TGFβ-responsive Genes Research Articles

Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9.

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.

Oxidative stress is responsible for maternal diabetes-impaired transforming growth factor beta signaling in the developing mouse heart

Oxidative stress plays a causal role in diabetic embryopathy. Maternal diabetes induces heart defects and impaired transforming growth factor beta (TGFβ) signaling, which is essential for cardiogenesis. We hypothesize that mitigating oxidative stress through superoxide dismutase 1 (SOD1) overexpression in transgenic (Tg) mice reverses maternal hyperglycemia-impaired TGFβ signaling and its downstream effectors. Day 12.5 embryonic hearts from wild-type (WT) and SOD1 overexpressing embryos of nondiabetic (ND) and diabetic mellitus (DM) dams were used for the detection of oxidative stress markers: 4-hydroxynonenal (4-HNE) and malondlaldehyde (MDA), and TGFβ1, 2, and 3, phosphor (p)-TGFβ receptor II (TβRII), p-phosphorylated mothers against decapentaplegic (Smad)2, and p-Smad3. The expression of 3 TGFβ-responsive genes was also assessed. Day 11.5 embryonic hearts were explanted and cultured exvivo, with or without treatments of a SOD1 mimetic (Tempol; Enzo Life Science, Farmingdale, NY) or a TGFβ recombinant protein for the detection of TGFβ signaling intermediates. Levels of 4-HNE and MDA were significantly increased by maternal diabetes, and SOD1 overexpression blocked the increase of these 2 oxidative stress markers. Maternal diabetes suppresses the TGFβ signaling pathway by down-regulating TGFβ1 and TGFβ3 expression. Consequently, phosphorylation of TβRII, Smad2, and Smad3, downstream effectors of TGFβ, and expression of 3 TGFβ-responsive genes were reduced by maternal diabetes, and these reductions were prevented by SOD1 overexpression. Treatment with Tempol or TGFβ recombinant protein restored high-glucose-suppressed TGFβ signaling intermediates and responsive gene expression. Oxidative stress mediates the inhibitory effect of hyperglycemia in the developing heart. Antioxidants, TGFβ recombinant proteins, or TGFβ agonists may have potential therapeutic values in the prevention of heart defects in diabetic pregnancies.

Transforming Growth Factor-Beta (TGF-β) Signaling in Paravertebral Muscles in Juvenile and Adolescent Idiopathic Scoliosis

Most researchers agree that idiopathic scoliosis (IS) is a multifactorial disease influenced by complex genetic and environmental factors. The onset of the spinal deformity that determines the natural course of the disease, usually occurs in the juvenile or adolescent period. Transforming growth factors β (TGF-βs) and their receptors, TGFBRs, may be considered as candidate genes related to IS susceptibility and natural history. This study explores the transcriptional profile of TGF-βs, TGFBRs, and TGF-β responsive genes in the paravertebral muscles of patients with juvenile and adolescent idiopathic scoliosis (JIS and AIS, resp.). Muscle specimens were harvested intraoperatively and grouped according to the side of the curve and the age of scoliosis onset. The results of microarray and qRT-PCR analysis confirmed significantly higher transcript abundances of TGF-β2, TGF-β3, and TGFBR2 in samples from the curve concavity of AIS patients, suggesting a difference in TGF-β signaling in the pathogenesis of juvenile and adolescent curves. Analysis of TGF-β responsive genes in the transcriptomes of patients with AIS suggested overrepresentation of the genes localized in the extracellular region of curve concavity: LTBP3, LTBP4, ITGB4, and ITGB5. This finding suggests the extracellular region of paravertebral muscles as an interesting target for future molecular research into AIS pathogenesis.

Abstract PD5-1: The type II TGFb receptor neutralizing antibody, TR1, abrogates the tumor-initiating cell (TIC) population in triple negative breast cancer (TNBC) via inhibition of SMAD and non-SMAD pathways

Abstract Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome. We have shown that the TGFb pathway plays a crucial role in the expansion of TICs and that inhibition of the type I TGFβ receptor (ALK5) decreases chemotherapy-resistant TICs. Further, a TGFβ-responsive gene signature is enriched in TNBCs upon treatment with chemotherapy and is predictive of poor prognosis (Bhola et al. JCI 2013). Herein we investigated the molecular and antitumor effects of TR1, a TGFβRII neutralizing antibody currently in phase I clinical trials, against TNBC. TR1 recognizes the TGFβ ligand binding epitope in the extracellular domain of TGFβRII; it blocks ligand binding in the nM range. Small molecule TGFβRI kinase inhibitors are another approach to inhibit TGFβ/SMAD signaling. However, they cross-react with other type I receptors (ALK1, 4, 7), some of which are important for normal physiology. Furthermore, TGFβRII can induce EMT in breast cancer cells independent of dimerization with TGFβRI. Therefore, we hypothesized that, by neutralizing TGFβRII, TR1 can abrogate the breast TIC population via inhibition of SMAD and non-SMAD signaling. Using the pCAGA12-luciferase reporter to assess SMAD-dependent transcriptional activity, TR1 decreased luciferase activity in TNBC cell lines (SUM159 and BT549) and steady-state levels of P-SMAD2, suggesting it inhibited autocrine TGFβ signaling. TR1 decreased the TIC fraction as identified by FACS analysis of cell line-specific TIC markers (ALDH for SUM159; CD44hi/PROCR+ for BT549; PROCR+/ESA+ for MDA231 cells). Although TR1 displayed no effects on TNBC growth in vitro, it decreased tumor growth in vivo. We used a phospho-kinase array (45 kinases) to elucidate the non-SMAD pathways altered by TR1. TR1 decreased phosphorylated EGFR (Tyr1068) and STAT3 (Tyr705) in TNBC cell lines. By flow cytometry, the cell-surface expression of EGFR was also lowered upon treatment with TR1. TR1 also reduced P-ERK1/2 and P-Akt (Ser473) levels, and lowered NFκB transcriptional activity as measured by luciferase reporter assay. Using a cytokine array (120 cytokines) and qRT-PCR we observed that TR1 decreased the expression and release of cytokines involved in TIC enrichment such as IL-6 and IL-8. Inhibition of EGFR (with gefitinib) and MEK1/2 (with selumetinib) decreased the TIC population while augmenting pCAGA luciferase activity. The combination of SMAD2 siRNA with each EGFR or MEK inhibitors significantly decreased the TIC population. We speculate based on these data that inhibition of both SMAD and non-SMAD signaling is necessary for complete abrogation of TICs. This may explain the efficacy of TR1 as a single agent. These findings suggest that targeting TGFβRII with TR1 exerts its anti-tumor effects via inhibition of both SMAD and non-SMAD (EGFR, ERK) pathways that are important to TIC survival. The results also encourage combination therapies of chemotherapy with TR1 to reduce tumor recurrences in patients with TNBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD5-1.

Hematopoietic Stem and Progenitor Cell Fate Decisions Are Regulated By a Cell-Autonomous p190-B/TGF-β Signaling Pathway

Hematopoietic stem cells (HSC) are undifferentiated cells that self-renew and produce all mature blood and immune cells in order to sustain life-long hematopoiesis. The molecular mechanisms that determine HSC fate decisions still remain poorly understood. Identifying these factors is of great biological and clinical importance, as alteration in HSC fate decisions will eventually lead to HSC depletion or malignant HSC expansion. We previously showed that p190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, is a critical regulator of HSC self-renewal. P190-B loss enhanced long-term engraftment during serial transplantation; but, surprisingly, without altering their survival, blood lineage differentiation and the balance between quiescence and proliferation (Xu et al, Blood). Therefore, we hypothesize that p190-B regulates HSC self-renewal by controlling a HSC fate decision to self-renew or to differentiate, so-called asymmetric versus symmetric self-renewal divisions. To test this, we compared patterns of single WT and p190-B-/- HSC (i.e. Lin-c-kit+Sca-1+CD150+CD48- [LSK-SLAM]) divisions over serial transplantation. First, we confirmed that the division rate of single LSK-SLAM was similar between the genotypes. Then, we performed the pair daughter cell assay to examine the ability of LSK-SLAM to generate daughter cells that retain multipotent lineage differentiation potential. We found that non-transplanted (CTL) HSC gave rise to 93% symmetric self-renewal divisions (i.e. both daughter cells are multipotent, neutrophil, erythroid, macrophage and megakaryocyte (nemM)), and 7% were asymmetric (only one daughter cell is multipotent). Interestingly, after 2 rounds of transplantation (2T), WT HSCs produced only 50% symmetric self-renewal divisions whereas p190-B-deficient HSCs maintained 92% symmetric self-renewal divisions. Hence, p190-B modulates asymmetric/symmetric self-renewal divisions to control HSC regeneration over serial transplantation. Using transcriptional profiling and chemical screening, we identified, quite unexpectedly, the transforming growth factor beta (TGFβ) pathway as a regulatory pathway mediating p190-B functions. Expression of TGFβ responsive genes (mRNA expression of tgif2, smurf2) were higher in 2T-WT than in 2T-p190-B–/–-HSCs and CTL HSC. To assess if increased TGFβ signaling affected HSC self-renewing decisions, we used the TGF-β inhibitor, SB431542, ex vivo. TGFβ inhibitor treatment of 2T-WT LSK-SLAM in pair daughter cell assay converted their fate decision to produce 90% symmetric self-renewal divisions; TGFβ inhibitor treatment ex vivo for 48h of WT LT-HSCs isolated from primary transplanted mice enhanced engraftment into secondary mice. Importantly, TGFβ inhibitor-treatment of mice over serial transplantation reversed transplant-related WT HSC decline as it enhanced HSC pool regeneration in BM and long-term HSC engraftment. Interestingly, it did not alter HSC cell cycle parameters or the relative heterogeneity of the HSC pool or blood lineage maturation. Mechanistically, p190-B appeared to regulate TGF-β signaling in a cell-autonomous manner by controlling bioactive TGF-β protein within HSCs. Finally we observed that TGFβ mediated its effect via non-canonical p38MAPK signaling. This study identifies a previously unknown p190-B/TGFβ signaling network that governs HSC fate decisions to self-renew or to differentiate, without altering HSC quiescence or development of the progeny. This also reveals an unexpected role for TGFβ as a regulator of HSC fate beyond its well-defined role in HSC hibernation. The clinical implications are important for the field of HSC expansion and bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

Genome-wide analysis reveals that Smad3 and JMJD3 HDM co-activate the neural developmental program

Neural development requires crosstalk between signaling pathways and chromatin. In this study, we demonstrate that neurogenesis is promoted by an interplay between the TGFβ pathway and the H3K27me3 histone demethylase (HDM) JMJD3. Genome-wide analysis showed that JMJD3 is targeted to gene promoters by Smad3 in neural stem cells (NSCs) and is essential to activate TGFβ-responsive genes. In vivo experiments in chick spinal cord revealed that the generation of neurons promoted by Smad3 is dependent on JMJD3 HDM activity. Overall, these findings indicate that JMJD3 function is required for the TGFβ developmental program to proceed.

Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs.

Transforming growth factor β1 (TGFβ1) and progesterone regulate matrix metalloproteinases (MMP) in human endometrial stromal cells.

Menstruation is preceded by progesterone withdrawal and endometrial matrix remodeling predominantly through induction of matrix metalloproteinases (MMP) and recruitment of invading neutrophils. Using endometrial tissues from women during various phases of the menstrual cycle, we found that MMP2, MMP9, and MMP11 were up-regulated in the late secretory phase/premenstrual phase. Because TGFβ-responsive genes were also up-regulated in endometrium during this time, we tested the hypothesis that TGFβ1 and progesterone regulate expression of MMP in human endometrial stromal cells (HESC). Treatment of HESC with TGFβ1 resulted in marked increases in MMP2 and MMP11 mRNA and pro- and active MMP2 activity. Progesterone inhibited TGFβ1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGFβ1 also decreased progesterone receptor (PR)-A and PR-B in HESC with a more pronounced effect on PR-A. These data support the hypothesis that TGFβ1 has endogenous anti-progestational effects in HESC and that the opposing effects of progesterone and TGFβ1 are important in regulation of matrix integrity in human endometrium.

Comparative analysis with collagen type II distinguishes cartilage oligomeric matrix protein as a primary TGFβ-responsive gene

This study aims to investigate the regulation of expression of Cartilage oligomeric matrix protein (COMP), which is predominately expressed by chondrocytes and functions to organize the extracellular matrix. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia and multiple epiphyseal dysplasia. The mechanism controlling COMP expression during chondrocyte differentiation is still poorly understood. Primary human bone marrow-derived stem cells were induced to differentiate into chondrocyte by pellet cultures. We then compared the temporal expression of COMP with the well-characterized cartilage-specific Type II collagen (Col2a1), and their response to transforming growth factor (TGF)β and Sox trio (Sox5, 6, and 9) stimulation. COMP and Col2a1 expression are differentially regulated by three distinct mechanisms. First, upregulation of COMP mRNA precedes Col2a1 by several days during chondrogenesis. Second, COMP expression is independent of high cell density but requires TGF-β1. Induction of COMP mRNA by TGF-β1 is detected within 2h in the absence of protein synthesis and is blocked by specific inhibitors of the TGFβ signaling pathway; and therefore, COMP is a primary TFGβ-response gene. Lastly, while Col2a1 expression is intimately controlled by the Sox trio, overexpression of Sox trio fails to activate the COMP promoter. COMP and Col2a1 expression are regulated differently during chondrogenesis. COMP is a primary response gene of TGFβ and its fast induction during chondrogenesis suggests that COMP is suitable for rapidly accessing the chondrogenic potential of stem cells.

Spatial control of cell fate using synthetic surfaces to potentiate TGF-β signaling

In organisms, cell-fate decisions result from external cues presented by the extracellular microenvironment or the niche. In principle, synthetic niches can be engineered to give rise to patterned cell signaling, an advance that would transform the fields of tissue engineering and regenerative medicine. Biomaterials that display adhesive motifs are critical steps in this direction, but promoting localized signaling remains a major obstacle. We sought to exert precise spatial control over activation of TGF-β signaling. TGF-β signaling, which plays fundamental roles in development, tissue homeostasis, and cancer, is initiated by receptor oligomerization. We therefore hypothesized that preorganizing the transmembrane receptors would potentiate local TGF-β signaling. To generate surfaces that would nucleate the signaling complex, we employed defined self-assembled monolayers that present peptide ligands to TGF-β receptors. These displays of nondiffusible ligands do not compete with the growth factor but rather sensitize bound cells to subpicomolar concentrations of endogenous TGF-β. Cells adhering to the surfaces undergo TGF-β-mediated growth arrest and the epithelial to mesenchymal transition. Gene expression profiles reveal that the surfaces selectively regulate TGF-β responsive genes. This strategy provides access to tailored surfaces that can deliver signals with spatial control.

Abstract 996: SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Abstract Metastasis remains the most deadly aspect of breast cancer. We previously published that when the Wnt/β-catenin signaling antagonist, secreted frizzled-related protein-1 (SFRP1), is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer. Interestingly, the observed phenotypic and genotypic changes that occur in response to loss of SFRP1 define the EMT process. Among the genes analyzed, the drastic increase in ZEB2 expression was of particular interest to us. This protein is a zinc-finger transcription factor that represses the expression of E-cadherin, which is the mechanism by which ZEB2 is thought to contribute to EMT. Additionally, ZEB2 has been shown to interact with downstream mediators of Transforming Growth Factor (TGF)-β signaling (SMADs). TGF-β signaling is known to actively contribute to the acquisition and development of metastatic phenotypes in part through its ability to stimulate EMT and we wondered whether this cytokine plays a role in the EMT observed in response to SFRP1 loss. Therefore, we tested the hypothesis that TGF-β signaling is augmented due to SFRP1 reduction. First, we used luciferase assays and real-time PCR analysis to show that in response to TGF-β treatment, the expression of TGF-β responsive genes are elevated in TERT-siSFRP1 cells. These downstream targets were also measured after cells were treated with a TGF-βR antagonist (LY364947) to ensure these effects were specifically due to the TGF-β signaling pathway. TGF-β signaling has also been shown to increases the phosphorylation of ERK1/2 which in turn enhances cellular migration. We found that the migratory TERT-siSFRP1 cells exhibit a marked increase in ERK1/2 phosphorylation that is readily blocked by the addition of LY364947. Furthermore, transwell assays revealed that LY364947 blocks cellular migration and this phenomenon is likely due to the inhibition of ERK1/2 because the ERK1/2 inhibitor (U0126) similarly blocks cellular migration. To investigate the contribution of ZEB2, we used siZEB2 oligonucleotides to knock down the expression of this protein. Reduced levels of ZEB2 lowered the expression of exogenous and endogenous TGF-β transcriptional targets and hindered cellular migration. We can conclude from these data that the TGF-β signaling pathway is aberrantly activated in TERT-siSFRP1 cells due to the significant up-regulation of the SMAD-interacting protein, ZEB2. This work has been supported in part by the Rays of Hope Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2011-996

First randomized trial comparing balloon kyphoplasty (BKP) to non-surgical management among cancer patients with vertebral compression fractures

but overexpression of type II receptor did not rescue Hfg inhibition of TGF-β-responsive promoter activity. Further, Hfg increased mRNA expression of the inhibitory Smad, Smad7 but knockdown of Smad7 did not rescue Hfg inhibition of TGF-β responsive genes. These data suggested that the effect of Hfg was not completely TGF-βdependent. We next studied the effect of Hfg on BMP signaling. Hfg reduced BMP–Smad-reporter (BRE)4 in MDA-MB-231 or PC-3 cells treated with BMP7, as well as significantly reduced phosphoSmad2/ 3 and phospho-Smad1/5/8 proteins. Further Hfg reduced ID1 mRNA, a BMP-regulated gene. These data suggest that Hfg is an effective therapy for bone metastases through both TGF-β-dependent and independent effects.

Poly(I:C) Drives Type I IFN- and TGFβ-Mediated Inflammation and Dermal Fibrosis Simulating Altered Gene Expression in Systemic Sclerosis

Immune activation of fibrosis likely has a crucial role in the pathogenesis of systemic sclerosis (SSc). The aim of this study was to better understand the innate immune regulation and associated IFN- and transforming growth factor-β (TGFβ)-responsive gene expression in SSc skin and dermal fibroblasts, in particular the effect of different Toll-like receptor (TLR) ligands. To better understand the relationship between inflammation and fibrosis in vivo, we developed a murine model for chronic innate immune stimulation. We found that expression of both IFN- and TGFβ-responsive genes is increased in SSc skin and SSc fibroblasts when stimulated by TLR ligands. In contrast, cutaneous lupus skin showed much more highly upregulated IFN-responsive and much less highly upregulated TGFβ-responsive gene expression. Of the TLRs ligands tested, the TLR3 ligand, polyinosinic/polycytidylic acid (Poly(I:C)), most highly increased fibroblast expression of both IFN- and TGFβ-responsive genes as well as TLR3. Chronic subcutaneous immune stimulation by Poly(I:C) stimulated inflammation, and IFN- and TGFβ-responsive gene expression. However, in this model, type I IFNs had no apparent role in regulating TGFβ activity in the skin. These results suggest that TLR agonists may be important stimuli of dermal fibrosis, which is potentially mediated by TLR3 or other innate immune receptors.

Transforming growth factor-β inhibits nephronectin-induced osteoblast differentiation

We used cDNA microarray to identify transforming growth factor beta (TGF-β) responsive target genes during osteoblast development and found that nephronectin (Npnt) is one such gene that is significantly down-regulated. Here we report the role of TGF-β in regulating Npnt-mediated osteoblast differentiation. We found that the effect of TGF-β on Npnt expression is associated with a change in cell morphology in a dose-dependent manner. Npnt-induced osteoblast differentiation was also inhibited by TGF-β, which changed cell morphology from cuboidal to fibroblastic, an indication that osteoblast differentiation was disrupted. Furthermore, TGF-β inhibited differentiation of osteoblasts transfected with various truncated Npnt constructs, suggesting that TGF-β can exert a down-stream effect on Npnt function. Our results suggest that TGF-β can inhibit osteoblast differentiation through various mechanisms.

Estrogen Inhibits Transforming Growth Factor β Signaling by Promoting Smad2/3 Degradation

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

Abstract SY35-03: eIF4E in breast cancer is a master regulator of tumor cell invasion through increased translation of beta-1 integrin mRNA and TGF-beta activation

Abstract eIF4E is frequently overexpressed in tumors of the breast, colon, prostate, and lung, and expression correlates with disease progression, increased tissue invasion and metastasis. It has been suggested that overexpression of eIF4E contributes to malignancy by selectively increasing translation of mRNAs containing significant secondary structure in the their 5' noncoding regions. A limited group of mRNAs encode key proteins involved in cellular growth, survival, angiogenesis and malignancy and contain long GC-rich highly structured 5' noncoding regions. The effect of increased levels of eIF4E in tumor cells, however, is still not well understood, nor is the role of its mTOR regulated repressor, 4E-BP1. Here we demonstrate the surprising finding that overexpression of eIF4E acts primarily to increase breast cancer cell invasion by significantly increasing translation of a single mRNA: β1 integrin. eIF4E overexpression at levels found in breast cancers (3-4 fold) results in a 10-fold increased level of tumor cell invasion. Analysis of polysomal mRNA found increased translation of β1 integrin mRNA with physiological levels of eIF4E overexpression observed in breast cancers. We found that increased β1 integrin expression was due to increased read-through of the complex GC-rich 5' noncoding region of β1 integrin mRNA following eIF4E overexpression, the first time this has been described physiologically, and that this effect is blocked by co-overexpression of 4E-BP1. The modest increase in β1 integrin protein levels is amplified by a disproportionately large increase in the formation of the cell surface heterodimer α3β1, an integrin frequently implicated in breast tumor invasion. These and other results can account for the large increase in invasive ability in eIF4E overexpressing breast cancer cells, suggesting that β1 integrin levels may be limiting in the cell. In addition, we found that increased expression of eIF4E results in increased transcription and consequent protein levels of many canonical TGFβ-responsive genes, including β1 integrin. TGFβ is partially responsible for the increased invasion seen with eIF4E overexpression, although cells are still 4.5 fold more invasive even in the absence of the cytokine, consistent with the TGFβ-independent increase in β1 integrin levels observed with eIF4E overexpresison. The TGFβ transcriptional effect is likely due to an increased rate of activation of latent TGFβ (typically present in serum or the stroma), mediated by the eIF4E-driven increased levels of β1 integrin and its subsequent heterodimerization with α integrins capable of activating TGFβ, including αvβ1. TGFβ transcriptional effects are due to TGFβ ligation with its receptor, and not eIF4E-mediated increased translation of a downstream TGFβ effector, as they are blocked with the use of a TGFβ receptor I kinase inhibitor. Additionally, eIF4E overexpression does not facilitate the translation of TGFβ mRNA, as protein levels of TGFβ are not increased with elevated levels of eIF4E. Co-overexpression of 4E-BP1 blocks all TGFβ effects. Our results suggest a new but prevalent model whereby eIF4E level is a master regulator that determines the direction of TGFβ activity. Increased expression of eIF4E at typical human breast cancer levels mediates increased tumor cell invasion through the selective increased translation of β1 integrin mRNA. Higher levels of β1 integrin promotes increased heterodimerization with α-integrins which activate latent TGFβ present in the breast stroma, thereby directing TGFβ to pro-invasive function. Citation Format: Celine Mestel, Mary-Helen Barcelos-Hoff, Jiri Zavadil, Silvia C. Formenti, Robert J. Schneider. eIF4E in breast cancer is a master regulator of tumor cell invasion through increased translation of beta-1 integrin mRNA and TGF-beta activation [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY35-03

YB-1 Mediates a Crosstalk between ERα and TGFβ Signaling in Breast Cancer Cells.

Abstract The multifunctional Y-box binding protein-1 (YB-1) belongs to the highly conserved family of cold shock proteins being involved in transcriptional/translational regulation, DNA repair as well as stress response. Additionally, it is highly expressed in many malignant tissues which seems to correlate with a high proliferation level, drug resistance and a poor prognosis in a variety of tumors including breast cancer.Using quantitative immunohistochemistry we now confirm the previously described negative prognostic impact of YB-1 on survival in a clinical study with tumor samples from 199 breast cancer patients (p=0.0146; median follow up 79 months).Treatment of the estrogen receptor α (ERα) positive human breast cancer cell line MCF-7 with antiestrogens (4-hydroxytamoxifen, faslodex) resulted in a time and dose dependent decrease of YB-1 expression on the RNA- as well as on the protein level. In contrast, antiestrogen treatment of the ERα negative cell line MDA-MB-231 does not have any effect indicating that the antiestrogen effect on YB-1 is mediated by ERα. We have already demonstrated that the action of all antiestrogens is at least partially mediated by transforming growth factor β (TGFβ) (e.g. Breast Cancer Res. Treat. 107: 15-24, 2008). Using TGFβ-responsive reporter gene assays we now show that overexpression of YB-1 in MCF-7 cells enhances the induction of p3TPlux activity by TGFβ suggesting a crosstalk between YB-1 and TGFβ signaling pathways. Moreover, YB-1 also increases the antiestrogen induction of the TGFβ2-mRNA level coming along with a YB-1 regulated enhancement of the antiestrogen effect on cell growth.Very recent findings of our group point towards a ligand-independent influence of ERα on TGFβ signaling (Breast Cancer Res. Treat., in press). Due to the antiestrogen regulation of YB-1 via ERα and its crosstalk with the TGFβ pathway, the potential role of YB-1 as a mediator of the interaction between ERα and TGFβ signaling was analyzed. Performing pull-down assays with recombinant GST-ERα and whole cell lysates of MCF-7 cells confirms a protein-protein interaction between YB-1 and ERα. The physiological relevance of this interaction could be demonstrated, as the TGFβ-induced p3TPlux activity is clearly modulated by both, YB-1 and ERα.In conclusion, we present evidence that YB-1 and TGFβ signaling are functionally linked in MCF-7 cells. Combined with the detection of a physical interaction between YB-1 and ERα, our findings shed new light on a potential role of YB-1 as a part of the mechanism responsible for switching TGFβ from its anti-proliferative role in early tumor stages to tumor promoting effects in late-stage disease, most likely via a YB-1 regulated shift of TGFβ-signaling to pro-invasive pathways. This would also explain why high levels of YB-1 correlate with a poor prognosis in breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4172.

A TGFβ-Responsive Gene Signature Is Associated with a Subset of Diffuse Scleroderma with Increased Disease Severity

Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming growth factor-beta (TGFbeta) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFbeta-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal fibroblasts from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFbeta, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFbeta responsive. Expression of the TGFbeta-responsive signature was examined in skin biopsies from 17 dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFbeta-responsive signature was expressed in 10 out of 17 dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFbeta-responsive signature stratifies patients into two major groups, one of which corresponds to the "diffuse-proliferation" intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFbeta-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.

Heterogeneous activation of the TGFβ pathway in glioblastomas identified by gene expression-based classification using TGFβ-responsive genes

BackgroundTGFβ has emerged as an attractive target for the therapeutic intervention of glioblastomas. Aberrant TGFβ overproduction in glioblastoma and other high-grade gliomas has been reported, however, to date, none of these reports has systematically examined the components of TGFβ signaling to gain a comprehensive view of TGFβ activation in large cohorts of human glioma patients.MethodsTGFβ activation in mammalian cells leads to a transcriptional program that typically affects 5–10% of the genes in the genome. To systematically examine the status of TGFβ activation in high-grade glial tumors, we compiled a gene set of transcriptional response to TGFβ stimulation from tissue culture and in vivo animal studies. These genes were used to examine the status of TGFβ activation in high-grade gliomas including a large cohort of glioblastomas. Unsupervised and supervised classification analysis was performed in two independent, publicly available glioma microarray datasets.ResultsUnsupervised and supervised classification using the TGFβ-responsive gene list in two independent glial tumor gene expression data sets revealed various levels of TGFβ activation in these tumors. Among glioblastomas, one of the most devastating human cancers, two subgroups were identified that showed distinct TGFβ activation patterns as measured from transcriptional responses. Approximately 62% of glioblastoma samples analyzed showed strong TGFβ activation, while the rest showed a weak TGFβ transcriptional response.ConclusionOur findings suggest heterogeneous TGFβ activation in glioblastomas, which may cause potential differences in responses to anti-TGFβ therapies in these two distinct subgroups of glioblastomas patients.

Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline

Mutations in the gene for the transforming growth factor (TGF)-beta superfamily receptor, bone morphogenetic protein receptor II, underlie heritable forms of pulmonary arterial hypertension (PAH). Aberrant signaling via TGF-beta receptor I/activin receptor-like kinase 5 may be important for both the development and progression of PAH. We investigated the therapeutic potential of a well-characterized and potent activin receptor-like kinase 5 inhibitor, SB525334 [6-(2-tert-butyl-5-{6-methyl-pyridin-2-yl}-1H-imidazol-4-yl)-quinoxaline] for the treatment of PAH. In this study, we demonstrate that pulmonary artery smooth muscle cells from patients with familial forms of idiopathic PAH exhibit heightened sensitivity to TGF-beta1 in vitro, which can be attenuated after the administration of SB525334. We further demonstrate that SB525334 significantly reverses pulmonary arterial pressure and inhibits right ventricular hypertrophy in a rat model of PAH. Immunohistochemical studies confirmed a significant reduction in pulmonary arteriole muscularization induced by monocrotaline (used experimentally to induce PAH) after treatment of rats with SB525334. Collectively, these data are consistent with a role for the activin receptor-like kinase 5 in the progression of idiopathic PAH and imply that strategies to inhibit activin receptor-like kinase 5 signaling may have therapeutic benefit.