ALK5 Inhibitor Research Articles

5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors

A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC50=5.5nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC50=36nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).

Abstract 660: Inducible Ablation Of Emilin1 In Smooth Muscle Cells Increases Blood Pressure And Myogenic Tone By Enhancing Tgf-beta/alk5 Signaling.

Emilin1 (E1), a protein of the elastic extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, precursor to the LAP/TGFβ complex, that releases the growth factor. E1 null mice are hypertensives, with an increased activation of TGFβ. As E1 is expressed in blood vessels starting from early embryonic life to adulthood, is still unknown whether the E1 null phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER T2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain ( Smmhc ) promoter. TAM administration induced Cre activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1 flox/flox mice carrying the Smmhc -CreER T2 ( Smmhc -CreER T2 E1 flox/flox ) were given TAM for 7 days, E1 completely disappeared in 10 days from start of treatment. After that, blood pressure significantly increased in Smmhc -CreER T2 E1 flox/flox as compared to E1 flox/flox (124±1 vs 106±1 mmHg, n=10), whereas no structural changes of both large and resistance arteries were detected. Interestingly, a treatment with anti-TGFβ antibodies rescued the hypertensive phenotype of Smmhc -CreER T2 E1 flox/flox mice. Then, we found that isolated resistance arteries of Smmhc -CreER T2 E1 flox/flox displayed and increased myogenic response to perfusion pressure as compared to E1 flox/flox (7±1 vs 16±2 %, n=10). Moreover, we further clarified that the increased myogenic response was rescued by resistance arteries exposure to TGFβ neutralizing antibody. Finally, we found that the inhibition of TGFβ receptor ALK5, obtained transfecting isolated vessels with an adenovirus carrying a dominant negative ALK5mutant, rescued as well the increased myogenic response of Smmhc -CreER T2 E1 flox/flox. . Our results demonstrate that E1 exerts a homeostatic control of blood pressure by regulating myogenic tone of resistance arteries, through TGFβ/ALK5 in SMCs, thus suggesting that this pathway should be carefully investigated in essential hypertension.

The Src family kinase inhibitors PP2 and PP1 effectively block TGF-beta1-induced cell migration and invasion in both established and primary carcinoma cells

We have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines, the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524, 2011). In this report, the ability of PP2 and PP1, the more specific Src inhibitor SU6656, and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1, Colo 357) and primary NSCLC (Tu459) cell lines were investigated. The effects of PP2, PP1, SU6656, and SB431542 on TGF-β1-dependent cell scattering/EMT, cell migration/invasion, and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR, respectively. In all three cell lines tested, PP1, PP2, and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT, migration, and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast, SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1, and to a greater extent PP2, also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant. These data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC.

Abstract 1918: Anti-metastasis effect of EW-7197, a novel ALK5 inhibitor in both breast cancer cells in vitro and mouse model in vivo

Abstract We have synthesized new ALK5 inhibitor, EW-7197 and investigated their effects of on breast cancer cells as well as tumor bearing MMTV/cNeu transgenic mouse, 4T1 or 4T1-Luc cell-orthotopic implanted mouse models. The level of ß-casein or the number of metastatic nodule in the lung of the breast tumor bearing MMTV/cNeu transgenic mice or 4T1 cell-orthotopic implanted mice, respectively, were decreased by EW-7197 treatment. The treatment of the EW-7197 also inhibited the luciferase activity in the lung of 4T1-Luc cell-orthotopic implanted mouse compared to that of untreated animal. The levels of MMP2 and MMP9 were decreased in the lung and breast tumor of the EW-7197 treated mice. To determine the inhibitory effect of EW-7197 on TGF-β1-induced transcriptional activation, we performed luciferase reporter gene assay in breast cancer cells. EW-7197 effectively blocked TGF-β1-induced luciferase activity in the cells in a dose-dependent manner. TGFß signaling, the phosphorylation of smad2/3 was decreased by EW-7197. TGFß-stimulated mesenchymal markers of epithelial mesenchymal transition (EMT) were inhibited by ALK5 inhibitor concomitant treatment. Furthermore, EW-7197 rescued TGF-β-induced loss of E-cadherin in NMuMG cells. And also, EW-7197 showed higher inhibition on cell motility and cell invasion based on wound healing assay or invasion assay than SB-505124 and LY-2157299. In summary, EW-7197 have anti-metastatic effect on breast cancer cells in vitro as well as breast cancer models in vivo. This data strongly argue that the ALK5 inhibitor have anti-metastasis effects via inhibiting ALK5 activity in vivo and in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1918. doi:1538-7445.AM2012-1918

Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFβ receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFβ induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.

Hypoxia/Reoxygenation (H/R) Stress Increases Organic Anion Transporting Polypeptide 1a4 (Oatp1a4)‐mediated Transcellular Transport at the Blood‐Brain Barrier (BBB): Relevance to CNS Drug Delivery

Our objective was to investigate effects of H/R stress, a component of ischemic stroke, on Oatp1a4‐mediated transcellular transport at the BBB. Rats (200–250 g) were subjected to normoxia (Nx, 21% O2, 60 min), hypoxia (Hx, 6% O2, 60 min) or H/R (6% O2, 60 min followed by 21% O2, 10 min). In rat brain microvessels, Oatp1a4 expression was increased following H/R but was unchanged by Nx or Hx. Uptake of Oatp1a4 substrates (i.e., taurocholate, atorvastatin) were increased following H/R. Oatp1a4 inhibitors (i.e., fexofenadine, estrone‐3‐sulfate) blocked taurocholate and atorvastatin brain uptake in H/R animals, further implicating Oatp1a4 in BBB transcellular transport of these drugs. Additionally, H/R reduced serum levels of transforming growth factor‐β1 (TGF‐β1) and decreased microvascular expression of TGF‐β receptor activin receptor‐like kinase (ALK)‐5. Pretreatment with SB431542, an ALK5 inhibitor, increased Oatp1a4 functional expression both in H/R and Nx animals. These novel data demonstrate that targeting an endogenous BBB transporter may be a viable approach for delivering drugs to the brain during cerebral ischemia. Our study also identifies a BBB signaling mechanism (i.e., TGF‐β/ALK5 pathway) that can be exploited to control expression of Oatp1a4 during H/R and, therefore, optimize CNS drug delivery. This work was supported by NIH R01 grant DA‐11271 and an AAPS New Investigator Grant.

Abstract No. 102: IN-1233, an ALK-5 inhibitor eluting covered metallic stent: an experimental study in a rabbit esophageal model

Abstract No. 102: IN-1233, an ALK-5 inhibitor eluting covered metallic stent: an experimental study in a rabbit esophageal model

Reduced Klotho expression level in kidney aggravates renal interstitial fibrosis

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-β(1) (TGF-β(1)) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-β(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β(1) activity and is a cause of renal fibrosis. On the other hand, TGF-β(1) reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression.

Abstract C10: Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression

Abstract TNF, a pro-inflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. There are few examples of a physiologic role for TNF induced apoptosis in vivo, but here we describe a novel role for TNF in prostate epithelial cell apoptosis following androgen withdrawal. We demonstrate, using high resolution serial MRI (magnetic resonance imaging) to measure mouse prostate volume changes over time, that castration induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes, but not mice deficient for TRAIL or Fas signaling. Wild type mice treated with soluble TNFR2 (to bind TNF and block signaling) prior to castration showed a similar reduction. Additionally, consistent with a paracrine role for TNF in prostate regression, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate following castration. Injection of soluble TNF restores normal levels of prostate regression of Tnf −/− mice. Together, these data indicate that uniquely among these extrinsic death signals, TNF is required for castration induced prostate regression. However, wild type mice receiving soluble TNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient, but acts in the context of additional castration induced signals. A preliminary investigation using ALK5 inhibitors prior to castration suggests that TGFβ signaling may also be important. Understanding the physiologic role for TNF, and the interaction with other signaling pathways active in prostate regression following androgen withdrawal may lead to novel therapies for prostate cancer. Citation Format: Jennifer S. Davis, Kent L. Nastiuk. Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C10.

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC50=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC50=32nM).

Differential Signalling Through ALK-1 and ALK-5 Regulates Leptin Expression in Mesenchymal Stem Cells

Leptin plays a central role in maintaining energy balance, with multiple other systemic effects. Despite leptin importance in peripheral regulation of mesenchymal stem cells (MSC) differentiation, little is known about its expression mechanism. Leptin is often described as adipokine, while it is expressed by other cell types. We have recently shown an in vitro leptin expression, enhanced by glucocorticoids in synovial fibroblasts (SVF). Here, we investigated leptin expression in MSC from bone marrow (BM-MSC) and umbilical cord matrix (UMSC). Results showed that BM-MSC, but not UMSC, expressed leptin that was strongly enhanced by glucocorticoids. Transforming growth factor β1 (TGF-β1) markedly inhibited the endogenous- and glucocorticoid-induced leptin expression in BM-MSC. Since TGF-β1 was shown to signal via ALK-5-Smad2/3 and/or ALK-1-Smad1/5 pathways, we analyzed the expression of proteins from both pathways. In BM-MSC, TGF-β1 increased phosphorylated Smad2 (p-Smad2) expression, while ALK-5 inhibitor (SB431542) induced leptin expression and significantly restored TGF-β1-induced leptin inhibition. In addition, both prednisolone and SB431542 increased p-Smad1/5 expression. These results suggested the ALK-5-Smad2 pathway as an inhibitor of leptin expression, while ALK-1-Smad1/5 as an activator. Indeed, Smad1 expression silencing induced leptin expression inhibition. Furthermore, prednisolone enhanced the expression of TGF-βRII while decreasing p-Smad2 in BM-MSC and SVF but not in UMSC. In vitro differentiation revealed differential osteogenic potential in SVF, BM-MSC, and UMSC that was correlated to their leptin expression potential. Our results suggest that ALK-1/ALK-5 balance regulates leptin expression in MSC. It also underlines UMSC as leptin nonproducer MSC for cell therapy protocols where leptin expression is not suitable.

Ligand‐independent transforming growth factor‐β type I receptor signalling mediates type I collagen‐induced epithelial–mesenchymal transition

Evidence suggests epithelial-mesenchymal transition (EMT) as one potential source of fibroblasts in idiopathic pulmonary fibrosis. To assess the contribution of alveolar epithelial cell (AEC) EMT to fibroblast accumulation in vivo following lung injury and the influence of extracellular matrix on AEC phenotype in vitro, Nkx2.1-Cre;mT/mG mice were generated in which AECs permanently express green fluorescent protein (GFP). On days 17-21 following intratracheal bleomycin administration, ~4% of GFP-positive epithelial-derived cells expressed vimentin or α-smooth muscle actin (α-SMA). Primary AECs from Nkx2.1-Cre;mT/mG mice cultured on laminin-5 or fibronectin maintained an epithelial phenotype. In contrast, on type I collagen, cells of epithelial origin displayed nuclear localization of Smad3, acquired spindle-shaped morphology, expressed α-SMA and phospho-Smad3, consistent with activation of the transforming growth factor-β (TGFβ) signalling pathway and EMT. α-SMA induction and Smad3 nuclear localization were blocked by the TGFβ type I receptor (TβRI, otherwise known as Alk5) inhibitor SB431542, while AEC derived from Nkx2.1-Cre;Alk5(flox/KO) mice did not undergo EMT on collagen, consistent with a requirement for signalling via Alk5 in collagen-induced EMT. Inability of a pan-specific TGFβ neutralizing antibody to inhibit effects of collagen together with absence of active TGFβ in culture supernatants is consistent with TGFβ ligand-independent activation of Smad signalling. These results support the notion that AECs can acquire a mesenchymal phenotype following injury in vivo and implicate type I collagen as a key regulator of EMT in AECs through signalling via Alk5, likely in a TGFβ ligand-independent manner.

TGF-Beta Type I Receptor (Alk5) Kinase Inhibitors in Oncology

The TGFβ type I receptor kinase (ALK5) is an attractive target for intervention in TGFβ signaling due to its druggability as well as its centrality and specificity in the pathway. A number of potent, selective ALK5 inhibitors have been discovered which interact with the ATP-binding site of ALK5. Crystallographic studies of these molecules bound to ALK5 have provided an understanding of potency and selectivity achieved by these inhibitors. ALK5 kinase inhibitors are potently active in models of cancer due to mechanisms of action similar to those for other TGFβ inhibitory agents. Recent insights into the function of TGFβ in human tumors as well as in preclinical models of cancer are helping to identify potential target patient populations and drug combinations for the development of ALK5 kinase inhibitors and other TGFβ- targeted therapeutics. Differences in the toxicological effects, pharmacokinetics and clinical side effects of ALK5 kinase inhibitors and other TGFβ-targeted agents provide a useful and differentiated set of TGFβ signaling inhibitory agents to investigate in clinical studies.

BMP-7/TGF-β1 signalling in myoblasts: Components involved in signalling and BMP-7-dependent blockage of TGF-β-mediated CTGF expression

We and others have recently described the antagonistic role of Bone morphogenetic protein-7 (BMP-7) in TGF-β signalling and myogenic differentiation. To specify the underlying mechanism(s), we here analysed the expression and function of the individual components mediating TGF-β1 and BMP-7 responses. We found that BMP-7 at a concentration of 25ng/ml induces signalling exclusively via ALK2 and ALK3 leading to the activation of Smad1 and Smad5 and subsequent expression of Id proteins. In contrast, low doses of TGF-β1 (0.1ng/ml) lead to an exclusive activation of ALK5 and phosphorylation of Smad2 and Smad3 that regulate specific target genes including connective tissue growth factor (CTGF). CTGF is rapidly induced by TGF-β1 already 1h after stimulation and reduced by BMP-7 application. Smad1/Smad5 or Id1/2 overexpression reduced the TGF-β1-mediated expression of CTGF. However, although siRNA-mediated knock down of Alk2/3 or Smad1/5 counteracts the BMP-7 effect on basal CTGF expression there was no consistent reversion of the observed BMP-7 effect on TGF-β1-mediated CTGF expression. Moreover, ALK5 inhibition using the SB431542 inhibitor significantly affected CTGF expression only at later time points whereas ERK1/2 inhibition completely abrogated CTGF expression. These findings point towards a regulatory role of BMP-7 that relies on modulation of Mitogen-activated protein kinases rather than mechanisms that are exclusively driven by differential Smad activation.

Induction of Heart Valve Lesions by Small-Molecule ALK5 Inhibitors

Aberrant signaling by transforming growth factor-β (TGF-β) and its type I (ALK5) receptor has been implicated in a number of human diseases and this pathway is considered a potential target for therapeutic intervention. Transforming growth factor-β signaling via ALK5 plays a critical role during heart development, but the role of ALK5 in the adult heart is poorly understood. In the current study, the preclinical toxicology of ALK5 inhibitors from two different chemistry scaffolds was explored. Ten-week-old female Han Wistar rats received test compounds by the oral route for three to seven days. Both compounds induced histopathologic heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells. The pathology was observed in all animals, at all doses tested, and occurred in all four heart valves. Immunohistochemical analysis of ALK5 in rat hearts revealed expression in the valves, but not in the myocardium. Compared to control animals, protein levels of ALK5 were unchanged in the heart valves of treated animals. We also observed a physeal dysplasia in the femoro-tibial joint of rats treated with ALK5 inhibitors, a finding consistent with a pharmacological effect described previously with ALK5 inhibitors. Overall, these findings suggest that TGF-β signaling via ALK5 plays a critical role in maintaining heart valve integrity.

EW-7195, a novel inhibitor of ALK5 kinase inhibits EMT and breast cancer metastasis to lung

Recently, researchers are actively pursuing efforts to develop potent and selective ALK5 (TβRI) kinase inhibitors for clinical development. In this study, the authors examined a novel small molecule inhibitor of ALK5, 3-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methylamino)benzonitrile (EW-7195) to determine if it has potential for cancer treatment. The inhibitory effects of EW-7195 on TGF-β-induced Smad signaling and epithelial-to-mesenchymal transition (EMT) were investigated in mammary epithelial cells using luciferase reporter assays, immunoblotting, confocal microscopy and wound healing assays. In addition, the suppressive effects of EW-7195 on mammary cancer metastasis to lung were examined using a Balb/c xenograft and MMTV/cNeu transgenic mice model system. The novel ALK5 inhibitor, EW-7195, inhibited the TGF-β1-stimulated transcriptional activations of p3TP-Lux and pCAGA12-Luc. In addition, EW-7195 decreased phosphorylated Smad2 levels and the nuclear translocation of Smad2 increased by TGF-β1. In addition, EW-7195 inhibited TGF-β1-induced EMT and wound healing of NMuMG cells. Furthermore, in xenografted Balb/c and MMTV/cNeu mice, EW-7195 inhibited metastasis to lung from breast tumours. The novel ALK5 inhibitor, EW-7195, efficiently inhibited TGF-β1-induced Smad signaling, EMT and breast tumour metastasis to the lung in vivo, demonstrating that EW-7195 has therapeutic potential for the breast cancer metastasis to the lung.

Screening and Identification of a Novel Class of TGF-β Type 1 Receptor Kinase Inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC(50) value of 0.7 µM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC(50) value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.

SMAD3 is essential for transforming growth factor-β1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-β1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-β1 signalling was interfered by the chemical inhibitor of the TGF-β1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-β1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-β1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-β1-induced uPA promoter transactivation. Moreover, Smad3−/− mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-β1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-β1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-β1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-β1 in transformed cells.

L-carnosine inhibits high-glucose-mediated matrix accumulation in human mesangial cells by interfering with TGF- production and signalling

Transforming growth factor beta is recognized as a major cytokine in extracellular matrix (ECM) pathobiology as occurs in diabetic nephropathy. While experimental studies have advanced a protective role of carnosine for diabetic complications, a link between carnosine, TGF-β and matrix accumulation remains to be elucidated. In the present study, we tested the hypothesis that L-carnosine inhibits TGF-β production and signalling, thereby reducing hyperglycaemia-associated ECM accumulation. Human mesangial cells (MC) were cultured in high-glucose (HG, 25 mM D-glucose) medium alone or in HG medium to which 20 mM L-carnosine was added. Collagen VI (Col6) and fibronectin (FN) deposition and messenger RNA expression were studied. In addition, TGF-β production and activation of Smad1/5/8 (ALK1) and Smad2/3 (ALK5) pathways were assessed. Under HG conditions, deposition of Col6 and FN were increased 1.4- and 1.6-fold. This was significantly inhibited on the protein and messenger RNA level by L-carnosine. TGF-β production increased under HG conditions but was completely normalized by addition of L-carnosine. Addition of exogenous TGF-β could not overcome the effect of L-carnosine on Col6 and FN expression, indicating additionally interference with TGF-β downstream signalling. Along the same line, L-carnosine reduced TGF-β-mediated Smad2 phosphorylation, suggesting an inhibitory effect on ALK5 signalling. ALK1 signalling remained unchanged. Under HG conditions, pharmacologic inhibition of ALK5 prevented Col6 accumulation but did not change FN deposition. L-carnosine can modulate matrix accumulation in two ways. Firstly, inhibition of TGF-β production might result in an overall inhibition of matrix accumulation and secondly, L-carnosine inhibits TGF-β-induced matrix accumulation, most likely via inhibition of the ALK5 pathway.

Highlights of This Issue

Tumor-associated myeloid cells can drive tumor progression in mice and human patients. Tumor microenvironment cues such as TNFα are capable of educating myeloid cells to transdifferentiate into a tumor-promoting population. Li and colleagues explored the mechanism by which TNFα mediates monocyte transdifferentiation into endothelial-like cells. Their work indicated that TNFα treatment of monocytes significantly increased expression of α5β1 integrin, a major fibronectin receptor. Fibronectin was a robust facilitator of endothelial differentiation of blood mononuclear cells. Blocking the interaction between α5β1 integrin and fibronectin reduced transdifferentiation both in vitro and in vivo. These findings implicate potential drug targets for anticancer treatment.Maspin, a novel serine protease inhibitor with tumor suppressive activity, has been shown to act as an endogenous inhibitor of histone deacetylase 1 (HDAC1). In the current study, Li and colleagues explored the possibility that maspin may also block or reverse DNA methylation-dependent gene silencing because histone deacetylation and DNA methylation can be mechanistically coupled. Data on the transcription regulation of glutathione s-transferase pi isoform in prostate cancer cell lines support this hypothesis and reveals a critical role of maspin in blocking pathologic DNA silencing in tumor progression.It is generally accepted that TGFβ1 is a growth inhibitor for early stages of epithelial cancer and that genetic inhibition of this pathway accelerates progression. Using primary keratinocytes expressing an inducible RAS oncogene, however, Markell and coworkers found that the response to pharmacologic inhibition of ALK5, the TGFβ type I receptor, distinguished two populations of keratinocytes linked to either induction of terminal differentiation or increased proliferation and escape from senescence. These results suggest that TGFβ1 signaling has divergent effects on subpopulations of RAS-initiated keratinocytes with potentially distinct tumorigenic potential.Targeting receptor tyrosine kinases (RTK)can yield successful cancer therapeutics. RTKs capture arrays revealed that receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, KIT, FGFR3, and HER3 are activated in a panel of early passage melanoma cell lines. Transcriptional analysis suggested that some RTKs, such as FGFR3, are activated via an autocrine loop. shRNA knockdown of a subset of receptors (IGF1R, HER3, and AXL) reduced melanoma cell proliferation and migration, regardless of NRAS/BRAF mutational status. This study reveals the landscape of RTK activations in melanoma and suggests new candidates for therapeutic targeting irrespective of NRAS and BRAF driver mutations.