Dual mTOR Inhibition Is Required to Prevent TGF-β-Mediated Fibrosis: Implications for Scleroderma

Extracellular matrix (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Normal human dermal fibroblasts Platelet derived growth factor Transforming growth factor-β TO THE EDITOR Transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) are central mediators of fibrosis, and their overexpression contributes to the pathophysiology of scleroderma, chiefly by inducing the overproduction of extracellular matrix proteins (ECM) by dermal fibroblasts (Gay et al., 1989Gay S. Jones Jr, R.E. Huang G.Q. et al.Immunohistologic demonstration of platelet-derived growth factor (PDGF) and sis-oncogene expression in scleroderma.J Investig Dermatol Symp Proc. 1989; 92: 301-303Abstract Full Text PDF Scopus (153) Google Scholar; Sargent et al., 2010Sargent J.L. Milano A. Bhattacharyya S. et al.A TGFbeta-responsive gene signature is associated with a subset of diffuse scleroderma with increased disease severity.J Investig Dermatol Symp Proc. 2010; 130: 694-705Abstract Full Text Full Text PDF Scopus (109) Google Scholar; Bhattacharyya et al., 2012Bhattacharyya S. Wei J. Varga J. Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities.Nat Rev Rheumatol. 2012; 8: 42-54Crossref Scopus (267) Google Scholar). TGF-β also promotes the differentiation of dermal fibroblasts into myofibroblasts, which are key mediators of scleroderma (Abraham et al., 2007Abraham D.J. Eckes B. Rajkumar V. et al.New developments in fibroblast and myofibroblast biology: implications for fibrosis and scleroderma.Curr Rheumatol Rep. 2007; 9: 136-143Crossref PubMed Scopus (129) Google Scholar). Thus, targeting this pathway is a reasonable strategy to treat a variety of fibrotic diseases including scleroderma, for which current treatment options are limited. Herein we explore the potential of novel mTOR inhibition as a means to block the pro-fibrotic effects of TGF-β. Recent studies have suggested a functional role of mTOR in fibrotic diseases and autoimmunity (Ong et al., 2007Ong C.T. Khoo Y.T. Mukhopadhyay A. et al.mTOR as a potential therapeutic target for treatment of keloids and excessive scars.Exp Dermatol. 2007; 16: 394-404Crossref PubMed Scopus (78) Google Scholar; Fried et al., 2008Fried L. Kirsner R.S. Bhandarkar S. et al.Efficacy of rapamycin in scleroderma: a case study.Lymphat Res Biol. 2008; 6: 217-219Crossref PubMed Scopus (26) Google Scholar; Su et al., 2009Su T.I. Khanna D. Furst D.E. et al.Rapamycin versus methotrexate in early diffuse systemic sclerosis: results from a randomized, single-blind pilot study.Arthritis Rheum. 2009; 60: 3821-3830Crossref PubMed Scopus (81) Google Scholar; Raychaudhuri and Raychaudhuri, 2014Raychaudhuri S.K. Raychaudhuri S.P. mTOR Signaling Cascade in Psoriatic Disease: Double Kinase mTOR Inhibitor a Novel Therapeutic Target.Indian J Dermatol. 2014; 59: 67-70Crossref PubMed Scopus (30) Google Scholar). This pathway is initiated by cytokines and growth factors that induce phosphorylation of Akt(Thr308) and its downstream mediators mTORC1, p70S6K1, and 4E-BP1. In addition to mTORC1, mTOR is now known to comprise a second multi-protein complex, mTORC2, which positively regulates the activity of mTOR through phosphorylation of AktSer473 (Bhagwat et al., 2011Bhagwat S.V. Gokhale P.C. Crew A.P. et al.Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.Mol Cancer Ther. 2011; 10: 1394-1406Crossref PubMed Scopus (159) Google Scholar). Blockade of mTORC1 has already been attempted as a treatment for scleroderma without much understanding about its molecular mechanism (Fried et al., 2008Fried L. Kirsner R.S. Bhandarkar S. et al.Efficacy of rapamycin in scleroderma: a case study.Lymphat Res Biol. 2008; 6: 217-219Crossref PubMed Scopus (26) Google Scholar; Su et al., 2009Su T.I. Khanna D. Furst D.E. et al.Rapamycin versus methotrexate in early diffuse systemic sclerosis: results from a randomized, single-blind pilot study.Arthritis Rheum. 2009; 60: 3821-3830Crossref PubMed Scopus (81) Google Scholar; Yoshizaki et al., 2010Yoshizaki A. Yanaba K. Yoshizaki A. et al.Treatment with rapamycin prevents fibrosis in tight-skin and bleomycin-induced mouse models of systemic sclerosis.Arthritis Rheum. 2010; 62: 2476-2487Crossref PubMed Scopus (109) Google Scholar). Such strategies focused only on mTORC1 and did not account for the contributions of mTORC2 (Bhagwat et al., 2011Bhagwat S.V. Gokhale P.C. Crew A.P. et al.Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.Mol Cancer Ther. 2011; 10: 1394-1406Crossref PubMed Scopus (159) Google Scholar). To increase the effectiveness of mTOR blockade, dual inhibitors targeting both mTORC1 and mTORC2 have now been developed (Bhagwat et al., 2011Bhagwat S.V. Gokhale P.C. Crew A.P. et al.Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.Mol Cancer Ther. 2011; 10: 1394-1406Crossref PubMed Scopus (159) Google Scholar). Herein, we demonstrate that dual mTOR blockade can more effectively inhibit the pro-fibrotic effects of TGF-β and PDGF. These have therapeutic implications for scleroderma and other TGF-β dominant fibrotic diseases. The antimitotic effects of mTOR inhibition were evaluated using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Normal human dermal fibroblasts (NHDFs) were incubated with PDGF, a known NHDF mitogen, and cultures were treated with either OSI-027 or the mTORC1 inhibitor rapamycin. OSI-027 was found to be more effective at inhibiting PDGF-induced NHDF proliferation compared with rapamycin (P<0.05) (Figure 1a). Apart from proliferating more, in response to TGF-β, NHDFs are known to differentiate into myofibroblasts and increase their production of ECM proteins. Thus, the expression of α-SMA (a marker of myofibroblasts) and the ECM-collagen I and III was determined by immunofluorescence microscopy (IF) following incubation with TGF-β (Abraham et al., 2007Abraham D.J. Eckes B. Rajkumar V. et al.New developments in fibroblast and myofibroblast biology: implications for fibrosis and scleroderma.Curr Rheumatol Rep. 2007; 9: 136-143Crossref PubMed Scopus (129) Google Scholar). In comparison with rapamycin, OSI-027 was more effective at inhibiting TGF-β-induced α-SMA (P<0.01), collagen I (P<0.01), and collagen III (P<0.05) expression (Figure 1b and c). Similarly, by quantitative PCR (qPCR), OSI-027 was found to be more effective at inhibiting TGF-β-induced upregulation of ACTA2 (P<0.005), COL1A1 (P<0.005), and COL3A1 (P<0.05) gene expression. OSI-027 was also able to reverse TGF-β-induced downregulation of MMP1 (P<0.05) (Figure 1d). The ability of PDGF and TGF-β to alter mTOR signaling was confirmed using a luminex-based cell-signaling assay. Figure 1e and f demonstrated that TGF-β and PDGF were able to induce significant phosphorylation of AktSer473 (representing mTORC2 activity) and mTORSer2448. As Smad signaling is critical for TGF-β-mediated induction of ECM (Bhattacharyya et al., 2012Bhattacharyya S. Wei J. Varga J. Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities.Nat Rev Rheumatol. 2012; 8: 42-54Crossref Scopus (267) Google Scholar), we determined the effect of OSI-027 on phosphorylation of Smad2. We found that TGF-β-induced phosphorylation of Smad2 was effectively inhibited by OSI-027 (P<0.05) (Figure 1g). This provides an underlying molecular mechanism for the antifibrotic effects of dual mTOR inhibition. On the basis of the in vitro data, we sought to evaluate whether mTOR was expressed in lesional skin of scleroderma patients. Immunohistochemical staining (Figure 2a) demonstrated elevated expression of pAktSer473, pmTORSer2448, and their respective total proteins in scleroderma compared with normal skin. Quantification of pAktSer473 was used as a surrogate for mTORC2 activity (Bhagwat et al., 2011Bhagwat S.V. Gokhale P.C. Crew A.P. et al.Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.Mol Cancer Ther. 2011; 10: 1394-1406Crossref PubMed Scopus (159) Google Scholar). Although there are few studies suggesting the antifibrotic properties of rapamycin, chiefly a mTORC1 inhibitor (Ong et al., 2007Ong C.T. Khoo Y.T. Mukhopadhyay A. et al.mTOR as a potential therapeutic target for treatment of keloids and excessive scars.Exp Dermatol. 2007; 16: 394-404Crossref PubMed Scopus (78) Google Scholar; Fried et al., 2008Fried L. Kirsner R.S. Bhandarkar S. et al.Efficacy of rapamycin in scleroderma: a case study.Lymphat Res Biol. 2008; 6: 217-219Crossref PubMed Scopus (26) Google Scholar; Su et al., 2009Su T.I. Khanna D. Furst D.E. et al.Rapamycin versus methotrexate in early diffuse systemic sclerosis: results from a randomized, single-blind pilot study.Arthritis Rheum. 2009; 60: 3821-3830Crossref PubMed Scopus (81) Google Scholar; Yoshizaki et al., 2010Yoshizaki A. Yanaba K. Yoshizaki A. et al.Treatment with rapamycin prevents fibrosis in tight-skin and bleomycin-induced mouse models of systemic sclerosis.Arthritis Rheum. 2010; 62: 2476-2487Crossref PubMed Scopus (109) Google Scholar; Tamaki et al., 2014Tamaki Z. Asano Y. Kubo M. et al.Effects of the immunosuppressant rapamycin on the expression of human alpha2(I) collagen and matrix metalloproteinase 1 genes in scleroderma dermal fibroblasts.J Dermatol Sci. 2014; 74: 251-259Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), to date, there have been no detailed mechanistic studies on mTOR’s role in TGF-β-mediated fibrotic pathways. Moreover, in cancer biology, it is becoming increasingly evident that targeting only mTORC1 does not completely inhibit this cascade (Figure 2b). To overcome this deficiency, dual mTOR (mTORC1/mTORC2) inhibitors have been developed––e.g. OSI-027 (Bhagwat et al., 2011Bhagwat S.V. Gokhale P.C. Crew A.P. et al.Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.Mol Cancer Ther. 2011; 10: 1394-1406Crossref PubMed Scopus (159) Google Scholar)––and are currently being evaluated in a phase I trial for solid cancers (https://clinicaltrial.gov/ct2/show/NCT00698243?term=OSI-027&rank=1). Herein using an in vitro system, we demonstrated the ability of dual mTOR inhibition to reverse TGF-β and PDGF-mediated fibrotic processes more efficiently compared with rapamycin and elucidated the underling molecular mechanism. Considering the crucial role of PDGF and TGF-β in the pathogenesis of scleroderma, our results support the development of dual mTOR inhibitors for scleroderma and other TGF-β-mediated fibrotic diseases. Third to sixth passage NHDFs (ATCC PCS-201-012) were cultured at 37 °C per 5% CO2 in DMEM containing 10% FBS and 1% antibiotic-antimycotic. Cells were incubated in the presence or absence of PDGF (40 ng ml-1), TGF-β (5 ng ml-1) with or without rapamycin (10 nM), and OSI-027 (5 uM) for 3 days, which corresponded to their optimal concentrations. To measure proliferation, the MTT reagent was added and incubated at room temperature for 2 hours. Plates were read at 570 nm (Datta-Mitra et al., 2013Datta-Mitra A. Mitra A. Ray R. et al.1,25-Dihydroxyvitamin D3-3-bromoacetate, a novel vitamin D analog induces immunosuppression through PI3K/Akt/mTOR signaling cascade.Int Immunopharmacol. 2013; 17: 744-751Crossref PubMed Scopus (17) Google Scholar). NHDFs were processed for IF as described (Luna et al., 2011Luna J.I. Ciriza J. Garcia-Ojeda M.E. et al.Multiscale biomimetic topography for the alignment of neonatal and embryonic stem cell-derived heart cells.Tissue Eng Part C Methods. 2011; 17: 579-588Crossref PubMed Scopus (69) Google Scholar). The data are represented as adjusted integrated density using Image J (NIH, Bethesda, MD). Expression of pAktSer473, pmTORSer2448, pSmad2Ser465/467, corresponding total proteins, and β-actin was determined using the Bio-Plex Pro cell signaling kit (Bio-Rad) (Lang and Sandoval, 2014Lang W.H. Sandoval J.A. Detection of PI3K inhibition in human neuroblastoma using multiplex luminex bead immunoassay: a targeted approach for pathway analysis.J Biomol Screen. 2014; 19: 1235-1245Crossref PubMed Scopus (4) Google Scholar). The phospho-proteins were normalized to respective total-proteins and represented as adjusted median fluorescence intensity (MFI). RNA was extracted using the RNeasy plus mini kit (Qiagen) and integrity determined by Agilent 2200 TapeStation. Total RNA was reverse transcribed using iScript, and qPCR was performed using customized PrimePCR plates (Bio-Rad, Hercules, CA). Reference genes used for normalization were GAPDH and HPRT1. Data were analyzed using the Bio-Rad CFX manager software (Bio-Rad) and expressed as fold change. After obtaining IRB approved written informed consent, punch biopsies were obtained from three scleroderma patients with diffuse disease, and normal skin samples were obtained from discarded surgical skin. Serial sections were deparaffinized and rehydrated and then immersed in 10 mM sodium citrate buffer. After antigen retrieval, endogenous peroxidase activity was blocked, and 10% BSA was used to inhibit nonspecific binding. Sections were stained with antibodies with the following specificities––pAktSer473, total Akt, pmTORSer2448, and total mTOR. Stained tissues were incubated with a secondary antibody followed by ABC reagents and DAB (Vector Lab, Burlingame, CA). Toludine blue was used to counterstain. The authors state no conflict of interest. This work was supported by a grant from the NIH (No. 1DP2OD008752-01) awarded to EM. EM was also supported by career awards from the Burroughs Wellcome Fund and the Howard Hughes Medical Institute.