Th17 differentiation and uncovers Ski-Smad4 axis as a potential therapeutic target for treating Th17 related diseases.

PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis.

Tumor genetics and escape from immune surveillance concur in the poor prognosis of PDAC. In this study an experimental model was set up to verify whether SMAD4, deleted in about 55% PDAC and associated with poor prognosis, is involved in determining immunosuppression through Exosomes (Exo). Potential mechanisms and mediators underlying SMAD4-dependent immunosuppression were evaluated by studying intracellular calcium (Fluo-4), Exo-miRNAs (microarray) and Exo-proteins (SILAC). Two PDAC cell lines expressing (BxPC3-SMAD4+) or not-expressing (BxPC3) SMAD4 were used to prepare Exo-enriched conditioned media, employed in experiments with blood donors PBMCs. Exo expanded myeloid derived suppressor cells (gMDSC and mMDSC, flow cytometry) and altered intracellular calcium fluxes in an SMAD4 dependent manner. BxPC3-SMAD4+, but mainly BxPC3 Exo, increased calcium fluxes of PBMCs (p = 0.007) and this increased intracellular calcium trafficking characterized mMDSCs. The analysis of de-regulated Exo-miRNAs and transfection experiments revealed hsa-miR-494-3p and has-miR-1260a as potential mediators of SMAD4-associated de-regulated calcium fluxes. Eleven main biological processes were identified by the analysis of SMAD4-associated de-regulated Exo-proteins, including translation, cell adhesion, cell signaling and glycolysis. A reverse Warburg effect was observed by treating PBMCs with PDAC-derived Exo: BxPC3 Exo induced a higher glucose consumption and lactate production than BxPC3-SMAD4+ Exo. Conclusion: PDAC-derived Exo from cells with, but mainly from those without SMAD4 expression, create an immunosuppressive myeloid cell background by increasing calcium fluxes and glycolysis through the transfer of SMAD4-related differentially expressed miRNAs and proteins.

Smad4 is required to inhibit osteoclastogenesis and maintain bone mass.

Bone homeostasis is maintained as a delicate balance between bone-resorption and bone-formation, which are coupled to maintain appropriate bone mass. A critical question is how bone-resorption is terminated to allow bone-formation to occur. Here, we show that TGFβs inhibit osteoclastogenesis and maintain bone-mass through Smad4 activity in osteoclasts. We found that latent-TGFβ1 was activated by osteoclasts to inhibit osteoclastogenesis. Osteoclast-specific Smad4 conditional knockout mice (Smad4-cKO) exhibited significantly reduced bone-mass and elevated osteoclast formation relative to controls. TGFβ1-activation induced expression of Irf8 and Bcl6, both of which encode factors inhibiting osteoclastogenesis, by blocking their negative regulator, Prdm1, in osteoclasts in a Smad4-dependent manner. Reduced bone-mass and accelerated osteoclastogenesis seen in Smad4-cKO were abrogated by Prdm1 deletion. Administration of latent-TGFβ1-Fc to wild-type mice antagonized LPS-induced bone destruction in a model of activated osteoclast-mediated bone destruction. Thus, latent-TGFβ1-Fc could serve as a promising new therapeutic agent in bone diseases marked by excessive resorption.

SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC)-derived exosomes enrich the microenvironment in Myeloid Derived Suppressor Cells (MDSCs) in a Smad4-dependent manner through a new calcium related axis

Pancreatic ductal adenocarcinoma (PDAC)-derived exosomes enrich the microenvironment in Myeloid Derived Suppressor Cells (MDSCs) in a Smad4-dependent manner through a new calcium related axis

GATA-2 inhibits transforming growth factor-β signaling pathway through interaction with Smad4

GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway.

Stem Cell Marker Nestin Is Critical for TGF-β1-Mediated Tumor Progression in Pancreatic Cancer

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-β1/Smad4 pathway in PDAC. We examined nestin expression after TGF-β1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-β1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-β1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-β1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-β1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.

Dietary Supplementation with Genistein Increases Hepcidin Expression in Wild Type Mice

Abstract 3208Iron overload is an important cause of morbidity and death in patients with hemoglobinopathies, transfusion-dependent anemias, and hereditary hemochromatosis. As humans have no means of excreting iron, regulation of iron homeostasis depends on limiting intestinal iron absorption and optimizing iron release from macrophages to developing erythrocytes. Hepcidin, a peptide hormone produced in the liver, modulates intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. We conducted a small-scale chemical screen and found that the isoflavone genistein, a major dietary component of soybeans, enhanced Hepcidin transcript levels in zebrafish embryos. Furthermore genistein treatment increased Hepcidin transcript levels and Hepcidin promoter activity in human hepatocytes (HepG2 cells) in a Stat3 and Smad4-dependent manner. To evaluate genistein's effect in a mammalian model, we placed groups of 4 four-week old male C57BL/6 mice on an iron-sufficient, low soy diet (AIN93G containing 35 mg of iron/kg) supplemented with 0, 250, or 500 mg of genistein per kg of food for 7 weeks, and then sacrificed the animals for analysis. Plasma genistein levels (mean±SE) at the time of sacrifice were 0.015±0.015, 0.52±0.173, and 2.07±0.65 micromolar, respectively. Compared to mice not treated with genistein, the 250 mg/kg dose produced a significant increase in hepatic Hepcidin (HAMP1) transcript levels (1.49±0.10 vs 0.93±0.10, p=0.01), while the 500 mg/kg dose did not. Although liver iron content, spleen iron content, and weight gain were not significantly different among the groups, the ratio of Hepcidin expression to liver iron content was significantly increased in the animals treated with genistein 250 mg/kg compared to controls (0.013±0.0009 vs 0.0074±0.00068, p=0.0068). In conclusion, genistein is the first orally administered small molecule experimental drug shown to increase Hepcidin transcript levels in vivo. Future experiments will evaluate the effects of genistein on genetic models of iron overload syndromes. Disclosures:No relevant conflicts of interest to declare.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.

Proteomics of Smad4 regulated transforming growth factor-beta signalling in colon cancer cells

TGF-β signalling can play a paradoxical cell type specific role in cancer progression. Smad4 is a key mediator of the TGF-β pathway, and is mutated and/or deleted in many cancers. To investigate Smad4 regulated TGF-β signalling in colon cancer we conducted an iTRAQ mass spectrometry quantitative screen using wild type SW480 (Smad4 negative) colon carcinoma cells and stably restored Smad4 positive SW480 cells. In cells possessing a restored canonical TGF-β signalling pathway, 48 h TGF-β stimulation induced the expression of 15 proteins and repressed 1 protein, while in Smad4 wild type cells, TGF-β induced 7 proteins and repressed 2 proteins. The expression of several S100 protein family members (A2, A4, A10, and A11), transgelin-2 and AKAP12, amongst others, were shown to be regulated by TGF-β in a Smad4 dependent manner. We observed that S100 A4 could be repressed by TGF-β, independently of Smad4 expression, while other Smad4 independent TGF-β responses were restricted to induction of ribosomes and cytoskeletal proteins. Our proteomic screen has identified new Smad4 dependent and independent TGF-β responses in colon carcinoma cells.