Inhibition Of TGF-beta Signaling Research Articles

Elevated secretion of pro-collagen I-alpha and vascular endothelial growth factor as biomarkers of acetabular labrum degeneration and calcification in hip osteoarthritis: An explant study

BackgroundHip osteoarthritis (OA) involves structural degeneration of different joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular labrum. However, the molecular mechanisms underlying labrum degeneration in hip OA remain poorly understood. AimTo assess secretion of putative biomarkers for OA from explanted human labrum tissues under basal and inflammatory conditions and to determine whether these could differentiate between OA and calcification status compared to fracture controls. MethodsIntact labrum specimens were collected from patients undergoing joint arthroplasty for primary hip OA (n ​= ​15, mean age 70) or non-OA femoral neck fracture (n ​= ​5, mean age 64). Tissues were dissected in equal-sized samples and explanted for one week. To mimic activation of inflammatory signaling by endogenous damage-associated molecular patterns (DAMP) tissue were stimulated with a toll-like receptor 4 (TLR4) agonist (1 ​μg/mL LPS). The involvement of transforming growth factor-beta (TGF-beta) signaling was evaluated by treatment with a TGF-beta type 1 receptor inhibitor (10 ​μM SB-505124). Secretion of aggrecan (ACAN), pro-collagen-I alpha (Pro-Col-Iα), cartilage oligomeric matrix protein (COMP), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay (ELISA). Labrum calcification was evaluated by 3D whole mount fluorescent microscopy of ethyl cinnamate-based optically cleared tissues stained with Alcian blue/Alizarin red. ResultsWhole mount microscopy revealed non-OA fracture controls were non-calcified, whereas six OA labra (40%) were partially calcified or ossified. Basal secretion of Pro-Col-Iα and VEGF was increased four-fold in OA versus non-OA labra. Pro-Col-Iα levels were correlated with those of VEGF (r ​= ​0.65) and COMP (r ​= ​0.54). Stimulation of DAMP signaling through TLR4 affected secretion of IL-6, VEGF, COMP and Pro-Col-Iα, with distinct responses between non-OA and OA tissues. Inhibition of TGF-beta signaling specifically reduced elevated secretion of Pro-Col- Iα and VEGF in calcified OA labrum. ConclusionsSecretion of the putative OA biomarkers Pro-Col-Iα and VEGF is elevated in degenerated human acetabular labrum and may serve as indicators of OA and calcification status. Secretion of both factors was partially regulated by TGF-beta signaling in calcified OA labrum tissues.The Translational potential of this article:Our findings suggest that a biomarker panel consisting of Pro-Col-Iα/VEGF/COMP may be valuable for assessing subradiographic labrum degeneration and calcification in hip OA. Targeting TGF-beta signaling may offer a means to reduce vascular invasion and fibrosis in acetabular labrum tissue.

Magic Bullets: The Coming Age of Meaningful Pharmacological Control of the Corneal Responses to Injury and Disease.

Corneal injuries from chemical burns, mechanical trauma, infections, immunological rejections, surgical complications, and some diseases are commonly associated with persistent epithelial defects (PED), neurotrophic epitheliopathy, scarring fibrosis, corneal neovascularization (CNV), and/or corneal endothelial damage that lead to vision loss. Several Food and Drug Administration (FDA) approved medications have recently become available, are currently in clinical trials, or are likely to enter clinical trials in the near future. For example, a 2-week course of topical human recombinant nerve growth factor is frequently an effective treatment for corneal neurotrophic epitheliopathy associated with PEDs. Topical losartan, an angiotensin converting enzyme II receptor antagonist that also inhibits TGF beta signaling, has been shown to effectively decrease myofibroblast generation and scarring fibrosis in alkali burn injury and Descemetorhexis rabbit models. Small molecule topical tyrosine kinase inhibitors, such as sunitinib and axitinib, FDA approved as chemotherapeutic agents to treat specific cancers, have also been found to be effective topical inhibitors of CNV in animal and human trials. Rho-kinase inhibitors, such as ripasudil and netarsudil, that are currently approved agents for the treatment of glaucoma in some countries, have been shown to stimulate corneal endothelial proliferation in animal studies and human trials, and may accelerate the regeneration of Descemet's membrane. These agents, as well as other drugs in development, will be used in targeted combinations to treat corneal pathophysiology associated with epithelial healing disorders, stromal scarring fibrosis, CNV, and corneal endothelial injury during the next decade.

Trefoil Factor 1 Suppresses Epithelial-mesenchymal Transition through Inhibition of TGF-beta Signaling in Gastric Cancer Cells.

Gastric cancer is a malignancy with high incidence and mortality worldwide. In gastric cancer, epithelial-mesenchymal transition (EMT) and metastasis further increase the mortality rate. Trefoil factor 1 (TFF1) has been reported as a protective factor in the gastric mucosa. In this study, TFF1 inhibited the migration and invasive capability of gastric cancer cells. Elevated TFF1 levels induced the expression of E-cadherin, the epithelial marker, and reduced the expression of N-cadherin, vimentin, Snail, Twist, Zinc finger E-box binding homeobox (ZEB) 1 and ZEB2, well-known repressors of E-cadherin expression. In addition, the expression of matrix metalloproteinase (MMP)-2, MMP-7 and MMP-9, which are major markers of cancer metastasis, was suppressed by TFF1. Upregulation of TFF1 inhibited TGF-β, a major signaling for EMT induction, and the phosphorylation of Smad2/3 activated by TGF-β in AGS cells. In conclusion, TFF1 inhibits EMT through suppression of TGF-β signaling in AGS cells, which might be used in therapeutic strategies for reducing metastatic potential and invasiveness of these cells.

A Multi-Trait Approach Identified Genetic Variants Including a Rare Mutation in RGS3 with Impact on Abnormalities of Cardiac Structure/Function

Heart failure is a major cause for premature death. Given the heterogeneity of the heart failure syndrome, identifying genetic determinants of cardiac function and structure may provide greater insights into heart failure. Despite progress in understanding the genetic basis of heart failure through genome wide association studies, the heritability of heart failure is not well understood. Gaining further insights into mechanisms that contribute to heart failure requires systematic approaches that go beyond single trait analysis. We integrated a Bayesian multi-trait approach and a Bayesian networks for the analysis of 10 correlated traits of cardiac structure and function measured across 3387 individuals with whole exome sequence data. While using single-trait based approaches did not find any significant genetic variant, applying the integrative Bayesian multi-trait approach, we identified 3 novel variants located in genes, RGS3, CHD3, and MRPL38 with significant impact on the cardiac traits such as left ventricular volume index, parasternal long axis interventricular septum thickness, and mean left ventricular wall thickness. Among these, the rare variant NC_000009.11:g.116346115C > A (rs144636307) in RGS3 showed pleiotropic effect on left ventricular mass index, left ventricular volume index and maximal left atrial anterior-posterior diameter while RGS3 can inhibit TGF-beta signaling associated with left ventricle dilation and systolic dysfunction.

Correction: Inhibiting TGF-beta signaling preserves the function of highly activated, in vitro expanded natural killer cells in AML and colon cancer models.

[This corrects the article DOI: 10.1371/journal.pone.0191358.].

Inhibiting TGF-beta signaling preserves the function of highly activated, in vitro expanded natural killer cells in AML and colon cancer models.

Natural killer cells harnessed from healthy individuals can be expanded ex vivo using various platforms to produce large doses for adoptive transfer into cancer patients. During such expansion, NK cells are increasingly activated and more efficient at killing cancer cells. Adoptive transfer however introduces these activated cells into a highly immunosuppressive tumor microenvironment mediated in part by excessive transforming growth factor beta (TGF-beta) from both cancer cells and their surrounding stroma. This microenvironment ultimately limits the clinical efficacy of NK cell therapy. In this study, we examined the use of a TGF-beta receptor kinase inhibitor, LY2157299, in preserving the cytotoxic function of ex vivo expanded, highly activated NK cells following sustained exposure to pathologic levels of TGF-beta in vitro and in a liver metastases model of colon cancer. Using myeloid leukemia and colon cancer cell lines, we show that the TGF-beta driven impairment of NK cell cytotoxicity is mitigated by LY2157299. We demonstrate this effect using quantitative cytotoxicity assays as well as by showing a preserved activated phenotype with high NKG2D/CD16 expression and enhanced cytokine production. In a mouse liver metastases model of colon cancer, we observed significantly improved eradication of liver metastases in mice treated with adoptive NK cells combined with LY2157299 compared with mice receiving NK cells or TGF beta inhibition alone. We propose that the therapeutic efficacy of adoptive NK cell therapy clinically will be markedly enhanced by complementary approaches targeting TGF-beta signaling in vivo.

SMAD dependent signaling plays a detrimental role in a fly model of SMARCB1-deficiency and the biology of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant brain tumors arising in young children. The majority of ATRT is characterized by inactivation of the chromatin remodeling complex member SMARCB1 (INI1/hSNF5). Little is known, however, on downstream pathways involved in the detrimental effects of SMARCB1 deficiency which might also represent targets for treatment. Using Drosophila melanogaster and the Gal4-UAS system, modifier screens were performed in order to identify the role of SMAD dependent signaling in the lethal phenotype associated with knockdown of snr1, the fly homolog of SMARCB1. Expression and functional role of human homologs was next investigated in ATRT tumor samples and SMARCB1-deficient rhabdoid tumor cells. The lethal phenotype associated with snr1 knockdown in Drosophila melanogaster could be shifted to later stages of development upon additional knockdown of several decapentaplegic pathway members including Smox, and Med. Similarly, the transforming growth factor beta (TGFbeta) receptor type I kinase inhibitor SB431542 ameliorated the detrimental effect of snr1 knockdown in the fruit fly. Examination of homologs of candidate decapentaplegic pathway members in human SMARCB1-deficent ATRT samples revealed SMAD3 and SMAD6 to be over-expressed. In SMARCB1-deficent rhabdoid tumor cells, siRNA-mediated silencing of SMAD3 or SMAD6 expression reduced TGFbeta signaling activity and resulted in decreased proliferation. Similar results were obtained upon pharmacological inhibition of TGFbeta signaling using SB431542. Our data suggest that SMAD dependent signaling is involved in the detrimental effects of SMARCB1-deficiency and provide a rationale for the investigation of TGFbeta targeted treatments in ATRT.

Inhibiting TGF-Beta Signaling in the Tumor Micro-Environment Enhances the Cytotoxic Function of Adoptive NK Cells

Introduction: The tumor micro-environment poses a limitation to the efficacy of adoptive NK cell therapy due to several immunosuppressive cytokines. TGF-beta, produced in excess by tumor cells, regulatory T cells and stromal cells, facilitates epithelial to mesenchymal transformation thereby promoting metastasis and fibrosis. This high TGF-beta milieu in cancer patients also impairs innate natural killer (NK) cell mediated cancer immunity through various mechanisms.Adoptive transfer of healthy donor NK cells will have limited clinical efficacy when highly activated NK cells are introduced into the immunosuppressive TGF-beta rich tumor microenvironment of cancer patients. We are testing several small molecule inhibitors of the TGF-beta receptor in combination with healthy donor NK cells with a goal of enhancing adoptive NK cell therapy in various disease models.Methods: Human NK cells isolated from healthy donor peripheral blood were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540). Cell culture media was supplemented with IL-2 (50mU/mL).For in vitro assays, at the beginning of expansion week 3 the culture conditions for NK cells was continued unchanged, supplemented with TGF-beta 1 ligand (TGF-B1) at 5ng/mL or 10ng/mL either alone or in combination with EW7197 (a TGF-beta Type 1 receptor inhibitor) or with EW7197 alone. These NK cells were tested with Calcein-AM release cytotoxicity assays at various time points from addition of TGF-B1 (0h, 36h, 72h and 96h). Briefly, NK cells were co-cultured with OCI-AML cells labeled with Calcein-AM at a ratio of 5NK:1 OCI-AML. At the end of 4 hours co-incubation, cytotoxicity was measured by relative fluorescence of calcein release into the culture supernatant compared with Triton-X induced complete target cell lysis.In a murine liver metastases model using the colon cancer cell line HCT116, 105 HCT116 cells were surgically implanted into NSG mouse spleens (following hemi-splenectomy). Mice in 2 groups (3/group) subsequently received 5 x 106 NK cells each (tail vein), weekly for two weeks, starting 10 days post-op. Another control group received vehicle infusions alone while a fourth control group received the TGF-beta inhibitor LY2157299 by oral gavage twice daily at 75mg/kg for two weeks. One group of mice receiving NK cells also received twice daily LY2157299 for two weeks starting with the first NK cell infusion. Mice receiving NK cells also received IL2 (75,000U IP) three times a week for two weeks.Results: At TGF-beta levels similar to that found in AML patients (5ng/ml), NK cell killing of OCI cells was markedly impaired progressively from 36h to 96h exposure. This reduced cytotoxic activity correlated with a 3-4 fold decrease in expression of NKG2D most marked at 96h(Figure 1). CD16 expression was also significantly reduced by 2-3 fold on TGF-beta exposed NK cells after 96h. The TGF-beta inhibitor EW7197 maintained NK cell killing as well as NKG2D and CD16 expression in the presence of TGF-B1 (Figure 1).At 32 days post-splenic implantation, vehicle mice (those that received HCT116 either alone or in combination with LY2157299 alone) appeared moribund and had grossly distended abdomens with ascites. All mice were autopsied at this time point. All control mice had grossly enlarged livers with significant metastases and absence of viable liver tissue (Fig 2). In contrast, mice treated with two weekly infusions of NK cells and LY2157299 for 4 weeks had no ascites and predominantly healthy livers. All mice that received NK cells alone had some evidence of liver metastases with various degrees of disease burden. Composite H&E stains of liver sections from 3 mice/group showed <10% liver metastases microscopically in mice treated with a combination of NK cells and LY2157299. This was significantly reduced compared to 80-90% in the untreated or LY2157299 alone mice and 60-70% in the NK cell alone group (Fig 3).Discussion: Our preliminary results indicate that TGF-beta inhibition in combination with adoptive NK cell therapy can mitigate the immunosuppressive tumor microenvironment conferred by TGF-beta signaling. We are in the process of validating this approach in other preclinical models of both hematologic malignancies and solid tumors. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

Abstract 1878: Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape

Abstract Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumor accounting for 60-70% of all gliomas. Current achieved median patient survival ranges only 12-15 months, and improvements in survival over the last century can be measured only in weeks. A hallmark of this malignancy is the intrinsic resistance to current therapies. The Transforming Growth Factor beta (TGF-beta) signaling pathway plays a key role in GBM. Several inhibitors of different elements and regulators of the TGF-beta pathway have entered to clinical trials. Here, we analyzed the potential effect of P144, a TGF-beta pathway inhibitor peptide, over cell proliferation and apoptosis in commercial GBM cell lines (A172 and U-87 MG). We found that treatment with P144 significantly decreased cell proliferation of both cell lines analyzed. In addition, different apoptosis determinations such as ELISA and Acridine Orange/Ethidium Bromide (AO/EB) staining, confirmed a significant increase in apoptosis in both cell lines. Quantification of SMAD2 phosphorylation status and its nuclear translocation, by western blot, confirmed the inhibition of TGF-beta signaling by P144. These data support the correlation between TGF-beta pathway inhibition and the apoptotic process induction. Anoikis escape sustains invasiveness and metastatic potential in GBM, and has been established as a clear target against tumor progression. Due to P144 clear effect on apoptosis, we analyzed its influence on anoikis escape. The inhibition of anoikis escape by P144 was confirmed in A172 and U-87 MG cell lines under in vitro anchorage-independent culture conditions. The significant upregulation of BAX, Lamin A and Lamin C produced by P144 confirmed the induction of this apoptotic subtype process in the analyzed cell lines. We can conclude that P144 increases apoptosis and induces anoikis through inhibition of the TGF-beta signaling pathway. Our results highlight the therapeutic potential of P144 for the treatment against GBM. However, previous to further clinical development, additional studies are required in order to confirm the effect of P144 over GBM in the brain environment, as well as to explore the therapeutic potential of P144 in combination with current and emerging molecular based therapies. Citation Format: Gabriel Gallo-Oller, Javier Dotor, Xing Fan, Javier S. Castresana. Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1878.

Abstract POSTER-BIOL-1310: Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer

Abstract Tumor dormancy is an important research topic because of its potential implications in metastasis and acquired resistance to chemotherapy. Dormant tumor cells at the secondary site may persist after chemotherapy and regrow as metastatic recurrence. Seminal studies by Dr. Bissell and Dr. Hendrix have shown that the tissue microenvironment can reprogram tumor cells into dormancy. Extracellular matrix (ECM) proteins are key components of tumor microenvironment and may regulate dormancy, and disruption of ECM homeostasis may cause recurrence and metastatic growth. A major cytokine that regulates ECM remodeling is tumor growth factor-beta (TGF-beta), and it is implicated in modulating tumor cell dormancy. In the tissue microenvironment, serine protease HtrA1 was shown to inhibit TGF-beta signaling by targeting TGF-beta family ligands and receptors for proteolysis. Our lab showed that HtrA1 is down-regulated in ovarian cancer cells, and HtrA1 loss promotes resistance to anoikis and in vivo metastasis. Loss of HtrA1 is also associated with increased incidence of carcinomatosis. Finally, previous studies have shown that HtrA1 degrades ECM proteins. Therefore, we hypothesize that loss of HtrA1 in the ovarian tumor microenvironment enhances TGF-beta signaling and promotes ECM protein production by stromal cells, thereby contributing to ECM remodeling and tumor dormancy. Ovarian cancer cells with endogenous HtrA1 were transduced with HtrA1-targeting shRNA (sh1) to evaluate the effect of HtrA1 downregulation on invasion, tumor dormancy, and ECM remodeling. Cells transduced with non-targeting shRNA (NT) served as a control. Cancer cells (NT and sh1) were plated over confluent mesothelial cell layer with or without 10 nM TGF-beta for 24 hours and 48 hours. No significant difference in cell invasion was observed between those cells cultured with or without TGF-beta. In order to test which matrix proteins are critical for cell proliferation versus cell cycle arrest, MicroMatrix 36 arrays were used to culture NT and sh1 cells. Cells were cultured for 48 hours on matrix slides, then fixed and stained with anti Ki67. It was determined matrix containing combinations of fibronectin showed cells that stained positive for Ki67, when compared to those not containing fibronectin or collagen together. Changes in intracellular signaling in the presence and absence of matrix were also investigated using kinase and MAPK arrays. Mesothelial cells were cultured until confluent, and cells were removed from culture plates using 2.0 mM EDTA in PBS. Matrix produced from mesothelial cells remained on the plates. NT and sh1 cells were then plated in the presence and absence of matrix and were allowed to culture until cell attachment was observed (3 hours at 37oC, 5% CO2). Cells were lysed and lysates were cultured on arrays according to manufactures instructions. ERK and Akt pathways were observed to be activated in cells plated with matrix compared to those without matrix. Taken together the evidence thus far suggests that matrix composition is critical for cell proliferation. Currently, Stable Isotope Amino Acid labeling is being employed to investigate changes in matrix remodeling on a global scale. In addition, Imaging Mass Spectrometry will also be used to evaluate formalin fixed paraffin embedded tissue samples from patients to identify at a molecular level changes in extracellular matrix composition between tumor and normal tissues. Citation Format: Cooley, Megan K., Chien, Jeremy. Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1310.

Akt2/ZEB2 may be a biomarker for exfoliant cells in ascitic fluid in advanced grades of serous ovarian carcinoma

Ovarian cancers present a mild clinical course when diagnosed early but an aggressive pathway when diagnosed in the peri- and postmenopausal periods. However, the predictability of tumor progression is stochastic and is difficult to predict. In the present study, we hypothesized to examine the key pathways that are dysregulated to promote epithelial-mesenchymal transition in serous ovarian carcinoma. Examination of these steps would help to identify ascitic fluid with cells poised for metastasis or otherwise. We focused on examining the Akt2 expression, mainly because of its report as being overamplified in the aggressive variants of ovarian cancer, as well as TGFbeta-sensitivity of Akt2 that forms the key basis for metastasis initiation of most kinds of carcinoma. We obtained primary ovarian carcinoma samples as well as ascitic fluid and distantly metastatic ovarian carcinoma to examine the expression of Akt2. The results of the study demonstrated that in malignant exfoliated ovarian cancer cells, Smad4 expression was tremendously increased in the nuclei, suggesting nuclear translocation of Smad, which thereafter may have activated ZEB2, and thereafter genomically affected the expression of E-cadherin, myosin II, and vimentin, key components for initiating and sustaining metastasis. All of these may have been stimulated by increased cellular expression of Akt2 in metastatic variants of the serous ovarian carcinoma. The reliance on Akt2 and TGF beta signaling may also potentiate the case for Akt inhibitors or small molecule inhibitors of TGFbeta signaling like doxycycline as adjunct chemotherapy in serous ovarian carcinoma, especially the metastatic variants.

Impairment of AML Engraftment to the Bone Marrow Niche Following Inhibition of TGF-Beta Signaling

The cell fate of the HSC to either self-renew or differentiate is controlled by a complex interplay between cell-intrinsic and -extrinsic regulatory signals generated by the surrounding bone marrow microenvironment called the HSC niche. A balance exists within the “cross talk” between HSCs and the niche, which allows HSC dormancy, activation and differentiation. Any alterations of this balance may lead to uncontrolled cellular proliferation and ultimately the promotion of leukemia. However, it remains to be determined exactly how the hematopoietic microenvironment contributes to the deregulation of normal hematopoiesis and/or promotes the maintenance of leukemia cells as a “leukemic niche”.To investigate this, we have now performed micro-array analysis of MS5 stromal cells that were co-cultured with a panel of leukemic cell lines and acute myeloid leukemia (AML) patient samples. The most significantly up-regulated pathways as compared to MS5 cells cultured alone involved cytoskeleton remodeling, cell cycle, cell adhesion and development through cytokine signaling. Since transcript and protein levels of number of effectors of the TGF-beta (TGF-β) signaling pathway were up-regulated in the stroma co-cultured with leukemic cells, we next investigated inhibition of this pathway using a specific inhibitor of TGF-β receptor kinase, SB-431542 (10µM). Treatment with the inhibitor significantly reduced the cell number and increased the levels of apoptosis in the AML cells co-cultured on stromal cells, whilst having mininal effect on normal cells. Treatment with SB-431542 (10mg/kg), also significantly reduced the level of AML cell engraftment on treatment in vivo (n=3) (untreated- 68.65±6.95; 56.15±22.85; 84.35±5.75 and SB-treated- 45.5±11.6; 30.5±19.6; 54.1±4.9). In order to inhibit TGF-β signaling more specifically within the stromal compartment, we next used shRNA against TGF-β Receptor II (TGFBR2) in MS5 stromal cells and co-cultured them with AML cells within 3D scaffold models (n=4), which were implanted in vivo. A significant reduction in engraftment was observed as compared to controls (shRNA control- 64.65±32.65; 87.7±7.2; 23.55±4.35; 49.65±33.65 and TGFBR2 knockdown- 20.2±3; 62.95±4.05; 15.7±1.5; 20.385±17.415). The co-culturing of normal cord blood CD34+ or mononuclear cells on the TGFBR2 knockdown stroma had no significant effect both in vitro and in vivo (n=3). To investigate whether TGF-β inhibition had an effect on the interaction of AML cells to the niche, we used intravital microscopy to track the cells live in vivo. HL60 (AML cell line) cells were labeled with 2µM CFSE and pretreated ±SB-431542 (10µM) on stroma, before being sorted and transplanted into immunodeficient mice. Distance to the calvaria was measured at 16 hours and we observed that the SB-431542-treated cells were positioned significantly further away from the bone surface as compared to untreated control (p=0.0001). Since the TGF-β inhibited cells appeared to have impaired ability to adhere to the bone marrow, we next investigated the relationship between extracellular matrix molecules and TGF-β signaling. We saw that stromal cells that were co-cultured with AML cells had a significantly increased expression of laminins A1, A5, B1 and G1. This effect could be recapitulated by treatment of naïve stromal cells with TGF-β2 and 3. We also observed a reciprocal decrease in expression of laminins following both treatment of AML-stromal co-cultures with SB-431542 and within TGFBR2 knockdown stroma. Furthermore, we saw an increase in the laminin receptor, integrin alpha-6 (CD49f), in AML cells treated with TGF-β 1, 2 and 3 and a reciprocal decrease following treatment with SB-431542, thereby, indicating that the abrogation of this signaling axis may be, at least, partially responsible for the impaired engraftment of AML cells to their niche following inhibition of the TGF-β pathway.These data thus highlight the potential for the development of therapies directed at modifying the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1932: Parallel functions of the endoplasmic reticulum chaperone protein GRP78 in tumorigenesis and the induction of pluripotency

Abstract Increasing evidence supports the concept that instances of cancer recurrence may be due to a subpopulation of cells within a tumor that behave as stem cells. Previous studies have demonstrated that pathways critical in oncogenesis parallel those necessary for the induction of pluripotency, suggesting that similar mechanisms regulate both processes. By therefore understanding the mechanisms that govern reprogramming, we may gain insight into the methods by which cancer cells acquire and exploit stem cell properties, and enable more strategic targeting of these cell populations to prevent malignant relapse. Members of the TGF-beta superfamily (e.g. activin/Nodal), which regulate many important normal cellular responses including cell growth and differentiation, have also been shown to promote tumorigenesis. Cripto, a regulator of TGF-beta superfamily ligand signaling expressed on human embryonic stem cells, is overexpressed in many types of cancer, and has also been shown to promote tumor growth and metastasis. Previous work has demonstrated that Cripto regulates TGF-beta function in tumor cell lines by forming a complex with GRP78, a protein generally restricted to the endoplasmic reticulum in normal tissues, but expressed at the cell surface in many types of tumors. Targeting GRP78 in mouse models suppresses tumor growth, and cell surface GRP78 has been shown to be a molecular target on human tumor samples. Although these studies have suggested an important role for GRP78 in promoting tumorigenesis, the mechanisms are not yet fully understood. We have discovered that GRP78 expression is induced during reprogramming, and becomes localized to the cell surface in pluripotent cells, where it co-localizes with Cripto. Overexpression of GRP78 in somatic cells induced their reprogramming efficiency. We further found that a GRP78 antibody, that disrupts cell surface GRP78/Cripto binding and Cripto-mediated TGF-beta superfamily signaling, inhibited reprogramming. Treatment of pluripotent stem cell populations with this GRP78 antibody also decreased proliferation, but did not impact pluripotency. These combined findings suggest that GRP78 may be localized to the cell surface during reprogramming where it functions to inhibit TGF-beta signaling and promote stem cell proliferation and/or survival. Interestingly, overexpression of GRP78 in human breast cancer cell lines caused an increased resistance to cisplatin. Furthermore, inhibiting cell surface function of GRP78 in these breast cancer lines resulted in a higher susceptibility to cisplatin treatment, demonstrating a specific function for cell surface GRP78 in cisplatin resistance. These combined stem cell functions of GRP78 therefore parallel many of the functions of GRP78 for cancer cells, and thus provide insight into understanding how cancer cells acquire and exploit stem cell properties. Citation Format: Athanasia D. Panopoulos, Yuriko Hishida, Min-Zu Wu, Sergio Ruiz, Erika Batchelder, Tomoaki Hishida, Jonathan A. Kelber, Peter C. Gray, Juan Carlos Izpisua Belmonte. Parallel functions of the endoplasmic reticulum chaperone protein GRP78 in tumorigenesis and the induction of pluripotency. [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 1932. doi:10.1158/1538-7445.AM2014-1932

Acquired Choanal and Subglottic Stenosis Associated with MYHRE‐LAPS Syndrome: A Case Report and Literature Review

Objectives: MYHRE-LAPS syndrome (MLS) is a recently recognized disease caused by a mutation in the SMAD4 gene that alters the transforming growth factor (TGF)–beta pathway. This results in a wide range of pathology including laryngotracheal stenosis, arthropathy, prognathism and short stature, or LAPS syndrome. We present a unique case of MLS associated with both acquired choanal stenosis (CS) as well as subglottic stenosis (SGS) and perform a systematic review of all MLS associated airway pathology. Methods: Single case reviewed via the medical record. Literature review. Results: MLS was reported in 32 patients, and 5 had clinical airway symptoms. We added the case of a 43F who presented with 9 months of progressive nasal obstruction and was found to have bilateral CS and SGS on endoscopic examination. The patient had no recent airway instrumentation but had two prior operations requiring intubation. Genetic testing revealed SMAD4 mutation consistent with MLS and Losartan was initiated to inhibit TGF-beta signaling. Most patients with MLS develop airway symptoms as a late sequela of disease with a median onset of 22 years old. Attempts to surgically address airway stenosis invariably results in recurrence secondary to robust scar formation. All patients who have undergone surgical management of airway stenosis were tracheostomy dependent (4/4). Conclusions: MLS is a newly recognized disorder characterized by progressive systemic fibrosis. Airway management is complicated by prognathism, trismus, and limited neck extension as well as invariable restenosis of the airway after instrumentation. Caution should be exercised when surgical intervention is entertained in these patients.

Su1987 Inhibition of TGFbeta Signaling Promotes Collective Invasion of Esophageal Keratinocytes Through an Adamts1-Dependent Release of EGF

Su1987 Inhibition of TGFbeta Signaling Promotes Collective Invasion of Esophageal Keratinocytes Through an Adamts1-Dependent Release of EGF

Regulation of Renal Fibrosis by Smad3 Thr388 Phosphorylation

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.

Do all roads lead to Oct4? The emerging concepts of induced pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Breast cancer cells acquire stem cell-like properties upon contact with functionally impaired T cells

Breast cancer is the most common malignancy in women and prone to relapse. This has been ascribed to a small subpopulation of so-called tumor initiating or cancer stem cells (CSC) characterized by increased clonogenicity, sphere formation and tumor initiation capacity. We investigated interactions between T cells and breast cancer stem cells which can be recognized by the marker combination CD44highCD24low. Upon cell-cell-contact with polyclonally expanded and activated CD4+ and CD8+ T cells from healthy donors, the CSC population in MCF7 breast cancer cells is not only spared from killing and thus enriched by selection, it is further induced. This CSC-inducing effect was considerably enhanced when the degranulation of T cells was inhibited by pretreatment with Concanamycin A. Interestingly, serum from patients with metastasized breast cancer also promoted a non-lytic phenotype in T cells from healthy donors. After co-culture, MCF-7 cells displayed increased clonogenicity and spheroid formation in vitro. Gene expression analysis showed that this de-differentiation occurred via epithelial-to-mesenchymal transition and was sensitive to inhibition of TGF-beta signaling or the TNF-alpha-, hedgehog- and NF-κB pathways. Our results suggest that induction of a stem-cell-like phenotype is an alternative and undesirable outcome of the interaction between immune cells and tumor cells. However, modulation of this effect seems possible since several of the involved pathways represent potentially druggable targets for therapy. Further studies will now have to evaluate the distinct molecular interactions mediating this cell-contact-dependent CSC induction.