TGF-β Neutralizing Antibody Research Articles

Thrombin Activates Latent TGFβ1 via Integrin αvβ1 in Gingival Fibroblasts

Transforming growth factor β (TGFβ) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGFβ1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGFβ1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFβ1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFβ-neutralizing antibody, TGFβ type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits αv and β1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFβ1 levels; however, similar effects were not observed for integrins αvβ3 and αvβ5. These results suggest that protease-activated receptor 1–induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvβ1–induced latent TGFβ1 activation and subsequent TGFβ1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFβ1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFβ1 activation.

Expression of Wnt/β-Catenin Signaling Pathway and Its Regulatory Role in Type I Collagen with TGF-β1 in Scleral Fibroblasts from an Experimentally Induced Myopia Guinea Pig Model.

Background. To investigate Wnt/β-catenin signaling pathway expression and its regulation of type I collagen by TGF-β1 in scleral fibroblasts from form-deprivation myopia (FDM) guinea pig model. Methods. Wnt isoforms were examined using genome microarrays. Scleral fibroblasts from FDM group and self-control (SC) group were cultured. Wnt isoforms, β-catenin, TGF-β1, and type I collagen expression levels were examined in the two groups with or without DKK-1 or TGF-β1 neutralizing antibody. Results. For genome microarrays, the expression of Wnt3 in FDM group was significantly greater as confirmed in retinal and scleral tissue. The expression of Wnt3 and β-catenin significantly increased in FDM group and decreased significantly with DKK-1. TGF-β1 expression level decreased significantly in FDM group and increased significantly with DKK-1. Along with morphological misalignment inside and outside cells, the amount of type I collagen decreased in FDM group. Furthermore, type I collagen increased and became regular in DKK-1 intervention group, whereas it decreased and rearranged more disorder in TGF-β1 neutralizing antibody intervention group. Conclusions. The activation of Wnt3/β-catenin signaling pathway was demonstrated in primary scleral fibroblasts in FDM. This pathway further reduced the expression of type I collagen by TGF-β1, which ultimately played a role in scleral remodeling during myopia development.

TGF-β Pathway Inhibition Rescues the Function of Hematopoietic Stem and Progenitor Cells Derived from Patients with Fanconi Anemia

Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutation in one of eighteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA.Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice.Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs.Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. DisclosuresNo relevant conflicts of interest to declare.

Fibulin-4 deficiency increases TGF-β signalling in aortic smooth muscle cells due to elevated TGF-β2 levels.

Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-β signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-β pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-β neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-β signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-β isoforms. These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels. Significantly increased TGF-β2 levels were also detectable in plasma from homozygous Fibulin-4R/R mice, not in wild type mice. TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-β signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-β1, but especially TGF-β2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-β pathway regulation in the pathogenesis of aortic aneurysms.

Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGFβ1-Mediated Upregulation of Periostin Expression.

Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi-quantitative reverse-transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL-related genes, such as periostin, type-I collagen, and fibrillin-1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a-induced PDL-related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1-mediated upregulation of periostin expression.

Prostaglandin E2 and Transforming Growth Factor-β Play a Critical Role in Suppression of Allergic Airway Inflammation by Adipose-Derived Stem Cells.

BackgroundThe role of soluble factors in the suppression of allergic airway inflammation by adipose-derived stem cells (ASCs) remains to be elucidated. Moreover, the major soluble factors responsible for the immunomodulatory effects of ASCs in allergic airway diseases have not been well documented. We evaluated the effects of ASCs on allergic inflammation in asthmatic mice treated with a prostaglandin E2 (PGE2) inhibitor or transforming growth factor-β (TGF-β) neutralizing antibodies.Methods and FindingsAsthmatic mice were injected intraperitoneally with a PGE2 inhibitor or TGF-β neutralizing antibodies at approximately the same time as ASCs injection and were compared with non-treated controls. In asthmatic mice, ASCs significantly reduced airway hyperresponsiveness, the number of total inflammatory cells and eosinophils in the bronchoalveolar lavage fluid (BALF), eosinophilic inflammation, goblet cell hyperplasia, and serum total and allergen-specific IgE and IgG1. ASCs significantly inhibited Th2 cytokines, such as interleukin (IL)-4, IL-5, and IL-13, and enhanced the Th1 cytokine (Interferon-γ) and regulatory cytokines (IL-10 and TGF-β) in the BALF and lung draining lymph nodes (LLNs). ASCs engraftment caused significant increases in the regulatory T cell (Treg) and IL-10+ T cell populations in LLNs. However, blocking PGE2 or TGF-β eliminated the immunosuppressive effect of ASCs in allergic airway inflammation.ConclusionsASCs are capable of secreting PGE2 and TGF-β, which may play a role in inducing Treg expansion. Furthermore, treatment with a PGE2 inhibitor or TGF-β neutralizing antibodies eliminated the beneficial effect of ASCs treatment in asthmatic mice, suggesting that PGE2 and TGF-β are the major soluble factors responsible for suppressing allergic airway inflammation.

Inhibition of SET Domain-Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis.

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain-containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro. However, in vivo studies utilizing an experimental model of renal fibrosis are required to develop therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-β1 neutralizing antibody. TGF-β1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P<0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent.

Adamtsl2 deletion results in bronchial fibrillin microfibril accumulation and bronchial epithelial dysplasia--a novel mouse model providing insights into geleophysic dysplasia.

ABSTRACTMutations in the secreted glycoprotein ADAMTSL2 cause recessive geleophysic dysplasia (GD) in humans and Musladin–Lueke syndrome (MLS) in dogs. GD is a severe, often lethal, condition presenting with short stature, brachydactyly, stiff skin, joint contractures, tracheal-bronchial stenosis and cardiac valve anomalies, whereas MLS is non-lethal and characterized by short stature and severe skin fibrosis. Although most mutations in fibrillin-1 (FBN1) cause Marfan syndrome (MFS), a microfibril disorder leading to transforming growth factor-β (TGFβ) dysregulation, domain-specific FBN1 mutations result in dominant GD. ADAMTSL2 has been previously shown to bind FBN1 and latent TGFβ-binding protein-1 (LTBP1). Here, we investigated mice with targeted Adamtsl2 inactivation as a new model for GD (Adamtsl2−/− mice). An intragenic lacZ reporter in these mice showed that ADAMTSL2 was produced exclusively by bronchial smooth muscle cells during embryonic lung development. Adamtsl2−/− mice, which died at birth, had severe bronchial epithelial dysplasia with abnormal glycogen-rich inclusions in bronchial epithelium resembling the cellular anomalies described previously in GD. An increase in microfibrils in the bronchial wall was associated with increased FBN2 and microfibril-associated glycoprotein-1 (MAGP1) staining, whereas LTBP1 staining was increased in bronchial epithelium. ADAMTSL2 was shown to bind directly to FBN2 with an affinity comparable to FBN1. The observed extracellular matrix (ECM) alterations were associated with increased bronchial epithelial TGFβ signaling at 17.5 days of gestation; however, treatment with TGFβ-neutralizing antibody did not correct the epithelial dysplasia. These investigations reveal a new function of ADAMTSL2 in modulating microfibril formation, and a previously unsuspected association with FBN2. Our studies suggest that the bronchial epithelial dysplasia accompanying microfibril dysregulation in Adamtsl2−/− mice cannot be reversed by TGFβ neutralization, and thus might be mediated by other mechanisms.

Dimorphic effects of transforming growth factor-β signaling during aortic aneurysm progression in mice suggest a combinatorial therapy for Marfan syndrome.

Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFβ signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFβ neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFβ neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1(mgR/mgR) mice). Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1(mgR/mgR) mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFβ-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFβ neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFβ hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1(mgR/mgR) mice from ruptured TAA. By demonstrating that promiscuous AT1r and TGFβ drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFβ.

Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma.

Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.

Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression.

Influence of sirolimus-induced TGF-β secretion on mouse Treg cell proliferation.

We examined the effects of co-culturing CD4+ CD25+ Treg cells with sirolimus or cyclosporin A on Treg cell proliferation and differentiation and on transforming growth factor-β (TGF-β) and Foxp3 expression. CD4+ CD25+ Treg cells were harvested from mononuclear cells of spleens of C57BL/6 mice using immunomagnetic beads and divided into control, sirolimus, and cyclosporine groups. Following a 96-h co-culture, Treg cells were assayed by flow cytometry. FoxP3 and TGF-β mRNA levels and secretion were assayed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Smad protein of the TGF-β signaling pathway was assayed by western blot and its effect on CD4+ CD25+ FoxP3+ Treg cell proliferation was determined. Sirolimus-promoted differentiation and proliferation was examined using a TGF-β neutralizing antibody. Sirolimus-treated CD4+ T cell TGF-β secretion increased 2.5X over control levels (P < 0.01), but that of the cyclosporine group decreased marginally (P > 0.05). The CD4+ cell proportion decreased significantly (41.25 vs 69.22%, P < 0.01) and slightly (65.21 vs 69.22, P > 0.05) in the cyclosporine and sirolimus groups, respectively. T cell Foxp3 mRNA expression was significantly higher in the sirolimus-treated than in the cyclosporine (53.7 vs 40.2%, P < 0.05) and control groups (P < 0.01), but was significantly lower in the cyclosporine group than in controls (23.6 vs 40.2%, P < 0.01). Overall, sirolimus promoted CD4+ CD25+ Treg cell proliferation and growth in vitro, whereas cyclosporin A inhibited proliferation. Sirolimus might promote CD4+ CD25+ FoxP3+ regulatory T cell proliferation by inducing TGF-β secretion in vivo.

Transforming growth factor β1-mediated anti-inflammation slows progression of midbrain dopaminergic neurodegeneration in Parkinson's disease?

Transforming growth factor β1-mediated anti-inflammation slows progression of midbrain dopaminergic neurodegeneration in Parkinson's disease?

Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non-small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. TGF-β-mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation, or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.

Pro-Survival Effects of TGF-β1 Are Associated with Molecular Signaling Changes of ERK, FLI-1, and CD44 in AML Cells

Transforming growth factor β (TGF-β) is a key regulator of cell growth and survival. We have previously reported the pro-survival role of TGF-β1 and the anti-leukemic efficacy of TGF-β neutralizing antibody in the bone marrow (BM) microenvironment (Xu et al., Br J Haematol.2008, Tabe et al. PLoS One. 2013). FLI-1 is a transcription factor that plays a pivotal role in the regulation of extracellular matrix genes under negative regulation by TGF-β1–dependent acetylation. We reported that patients with acute myeloid leukemia (AML) expressing a low level of FLI-1 achieved high rates of remission, but their remission duration and survival were curtailed (Kornblau et al., Blood, 2011). Moreover, treatment with rhTGF-β1 induced expression of matrix metalloproteinase-1 (MMP-1) mRNA in AML cells, which inversely correlated with FLI-1 expression (Tabe et al. ASH 2012), concordant with a previous report that FLI-1 downregulates the expression of MMP-1 promoter activity.In this study, we investigated the molecular mechanisms by which TGF-β induces AML cell survival. In AML cell lines NB4, U937, and OCI-AML3, rhTGF-β1 (2ng/mL) induced the TGF-β downstream targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and Smad2 phosphorylation (p-Smad2). These effects were reversed by anti–TGF-β neutralizing antibody 1D11 but not by the isotype control antibody 13C4. Treatment with rhTGF-β1 significantly inhibited serum starvation–induced apoptosis and promoted G0/G1 cell cycle arrest in NB4 and U937 cells but not in OCI-AML3 cells (subG1% vs controls at 72 h: NB4, 18.9 ± 1.7 vs 12.8 ± 1.3; U937, 51.9 ± 2.8 vs 29.1 ± 2.4; OCI-AML3, 8.8 ± 0.5 vs 7.7 ± 0.5; G0/G1 % vs controls at 72 h:,NB4, 39.2 ± 3.5 vs 55.6 ± 5.6; U937, 16.0 ± 1.8 vs 37.6 ± 2.1; OCI-AML3, 68.0 ± 4.2 vs 55.1 ± 9.9). These changes were effectively reversed by 1D11, indicating that rhTGF-β1 facilitated pro-survival effects along with transition into a quiescent G0 state in NB4 and U937 cells but not in OCI-AML3 cells.To investigate the differences in TGF-β downstream signaling activation in these cells, we focused on ERK phosphorylation and FLI-1 expression. TGF-β is known to activate ERK downstream of the Ras pathway, which cross-talks with Smad signaling and associates with FLI-1 activation. In U937 and NB4 cells, after rhTGF-β1 treatment, ERK phosphorylation was upregulated in the early phase (0.5-3 h) and then downregulated (24 h), and FLI-1 expression was downregulated (mRNA and protein, 24 h). Protein phosphatase 2A (PP2A), a modulator of TGF-β and a negative regulator of p-ERK, was downregulated by rhTGF-β1. ERK is known to participate in multiple feedback loops that include its upstream regulator Ras, which is important for the reduction of ERK activity at a late stage after stimulation. Indeed, p-Raf, a direct upstream regulator of p-ERK, demonstrated concordant expression changes with p-ERK after rhTGF-β1 treatment. No changes in p-ERK and FLI-1 expression were detected in OCI-AML3 cells. rhTGF-β1 induced significant upregulation of anti-apoptotic Bcl-2 in NB4 and U937 but not in OCI-AML3 cells.We next investigated the rhTGF-β1–induced expression of CD44 and its variant CD44v. CD44 is recognized as a marker of cancer stem cells, and CD44v regulates redox status in stem-like cancer cells. In several types of cancer cells, CD44 is known to mediate cell migration via interaction with TGF-β type I receptor and close association with ERK signaling. Treatment with rhTGF-β1 increased both total and variant CD44 mRNA in NB4 cells but caused no change in OCI-AML cells (fold change of CD44/CD44v mRNA compared to controls: NB4, 3.0/3.7; OCI-AML3, 1.2/0.9). These changes were inversely correlated with FLI-1 expression in NB4 cells.Taken together, these results show that TGF-β–induced ERK activation and its subsequent negative feedback on the Ras/Raf pathway are associated with FLI-1 downregulation and other responses, including CD44 upregulation, in AML in a cell-dependent manner (Figure 1). These molecular changes might contribute to maintenance of the stem cell–like properties, support survival of AML cells, and play pathologic roles in the TGF-β–abundant BM microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 13392: Deficiency of Microrna-378 Contributes to the Development of Cardiac Fibrosis Involving a Tgfβ1-dependent Paracrine Mechanism

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during the development of heart failure. Previous studies implicated that microRNA-378 is primarily expressed in cardiomyocytes, and it is down-regulated during heart failure. To understand the consequence of miR-378 depletion during cardiac remodeling, the present study employed a LNA-modified-antimiR to target miR-378 in vivo. Results showed that loss of miR-378 function in mouse hearts led to the development of cardiomyocyte hypertrophy and fibrosis. Upon evaluation of the mechanism of profibrotic response of miR-378 inhibition, we found that antimiR treatment induced TGFβ1 expression in mouse hearts as well as in cultured cardiomyocytes, whereas its expression in cardiomyocytes abolished AngII-stimulated induction of TGFβ1 mRNA. Among various secreted cytokines, only TGFβ1 levels were found to be increased in the conditioned-media of miR-378 depleted cardiomyocytes. Treatment of cardiac fibroblasts with the conditioned-media of miR-378 depleted myocytes activated pSMAD2/3, a critical step in TGFβ-signaling, and induced fibrotic gene expression. This effect of miR-378 depletion was counteracted by including a TGFβ1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, antimiR-mediated stimulation of TGFβ1 mRNA was correlated with the increased expression of c-fos and c-jun. Adenovirus expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFβ1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. These results demonstrate that reduction in miR-378 levels during pathological conditions participate in the process of cardiac remodeling through paracrine release of a profibrotic cytokine, TGFβ1, from cardiomyocytes. Our data imply that the presence of miR-378 in cardiomyocytes plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.

TGFβ1 mediates alcohol-induced Nrf2 suppression in lung fibroblasts.

BackgroundChronic alcohol ingestion induces the expression of transforming growth factor beta-1(TGFβ1), inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated activation of the antioxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study, we examined the mechanistic relationship between TGFβ1 and Nrf2-ARE signaling in the experimental alcoholic lung.MethodsWild-type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (SFP; an activator of Nrf2), ±TGFβ1, ±TGFβ1 neutralizing antibody, and/or ±activin receptor-like kinase 5 inhibitors (to block TGβ1 receptor signaling) and then analyzed for the expression of Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and TGFβ1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent antioxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, silencing RNA (siRNA) of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFβ1 expression.ResultsAlcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Furthermore, alcohol exposure increased TGFβ1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with SFP; in contrast, Nrf2 SiRNA expression exacerbated alcohol-induced TGFβ1 expression. Finally, TGFβ1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFβ1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity.ConclusionsAlcohol-induced oxidative stress is mediated by TGFβ1, which suppresses Nrf2-ARE-dependent expression of antioxidant defenses and creates a vicious cycle that feeds back to further increase TGFβ1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.

Abstract LB-175: Concomitant radiotherapy (RT) and TGFβ neutralizing antibodies alters tumor microenvironment and promotes tumor regression

Abstract Transforming growth factor-β (TGFβ) has a broad range of pro-tumorigenic activities and immunosuppressive functions that have drawn considerable interest in cancer, especially since pharmaceutical inhibitors are in clinical trials. It is not clear how best to release the considerable potential of TGFβ inhibitors. Radiation induces TGFβ activity that promotes effective response to DNA damage, which protects cancer cells from radiation-induced cell death. Radition therapy is quite effective in eliciting immunogenic signals but is itself a source of immunosuppressive effects in the tumor microenvironment. Here we demonstrate that inhibition of TGFβ in pre-clinical models of breast and lung cancer creates a systemic environment that relieves the tumor-driven immune-suppression and creates a tumor microenvironment that promotes tumor regression. Balb/c and C57/BL6 mice were inoculated with TSA breast cancer cells and Lewis lung cancer cells respectively into the flank and injected with 10mg/kg i.p preclinical TGFβ neutralizing antibody 1D11 or control antibody 13C4, both provided by Genzyme, Inc., beginning on day 13 post tumor cell implantation and continuing on alternate days to experiment termination. Radiotherapy (RT) was initiated on day 14 in 5 daily, 6Gy fractions. Tumor growth was caliper-measured thrice/week. Flow cytometry analysis and ex vivo cultures were used to assess frequency and functional characteristics of immune cells. TGFβ neutralizing antibody monotherapy had no effect on tumor growth, however when combined with RT, tumor growth was substantially decreased relative to radiation treatment alone in both tumor models (p&amp;lt; 0.001). Notably, more than half of mice achieved complete tumor regression in the breast cancer model, suggesting activation of tumor immunity. TGFβ blockade decreases a splenic reservoir of CD11b+ myeloid-derived suppressor cells that mediate immunosuppression in the tumor microenvironment as well as CD11b+Gr1+/intF4/80+ precursors of tumor-associated macrophages in both tumor models. Immunoactivating IL12 was upregulated upon TGFβ inhibition while immunosuppressive IL10 and pro-angiogenic CXCL1 decreased. The number of splenic CD11c+ MHC Class I+ antigen presenting cells was dramatically reduced from either irradiated TSA and LLC tumor-bearing mice, but unaffected in mice receiving 1D11 antibody concomitant with RT. Tumor cell MHC Class I overexpression, which is induced by in vitro irradiation, was impaired after co-culture with splenocytes from irradiated mice but not from mice receiving the dual treatment or 1D11 alone. Thus, TGFβ blockade, which sensitizes tumor cells to RT, also alleviates tumor suppressive consequences of radiation systemically and creates a pro-immunogenic signal. Together, these actions can achieve persistent tumor control. Citation Format: Ilenia Pellicciotta, Shisuo Du, Silvia Formenti, Mary Helen Barcellos-Hoff. Concomitant radiotherapy (RT) and TGFβ neutralizing antibodies alters tumor microenvironment and promotes tumor regression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-175. doi:10.1158/1538-7445.AM2014-LB-175

Abstract 633: Inhibition of TGFβ as a strategy to convert the irradiated tumor into in situ individualized vaccine

Abstract Accumulating data support the concept that ionizing radiation therapy (RT) has the potential to convert the tumor into an in situ, individualized vaccine; however this potential is rarely realized by RT alone. Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that is activated by RT and inhibits the antigen-presenting function of dendritic cells and the differentiation of effector CD8+ T cells. Here we tested the hypothesis that TGFβ hinders the ability of RT to promote anti-tumor immunity. Development of tumor-specific immunity was examined in two pre-clinical models of metastatic breast cancer and analyzed in patients with metastatic breast cancer treated with local radiotherapy and the TGFβ-neutralizing antibody Fresolimumab. Mice bearing established 4T1 and TSA mouse mammary carcinomas treated with pan-isoform specific TGFβ neutralizing antibody, 1D11, showed significantly improved control of the irradiated tumor and non-irradiated metastases, but no effect in the absence of RT. Notably, whole tumor transcriptional analysis demonstrated the selective upregulation of genes associated with immune-mediated rejection only in tumors of mice treated with RT+TGFβ blockade. Mice treated with RT+TGFβ blockade exhibited cross-priming of CD8+ T cells producing IFNγ in response to three tumor-specific antigens in tumor-draining lymph nodes, which was not evident for single modality treatment. Likewise, HLA-A2.1+ metastatic breast cancer patients (n=8) enrolled in NCT01401062 trial of local RT and fresolimumab were examined for CD8+ T cells specific for the tumor antigen survivin by tetramer staining. Three patients developed increased frequencies of survivin-specific CD8+ T cells in the blood during treatment, while two patients negative at baseline became positive. Analysis of the immune infiltrate in mouse tumors showed a significant increase in CD4+ and CD8+ T cells only in mice treated with the combination of RT+TGFβ blockade. Depletion of CD4+ or CD8+ T cells abrogated the therapeutic benefit of RT+TGFβ blockade. These data identify TGFβ as a master inhibitor of the ability of RT to generate an in situ tumor vaccine, which supports testing inhibition of TGFβ during radiotherapy to promote therapeutically effective anti-tumor immunity. Supported by DOD BCRP Multi-Team Award BC100481. Citation Format: Claire I. Vanpouille-Box, Julie M. Diamond, Jiri Zavadil, James Babb, Dörthe Schaue, Mary Helen Barcellos-Hoff, William H. McBride, Silvia C. Formenti, Sandra Demaria. Inhibition of TGFβ as a strategy to convert the irradiated tumor into in situ individualized vaccine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 633. doi:10.1158/1538-7445.AM2014-633

Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.