Targeting the TGFβ pathway for cancer therapy Cindy Neuzillet, Annemila¨ı Tijeras-Raballand, Romain Cohen, J´erˆome Cros, Sandrine Faivre, Eric Raymond, Armand de Gramont PII: DOI: Reference:

S0163-7258(14)00201-0 doi: 10.1016/j.pharmthera.2014.11.001 JPT 6726

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Neuzillet, C., Tijeras-Raballand, A., Cohen, R., Cros, J., Faivre, S., Raymond, E. & de Gramont, A., Targeting the TGFβ pathway for cancer therapy, Pharmacology and Therapeutics (2014), doi: 10.1016/j.pharmthera.2014.11.001

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P&T # 22632 Targeting the TGFβ pathway for cancer therapy

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Cindy Neuzillet1, Annemilaï Tijeras-Raballand2, Romain Cohen2, Jérôme Cros3, Sandrine

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Faivre1, Eric Raymond4, Armand de Gramont4

Affiliations:

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1. INSERM U728 & U773 and Department of Medical Oncology, Beaujon University Hospital (AP-HP – PRES Paris 7 Diderot), 100 boulevard du Général Leclerc, 92110

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Clichy, France

2. AAREC Filia Research, Translational Department, 1 place Paul Verlaine, 92100 Boulogne-Billancourt, France

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3. Department of Pathology, Beaujon University Hospital (AP-HP – PRES Paris 7 Diderot),

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100 boulevard du Général Leclerc, 92110 Clichy, France

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4. New Drug Evaluation Laboratory, Centre of Experimental Therapeutics and Medical Oncology, Department of Oncology, Centre Hospitalier Universitaire Vaudois (CHUV),

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Lausanne, Switzerland

Correspondence:

Armand de Gramont, Ph.D., New Drugs Evaluation Laboratory, Centre of Experimental Therapeutics, Department of Oncology, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland ; E-mail: [email protected]

Details of the manuscript: Number of pages Abstract Text Tables/Figures

39 Pages 209 Words, max of 250 5623 Words 1/2 Tables/Figures

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References

91 References

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Running Title: Targeting TGFβ for cancer therapy

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Abstract The TGFβ signaling pathway has pleiotropic functions regulating cell growth, differentiation,

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apoptosis, motility and invasion, extracellular matrix production, angiogenesis, and immune

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response. TGFβ signaling deregulation is frequent in tumors and has crucial roles in tumor

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initiation, development and metastasis. TGFβ signaling inhibition is an emerging strategy for cancer therapy. The role of the TGFβ pathway as a tumor-promoter or suppressor at the

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cancer cell level is still a matter of debate, due to its differential effects at the early and late stages of carcinogenesis. In contrast, at the microenvironment level, the TGFβ pathway

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contributes to generate a favorable microenvironment for tumor growth and metastasis throughout all the steps of carcinogenesis. Then, targeting the TGFβ pathway in cancer may

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be considered primarily as a microenvironment-targeted strategy. In this review, we focus on the TGFβ pathway as a target for cancer therapy. In the first part, we provide a

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comprehensive overview of the roles played by this pathway and its deregulation in cancer, at the cancer cell and microenvironment levels. We go on to describe the preclinical and

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clinical results of pharmacological strategies to target the TGFβ pathway, with a highlight on the effects on tumor microenvironment. We then explore the perspectives to optimize TGFβ inhibition therapy in different tumor settings.

Key Words (max of 6): angiogenesis, fibrosis, metastasis, microenvironment, TGFβ inhibitors, tumor-stroma interactions.

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Table of Contents

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1. INTRODUCTION ............................................................................................................. 7

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2. ROLES OF TGFΒ IN CANCER ............................................................................................ 8

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2.1. At the cancer cell level: dual tumor-promoter and suppressor role of TGFβ signaling... 8 2.2. At the microenvironment level: a key thread of pro-tumoral activities ...................... 11

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3. TGFΒ PATHWAY INHIBITORS: PRECLINICAL AND CLINICAL RESULTS.............................. 16 3.1. Classification of agents targeting the TGFβ pathway for cancer therapy ................... 16

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3.2. Preclinical results: proof of concept .......................................................................... 17 3.3. Clinical results .......................................................................................................... 21

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4. SUMMARY AND PERSPECTIVES ................................................................................... 25

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Abbreviations bFGF: basic fibroblast growth factor

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bi-shRNAi: bi-functional short hairpin RNAi

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CAF: cancer-associated fibroblasts

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CR: complete response CTGF: connective tissue growth factor

EMT: epithelial-to-mesenchymal transition HCC: hepatocellular carcinoma

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HGF: hepatocyte growth factor HSC: hepatic stellate cell

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I-SMAD: inhibitory SMAD

IL: interleukin

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LOH: loss of heterozygosity

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IFN: interferon

MCP-1: chemoattractant protein-1 MMP: matrix metalloproteinase

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MSI: microsatellite instability NK: natural killer

NOX: NADPH oxidase NSCLC: non-small cell lung cancer PBMC: peripheral blood mononuclear cell PDAC: pancreatic ductal adenocarcinoma PDGF: platelet-derived growth factor PR: partial response PSC: pancreatic stellate cell OS: overall survival ROS: reactive oxygen specie

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ECM: extracellular matrix

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SD: stable disease TGFβ: transforming growth factor-β

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TNFα: tumor necrosis factor-α

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Th: T-helper

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T-reg: T-regulatory cell

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VEGF: vascular endothelial growth factor

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1. Introduction The transforming growth factor-β (TGFβ) superfamily consists of more than 30 different

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members including the TGFβs (comprising three highly homologous isoforms, TGFβ1, TGFβ2,

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and TGFβ3), activins, NODAL, bone morphogenetic proteins (BMP), growth and differentiation factors (GDF) and anti-Müllerian hormone (AMH) (Wakefield & Hill, 2013). They share common properties regarding their structure, synthesis, signal transduction

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mechanisms and regulation [Figure 1] (Bierie & Moses, 2006; Ikushima & Miyazono, 2010;

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Sakaki-Yumoto, Katsuno, & Derynck, 2013; Wakefield & Hill, 2013; Watabe & Miyazono, 2009).

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The TGFβ superfamily signaling pathways have pleiotropic functions regulating cell growth,

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differentiation, apoptosis, motility and invasion, extracellular matrix (ECM) production,

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angiogenesis, and immune response. They play essential roles in early embryonic development and in regulating tissue homeostasis in adults (Wakefield & Hill, 2013). Deregulated signaling by many of these members has crucial roles in both the development

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of tumors and metastasis. Their influence extends beyond the cancer cells themselves, to the tumor microenvironment and the whole organism. Furthermore, these signaling pathways are emerging therapeutic targets. Among the TGFβ-superfamily members, the TGFβs themselves have been the most studied and inhibitors of this pathway are the most advanced in their clinical development. The role of the TGFβ pathway as a tumor-promoter or suppressor at the cancer cell level is still a matter of debate, due to its differential effects at the early and late stages of carcinogenesis. In contrast, at the microenvironment level, the TGFβ pathway contributes to generate a favorable microenvironment for tumor growth and

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metastasis throughout all the steps of carcinogenesis. Then, targeting the TGFβ pathway for

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cancer therapy may be considered primarily as a microenvironment-targeted strategy.

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In this review, we focus on the TGFβ pathway as a target for cancer therapy. In the first part,

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we provide a comprehensive overview of the roles played by this pathway and its deregulation in cancer, at the cancer cell and microenvironment levels. We go on to describe the preclinical and clinical results of pharmacological strategies to target the TGFβ pathway,

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with a highlight on the effects on tumor microenvironment. We then explore the

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2. Roles of TGFβ in cancer

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perspectives to optimize TGFβ inhibition therapy in different tumor settings.

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2.1. At the cancer cell level: dual tumor-promoter and suppressor role of TGFβ signaling Hijacking crucial biological functions by deregulating the TGFβ signaling pathway by cancer cells has recently emerged as a leading area of preclinical and clinical cancer research. TGFβ

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expression has been studied in a large panel of cancer types, including prostate, breast, lung, colorectal, pancreatic, liver, skin cancers, and gliomas (Padua & Massague, 2009). In earlystage tumors, levels of TGFβ are positively associated with a favorable prognosis, while in advanced tumors, they are positively associated with tumor aggressiveness and poor prognosis (Padua & Massague, 2009). Through these observations, it has emerged that TGFβ pathway has both anti- and pro-tumoral activities (Inman, 2011; Principe, et al., 2014). In early-stage tumors, the TGFβ pathway promotes cell cycle arrest and apoptosis (Drabsch & ten Dijke, 2012; Jakowlew, 2006; Tian, Neil, & Schiemann, 2011). In contrast, at advanced stages, by promoting cancer cell motility, invasion, epithelial-to-mesenchymal transition

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(EMT), and cell stemness, the TGFβ pathway promotes tumor progression and metastasis (Drabsch & ten Dijke, 2012; Jakowlew, 2006; Tian, et al., 2011). This functional switch is

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known as the first “TGFβ paradox” (Wendt, Tian, & Schiemann, 2012).

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The current paradigm for the role of TGFβ in carcinogenesis at the cancer cell level is that accumulation of genetic alterations in the TGFβ pathway drives the pathway’s evolution from tumor-suppressive to tumor-promoting activities (Jakowlew, 2006; Neuzillet, et al.,

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2014). Indeed, tumor progression requires shutting down the tumor-suppressive effects of

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the TGFβ signaling, which can be reached by either of two general mechanisms. The first one consists in “decapitation” of the pathway by inactivation, either through mutation or

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through allelic loss of heterozygosity (LOH), of its core components: the receptors (TGFBR2,

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TGFBR1) or the SMAD transcription factors (SMAD2, SMAD4/DPC4) (Padua & Massague,

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2009). For example, TGFBR2-inactivating mutations are frequently found in cancers associated with microsatellite instability (MSI), which arises from defects in the mismatch repair system, and are associated with high CpG island methylator phenotype (Markowitz, et

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al., 1995; Ogino, et al., 2007; Shima, et al., 2011). The TGFBR2 gene contains a 10-base pair poly-adenine repeat, which is exposed to replication errors leading to gene inactivation specifically in MSI+ cancers, including gastric, colorectal, biliary and lung adenocarcinomas. Alternatively, SMAD4/DPC4 is found inactivated in about 50% of pancreatic ductal adenocarcinoma (PDAC), either by 18q LOH or mutation, and in colon and oesophagus cancers, (Padua & Massague, 2009). These genetic inactivations of core components of the TGFβ pathway result in elimination of most or all of TGFβ responses including tumorsuppressive activities, and cooperate with other genetic alterations to promote tumor initiation and malignant progression. Mice models harboring oncogenic mutation (APC for

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colon cancer, KRAS for PDAC) combined with SMAD4 or TGFBR2 inactivation display accelerated cancer development with more aggressive behaviour compared with mice

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without combined TGFβ signaling alteration (Datto & Wang, 2000; J. P. t. Morris, Wang, &

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Hebrok, 2010). In contrast, breast, prostate cancers, melanomas, and gliomas, frequently retain functional TGFβ signaling but selectively “amputate” the tumor suppressor arm downstream these core components, for example through P15INK4B deletion or C/EBPβ

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inhibition (Padua & Massague, 2009). These tumors can benefit from the remaining TGFβ

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activities promoting tumor progression and metastasis, such as invasion and EMT. Similarly, in HCC, anti-proliferative effects of TGFβ are bypassed via mitogenic signals or impaired

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sensitivity to anti-growth signals (Neuzillet, et al., 2014). Coulouarn et al. (Coulouarn, Factor,

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& Thorgeirsson, 2008) described the switch from a tumor-suppressive “early TGFβ signature”, with low endogenous levels of TGFβ and SMAD7 and strong transcriptional

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SMAD3 activity, and which was correlated with better outcome in HCC patients, to a “late TGFβ signature”, with high amounts of TGFβ and SMAD7 and reduced SMAD3 signaling,

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associated with invasive phenotype and increased tumor recurrence. The early TGFβ signature is also characterized by expression of the DNA damage gene family Gadd45, which is involved in cell cycle arrest and apoptosis (Coulouarn, et al., 2008; Dooley & ten Dijke, 2012). Given this strong induction of anti-tumorigenic genes, tumor promoting activity of TGFβ requires a cellular context with imbalanced sensitivity towards pro- and anti-growth signals (Neuzillet, et al., 2014). For example, p16INK4 gene alterations are present in up to 90% of HCCs and favour insensitivity to anti-growth signals by relieving cyclin D/CDK4,6 complex inhibition and lowering p53 activation (Neuzillet, Hammel, Tijeras-Raballand, Couvelard, & Raymond, 2013). Once the tumor-suppressive effects of the TGFβ signaling

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shut down, it can exert its pro-tumoral and pro-metastatic activities in late steps of

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carcinogenesis.

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To summarize, the TGFβ pathway has dual anti- and pro-tumoral roles at the cancer cell

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level, depending on tumor stage and genetic alteration background, with mechanistic differences between cancer models. This complexity, combined with intratumor genetic heterogeneity, makes the resulting effects of TGFβ inhibition on cancer cell compartment

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difficultly predictable. Moreover, the fact that TGFβ induces pro-tumoral effects although its

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signaling is shut down in cancer cells represents a second “paradox” that leads to shift attention to the microenvironment surrounding cancer cells.

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2.2. At the microenvironment level: a key thread of pro-tumoral activities

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The TGFβ pathway exerts most of its pro-tumoral effects by mediating tumor-stroma

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interactions and remodeling tumor microenvironment (Neuzillet, et al., 2014). The stroma is a complex structure composed of ECM proteins (mainly, type I collagen) and various cell

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types including mesenchymal cells (cancer-associated fibroblasts [CAF]), endothelial cells and pericytes, nerve cells, immune cells, and bone marrow-derived stem cells. These cell types express TGFβ receptors, and the TGFβ pathway can thus impacts microenvironment fibrosis, angiogenesis, and immune cell infiltration (Neuzillet, et al., 2014). The TGFβ pathway activation contributes both to the creation, from a non-tumoral environment, and to the maintenance of a favorable tumoral microenvironment. Effect on tumor initiation SMAD4/DPC4 germline inactivating mutation is genetically responsible for familial juvenile polyposis, an autosomal dominant inherited condition characterized by the development of

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multiple hamartomatous tumors in the gastrointestinal tract and predisposition to cancer. Animal models with a loss of SMAD function have provided insight into the role of SMADs in

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a variety of physiologic systems and tumors (Datto & Wang, 2000; Takaku, et al., 1998).

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Using genetically engineered mice models of familial juvenile polyposis in which the SMAD4 gene is either specifically deleted in T-cells or broadly within epithelia including the intestinal mucosa, Kim et al. (B. G. Kim, et al., 2006) showed that selective disruption of SMAD4 within

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the T-lineage in mice is sufficient to induce the formation of hamartomatous lesions and

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cancer within the gastrointestinal mucosa, contrary to epithelial-specific deletion of the SMAD4 gene. Then, SMAD4-dependent signaling in T-lymphocytes is required to maintain

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immune homeostasis and cancer suppression within the gastrointestinal mucosa, and

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alterations of the TGFβ signaling in the microenvironment contribute to create a favorable

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context for cancer development.

About 15% of human cancers emerge on underlying chronic inflammatory diseases, e.g. colorectal cancer on colitis/Crohn's disease, gastric cancer on chronic gastritis, HCC on liver

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chronic hepatitis/cirrhosis, PDAC on chronic pancreatitis (Coussens & Werb, 2002). As described in the physiological response in injured organs, TGFβ1 is produced in response to pro-inflammatory cytokines (e.g., tumor necrosis factor [TNFα], interleukin [IL]-1) in tumors, particularly by stromal cells, as an attempt to control the inflammatory reaction (LopezNovoa & Nieto, 2009). The activation of the TGFβ pathway results in increased production and reduced degradation of ECM components, in particular type I collagen, as well as mesenchymal cell proliferation and differentiation into myofibroblasts with migration and contractile capabilities (Lopez-Novoa & Nieto, 2009; Pohlers, et al., 2009; Van De Water, Varney, & Tomasek, 2013). TGFβ stimulates reactive oxygen species (ROS) production by

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various mechanisms (including activation of NADPH oxidases [NOX] family members) that, in turn, engage downstream signaling pathways (e.g., SMADs, EGFR, Src and MAPK family)

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resulting in expression of a subset of pro-fibrotic genes (e.g., PAI-1, connective tissue growth

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factor [CTGF], TGFβ1, angiotensiogen) (Samarakoon, Overstreet, & Higgins, 2013). TGFβ overproduction, as a driver of the fibrotic process of chronic phases of inflammatory diseases, precedes tumor formation and prepares a favorable microenvironment for cancer

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cells (Jakowlew, 2006; Lopez-Novoa & Nieto, 2009).

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Effect on tumor maintenance and progression

Some established tumors are characterized by a dense fibrotic stroma (e.g., PDAC) . CAFs

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and stellate cells are responsible for excess ECM production and are engaged through

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signaling pathways in an “unholy alliance” with cancer cells (Coulouarn & Clement, 2014;

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Vonlaufen, et al., 2008). TGFβ1 is a key mediator of this dialogue between CAFs or stellate cells and cancer cells. Cancer cells release mitogenic and fibrogenic stimulants (including

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TGFβ, PDGF, and sonic hedgehog), which activate surrounding CAFs and stellate cells. Activated CAFs and stellate cells in turn secrete various factors (including EGF, IGF-1, PDGF, FGF, MMP, and type I collagen) that promote tumor growth, invasion, metastasis, and resistance to chemotherapy (Duner, Lopatko Lindman, Ansari, Gundewar, & Andersson, 2010). They also contribute to create a hypoxic microenvironment exerting a selection pressure toward a more invasive cancer cell phenotype (Duner, et al., 2010). Overall, the stroma may act either as a barrier or a facilitator to metastatic dissemination depending on collagen I structure (Levental, et al., 2009).

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The TGFβ pathway also plays a pro-tumoral role by promoting angiogenesis (Neuzillet, et al., 2014; ten Dijke & Arthur, 2007). It cooperates in an autocrine/paracrine fashion with other

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signaling cascades, including vascular endothelial growth factor (VEGF), basic fibroblast

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growth factor (bFGF), platelet-derived growth factor (PDGF), CTGF, angiopoietin, and Notch, to regulate angiogenesis through direct or indirect effects on quiescence, migration, and proliferation of endothelial cells (Sakurai & Kudo, 2011). TGFβ has both pro-angiogenic and

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anti-angiogenic properties, depending on its expression level. Low levels of TGFβ contribute

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to angiogenesis indirectly by upregulating expression and activity of angiogenic factors (VEGF, bFGF, CTGF) and proteases, while high levels of TGFβ stimulate basement membrane

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reformation, recruit smooth muscle cells, increase differentiation, and inhibit endothelial cell

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growth (Sakurai & Kudo, 2011; Sanchez-Elsner, et al., 2001). TGFβ plays a crucial role in angiogenesis regulation in hypervascularized tumors such as HCCs or gliomas (Ito, et al.,

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1995; Roy, Poirier, & Fortin, 2014).

Many lines of preclinical evidence suggest that TGFβ plays a central role in immune

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regulation (Teicher, 2007; Wojtowicz-Praga, 2003; L. Yang, 2010). The immune system is responsible for the early detection and destruction of cancer cells. These latters can escape immune surveillance either by becoming immunologically invisible (i.e., cancer cell “hiding”) or by secreting cytokines that “blind” the immune system to the presence of abnormal antigens at the cancer cell surface. TGFβ1 is the most potent immunosuppressor and plays a crucial role in this process, along with IL-10 . Tumor-associated TGFβ1 downregulates the host immune response via several mechanisms: it (1) drives the T-helper (Th)1/Th2 balance toward the Th2 immune phenotype (i.e., humoral immunity, without cytotoxic activity against cancer cells) via IL-10 as an intermediate; (2) directly inhibits anti-tumoral Th1-type

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responses (i.e., cell-mediated immunity) and M1-type macrophages; (3) suppresses cytotoxic CD8+ T-lymphocytes, natural killer (NK) lymphocytes and dendritic cells functions; (4)

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generates CD4+CD25+ T-regulatory cells (T-reg) that suppress activity of other lymphocyte

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populations; (5) promotes M2-type macrophages with pro-tumoral activities (Achyut & Yang, 2011; Teicher, 2007; Truty & Urrutia, 2007; Wojtowicz-Praga, 2003; L. Yang, 2010; L. Yang, Pang, & Moses, 2010). This feature is of particular interest in tumors expressing

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immunogenic antigens such as melanoma (Perrot, Javelaud, & Mauviel, 2013). TGFβ was

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shown to increase the expression of monocyte chemoattractant protein-1 (MCP-1) and IL-10 in melanoma cells, enhancing tumor infiltration and immunosuppression (Diaz-Valdes, et al.,

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2011).

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Solid tumors display a non-random metastatic tropism, suggesting that cancer cells need to

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be adapted to distant site to engraft and proliferate (Bhowmick, 2012). The microenvironment of both the primary and the metastatic tumor sites can determine the ability for a disseminated tumor to progress. For example, TGFβ signaling contributes to

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colon cancer metastasis into the liver and lungs. Calon et al. (Calon, et al., 2012) demonstrated that constitutively TGFβ-pathway inactive KM12L4a colorectal cancer cells (homozygote TGFBRII mutation) were unable to develop metastasis in an orthotopic xenograft mouse model, while KM12L4a engineered to secrete TGFβ developed lung and/or liver metastasis without any autocrine activation of the TGFβ pathway in tumor cells. In addition, subcutaneous inoculation of patients’ purified colorectal cancer stem cells expressing TGFβ in immunodeficient mice generated tumors that ellicited a strong TGFβ pathway activation in stromal cells at both primary and metastatic sites.

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Finally, the role of stromal neural cells as a promoter of tumor growth and metastasis is receiving growing attention (Demir, Friess, & Ceyhan, 2012). There is emerging data

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regarding the role of TGFβ signaling in the interactions between nerve cells and cancer cells

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(Aigner & Bogdahn, 2008; Haas, et al., 2009). For example, TGFβ has been shown to induce nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway (Haas, et al., 2009).

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To summarize, at the microenvironment level, the TGFβ pathway mediates a broad

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spectrum of tumor-stroma interactions in both early and late tumor stages, creating a favorable microenvironment for tumor initiation, cancer cell growth and metastasis, and

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establishing a key thread of pro-tumoral activities throughout the steps of carcinogenesis.

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This establishes interpendencies between the cancer cells that display altered or no

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response to TGFβ, and the TGFβ-stimulated and responsive stromal cells. Thus, TGFβ targeted therapy may affect cancer cells by indirect, microenvironment-mediated,

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mechanisms throughout all the steps of carcinogenesis.

3. TGFβ pathway inhibitors: preclinical and clinical results 3.1. Classification of agents targeting the TGFβ pathway for cancer therapy Many TGFβ pathway inhibitors have been investigated in the preclinical setting, some of which are now in clinical development. Schematically, TGFβ pathway inhibition can be realized at three levels: (i) the ligand level: antisense oligonucleotides delivered directly intravenously or engineered into immune cells to prevent TGFβ synthesis (for example, trabedersen [AP12009], an antisense oligonucleotide targeting TGFβ2; and Lucanix®

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[belagenpumatucel-L], a TGFβ2 antisense gene-modified allogeneic cancer cell vaccine); (ii) the ligand-receptor level: ligand-traps (TGFβ-neutralizing monoclonal antibodies and soluble

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receptors) and anti-TGFβ-receptor monoclonal antibodies to prevent ligand-receptor

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interaction (for example, fresolimumab, a pan-TGFβ antibody; disitertide [P144], a peptidic TGFβ1 inhibitor specifically designed to block the interaction with its receptor; and IMC-TR1 [LY3022859], a monoclonal antibody against TGFβRII) ; and (iii) intracellular level: TGFβ

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receptor kinase inhibitors to prevent signal transduction (for example, galunisertib

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[LY2157299], a small molecule inhibitor of TGFβRI, which is to date the most advanced TGFβ signaling inhibitor under clinical development) (Katz, et al., 2013; Smith, Robin, & Ford,

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2012). Molecules under clinical development in oncology are summarized in Table 1.

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Effect on cancer cells

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3.2. Preclinical results: proof of concept

TGFβ pathway inhibitors display limited anti-proliferative activity in vitro despite efficient

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blockade of both canonical and non-canonical pathway. Indeed, for example, galunisertib displayed potent inhibition of canonical (inhibition of phospho-SMAD2) and non-canonical (MAPK or PI3K/AKT/mTOR) pathways in a variety of in vitro HCC cell lines models, of epithelial and mesenchymal phenotypes and regardless of TGFβ pathway protein expression (Dituri, et al., 2013; Dzieran, et al., 2013) (Serova, et al., 2013). The effects of TGFβ and galunisertib on proliferation were not correlated with signal inhibition and were dependent on the cellular context as defined by Coulouarn et al. (Coulouarn, et al., 2008; Dituri, et al., 2013; Dzieran, et al., 2013) [Serova et al., in press]. In the “late TGFβ signature” cell group (SK-HEP1, SK-Suni, SK-Sora, JHH6, HLE, HLF, and FLC-4 cell lines), no effects on proliferation were observed with galunisertib with or without TGFβ, while In the “early TGFβ signature”

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cell group (HepG2, Hep3B, HuH7, and PLC/PRF/5 cell lines), cancer cells were sensitive to TGFβ dependent growth inhibition and displayed limited sensitivity to galunisertib (Dzieran,

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et al., 2013) [Serova et al., in press]. Despite limited anti-proliferative activity in vitro,

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galunisertib had potent anti-invasive activity (Dituri, et al., 2013; Dzieran, et al., 2013) [Serova et al., in press]. Moreover, galunisertib displayed anti-proliferative activity in ex vivo models representing a more physiological model, suggesting that the TGFβ inhibition can

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(Dituri, et al., 2013) [Serova et al., in press].

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exert anti-tumoral effects and that these effects are mediated by tumor microenvironment

Noticeably, the interdependencies between cell types and the crosstalk between TGFβ and

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other signaling pathways may lead to unexpected effects of TGFβ inhibition. For example,

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TGFβ inhibition by various agents (LY2109761, SD-208, and trabedersen) reduced PDAC cells

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invasion in vitro and metastasis in vivo (Gaspar, et al., 2007; Melisi, et al., 2008; Schlingensiepen, et al., 2011). However, Oyanagi et al. (Oyanagi, et al., 2014) reported that inhibition of TGFβ signaling by galunisertib or SB431542 potentiated PDAC cell invasion into

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collagen matrix in 3D co-culture with fibroblasts. This was due to crosstalk between the TGFβ and the hepatocyte growth factor (HGF) pathway, another major invasion-promoting factor in PDAC. TGFβ inhibition increased the secretion of HGF by fibroblasts by blocking the HGF-suppressing activity of cancer cell-derived TGFβ. These results from a simplified tumorstroma model suggest that TGFβ signaling inhibitors may promote tumor progression under some pathological conditions. Effect on fibrosis PDAC displays the most prominent desmoplastic stromal reaction of all epithelial tumors, often greater than the epithelial component of the tumor itself (Neuzillet, et al., 2014).

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Activated fibroblastic pancreatic stellate cells (PSC) are responsible for excess ECM production in PDAC, and TGFβ1 mediates the dialogue between PSCs and PDAC cancer cells

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(Apte, et al., 2004). Interestingly, a study in the Panc-1 orthotopic PDAC model, showed that

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TGFβRI inhibition by SD-208 treatment significantly decreased tumor growth and was associated with reduced fibrosis in the tumor microenvironment (Medicherla, et al., 2007). The Gianelli group (Mazzocca, et al., 2010) reported similar observation in a HCC xenograft

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mouse model. They showed that HCC cell lines producing high levels of CTGF generated high

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stromogenic tumors, which was reversed by CTGF knockdown. Upon TGFβ1 stimulation, lowCTGF HCC cells formed tumors with a high stromal content and CTGF expression, which was

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inhibited by treatment with LY2109761. Blocking TGFβ signaling with LY2109761 or

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galunisertib inhibited CTGF synthesis and release by HCC cells and reduced tumor stromal

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content by inhibiting fibroblastic cell proliferation (Mazzocca, et al., 2010). Effect on angiogenesis

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HCCs are typically hypervascularized tumors with predominant arterial perfusion, and TGFβ plasma levels are positively correlated with tumor vascularity (Ito, et al., 1995). TGFβ stimulate HCC cancer cells to produce VEGF. Mazzocca et al. (Mazzocca, Fransvea, Lavezzari, Antonaci, & Giannelli, 2009) demonstrated that the TGFβRI inhibitor LY2109761 decreased microvessel density (CD31 immunostaining) in a HCC xenograft model. Mechanistically, LY2109761 blocked VEGF gene expression in HCC cells and paracrine crosstalk with endothelial cells, and consequently the formation of tumoral blood vessels. This antiangiogenic effect required functional SMAD2/3 signaling in cancer cells. Interestingly, the anti-angiogenic and anti-tumoral effects of LY2109761 were more potent than those of bevacizumab, a specific anti-VEGF monoclonal antibody. Similarly, reduced vessel density

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after treatment with TGFβ pathway inhibitor was reported in colorectal cancer with SD-208 (Akbari, et al., 2014), and in glioblastoma models using LY2109761 (M. Zhang, Herion, et al.,

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2011; M. Zhang, Kleber, et al., 2011). The angiogenic effect of TGFβ1 in this latter model may

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be mediated by JNK pathway and macrophage infiltration (X. J. Yang, et al., 2013). Effect on immune infiltration

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Some tumors express immunogenic antigens but secrete a variety of immunosuppressive cytokines, including TGFβ or IL-10, to outgrow and evade host immune surveillance. The

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typical example of this immune tolerance induction is melanoma. This gave the rational for the development of immunotherapy, aiming to reactivate the immune system against these

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tumor antigens. TGFβ pathway inhibitors may contribute to reverse this microenvironment-

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induced immune suppression, by inhibiting T-reg and restoring natural killer and T cell-

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mediated cytotoxicity (Teicher, 2007). A large variety of TGFβ pathway inhibitors (small molecules SB-431542, SD-108, SX-007, and SM-16; anti-TGFβ antibodies; adenovirus

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expressing TGFβ1 or TGFβ2 shRNA; nanoscale liposomal polymeric gels releasing TGFβ inhibitor; P144 and P17 synthetic small peptides inhibiting TGFβ1 and TGFβ2) have been tested in preclinical models as monotherapy or in combination with other immunotherapy (agonistic anti-TNFα receptor or anti-CD40 antibodies, adenovirus expressing interferon (IFN)β, IL-2, anti-tumor vaccine) and were shown to restore immune response and increase the efficacy of combined immunotherapy (Garrison, et al., 2012; Gil-Guerrero, et al., 2008; S. Kim, et al., 2008; Llopiz, et al., 2009; Oh, et al., 2013; Park, et al., 2012; Tanaka, et al., 2010; Tran, et al., 2007; Uhl, et al., 2004; Wilson, et al., 2011; Xu, Wang, Zhang, & Huang, 2014). For example, in a model of advanced melanoma, TGFβ down-regulation by means of a siRNA boosted the vaccine efficacy and inhibited tumor growth by 52% compared with vaccine

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treatment alone, as a result of increased level of tumor infiltrating CD8+ T cells and decreased level of T-reg (Xu, et al., 2014). Similarly, combination delivery of TGFβ inhibitor

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and IL-2 by nanoscale liposomal polymeric gels significantly delayed tumour growth,

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increased survival of melanoma tumor-bearing mice, and increased the activity of NK cells and of intratumoral-activated CD8+ T-cell infiltration (Park, et al., 2012).

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Effect on metastasis

A TGFβ-activated microenvironment facilitates colon cancer cell metastasis into the liver and

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lungs. Using an experimental model for liver metastasis by splenic injection of colorectal CT26 cancer cells constitutively expressing firefly luciferase in Balb/c mice, Zhang et al. (B.

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Zhang, Halder, Zhang, & Datta, 2009) showed that LY2109761 significantly reduced liver

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metastases as monitored by bioluminescence imaging when compared with vehicle control.

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The mean survival of LY2109761-treated mice was significantly prolonged (35.2 versus 24.5 days in control mice, p < 0.001). The overall survival at 30 days was 85.71% in LY2109761-

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treated mice and 0% in control mice. Similarly, Calon et al. (Calon, et al., 2012) demonstrated that galunisertib reduced phospho-SMAD2 stromal positivity and inhibited the formation of subcutaneous tumors by primary colorectal cancer stem cells. The authors suggest that a TGFβ-stimulated stromal cell response, involving IL-11, potentiates colon cancer engraftment and growth at liver and lung metastatic sites through activation of the STAT3 pathway in cancer cells. This illustrates how targeting TGFβ signaling can impact cancer cells by indirect mechanisms due to the interdependencies with stromal cells, through the remodeling of primary tumor and metastasis microenvironment. The effects of TGFβ signaling at the microenvironment level are summarized in Figure 2. 3.3. Clinical results

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Despite limited information on the microenvironmental effects of TGFβ inhibitors in the

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published early phase clinical trials, some information can be highlighted.

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A series of phase I and II trials targeting TGF2 or TGF1/2 expression used a vaccine-based

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strategy (Belagenpumatucel-L and FANG vaccines), and thus aimed to restore immune response in the tumor microenvironment. Belagenpumatucel-L, a TGF2 antisense genemodified non-viral based allogenic tumor cell vaccine, has been explored in a phase II trial in

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NSCLC at different stages (Nemunaitis, et al., 2006). Belagenpumatucel-L had an acceptable

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safety profile and the survival compared favorably with historical data, with a two-year overall survival (OS) rate of 47% in stage IIIB and stage IV patients who received the higher

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doses of the vaccine. Authors showed that immune activation may have contributed to a

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favorable clinical outcome. Patients who achieved partial responses (PR) or stable diseases

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(SD) were more likely to display elevated IFN-, IL-4, and IL-6 cytokine production by peripheral blood mononuclear cells (PBMC), as well as anti-HLA seroconversion to vaccine haplotypes. Based on previous positive clinical data with GM-CSF-secreting allogeneic tumor

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cells, which activity may be impaired by TGF expression, the authors proposed to combine the two approaches by creating a TAG vector co-expressing GM-CSF and TGF2 antisense transgenes in an allogenic tumor cell vaccine strategy (Olivares, et al., 2011). Twenty-three patients with advanced solid tumors received at least one vaccine injection in a phase I trial (Olivares, et al., 2011). The majority of patients experienced SD as their best response and one patient had a prolonged complete response (CR); the one-year OS rate was 35%. Similarly, patients with prolonged SD or CR displayed a positive response to autologous TAG as shown by the increased expression of IFN- in ELISPOT assay. As TGF1 and TGF2 isoforms have redundant functions, a vector combining GM-CSF gene expression with the

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expression of a bi-functional short hairpin RNAi (bi-shRNAi) targeting the furin convertase, which is involved in both TGF1 and TGF2 maturation, was developed. The resulting FANG

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vaccine was evaluated in a phase I study with similar benefits than in the previous trials, i.e.

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FANG vaccine displayed a good safety profile and was able to induce an immune response that was associated with prolonged disease control (Senzer, et al., 2012). Fresolimumab, a human anti-TGF1/2/3 monoclonal antibody has been tested in a phase I

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study involving mostly patients with advanced melanoma (n = 28/29) (J. C. Morris, et al.,

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2014). Fresolimumab was well tolerated; six patients had prolonged SD and one patient achieved PR. Various exploratory biomarkers related to the TGF pathway (plasma levels of

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TGFand VEGF, tumor levels of TGFand TGF receptors, and PBMC phospho-SMAD) were

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analyzed, but none correlated with tumor characteristics or clinical outcome; this may be

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due to the small sample size.

TβM1, a human anti-TGF1 monoclonal antibody, has been tested in 18 patients in a phase I

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study (Cohn, et al., 2014). It showed a good safety profile. Despite no valuable information on tumor response, a number of potential biomarkers were investigated. At specific TβM1 doses, VEGF and bFGF plasma levels tended to decrease. Gene expression profiling showed that TGFpathway was activated in cancer patients’ whole blood compared to normal patients. However, the short dosing duration did not translate into significant antitumor effects in the small number of patients investigated in this study. PF-03446962, a fully human monoclonal antibody against ALK1, has been tested in a phase I study, and showed some activities that suggested potential development in patients that failed anti VEGF/VEGFR therapy (Goff, et al., 2011). In a following phase II study as a single

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agent in platimum pre-treated patients with urothelial tumors, PF-03446962 was considered

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inactive and the trial was stop at the first interim analysis for futility (Necchi, et al., 2014).

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In a phase IIb trial two doses of trabedersen (10 µM and 80 µM), a synthetic TGF2 antisens

procarbazine/lomustine/vincristine) in

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oligodeoxynucleotide, were compared to standard chemotherapy (temozolomide or 145 patients with

recurrent

or

refractory

glioblastoma multiforme or anaplastic astrocytoma (Bogdahn, et al., 2011). It did not show

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superiority of trabedersen in controlling tumor growth at six months, which was the primary

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endpoint. However, delayed responses were observed even after treatment discontinuation, and 10 µM trabedersen tended to be superior to chemotherapy in term of two-year survival

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rate (p = 0.10). Median OS for 10 µM trabedersen was 39.1 months compared with 35.2

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months for 80 µM trabedersen and 21.7 months for chemotherapy (not significant).

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Preliminary results of the first phase II study of galunisertib as second-line treatment in HCC after sorafenib (NCT01246986) were presented at the ILCA 2013 and ASCO GI 2014 meetings

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(Sandrine J. Faivre, 2013); 106 patients were randomized to receive galunisertib at 160 or 300 mg/day. Galunisertib safety profile was suitable for patients with Child-Pugh A/B7 HCC. Median time to progression was 12 weeks. galunisertib treatment was associated with AFP responses in 24% of patients, reduction in TGFβ1 and soluble E-cadherin levels, and time to tumor progression was increased in patients with AFP and TGFβ1 levels reduction from baseline. Interestingly, for patients with AFP concentrations above 200 UI/mL, the difference in time to tumor progression (TTP) and overall survival (OS) between responders and nonresponders remained highly significant, suggesting that the effects of galunisertib might be more pronounced in poor prognostic patients with elevated AFP at baseline. Further analysis is expected to launch phase III clinical trials.

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Altogether, these results highlight that TGF inhibition may achieve prolonged disease control and that response to TGFβ inhibitors may be delayed, as it has long been observed

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with immunomodulatory agents. Identification of reliable predictive biomarkers to identify

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which patients are the most likely to beneficiate from these treatments is a critical issue.

4. Summary and perspectives

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TGFβ signaling inhibition is an emerging strategy for cancer therapy. As most cancer cells display altered or non-functional TGFβ signaling, TGFβ inhibitors have limited effects on

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these cells and exert their anti-tumoral activity mainly by affecting TGFβ responsive cells (fibroblastic, endothelial, and immune cells) in the tumor microenvironment. TGFβ signaling

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influences tumor microenvironment by promoting fibrosis, angiogenesis, and metastasis, and suppressing immune-related host response. TGFβ inhibition may be considered

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primarily to normalize tumor microenvironment homeostasis by down-regulating stromal stimulation resulting from excess TGFβ production by tumor and tumor-related tissues, with

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an indirect impact on cancer cells and limited expected side effects on normal tissues. This mechanism of action illustrates how the interdepencies between cancer and stromal cells can provide new therapeutic targets. There are many clinical challenges to developing TGFβ inhibitors, notably timing of treatment and predictive biomarkers for patient selection, in order to define in what kind of tumor microenvironment TGFβ inhibition may be more beneficial . TGFβ inhibition may be of particular interest in the early setting as a preventive strategy in tumors arising on chronic inflammation and fibrosis, in which TGFβ overproduction precedes tumor formation and create a favorable microenvironment for cancer cells. This may be useful both in the primary

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prevention setting and as adjuvant treatment after complete resection of tumor. Of note, there may be a potential hazard of stimulating synchronous occult tumors through the

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inhibition of TGFβ-induced tumor suppression (particularly, with inhibitors of TGFβ

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receptors), depending on the presence of preneoplastic condition (e.g., chronic inflammation) and genetic alteration background. However, clinical results of TGFβ inhibition in the phase II study in HCC patients are reassuring, without evidence of malignant

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transformation from underlying cirrhotic livers (Jakowlew, 2006; Sandrine J. Faivre, 2013).

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Alternatively, TGFβ inhibition (i.e., by inhibitors of TGFβ ligands) may be considered to downregulate excess TGFβ production that arises as a consequence of tumor development. Thus,

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TGFβ inhibitors may be preferentially used in the advanced setting.

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As TGFβ inhibitors are mainly targeting the tumor microenvironment, with little or dual

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effect on cancer cell proliferation, they should be used in combination with cytotoxic agents to kill these latter cells. In addition, radiotherapy and chemotherapy can induce TGFβ activity, possibly promoting metastatic progression, and high levels of TGFβ are associated

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with resistance to anticancer treatments (Biswas, et al., 2007; Drabsch & ten Dijke, 2012). Then, combined TGFβ inhibition may enhance tumor sensitivity to chemotherapy and radiotherapy (Drabsch & ten Dijke, 2012). There is also a rationale for combination with other therapies targeting tumor microenvironment. For example, TGFβ cooperates with hypoxia to induce EMT and VEGF signaling through HIF-1α induction (Copple, 2010; Drabsch & ten Dijke, 2012; Mimeault & Batra, 2013). This provides a rationale for the use of TGFβ pathway inhibitors in combination with or after failure of anti-angiogenic agents (tyrosine kinase inhibitors such as sunitinib or sorafenib, or antibodies such as bevacizumab), or hypoxia-inducing procedures such as arterial embolization. We also described that

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combination with immunotherapies may be an option through the restoration of the

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immune response by TGFβ inhibitors (Drabsch & ten Dijke, 2012; L. Yang, 2010).

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Identification of reliable predictive biomarkers of response to TGFβ inhibitors is also a critical

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issue. AFP, TGFβ1, and soluble E-cadherin levels have been suggested to predict response in HCC (Sandrine J. Faivre, 2013). More so than cancer cell characteristics, predictive power of biomarkers in terms of TGFβ inhibitor efficacy may be related to the tumor

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microenvironment or a patient’s overall blood biomarker profile (Neuzillet, et al., 2014). For

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example, patients with high intra-tumoral and/or circulating levels of TGFβ may be more likely to respond to specific TGFβ inhibitors.

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In conclusion, microenvironment remodeling by TGFβ, in space and time, generates a

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favorable microenvironment for tumor growth and metastasis. TGFβ inhibitors have entered

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clinical development in cancer patients with encouraging first clinical results. The future of TGFβ inhibitors in cancer therapy as tumor microenvironment targeting agents is promising

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and opens new challenges in terms of biomarkers and patient selection.

Acknowledgments: We acknowledge the Foundation Nelia & Amadeo Barleta (FNAB) and the Association pour l’Aide à la Recherche & l’Enseignement en Cancérologie (AAREC). Conflicts of Interest: Sandrine Faivre is a consultant for Merck, Pfizer, Novartis, Bayer, and Lilly; and Eric Raymond is a consultant for Pfizer, Novartis, Bayer, and Lilly. Other authors have no conflict of interest.

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Table 1. TGFβ pathway inhibitors in development in cancer Name TGFβ ligand inhibitors Lerdelimumab (CAT-152) Genzyme® Metelimumab Genzyme® Fresolimumab (GC1008) Genzyme®/Aventis®

Targets

LY2382770 Eli Lilly® Trabedersen (AP12009) Antisens Pharma®

TGFβ1

Current Status Development stopped

TGFβ1

Development stopped NCT00356460 NCT00923169 NCT01472731 NCT01112293 NCT01401062

In progress outside oncology

NCT00844064 NCT00431561 NCT00761280

Results in glioma, PDAC, CRC, melanoma and glioblastoma

NCT01058785 NCT00676507

Results in glioma and NSCLC; combination phase I in progress

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TGFβ2

NCT01061840 NCT01309230 NCT01505166 NCT01453361

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TGFβ1

TGFβ receptor inhibitors Galunisertib TGFβRI (LY2157299) Eli Lilly®

TEW-7197 MedPacto® PF-03446962 Pfizer®

TGFβRI

IMC-TR1 (LY3022859) Eli Lilly®

TGFβRII

Results in RCC, melanoma, mesothelioma and glioma; combination phase I/II in progress in breast cancer

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TGFβ1, -β2, -β3

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TGFβ2

Lucanix TGFβ2 (Belagenpumatucel-L) NovaRx Corporation® FANG™ Vaccine TGFβ1, -β2 (rhGMCSF/shRNAfurin) Gradalis® Disitertide (P144) Digna Biotech®

Trial identifier

ALK-1 (TGFβRI)

In progress in melanoma, CRC and ovarian cancer

In progress outside oncology

NCT01246986 NCT01373164 NCT01220271 NCT02178358 NCT01582269 NCT02160106

Phase II in progress in PDAC, HCC, glioma and glioblastoma

NCT00557856 NCT01337050 NCT01911273 NCT01486368 NCT01620970 NCT02116894 NCT01646203

Results of phase I; phase II results pending in HCC and in progress in malignant pleural mesothelioma and refractory urothelial carcinoma; combination phase I in progress with regorafenib in CRC

Phase I in progress

Phase I in progress

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CRC: colorectal carcinoma ; HCC: hepatocellular carcinoma; NSCLC: non-small cell lung

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carcinoma; PDAC: pancreatic ductal adenocarcinoma; RCC:rRenal cell carcinoma

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Figure legends

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Figure 1: Overview of the TGFβ family pathways

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All of the ligands of the TGFβ family are initially synthesized and secreted as latent

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precursors that need to be proteolytically processed by extra-cellular pro-protein convertases to produce biologically active dimeric ligands. The signal transduction

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mechanism for all of them is similar. It requires ligand binding to one of the five distinct members of constitutively activated membranous type II serine/threonine kinase receptors,

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and subsequent recruitment and transphosphorylation of one of the seven type I serine/threonine kinase receptors, also known as activin receptor-like kinases (ALK1-7)

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(Wakefield & Hill, 2013). For some ligands, additional co-receptors are required for optimal

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ligand binding and activation of the type I-type II receptor heterodimer. After

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phosphorylation, the type I receptor kinase domain becomes activated and can then activate the canonical (SMAD-dependent) pathway through phosphorylation of the receptor-

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regulated SMAD proteins (R-SMAD). It is classically admitted that TGFβs, activins, and NODAL signal through SMAD2 and SMAD3, and BMPs and GDFs through SMAD1, SMAD5, and SMAD8, but TGFβs can also induce phosphorylation of SMAD1 and SMAD5 (Wakefield & Hill, 2013). The R-SMADs form heteromeric complexes with SMAD4 in early endosomes, through a clathrin-dependent internalization pathway that requires accessory proteins such as SARA (Bierie & Moses, 2006; Ikushima & Miyazono, 2010). These complexes then translocate and accumulate into the nucleus and bind to site-specific recognition sequences within the promoter regions of hundreds of target genes to directly regulate their transcription, both positively and negatively. Various other DNA binding co-factors such as p300, CBP, and FOXH1 can interact with SMAD complexes to amplify SMAD-dependent gene

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transcription. The TGFβ superfamily pathway activities are subject to numerous levels of regulation: interaction of the ligands with extracellular antagonists that prevent their binding

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to receptors; modulation of R-SMAD stability through phosphorylation by MAPKs, glycogen

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synthase kinase 3β (GSK3β) and cyclin-dependent kinases (CDKs); Smurf (SMADubiquitination-regulatory factor)-dependent degradation; or expression of inhibitory SMAD proteins (I-SMAD), SMAD6 and SMAD7 (Wakefield & Hill, 2013). In the non-canonical

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pathway, TGFβ signaling activates SMAD-independent pathways such as PI3K/AKT, MAPK

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pathways (ERK, JNK, and p38 MAPK), c-Src, NF-κB, FAK, Abl, or small GTPases such as RhoA, Rac1 and Cdc42. Moreover, transversal signaling, especially at the SMAD level, allows TGFβ

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pathway activation to integrate signals from integrins, Wnt/β-catenin, Notch, Hedgehog,

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TNF-α, or EGF-dependent pathways (Sakaki-Yumoto, et al., 2013; Watabe & Miyazono,

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2009).

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Figure 2: Summary of the effects of TGFβ at the microenvironment level bFGF: basic fibroblast growth factor; CTGF: connective tissue growth factor; EMT epithelialto-mesenchymal transition; IL-11: interleukin-11; VEGF: vascular endothelial growth factor Fibrosis: TGFβ mediates the dialogue between cancer cells and cancer-associated fibroblastic cells (fibroblasts and stellate cells) by stimulating cancer cells to produce CTGF that has pro-fibrotic effects. Angiogenesis: TGFβ has pro-angiogenic activity by cooperating with and upregulating the expression of angiogenic factors (VEGF, bFGF, CTGF) by cancer cells, with direct or indirect effects on quiescence, migration, and proliferation of endothelial cells.

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Metastasis: TGFβ promotes metastasis by enhancing EMT and cancer cell motility and invasion. It also creates a favorable microenvironment for cancer cell engraftment and

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growth at liver and lung metastatic sites through modulation of cytokines such as IL-11.

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Immunity: TGFβ induces a pro-tumoral state of immune tolerance by deregulating the immune balance towards immunosuppression and inflammation, with a predominant Th2

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profile.

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