Targeting the TGFβ signalling pathway in disease

Inhibition of cell proliferation

TGFβ strongly inhibits the growth of many cell types, including epithelial, endothelial, haematopoietic and immune cells^37,38. TGFβ also has pro-apoptotic and differentiation-inducing actions on epithelial cells; together, these actions result in tumour suppression in the context of cancer³⁴. TGFβ in epithelial cells activates transcription of cyclin-dependent kinase inhibitor 1A (CDKN1A) and CDKN2A (which encode p21^CIP1 and p15^INK4B, respectively) to mediate cell cycle arrest at the G1 phase³⁹. Conversely, TGFβ represses the transcription of MYC, which encodes a potent transcriptional activator of genes that is required for cell proliferation and growth, and inhibitor of DNA binding (ID) family genes, which encode transcription factors that promote cell differentiation and determination⁴⁰. In oncology, many tumours attenuate TGFβ growth-inhibitory effects but respond to this ligand in a pro-tumorigenic manner. Thus, depending on the tumour type and the stage of tumour progression, TGFβ may provide potent tumour-suppressive or tumour-promoting functions directly on the tumour cell, presumably by mediating differential gene expression programmes (Fig. 3).

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Figure 3

Biphasic activities of the TGFβ signalling pathway during tumorigenesis: from the tumour suppressor to the tumour promoter

Transforming growth factor-β (TGFβ) has biphasic actions during tumorig enesis, suppressing tumorigenesis at early stages but promoting tumour progression later on, which is the underlying paradigm for the action of TGFβ during disease progression in general and thus complicates the development of therapies targeting TGFβ signalling. The light grey arrows indicate a positive feedforward loop resulting in higher levels of TGFβ, which is a feature of non-neoplastic as well as neoplastic diseases. The current goal in cancer therapy is to downmodulate excessive levels of TGFβ ligands and to target the tumour-progressing versus the tumour-suppressing arm of TGFβ action; the latter goal will almost certainly require more-specific second-generation drugs. CTGF, connective tissue growth factor; EMT, epithelial-mesenchymal transition; IL, interleukin; PTHRP, parathyroid hormone-related protein; TAMs, tumour-associated macrophages; TANs, tumour-associated neutrophils; VEGF, vascular endothelial growth factor.

Unlike the role of TGFβ signalling during tumorigenesis, the contribution of TGFβ to vascular disease is more complex and confusing. Studies on clinical samples from vascular disorders, such as atherosclerosis, hypertension and pulmonary hypertension, often find signatures of both upregulation and downregulation of TGFβ signalling, as well as complex interactions between this pathway and other ligands of the TGFβ family, such as BMPs (Box 3). This has been confirmed by in vitro studies, demonstrating the contradictory effects of TGFβ in the regulation of vascular cells^36,41. Furthermore, the TGFβ pathway often exhibits contrasting effects in different vascular cell types, such as endothelial versus vascular smooth muscle cells³⁶. The promiscuous and cell type-specific action of the TGFβ pathway on vascular cells makes the application of targeted TGFβ signalling therapies for cardiovascular disease a particular challenge.

Induction of epithelial-mesenchymal transition and the myofibroblast phenotype

TGFβ can induce an EMT of both epithelial and endothelial cells. This has consequences for disease progression in both cancer and fibrosis³. EMT enhances cellular migration and invasive properties, as cell migration requires loss of cell-cell contacts and acquisition of fibroblastic characteristics. E-cadherin is commonly downregulated in many cancers, and its overexpression can suppress invasion by tumour cells. The TGFβ-SMAD pathway mediates the expression of high mobility group AT-hook 2 (HMGA2), which is important for the induction of SNAIL (also known as SNAI1) and SLUG (also known as SNAI2): two zinc-finger transcription factors that are known to repress the E-cadherin gene³³. In breast and skin cancer, tumour cell EMT contributes to cancer progression as cells consequently become more migratory and invasive, and they can ultimately transition to a myofibroblastic phenotype³. The myofibroblast further modulates the basic biology of the tumour by increasing ECM elaboration and eliciting a tissue contraction process, which results in increased interstitial fluid pressure (IFP). This has consequences for the efficiency of drug delivery to the tumour⁴², as drugs cannot penetrate tissue under positive IFP. EMT can also polarize carcinoma cells towards ‘stem cell-like’ properties, such as increased tumour-initiating capacity and tumour cell drug resistance⁴³. Blocking the TGFβ pathway can thus have a threefold benefit: the reduction of tumour invasion and metastasis; the suppression of cancer stem cell-like properties; and the restoration of negative IFP to enhance chemotherapeutic drug delivery⁴⁴.

In fibrotic conditions, excessive TGFβ production induced in the diseased state contributes to EMT elaboration, which can further exacerbate fibrosis, as seen in pulmonary^45,46, cardiac⁴⁷ and renal^48,49 fibrosis, and in arterial restenosis following surgical trauma⁵⁰. TGFβ can also promote a proliferative and/or migratory phenotype on smooth muscle cells that can aggravate some vascular diseases, including neointimal formation following vascular surgery^51-53.

Extracellular matrix regulation

The ECM is a complex structure that surrounds mammalian cells. It is the major component of connective tissue and is composed of multiple proteins, such as collagen, elastin, fibrillin, fibronectin, lamin and proteoglycans. Fibrosis is characterized by the accumulation of fibroblasts, which secrete excessive amounts of ECM. As TGFβ is widely documented to increase collagen synthesis and deposition by fibroblasts, TGFβ has become a central therapeutic target for different types of fibrosis. TGFβ activity and the synthesis of ECM proteins are mutually regulated. Several genes encoding ECM proteins that are known to be important in driving fibrosis are directly regulated by TGFβ-SMAD signalling pathways. There is a reciprocal regulation of TGFβ by the ECM: latent TGFβ bound to ECM components, such as fibronectin and fibrillin, is inactive until physiological or pathological processes initiate its release. This is seen in MFS, in which the mutation of a fibrillin-encoding gene results in reduced fibrillin levels and a consequent increase in levels of unbound TGFβ; this, in turn, leads to the activation of TGFβ signalling, which is possibly responsible for the aetiology of many Marfanoid features^11,12.

Antisense oligonucleotides and antisense RNA

Antisense Pharma uses the strategy of targeting mRNA translation using ASOs to downregulate ligand synthesis^70,83. Its focus has been on targeting TGFβ2, which is produced in excessive quantities by glioblastoma and pancreatic carcinoma cells. Trabedersen (AP12009), a synthetic 18-mer phosphorothioate ASO, binds specifically to human TGFB2 mRNA, and this drug has progressed to a Phase III clinical trial for oncology applications (Box 4). One of the challenges of this drug is delivering it directly to the tumour to avoid the off-target toxicity associated with systemic delivery of first-generation ASOs. In the case of glioblastoma, this was achieved using intrathecal catheter delivery directly into the tumour⁷⁴. More recently, the company has started developing intravenous delivery approaches for pancreatic cancer, which appear to be effective in mouse models⁷³ and were recently shown to be safe in humans⁸⁴.

An anti-TGFB2 antisense strategy has also been used to generate augmented tumour vaccines. Belagenpumatucel-L (Lucanix; NovaRx) is such a drug, in which an ~900-nucleotide TGFB2 antisense construct is transfected into allogeneic non-small-cell lung cancer (NSCLC) cells, which are then used as a tumour vaccine. Here, drug delivery is not an issue as the ‘drug’ is in fact genetically engineered NSCLC tumour cell lines. This tumour vaccine has superior activity compared to conventional tumour vaccination approaches^85,86. A significant dose-related survival difference was seen in patients who received 2.5 × 10⁷ cells per injection, allowing progression to a Phase III clinical trial⁸⁷.

Monoclonal antibodies

The advantages of monoclonal antibodies are their specificity and extracellular mechanism of action — an advantage when trying to mop up excess extracellular ligand. This is tempered by the less con venient intravenous mode of delivery. However, prolonged pharmacokinetic stability permits infrequent drug administration. Cambridge Antibody Technologies and Genzyme developed humanized (or murinized for preclinical studies) monoclonal antibodies specific to individual ligands, such as lerdelimumab (CAT-152)^88,89 and metelimumab (CAT-192)⁹⁰, or with pan-ligand specificity, such as fresolimumab (GC-1008)^91-93. These antibodies have proceeded through various stages of preclinical and clinical development. Of these three humanized antibodies, fresolimumab has progressed furthest in the clinic for both neoplastic and non-neoplastic applications. This drug was found to be well tolerated and safe at 15 mg per ml in Phase I trials for metastatic melanoma (MetM) plus renal cell carcinoma⁹³ and at 1 mg per ml for the fibrotic disorder focal segmental glomerulosclerosis⁹². Lerdelimumab^88,89 and metelimumab⁹⁰, despite passing safety tests, failed to show efficacy in fibrotic models of corneal scarring and systemic sclerosis, respectively, and were therefore discontinued⁹⁰. Despite a promising Phase I oncology trial of fresolimumab, after Genzyme was acquired by Sanofi the company made the decision to focus on fibrotic applications of this drug.

Eli Lilly entered the monoclonal antibody arena with a TGFβ1 ligand-selective blocking antibody, LY2382770, which has progressed to Phase II trials for kidney fibrosis (Table 1). Since merging with ImClone, Eli Lilly has also developed a TβRII-blocking antibody, IMC-TR1 (Ref. 94), which has just entered clinical trials for breast and colon cancer (ClinicalTrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT01646203","term_id":"NCT01646203"}}NCT01646203). In addition, Biogen Idec and Stromedix have developed an anti-integrin β6 antibody that prevents the activation of TGFβ and has been used efficaciously in preclinical studies of fibrosis and cancer⁹⁵; it is in a Phase II trial for fibrosis (ClinicalTrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT01371305","term_id":"NCT01371305"}}NCT01371305).

Ligand traps and peptides

Genzyme developed a ligand trap by fusing Fcγ to the extracellular domain of TβRII, but this construct never reached clinical trials⁹⁶. However, an alternative ligand trap approach, pursued by Digna Biotech, using peptide mimetics of TβRIII (also known as betaglycan and TGFBR3), completed a Phase IIa clinical trial for scleroderma and skin fibrosis, showing safety and efficacy when topically applied to skin (Table 1; Box 4). This company has plans to extend to Phase IIb/III trials in 2013 (J. Dotor, personal communication). A peptide antagonist of TGFβ activation, LSKL (Leu-Ser-Lys-Leu), binds to a conserved sequence in the LAP region of the latent complex and has demonstrated efficacy in reducing TGFβ signalling in vitro⁹⁷. This antagonist is based on thrombospondin and specifically blocks TGFβ activation. The issue of peptide drug delivery is not a problem for topical application; however, to progress to systemic delivery, Digna Biotech has partnered with Flamel Technologies to investigate proprietary peptide delivery systems.

Small-molecule inhibitors

There are a plethora of SMIs that specifically target the type I receptor of TGFβ to inhibit the phosphorylation of SMAD2 and SMAD3 while keeping at least some non-canonical responses, such as TAK1 activation, intact. These drugs are generally ATP mimetics that bind competitively within the hydrophobic ATP binding pocket of the receptor kinase. The chemistry of these compounds has been extensively reviewed^98,99 and some molecular structures are shown in Fig. 6. The obvious advantages of these molecules over most others are their economical production, stability and ease of oral administration, set against a possible disadvantage of cross-inhibition of other kinases. The short half-life of these drugs provides the possibility of rapid drug withdrawal should adverse events arise. Many successful preclinical studies for metastatic cancer have been undertaken with these SMIs, as reviewed previously^100,101. However, the only company to continue pursuit of a TβRI-targeted SMI into clinical trials for oncology is Eli Lilly with LY2157299 (Ref. 82) (ClinicalTrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT01373164","term_id":"NCT01373164"}}NCT01373164).

Other approaches

A novel approach to the suppression of ligand production has been the preclinical development of pyrrole-imidazole polyamides that bind with sequence specificity to the TGFB1 gene promoter to attenuate gene expression^50,102,103. These large ~17 kDa polymeric molecules (Figs 5,​,6)6) bind within the minor groove of DNA to prevent transcription factor binding. Challenges associated with these drugs include the specificity of promoter binding, along with drug delivery issues owing to their large molecular size and the high local concentration required for activity. However, preclinical studies suggest that these molecules might be used in drug-eluting stents for the purpose of reducing restenosis after coronary or carotid artery surgery⁵⁰.

An alternative approach to suppress TGFβ signalling is gene transfer of antagonizing signalling molecules, such as the inhibitory SMAD7. This approach has been applied in model systems to treat or prevent various pathological conditions, including colonic and hepatic fibrosis, vascular remodelling and diabetic kidney disease^104,105. Such an application has the potential to be applied to many other systemic diseases to attenuate the activity of the TGFβ pathway, with the caveat that gene therapy is still far from being widely accepted as a therapeutic approach¹⁰⁶.

As an approach to stimulate immune destruction of cancer cells by tumour-infiltrating T cells, human tumour antigen-specific CTLs have been engineered to express DNRII using a clinical grade retrovirus vector. TGFβ-resistant CTLs were found to have a functional advantage over unmodified CTLs in clearing TGFβ-secreting Epstein-Barr virus (EBV)-positive lymphoma in vitro and in vivo¹⁰⁷, and this approach to therapy has progressed to a Phase I clinical trial for EBV-positive lymphoma. A further modification of the CTLs, by engineering in an HER2 (also known as ERBB2) chimeric receptor as well as a DNRII, allows the CTLs to target HER2-positive tumour cells^108-111. This approach is in a Phase I clinical trial for advanced HER2-positive lung malignancy, labelled the HERCREEM trial (ClinicalTrials. gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT00889954","term_id":"NCT00889954"}}NCT00889954).

Finally, Renova has developed a recombinant TGFβ3 ligand as an anti-scarring agent on the basis of the hypothesis that this ligand has activity that is independent of and antagonistic to TGFβ1 (Ref. 112). The drug, administered by injection around a surgical wound site, progressed to a Phase III clinical trial, but unfortunately it did not reach its primary or secondary efficacy end points.

Myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is characterized by abnormal myeloid and/or erythroid differentiation of bone marrow cells that results in various anaemias and cytopaenias. In one-third of MDS cases, a high-risk group of patients can progress to leukaemia. However, refractory cytopaenias are the major cause of morbidity and mortality in sufferers. It was recently shown that reduced expression of SMAD7, an inhibitor of TβRI, was a common and significant event observed in CD34⁺ myeloid progenitor cells in the bone marrow of patients with MDS¹⁶⁰. Indeed, low levels of myeloid SMAD7 expression were seen in most patients with MDS, regardless of the risk for progression to leukaemia. Downregulation of SMAD7 expression sensitized myeloid precursors to TGFβ such that even very low levels of the ligand elicited an increase in TGFβ responsiveness, as defined by P-SMAD2 levels and enhanced immune-suppressive effect, thus providing another opportunity to utilize TGFβ inhibitors for therapeutic utility in human disease.

Treatment of primary CD34⁺ haematopoietic stem cells with LY2157299 suppressed the activation of TβRI by its ligand. Moreover, in a liver-specific TGFβ1-overexpressing transgenic mouse model of MDS that exhibits severe anaemia, LY2157299 decreased P-SMAD2 levels in the bone marrow and significantly increased the haematocrit of these mice. Importantly, in ten out of ten primary bone marrow cultures from patients with MDS, administration of LY2157299 significantly increased erythroid (burst-forming unit (BFU-E)) and myeloid (colony-forming unit (CFU); granulocytic monocytic) colony numbers in vitro, harbouring great promise for the treatment of patients with MDS¹⁶⁰.

Fibrosis

IPF is a progressive, chronic and irreversible lung disease occurring in older adults, and has an unknown cause¹⁶¹. The main histological features of IPF are heterogeneous parenchyma, with areas of fibrosis and honeycombing alternating with areas of less-affected or normal parenchyma. IPF is characterized by a progressive reduction in lung function, with an estimated 20% survival prospect after 5 years, making it more lethal than many cancers. The progressive fibrotic reaction in IPF is associated with an epithelium-dependent fibroblast activation, in which TGFβ plays a major part¹⁶. TGFβ1, which is secreted by alveolar epithelial cells in patients with IPF, drives the process by promoting the migration, proliferation and differentiation of resident mesenchymal cells. αVβ6 integrin, which binds and activates latent TGFβ1 and TGFβ3, is highly induced following lung injury or fibrosis¹⁶². TGFβ activity¹⁶³ then promotes activation and differentiation of fibroblasts into myofibroblasts, which are specialized contractile cells that cause aberrant ECM deposition, leading to the destruction of lung architecture, scarring¹⁶² and reduced lung function. TGFβ also promotes pulmonary EMT that additionally contributes to the expansion of fibroblasts and myofibroblasts¹⁶⁴.

Pirfenidone, a novel compound that inhibits TGFβ activity in vitro, decreased the rate of decline in vital lung capacity and marginally increased progression-free survival in patients with IPF. Pooled data from two concurrent Phase III clinical trials in IPF indicated improvement in pulmonary function in the pirfenidone-treated group¹⁶⁵. Currently, there are no US Food and Drug Administration (FDA)-approved drugs for IPF, and pirfenidone is the first such drug to be approved for IPF in Europe. Other approaches to develop TGFβ-based therapies for IPF include gene transfer of a soluble TβRII construct (as a ligand decoy), which attenuated injury and fibrosis in bleomycin-induced IPF in mice¹⁶⁶. P144 (disitertide; Digna Biotech), a synthetic peptide that attenuates TGFβ activity and is derived from the extracellular domain of betaglycan, was also shown to reduce carbon tetrachloride-induced liver injury in mice⁷⁸. P17, another Digna Biotech anti-TGFβ peptide, has been shown to be efficacious in attenuation of injury and fibrosis in bleomycin-induced IPF in mice¹⁶⁷. Although P144 has been used clinically for skin fibrosis, drug delivery is an issue for the clinical development of P144 for IPF. However, Digna Biotech has recently partnered with Flamel Technologies to investigate the use of a proprietary drug delivery platform for this application (see the press release: ‘Flamel Technologies and Digna Biotech Announce Multiple Product Development Agreement’). Other anti-TGFβ therapies in clinical trials for IPF include the pan TGFβ-neutralizing antibody GC-1008 (Genzyme) and the αVβ6 integrin-blocking antibody STX-100 (Stromedix)^168,169.

Renal fibrosis has long been thought to be driven by excess TGFβ, which results in renal scarring and, ultimately, kidney failure¹⁷⁰. With the increasing incidence of diabetes and associated kidney damage in affluent countries, this is a clinical application of growing importance. Mice overexpressing an active form of TGFβ1 (from the liver) develop progressive liver and renal fibrosis¹⁷⁰. Interestingly, although mice overexpressing active TGFβ1 develop progressive renal injury, latent TGFβ1 also has a protective role in renal fibrosis through negative effects on inflammation⁴⁹. TGFβ1 mediates progressive renal fibrosis by stimulating the synthesis of ECM production while inhibiting its degradation⁴⁹. TGFβ1 also mediates renal fibrosis by inducing the transformation of tubular epithelial cells into myofibroblasts through EMT in a similar way to the process seen in IPF¹⁷¹. Blockade of TGFβ1 with neutralizing TGFβ antibodies prevents or ameliorates renal fibrosis in vivo and in vitro, demonstrating the functional role of TGFβ1 in EMT and renal fibrosis. A number of therapeutic interventions that block the action of TGFβ have resulted in various degrees of improvement in kidney structure and function in preclinical studies; such interventions include TGFβ ASOs, a neutralizing anti-TGFβ antibody, a soluble TGFβ receptor, blockade of TGFβ activation by decorin¹⁷², an SMI of TGFβ receptors, delivery of the inhibitor protein SMAD7 (Ref. 173) and a THBS1-blocking peptide that interferes with TGFβ activation⁹⁷. A Phase I/II trial with GC-1008, a pan-TGFβ-neutralizing antibody, exhibited encouraging efficacy in patients with focal segmental glomerulosclerosis⁹², and Eli Lilly is undertaking trials of its own anti-TGFβ1 monoclonal antibody, LY2382770, in diabetic kidney disease.

Cardiac fibrosis is a pathological feature that is common to a number of forms of heart disease, including myocardial infarction, ischaemic, dilated and hypertrophic cardiomyopathies and congestive heart failure³⁶. The cellular basis of cardiac fibrosis is the aberrant accumulation of collagens and other ECM proteins, which impair ventricular function and predispose to cardiac arrhythmias. Because TGFβ has pleiotropic effects in the cardiovascular system and as cardiac fibrosis is a multifactorial disease, the development of an effective therapy will require a detailed understanding of the role of the TGFβ signalling pathway in this pathogenesis. TGFβ, a potent stimulator of collagen production by cardiac fibroblasts, is induced in response to cardiovascular injury. The TGFβ-SMAD pathway activates the transcription of several key fibrotic genes, such as those encoding connective tissue growth factor (CTGF), fibronectin, collagens and plasminogen activator inhibitor 1 (PAI1)³⁶. TGFβ reduces collagenase production and stimulates the expression of tissue inhibitor of metalloproteinases (TIMPs), resulting in an overall inhibition of ECM degradation and leading to excessive ECM accumulation. P144 has been investigated in a preclinical model of cardiac fibrosis⁷⁷, and losartan can reverse fibrosis in a mouse model of hypertrophic cardiomyo pathy¹⁷⁴; however, no drug targeting the TGFβ pathway has yet reached clinical trials for this application. A recent study demonstrated that miR-21, which is regulated by SMADs upon TGFβ activation, is consistently induced by cardiac stress. As miR-21 plays a part in tumorigenesis by promoting cell proliferation, increased expression of miR-21 might contribute to the progression of fibrotic lesions¹⁷⁵. ASOs against miR-21 might therefore become a novel therapeutic approach for treating cardiac fibrosis.

Scleroderma

Scleroderma (progressive systemic sclerosis) is a systemic autoimmune disorder characterized by skin sclerosis, calcinosis and changes in microvasculature. Increased expression of TβRI and TβRII in sclerodermal fibroblasts suggests that increased production of type I collagen by autocrine TGFβ signalling leads to aberrant ECM deposition and scarring³⁶. Therapeutic approaches to scleroderma have included inhibition of TGFβ activity in sclerotic tissue. Unfortunately, CAT-192, a TGFβ1-neutralizing antibody, did not show evidence of efficacy in a study on the treatment of patients with early-stage systemic scleroderma⁹⁰; however, GC-1008 is now in a Phase I clinical trial for patients with diffuse systemic sclerosis. Furthermore, topical application of Digna Biotech’s P144 peptide inhibitor of TGFβ1 has shown some efficacy in reducing skin fibrosis in a Phase II clinical trial for systemic sclerosis (see the press release: ‘Flamel Technologies and Digna Biotech Announce Multiple Product Development Agreement’), with the caveat that clinical end points for quantifying skin fibrosis have not yet been standardized¹⁷⁶.

Restenosis following coronary artery bypass and angioplasty

The development of fibromuscular intimal hyperplasia following angioplasty and coronary artery bypass surgery is a major clinical problem and can lead to coronary artery graft failure. The success of coronary artery reconstructive procedures is limited by the high incidence of restenosis secondary to intimal hyperplasia. TGFβ1 is a major player in the early development of intimal hyperplasia in arteries and peripheral vein grafts. The exact mechanism of action of TGFβ signalling in intimal hyperplasia and subsequent graft failure is unclear, but it is speculated that TGFβ1 contributes at multiple steps, including EMT, promotion of fibroblast, endothelial and vascular smooth muscle cell proliferation, increased collagen synthesis and deposition, and induction of fibrosis¹⁷⁷. Soluble forms of the small, leucine-rich proteoglycans decorin and fibromodulin, which possess TGFβ-antagonist activity, exhibit potent intimal hyperplasia-suppressing effects in cultured human saphenous vein, offering the potential for therapeutic benefit after coronary artery bypass surgery^178-180. The novel pyrrole-imidazole polyamide drug class, targeted to suppress TGFB1 gene transcription, showed efficacy in reducing neointimal hyperplasia and stimulating re-endothelialization of carotid arteries in a preclinical model of arterial injury⁵⁰.

Marfan syndrome

MFS is a connective tissue disorder that affects the musculoskeletal, ocular and cardiovascular systems. It is caused by mutations in the gene encoding an ECM protein, fibrillin 1 (FBN1)¹⁸¹. Growing evidence suggests that FBN1 mutations perturb not only the general integrity and elasticity of tissues but also — probably more crucially — local TGFβ signalling¹¹. Normal fibrillin 1-containing microfibrils interact with the large latent TGFβ complex (LLC) to control the release of mature, active TGFβ¹⁸¹. Mutated fibrillin 1 fails to sequester latent TGFβ, leading to the promiscuous activation of TGFβ¹¹. Thus, MFS highlights the critical role of microfibrils in regulating local concentrations of TGFβ and in maintaining the homeostasis, morphogenesis and function of various organs¹⁸¹. Dilation of the aortic root, which leads to aortic rupture and sudden death, is a major clinical issue for patients with MFS and patients of the Loeys-Dietz spectrum who carry mutations in TGFBR1, TGFBR2 and SMAD3. A mouse model of MFS carrying a heterozygous Fbn1 mutation developed an aortic aneurism similar to that of patients with MFS. Administration of TGFβ antagonists, including a TGFβ-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker losartan, successfully rescued both cardiovascular and non-cardiovascular manifestations of MFS^12,182. As losartan is already in widespread clinical use for hypertension and has shown no adverse effects, this drug is currently being tested in Phase I/II clinical trials for MFS (ClinicalTrials.gov identifiers: {"type":"clinical-trial","attrs":{"text":"NCT00429364","term_id":"NCT00429364"}}NCT00429364, {"type":"clinical-trial","attrs":{"text":"NCT00593710","term_id":"NCT00593710"}}NCT00593710, {"type":"clinical-trial","attrs":{"text":"NCT00683124","term_id":"NCT00683124"}}NCT00683124, {"type":"clinical-trial","attrs":{"text":"NCT00723801","term_id":"NCT00723801"}}NCT00723801, {"type":"clinical-trial","attrs":{"text":"NCT00763893","term_id":"NCT00763893"}}NCT00763893, {"type":"clinical-trial","attrs":{"text":"NCT00782327","term_id":"NCT00782327"}}NCT00782327 and {"type":"clinical-trial","attrs":{"text":"NCT01145612","term_id":"NCT01145612"}}NCT01145612) and may plausibly reduce this life-threatening manifestation of MFS.

Patient selection for TGFβ inhibitors in oncology

The simplest biomarker for patient selection for TGFβ inhibitors in oncology is probably high circulating levels of TGFβ, as one major goal of this therapy is to reduce, but not totally ablate, TGFβ signalling¹⁹³. As indicated above, non-invasive biomarkers for predicting patient responsiveness and efficacy of TGFβ inhibitors have been developed on the basis of measuring P-SMAD2 levels in PMNCs. Indeed, PMNCs can be treated with drugs ex vivo to determine, and thus predict, individual patient responses to SMIs^188,193. As TGFβ inhibitors certainly act to reduce tumour metastasis, the assay of circulating tumour cell number may also be a useful indicator for therapeutic response. Moreover, as many of the pro-tumorigenic effects of TGFβ are mediated by immune system modulation, it might be possible to monitor blood cell or plasma protein profiles for patient selection and for surrogate monitoring of patient responses.

In cancer (as well as non-malignant diseases), the outcome of reduced TGFβ signalling may be highly dependent on the innate genetic background of the individual, especially when considering tumour microenvironment effects, such as immune surveillance. Elucidating specifically which genetic variants influence signalling output will not only be useful for dissecting the intricacies of this signalling pathway in vivo, but may also provide predictive markers for the outcome (desired or undesired) of TGFβ signal inhibition. However, for cancer therapeutics, patient selection may be more complex, as the response of both the tumour cell and host (tumour microenvironment and normal patient tissue) to TGFβ blockade needs to be considered. Tumour biopsy and genetic analysis (for example, loss of TGFBR2, SMAD2 or SMAD4)^194,195 might predict whether the tumour retains growth sensitivity to TGFβ. Molecular and histological analyses may also contribute to the prediction of tumour responses to TGFβ inhibition. The activation of alternative intracellular signalling pathways and transcription factor profiles has been associated with the switch from tumour suppression — by TGFβ — to tumour progression. These include an increased ratio of liver-enriched inhibitory protein (LIP) to liver-enriched activating protein (LAP), which are isoforms of CCAAT/enhancer-binding protein-β (C/EBPβ), a central transcription factor that binds within the SMAD transcription factor complex to elicit TGFβ-mediated cytostatic responses¹⁹⁶. Upregulation of SIX1 in breast cancer has also been shown to be pivotal in the growth-suppressive to tumour-progressive switch¹⁹⁷, as has downregulation of DAB2 (Ref. 198). Any of these tumour markers, possibly in combination, may be used in the future to predict tumour responses to TGFβ inhibition.

Finally, the effects of interactions with other anticancer drugs will need to be considered, as some drugs may resurrect the growth-inhibitory arm of the TGFβ signalling pathway, which would then counter-indicate their combinatorial use with TGFβ inhibitors¹⁹⁹. Indeed, the ability to specifically target the pro-tumorigenic versus the tumour-suppressive effects of TGFβ on the tumour cell per se will require the development of next-generation drugs focusing on these downstream pathways. In the meantime, much research still remains to be undertaken to make inroads into the area of informed patient selection for oncology applications of TGFβ inhibitors.

The authors give thanks to J. Dotor, J. McPherson and J. Yingling for useful discussions. Work in the authors’ laboratories is funded by grants from the US National Cancer Institute, the US National Heart, Lung and Blood Institute (NHLBI), the US National Institute of Arthritis and Musculoskeletal and Skin Diseases, the US National Institute of General Medical Sciences, the Bouque Foundation and the Helen Diller Family Comprehensive Cancer Center to R.J.A., and from the NHLBI, the American Heart Association and the LeDucq Foundation to A.H. We apologize to all colleagues whose work could not be cited owing to space restrictions.