Journal of
    Clinical Medicine
    Review
    Stromal Modulators of TGF- in Cancer
    Brunella Costanza 1,† , Ijeoma Adaku Umelo 1,† , Justine Bellier 1,† , Vincent Castronovo 1
    and Andrei Turtoi 1,2, *
    1
    2
    *
    †
    Metastasis Research Laboratory, GIGA-Cancer, University of Liege, 4000 Liege, Belgium;
    brunellacostanza@gmail.com (B.C.); iumelo@ulg.ac.be (I.A.U.); justine.bellier@ulg.ac.be (J.B.);
    vcastronovo@ulg.ac.be (V.C.)
    Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194,
    Université Montpellier, Institut Régional du Cancer de Montpellier, 34298 Montpellier, France
    Correspondence: andrei.turtoi@inserm.fr; Tel.: +33-467-61-3746
    These authors contributed equally to this work.
    Academic Editor: Emanuel F. Petricoin
    Received: 4 November 2016; Accepted: 23 December 2016; Published: 6 January 2017
    Abstract: Transforming growth factor- (TGF- ) is an intriguing cytokine exhibiting dual activities
    in malignant disease. It is an important mediator of cancer invasion, metastasis and angiogenesis,
    on the one hand, while it exhibits anti-tumor functions on the other hand. Elucidating the precise role
    of TGF- in malignant development and progression requires a better understanding of the molecular
    mechanisms involved in its tumor suppressor to tumor promoter switch. One important aspect of
    TGF- function is its interaction with proteins within the tumor microenvironment. Several stromal
    proteins have the natural ability to interact and modulate TGF- function. Understanding the
    complex interplay between the TGF- signaling network and these stromal proteins may provide
    greater insight into the development of novel therapeutic strategies that target the TGF- axis.
    The present review highlights our present understanding of how stroma modulates TGF- activity in
    human cancers.
    Keywords: cancer-associated fibroblasts; proteases; proteoglycans; TGF- ; stroma
    1. Introduction
    The transforming growth factor- ligands (TGF- 1, TGF- 2 and TGF- 3) are members of a super
    family of secreted cytokines that regulate a variety of physiological cellular processes, including
    proliferation, differentiation, migration, survival and immunity [1–3]. In its active form, TGF- signals
    to the nucleus mainly through its cognate receptors, TGF- type I and type II receptors (TGF RI
    and TGF RII), which phosphorylate canonical SMAD2/3 downstream transducers (Figure 1) [4,5].
    In addition, several factors, such as various mitogen-activated protein kinases (MAPKs; namely,
    the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK)and p38 MAPK),
    phosphatidylinositide 3-kinase (PI3K)/AKT, Rho-like GTPases (Rho) and TNF receptor-associated
    factor 4/6 (TRAF 4/6), can be activated by TGF- via non-canonical signaling cascades (Figure 1) [6].
    Of note, emerging evidence has revealed that both TGF- canonical and non-canonical signaling
    cascades can simultaneously occur through crosstalk of core pathway components and combined
    utilization of SMAD/non-SMAD transcription factors [7] (Figure 1). A number of excellent reviews
    have extensively covered TGF- signal transduction [8–11].
    J. Clin. Med. 2017, 6, 7; doi:10.3390/jcm6010007
    www.mdpi.com/journal/jcm
    J. Clin. Med. 2017, 6, 7
    J. Clin. Med. 2017, 6, 7 2 of 25
    2 of 24 Figure 1. Canonical and non-canonical
    non‐canonical TGFTGF‐β signaling
    signaling pathways.
    pathways. (A) canonical signaling
    signaling Figure 1.
    Canonical and
    (A) In In the the canonical
    pathway, biologically active TGF‐β ligands bind to TGFβRII, which in turn activates pathway, biologically active TGF- ligands bind to TGF RII, which in turn activates TGFβRI. TGF RI.
    TGFβRI‐regulated and TGF
    RI-regulated SMAD2/3 SMAD2/3proteins proteins are are phosphorylated phosphorylated at at their their C‐terminal C-terminal serine serine residues residues and
    a number of biological processes through form complexes with SMAD4 (co‐SMAD), initiating form complexes with SMAD4 (co-SMAD), initiating a number of biological processes through
    transcriptional target genes.
    genes. (B) the non-canonical
    non‐canonical signaling
    signaling pathways,
    pathways, the
    the TGFTGF‐β transcriptional regulation regulation of of target
    (B) In In the
    receptor complex transmits its signal through other factors, such as the mitogen‐activated protein receptor complex transmits its signal through other factors, such as the mitogen-activated protein
    kinases (MAPKs), phosphatidylinositide 3‐kinase TNF receptor‐associated factor 4/6 kinases (MAPKs),
    phosphatidylinositide
    3-kinase
    (PI3K),(PI3K), TNF receptor-associated
    factor 4/6 (TRAF4/6)
    (TRAF4/6) and Rho family of small GTPases. Activated MAPKs can exert transcriptional regulation and Rho family of small GTPases. Activated MAPKs can exert transcriptional regulation either through
    either through direct interaction with the nuclear SMAD protein complex or via other downstream direct interaction with the nuclear SMAD protein complex or via other downstream proteins. Moreover,
    proteins. Moreover, activated JNK/p38/ERK act in concert with SMADs to regulate cellular apoptosis activated JNK/p38/ERK act in concert with SMADs to regulate cellular apoptosis and proliferation,
    and proliferation, whereas they mediate metastasis, angiogenesis and cellular growth through other whereas
    they mediate metastasis, angiogenesis and cellular growth through other transcription factors,
    transcription factors, such as c‐JUN and ATF. RhoA/ROCK can be activated by TGF‐β to induce actin such as c-JUN and ATF. RhoA/ROCK can be activated by TGF- to induce actin stress fiber formation
    stress fiber formation during EMT via a non‐transcriptional mechanism. TGF‐β can activate PI3K and during EMT via a non-transcriptional mechanism. TGF- can activate PI3K and AKT by inducing a
    AKT by interaction
    inducing a between
    physical the
    interaction the PI3K p85 subunit and the receptor complex physical
    PI3K p85between subunit and
    the receptor
    complex
    leading
    to translational
    activation. TGF‐β activation of the nuclear
    TRAF leading to via
    translational responses via mTOR/S6kinase responses
    mTOR/S6kinase
    activation.
    TGF- activation
    of the TRAF
    proteins
    can initiate
    proteins initiate nuclear factor‐κB (NF‐κB) activity, leading to the inflammatory factor-B can (NF-B)
    signaling
    activity,
    leading
    to thesignaling inflammatory
    response
    among
    other
    processes.
    response among other processes. The arrows indicate activation/signaling direction of the respective The
    arrows indicate activation/signaling direction of the respective pathway.
    pathway. Cancer cells use TGF- in order to enhance their characteristic properties and features [2,12–14].
    Cancer cells use TGF‐β in order to enhance their characteristic properties and features [2,12–14]. In the
    transition from a physiological to pathological phenotype, TGF- can induce intrinsic
    In the transition from a reflect
    physiological to pathological TGF‐β can induce Although
    intrinsic dichotomous
    effects,
    which
    both its tumor
    suppressivephenotype, and tumor promoting
    function.
    dichotomous reflect both understood
    its tumor [15,16],
    suppressive and tumor function. this dual role ofeffects, TGF- which in cancer
    is poorly
    it is known
    that thepromoting stage of progression
    Although this dual role of TGF‐β in cancer is poorly understood [15,16], it is known that the stage of and cellular context are key factors. In epithelial cells and during tumor initiation, TGF- acts as
    progression and cellular context are key factors. In epithelial and during tumor initiation, a tumor suppressor
    by inhibiting
    the growth
    of malignant
    cells cells via canonical
    SMAD2/3
    signaling
    TGF‐β acts as a tumor suppressor by inhibiting the growth of malignant cells via canonical activity [8,17]. Evidence also suggests that the pro-apoptotic effects of TGF- contribute to its observed SMAD2/3 signaling activity [8,17]. Evidence also suggests that the apoptosis
    pro‐apoptotic effects of TGF‐β cytostatic features
    during
    early
    tumor
    formation.
    TGF- -induced
    has been
    observed
    to
    contribute to its observed cytostatic features during early tumor formation. TGF‐β‐induced occur with SMAD [18], JNK [19] and p38 [20] signaling activity in neoplastic epithelium. In later
    apoptosis has been observed to occur with SMAD [18], JNK [19] and p38 [20] signaling activity in stages of malignant progression, however, cells can induce the loss of the tumor suppressive activities
    neoplastic epithelium. In later stages of malignant progression, however, cells can induce the loss of of TGF- signaling by acquiring mutations or alterations in canonical target genes. This switch in
    the tumor activities of accompanied
    TGF‐β signaling by acquiring mutations transition
    or alterations in response
    to suppressive TGF- by tumor
    cells is
    by epithelial-to-mesenchymal
    (EMT),
    canonical target genes. This switch in response to TGF‐β by tumor cells is accompanied by as well as the enhancement of a number of cancer cell hallmarks, including angiogenesis, invasion
    epithelial‐to‐mesenchymal transition (EMT), as well as the enhancement of a number of cancer cell and metastasis [8,21]. Accordingly, pre-clinical and clinical observations support the prevailing
    hallmarks, including invasion effects
    and metastasis [8,21]. and
    Accordingly, pre‐clinical and hypothesis that
    TGF- angiogenesis, exhibits two opposing
    in tumor control
    progression.
    Experimental
    clinical observations support the prevailing hypothesis that TGF‐β exhibits two opposing effects in evidence suggests that the TGF- signaling network can indeed switch from tumor suppressor to
    tumor control and progression. Experimental evidence suggests that the TGF‐β signaling network tumor promoter, especially in the presence of oncogenic events and epigenetic perturbations [17].
    can indeed switch from tumor suppressor to tumor promoter, especially in the presence of oncogenic events and epigenetic perturbations [17]. Recently, PEAK1 (a novel non‐receptor tyrosine J. Clin. Med. 2017, 6, 7
    3 of 25
    Recently, PEAK1 (a novel non-receptor tyrosine kinase), highly expressed in invasive breast cancer,
    has been described as relevant for switching TGF- from a tumor suppressor to a tumor-promoting
    factor [22]. Agajanial et al. provide preclinical evidence that PEAK1 potentiates TGF- -mediated
    proliferation and tumor progression in a fibronectin-dependent fashion. In the presence of fibronectin,
    PEAK1 causes a switch from canonical TGF- SMAD2/3 signaling to non-canonical Src and MAPK
    signaling [22]. In line with this, other pre-clinical findings support the idea that a balance shift
    between TGF- -induced SMAD-dependent and -independent signaling activity is the underlying basis
    for TGF- -induced tumor progression [7]. To this already complex dual role, an additional level of
    complexity is added by stromal-derived enhancer and suppressor molecules that can directly modulate
    TGF- activity [23]. Tumor stroma, an abundant source of TGF- , comprises a large array of epithelial,
    fibroblast, endothelial and inflammatory cell populations that coordinate together to regulate tumor
    growth and progression [24]. Of these, cancer-associated fibroblasts (CAFs) are a unique cellular subset
    due to their predominantly complex interaction with cancer cells. This complexity is further reflected in
    the dual function that CAFs can exhibit during the course of tumor progression, in either maintaining or
    inhibiting a variety of cancer cell-related processes [25]. Much of this interaction is transmitted through
    CAF-mediated TGF- signaling, where CAFs themselves are significant contributors of overall levels
    of TGF- in the tumor [26,27]. It is worth noting, however, that the concentration of TGF- in the tumor
    or the production of TGF- by cancer cells or CAFs does not directly translate to the bioavailability
    of this cytokine and, hence, its activity. Indeed, TGF- is secreted in the microenvironment as an
    inactive form called small latent complex (SLC) [28]. Latency occurs due to the non-covalent interaction
    between the functional TGF- ligand and its cleaved pro-domain, latency-associated peptide (LAP).
    In the extracellular matrix (ECM), TGF- activators release the active cytokine from its interaction
    with LAP through proteolytic cleavage or structural modifications, such as deglycosylation [2,28,29].
    To date, the identity and function of stromal factors that regulate the conversion of latent TGF- to
    active TGF- [30] or inhibit TGF- activity by binding to the cytokine or its cognate receptors have
    been constantly emerging. These factors are critical in understanding TGF- function as it relates to
    tumor formation and progression and, as such, represent the main subject of the present review.
    2. Cancer Cell/Stroma Crosstalk and TGF- Activity
    Clinical observations have demonstrated that elevated expression of stromal-derived TGFis associated with poor prognosis and locally-advanced disease in breast cancer [31], colorectal
    cancer [26,27,32] and prostate cancer [33]. Stromal-derived TGF- can activate resident stromal cells
    via autocrine signaling, giving rise to tumor-promoting stromal cells, such as CAFs and myofibroblasts.
    These activated stromal cells can in turn induce the upregulation of paracrine factors, such as HGF [34],
    CXCL1 and CXCL16 [35], which promote EMT and the induction of epithelial invasion in adjacent
    epithelial cells [36]. In addition, other studies have provided supportive evidence that TGF- secreted
    by epithelial cancer cells exerts a paracrine influence on stromal cells resulting in increased production
    of ECM and enhanced tumor proliferation [37,38]. Accordingly, given this pleiotropic role of TGF- in
    malignant progression, we examine in this section its impact on some key cancer hallmarks, focusing
    in particular on cancer cell-stroma cell interactions.
    2.1. Metastasis
    TGF- plays an important role in cancer metastasis [8,26,39], essentially by stimulating the
    invasive and metastatic potential of epithelial cells. This occurs in part through its induction of classical
    EMT mechanisms and by driving the intravasation and extravasation of malignant cells to distal sites.
    TGF- -induced EMT has been reported to integrate both SMAD- and non-SMAD-dependent signaling
    elements, requiring crosstalk between PI3K/AKT and SMAD signaling proteins (Figure 1) [40].
    Importantly, collective data have described the transcription factor SNAIL as a key mediator in
    TGF- -induced EMT [40–42]. TGF- 1-induced activation of SNAIL has also been reported to promote
    mesenchymal transition in colon cancer cells [43] breast cancer cells [44], lung cancer cells [45] and
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    endothelial
    cells [46]. TGF- exerts its influence on SNAIL in a two-step process, by increasing
    J. Clin. Med. 2017, 6, 7 4 of 24 its
    transcription and repressing E-cadherin through the recruitment of SMAD proteins [40]. Inhibition
    two‐step process, by increasing its transcription and repressing E‐cadherin through the recruitment of SNAIL
    with anti-sense oligonucleotides blocks TGF- -induced EMT and AKT phosphorylation,
    of SMAD proteins [40]. Inhibition of SNAIL with anti‐sense oligonucleotides blocks TGF‐β‐induced suggesting that SNAIL participates in TGF- -induced EMT by acting upstream of AKT [40]. In lung
    EMT and AKT phosphorylation, suggesting that SNAIL participates in TGF‐β‐induced EMT by cancer, the functional loss of E-cadherin and the acquisition of an EMT phenotype in epithelial cells
    acting upstream of AKT [40]. In lung cancer, the functional loss of E‐cadherin and the acquisition of have been reported to occur through TGF- 1-induced transcription of SNAIL via collaboration of
    an EMT phenotype in epithelial cells have been reported to occur through TGF‐β1‐induced the IL-6/STAT3
    (Figureof 2A)
    Other investigations
    have
    demonstrated
    transcription signaling
    of SNAIL pathway
    via collaboration the [45].
    IL‐6/STAT3 signaling pathway (Figure 2A) [45]. that
    genesOther upregulated
    by
    stromal-derived
    TGF(e.g.,
    JAG1,
    CTGF
    and
    TNC)
    are
    predictors
    of recurrent
    investigations have demonstrated that genes upregulated by stromal‐derived TGF‐β (e.g. and metastatic
    disease
    in colorectal
    cancerrecurrent and [26]. The investigators
    also in reveal
    that the
    initiation of
    JAG1, CTGF and TNC) are predictors of metastatic disease colorectal cancer [26]. The investigators also reveal that the of metastasis by TGF‐β‐induced stromal cells cells
    is via
    metastasis
    by TGF- -induced
    stromal
    cellsinitiation is dependent
    on GP130/STAT3
    signaling
    in cancer
    dependent on GP130/STAT3 signaling in cancer cells via paracrine of IL‐11 (Figure a2A). paracrine
    secretion
    of IL-11 (Figure
    2A). This
    tumor-stroma
    crosstalk secretion consequently
    provides
    survival
    This tumor‐stroma crosstalk consequently a survival metastatic cells and advantage
    to metastatic
    cells and
    correlates provides with a high
    risk of advantage treatmentto failure
    and relapse
    in the
    correlates with a high risk of treatment failure and relapse in the metastatic setting. In addition, in metastatic setting. In addition, in xenografts of breast cancer, the presence of TGF- has been shown
    xenografts of breast cancer, the presence of TGF‐β has been shown to promote metastasis by to promote metastasis by enhancing the motility of stromal cells expressing ↵ smooth muscle actin
    enhancing the motility of stromal cells expressing α smooth muscle actin (α‐SMA) and vimentin (↵-SMA) and vimentin [47].
    [47]. Figure 2. TGF‐β‐mediated cancer cell/stromal cell crosstalk. (A) TGF‐β can activate resident stromal Figure
    2. TGF- -mediated cancer cell/stromal cell crosstalk. (A) TGF- can activate resident
    cells giving rise to cancer‐associated fibroblasts (CAFs). In cancer cells, TGF‐β promotes the stromal cells giving rise to cancer-associated fibroblasts (CAFs). In cancer cells, TGF- promotes
    transcription of SNAIL, the functional loss of E‐cadherin, the acquisition of an EMT phenotype and the transcription of SNAIL, the functional loss of E-cadherin, the acquisition of an EMT phenotype and
    the recruitment of SMAD/AKT signaling proteins. The process of metastasis is further supported by the recruitment
    of SMAD/AKT signaling proteins. The process of metastasis is further supported by
    activated CAFs through secretion of IL‐11 or IL‐6, which further promotes STAT3 signaling in cancer activated
    CAFs through secretion of IL-11 or IL-6, which further promotes STAT3 signaling in cancer
    cells. (B) TGF‐β can trigger angiogenesis in endothelial cells through activation of VEGFR2 by VEGF. cells.The TGF‐β‐mediated angiogenic effect on cancer cells is regulated by TGFβRII/SMAD3‐dependent (B) TGF- can trigger angiogenesis in endothelial cells through activation of VEGFR2 by VEGF.
    The TGF-mediated angiogenic effect on cancer cells is regulated by TGF RII/SMAD3-dependent
    upregulation of fibroblast growth factor‐2 (FGF2) expression and release in the stroma. (C) Cancer cells via of
    the fibroblast
    induction growth
    of aberrant TGF‐β signaling can induce the down‐regulation in cells
    upregulation
    factor-2
    (FGF2)
    expression
    and release
    in the stroma.of (C)CAV1 Cancer
    adjacent fibroblasts leading to a CAF phenotype. The loss of CAV1 has been observed to lead to an via the
    induction of aberrant TGF- signaling can induce the down-regulation of CAV1 in adjacent
    increase in oxidative stress, activation of HIF‐1α and the induction of aerobic glycolysis. Under these fibroblasts
    leading to a CAF phenotype. The loss of CAV1 has been observed to lead to an increase in
    conditions, CAF have been reported to produce and secrete lactate, which is used as fuel by cancer oxidative
    stress, activation of HIF-1↵ and the induction of aerobic glycolysis. Under these conditions,
    cells. Blue arrows indicate proteins secreted by cancer cells. Magenta arrows indicate proteins CAF have been reported to produce and secrete lactate, which is used as fuel by cancer cells. Blue arrows
    secreted by stromal cells. Black arrows indicate overexpression (upward pointing) and indicate proteins secreted by cancer cells. Magenta arrows indicate proteins secreted by stromal cells.
    down‐regulation (downward pointing) of target proteins. Black arrows indicate overexpression (upward pointing) and down-regulation (downward pointing) of
    target
    proteins.
    2.2. Angiogenesis In later stages of cancer development, TGF‐β potently stimulates angiogenesis, mainly through its canonical signaling cascades [48]. In addition, TGF‐β knock‐out mice are not viable and display a phenotype defective in vasculogenesis and angiogenesis [48,49]. The role of TGF‐β in through
    the In
    later stages
    of cancer
    development, TGFpotently stimulates
    angiogenesis,
    mainly
    angiogenesis is multimodal. For example, the TGF‐β‐mediated angiogenic effect on xenografts of its canonical signaling cascades [48]. In addition, TGF- knock-out mice are not viable and display
    a
    prostate cancer is regulated by TGFBRII/SMAD3‐dependent upregulation of fibroblast growth phenotype defective in vasculogenesis and angiogenesis [48,49]. The role of TGF- in the angiogenesis
    factor‐2 (FGF2) expression and release in prostate stroma (Figure 2B) [50]. In addition to FGFD2, 2.2. Angiogenesis
    is multimodal. For example, the TGF- -mediated angiogenic effect on xenografts of prostate cancer
    is regulated by TGFBRII/SMAD3-dependent upregulation of fibroblast growth factor-2 (FGF2)
    J. Clin. Med. 2017, 6, 7
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    expression and release in prostate stroma (Figure 2B) [50]. In addition to FGFD2, TGF- affects
    the expression of other stromal-derived angiogenic factors, including vascular endothelial growth
    factor (VEGF) [51,52], connective tissue growth factor (CTGF) [53] and platelet-derived growth
    factor (PDGF) [54]. Interestingly, TGF- -induced endothelial cell apoptosis has been reported to
    trigger angiogenesis through paracrine and autocrine activation of VEGFR2 by VEGF (Figure 2B) [55].
    On the other hand, other lines of pre-clinical research have presented TGF- as an anti-angiogenic
    factor. Recent evidence in experimental models of CRC has revealed that TGF- -mediated
    signaling under hypoxic stress conditions promotes decreased VEGFA expression, thus reducing
    VEGFA-induced angiogenesis (Figure 2B). The investigation also indicates that TGF- regulates
    VEGFA at the post-transcriptional level by decreasing VEGFA protein stability through ubiquitination
    and degradation [56]. While the specific mechanism of TGF- -mediated control of pro-angiogenic
    and anti-angiogenic processes needs to be fully explored, preclinical evidence suggests that it may
    be dependent on the concentration of TGF- in the endothelium and distinct SMAD signaling
    cascades [48,57,58]. In endothelial cells, TGF- -mediated inhibition of angiogenesis occurs through the
    TGF RI/ SMAD2/3 signaling cascade [58,59], while its stimulatory influence on angiogenesis arises
    via plasminogen-dependent activation of TGF RI/ SMAD1/5 [58].
    2.3. Metabolism
    TGF- promotes metabolic alterations in the tumor microenvironment via “metabolic
    reprogramming” of CAFs through aberrant TGF- signaling and loss of stromal caveolin-1 (CAV1) [37].
    Moreover, pre-clinical investigations have demonstrated that epithelial cancer cells can induce the
    down-regulation of CAV1 in adjacent fibroblasts, leading to a specific, tumor-supportive, CAF
    phenotype (Figure 2C). While the precise mechanism is still not well understood, loss of CAV1
    has been observed to lead to increased oxidative stress, activation of HIF1↵ and the induction of
    aerobic glycolysis/the Warburg effect in the tumor microenvironment [60,61]. Pre-clinical evidence
    has also demonstrated that the loss of CAV1 regulates stromal TGF- via induction of aberrant
    TGF- signaling [62]. Similarly, overexpression of TGF- in stromal cells alters the CAF phenotype
    and promotes tumorigenesis by causing a shift towards catabolic metabolism [37]. These metabolic
    alterations in CAFs can result in an increased production of high-energy metabolites, such as
    L-lactate and ketone bodies [37,62], potentially further fueling the anabolic growth of adjacent cancer
    cells [63].Additional experimental evidence has also shown that TGF- -induced influence of activated
    CAFs on cancer cells can also enhance their mitochondrial activity [37].
    3. Stromal Activators of TGF- in Cancer
    It is well documented that the interaction between the latent TGF- complex, TGFactivators and components of the ECM exerts a regulatory impact on active TGF- levels [64–66].
    Several stromal-derived factors, including proteases, integrins and reactive oxygen species (ROS),
    have been implicated in the activation of latent TGF- . In this section, we examine known stromal
    activators of TGF- , although many of the described molecules are not exclusively stromal-derived,
    but also produced by cancer cells themselves.
    3.1. Matrix Metalloproteinases
    The matrix metalloproteinases (MMPs) are a multi-gene family of zinc-dependent proteases
    produced by malignant epithelial and adjacent stromal cell populations. These enzymes are involved in
    the proteolysis of ECM components and actively participate in several steps of malignant progression,
    such as tissue remodeling and cell migration. The role of MMPs in tumor progression has been
    summarized in a number of recent reviews [67–70].
    Besides their established pro-tumorigenic function, a number of MMPs, such as membrane type 1
    matrix metalloproteinase (MT1-MMP), MMP2, MMP3, MMP9 and MMP13, have been described as
    key elements in the stromal activation of latent TGF- (Figure 3A) [71–75]. Studies have demonstrated
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    that MT1-MMP, MMP2 and MMP9 release latent TGF- 1 from the ECM by proteolytic cleavage of the
    largeJ. Clin. Med. 2017, 6, 7 latent TGF- 1 binding protein-1 (LTBP1) [76]. Moreover, a preclinical investigation by 6 of 24 Tatti et al.
    has demonstrated the particular importance of the MMPs in LTBP1 cleavage [77]. The authors have
    found
    that shRNA-mediated knockdown of MMP expression or potent pharmacological inhibition
    preclinical investigation by Tatti et al. has demonstrated the particular importance of the MMPs in LTBP1 cleavage [77]. The authors have found that shRNA‐mediated knockdown of MMP expression of MMP
    activity prevents the release of LTBP1 from the ECM [77], thus limiting the availability of
    or potent pharmacological inhibition of MMP activity prevents the release of LTBP1 from the ECM soluble TGF- . In addition, it has recently been shown that the LH3 receptor acts as a docking
    [77], thus limiting the availability of soluble TGF‐β. In addition, it has recently been shown that the site in
    recruiting MMP9 to the surface of fibroblasts, which can trigger TGF- activation and
    LH3 receptor acts as a docking site in recruiting MMP9 to the surface of fibroblasts, which can myofibroblast differentiation [78]. Myofibroblasts, in turn, enhance tumor progression by remodeling
    trigger TGF‐β activation and myofibroblast differentiation [78]. Myofibroblasts, in turn, enhance the stroma. In comparison, MMP9 localized on the surface of cancer cells can also activate latent
    tumor progression by remodeling the stroma. In comparison, MMP9 localized on the surface of TGF-cancer cells can also activate latent TGF‐β, albeit in a CD44‐dependent manner [75].CD44 provides a , albeit in a CD44-dependent manner [75].CD44 provides a cell surface docking receptor for
    proteolytically-activeMMP9,
    resulting
    TGF- -induced myofibroblasts
    formation
    [75]. Proteolytic
    cell surface docking receptor for inproteolytically‐activeMMP9, resulting in TGF‐β‐induced cleavage
    of mature TGF- from LAP by MMP2, MMP3, MMP9 and MMP13 has also been reported
    myofibroblasts formation [75]. Proteolytic cleavage of mature TGF‐β from LAP by MMP2, MMP3, to release
    active
    TGF- has in the
    [66]. Maeda
    et al. in have
    a model
    where
    MMP9 and MMP13 also ECM
    been (Figure
    reported 3B)
    to release active TGF‐β the proposed
    ECM (Figure 3B) [66]. Maeda et al. have proposed a model where MMP3‐mediated activation of latent TGF‐β1 consists of MMP3-mediated activation of latent TGF- 1 consists of several steps, including proteolytic cleavage
    several steps, including proteolytic cleavage several novel sites of the LTBP1 molecule of several
    novel
    sites
    of the LTBP1
    molecule
    and of subsequent
    dissociation
    of the
    mature
    TGF- and protein
    subsequent dissociation of the mature TGF‐β protein from the LAP propeptide [73]. The authors also from the LAP propeptide [73]. The authors also demonstrate that treatment with an anti-MMP3
    demonstrate that treatment with an anti‐MMP3 antibody results in the dose‐dependent decrease of antibody results in the dose-dependent decrease of active TGF- 1. It is important to note, however,
    active TGF‐β1. It is important to note, however, that once activated, TGF‐β can also regulate the that once activated, TGF- can also regulate the secretion, expression and activation of all of the
    secretion, expression and activation of all of the aforementioned MMPs, resulting in a bidirectional aforementioned
    MMPs, resulting in a bidirectional regulatory loop [64,67].
    regulatory loop [64,67]. Figure
    3. Stromal
    activators
    ofof TGFmicroenvironment.
    MT1-MMP,
    MMP2
    Figure 3. Stromal activators TGF‐β in
    in the
    the tumor
    tumor microenvironment. (A) (A)
    MT1‐MMP, MMP2 and MMP9 proteolytically cleave latent transforming growth factor‐β binding protein (LTBP), and MMP9 proteolytically cleave latent transforming growth factor- binding protein (LTBP),
    thereby releasing latent TGF‐β from the extracellular matrix. Plasmin, thrombin, BMP1 and fibulin‐2 thereby
    releasing latent TGF- from the extracellular matrix. Plasmin, thrombin, BMP1 and fibulin-2
    MMP3, MMP9 also activate through cleavage or interaction LTBP1. (B) MMP2, also activate
    TGF-TGF‐β through
    cleavage
    or interaction
    withwith LTBP1.
    (B) MMP2,
    MMP3,
    MMP9
    andand MMP13
    MMP13 activate latent TGF‐β via proteolytic cleavage of the latency‐associated peptide (LAP), while activate latent TGF- via proteolytic cleavage of the latency-associated peptide (LAP), while integrins
    integrins expressed on fibroblasts (αvβ3, αvβ5 and αvβ8) bind to the large latent complex (LLC) and expressed on fibroblasts (↵v 3, ↵v 5 and ↵v 8) bind to the large latent complex (LLC) and activate
    activate latent TGF‐β through MT1‐MMP‐dependent cleavage of LAP. (C) Integrins αvβ‐1 and 5 bind latent TGF- through MT1-MMP-dependent cleavage of LAP. (C) Integrins ↵v -1 and 5 bind to the
    to the LLC and induce conformational changes in the latent complex via contractile action from LLC and induce conformational changes in the latent complex via contractile action from activated
    activated fibroblasts. (D) ROS produced by activated fibroblasts via the induction of oxidative stress fibroblasts.
    (D) ROS produced by activated fibroblasts via the induction of oxidative stress from
    from adjacent cancer cells can lead to the oxidation of the LAP domain and induce allosteric changes adjacent
    cancer
    canTGF‐β lead to
    theLAP. oxidation
    of of theCAV1 LAP expression domain and
    induce allosteric
    changes
    that release cells
    mature from The loss in activated fibroblasts is also that
    release
    mature
    TGFfrom
    LAP.
    The
    loss
    of
    CAV1
    expression
    in
    activated
    fibroblasts
    is
    also
    associated
    associated with enhanced oxidative stress and increased production of ROS. (E) Thrombospondin‐1 with (TSP‐1) directly interacts with the LAP domain, inducing conformational rearrangement of LAP and enhanced oxidative stress and increased production of ROS. (E) Thrombospondin-1 (TSP-1)
    altering the interaction of LAP with the mature domain of TGF‐β. The mature (active) form of TGF‐β directly
    interacts with the LAP domain, inducing conformational rearrangement of LAP and altering
    can then bind to its cognate receptor and exert its tumor promoting and tumor suppressive the interaction
    of LAP
    with
    the mature
    domain
    of TGF. The
    mature (active)
    form
    of TGF- can
    then properties. Dashed arrow indicates recruitment of the mature TGF‐β protein to its cognate receptor. bind to its cognate receptor and exert its tumor promoting and tumor suppressive properties.
    Dashed arrow indicates recruitment of the mature TGF- protein to its cognate receptor.
    J. Clin. Med. 2017, 6, 7
    7 of 25
    3.2. Integrins
    The integrins are a family of cell surface adhesion molecules that are known to play an important
    role in cell-ECM interactions and fibrosis [79]. These molecules contribute to the stromal activation of
    latent TGF- mainly by two non-mutually-exclusive mechanisms: (i) protease-dependent interaction
    of latent TGF- and MMPs (Figure 3A,B) [80]; or (ii) induction of conformational changes in the latent
    TGF- complex due to the generation of contractile forces (Figure 3C) [81].
    The first integrin identified as an activator of TGF- 1 and TGF- 3 was ↵v 6, an integrin
    solely expressed in epithelial cells [82]. However, in fibroblasts, integrin ↵v 8 has been reported
    to mediate the activation of various TGF- isoforms through binding with high affinity to the integrin
    binding motif (RGD) of the latent TGF- LAP domain [74]. This coordinated binding leads to
    MT1-MMP-dependent release of active TGF- at the cell surface [74]. Further to this, integrins
    can also contribute to TGF- activation independent of MMP-mediated proteolysis. Indeed, a recent
    study by Sheppard and collaborators has demonstrated the putative role of fibroblast-derived ↵v 1
    in the activation of TGF- 1 in lung and liver fibrosis. The investigation shows that ↵v 1 is able to
    directly bind the LAP domain of TGF- 1, which leads to the release of mature TGF- 1by contractile
    forces [83]. The experimental data also suggest that targeting ↵v 1 with a specific inhibitor could be a
    potential therapeutic component in the treatment of lung and liver fibrosis [83]. Wipff et al. have also
    recently demonstrated that integrin ↵v 5 can activate TGF- 1 on the surface of myofibroblasts, in the
    absence of proteases [81]. Mechanistically, the authors demonstrate that myofibroblast contraction can
    lead to this activation of latent TGF- 1. The cell traction forces that myofibroblasts transmit to the LAP
    domain generate allosteric changes in the latent complex and the subsequent release of the active form
    of TGF- 1 [81].
    An additional line of evidence has also shown that integrins ↵v 3 and ↵v 5 can contribute to the
    establishment of an autocrine TGF- loop through the activation of TGF- 1, in an MMP-independent
    fashion [84]. Integrins ↵v 3 and ↵v 5 are overexpressed in scleroderma fibroblasts compared
    to normal fibroblasts, where this altered expression increases the promoter activity of human
    collagen ↵-2 [84]. Systemic sclerosis is an immune-mediated, multi-system disorder characterized
    by microvasculature damage, circulating auto-antibodies and fibroblast activation. The findings also
    reveal that pre-treatment with anti-↵v 3 and anti-↵v 5 antibodies can reverse the myofibroblastic
    phenotype of scleroderma-derived fibroblasts, indicating that these integrins are crucial players in
    sclerosis pathogenesis [85]. A meta-analysis involving more than 7000 patients has estimated that
    sclerodermic patients have a 75% increased risk of developing cancers of the lung compared to the
    general population [86].
    3.3. Reactive Oxygen Species
    In cancer, the excessive production of reactive oxygen species (ROS) can induce chemical damage
    in proteins, lipids and DNA contributing to diverse tumorigenic effects [87]. While ROS are not a
    specific feature of stromal cells, cancer cells induce increased ROS production in CAFs. This in turn
    mediates CAF senescence and metabolic reprogramming. TGF- has been implicated in a number
    of reactive oxygen-mediated tissue processes, particularly inflammation [88]. Importantly, radiation
    exposure, known to generate ROS, has also been demonstrated as a factor in TGF- 1 activation [89].
    Barcellos-Hoff et al. have shown that irradiation of the latent TGF- complex can significantly increase
    TGF- activity. The authors further propose that ROS can oxidize specific amino acids in the LAP
    domain of the latent TGF- complex, inducing conformational changes that release active TGF- from
    the ECM (Figure 3D) [89].
    As described earlier in this review, deregulated metabolism and oxidative stress in CAFs is
    associated with the loss of CAV1 expression [37]. This loss of CAV1 also correlates with an increase in
    ROS production, leading to enhanced TGF- 1 secretion and aberrant activation of the TGF- signaling
    pathway [37]. In support of these observations, a number of independent studies has highlighted the
    direct role of CAV1 in the TGF- signaling axis. For example, Ayala et al. have shown that silencing
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    of CAV1 in prostatic fibroblasts results in increased TGF- 1 gene expression in tumor cells [90].
    Similarly, other groups have established that inhibition of endogenous CAV1 in murine fibroblasts
    increases TGFbRII gene expression, whereas its sustained expression suppresses TGF- -mediated
    cellular processes [91]. Recently, Jain et al. have demonstrated that mitochondrial-derived ROS plays
    an important role in regulating TGF- in normal human lung fibroblasts [92]. Their results further
    reveal that the use of anti-oxidants targeting mitochondrial ROS significantly attenuates TGF- gene
    expression, without affecting SMAD phosphorylation or nuclear translocation [92]. However, only the
    latent form of TGF- 1 seems to be sensitized by redox-mediated activation. Another investigation has
    determined that sensitivity to ROS activation depends on a conserved methionine residue in LAP- 1.
    The study has also demonstrated that altering this conserved residue with a site-specific mutation
    disrupts the sensitivity of LAP- 1 to ROS-mediated activation [93].
    3.4. Other Stromal Activators of TGF-b
    In addition to the MMPs, integrins and ROS, several other stromal-derived molecules are
    also known for their ability to activate TGF- . Experimental evidence has revealed that the ECM
    protein, thrombospondin-1 (TSP-1), can activate latent TGF- secreted by several cell types, including
    fibroblasts and mammary epithelial cells [94]. TSP1 can bind to both the LLC and the SLC forms of latent
    TGF- [95]. Moreover, Ribeiro et al. have revealed that TSP1-mediated activation of latent TGF- 1
    occurs by direct interaction of complementary residues in TSP1 and LAP, leading to the conformational
    rearrangement of LAP and the modulation of its interaction with the mature domain of TGF(Figure 3E) [96]. Interestingly, TSP1-null and TGF- 1-null mice demonstrate a phenotypic overlap with
    strikingly similar developmental abnormalities [97]. In addition, a co-operative mechanism for TGFactivation, involving TSP-1 and plasmin, has been described. The mechanism was demonstrated
    in a bleomycin-dependent model of pulmonary fibrosis [98]. TSP-1 is, therefore, able to activate
    latent TGF- through different mechanisms in different circumstances with beneficial or deleterious
    consequences. Indeed, while TSP1 expression in CRC tumor stroma was shown to inhibit angiogenesis
    and tumor growth by activating TGF- 1 [99], another group of researcher demonstrated that the
    over-expression of TSP1 and TGF- was responsible for more aggressive forms of glioma [100].
    Another key player in the stromal activation of TGF- is the bone morphogenetic protein 1
    (BMP1). Using a mouse model of embryonic fibroblasts, Ge et al. report that BMP-1 directly cleaves
    LTBP1 at its N- and C-terminal sites, resulting in the release of the LLC from the ECM [72]. Their data
    also demonstrate that the released latent TGF- complex is further processed via MMP2-mediated
    proteolysis of the LAP domain (Figure 3A) [72]. TGF- can also induce the expression of BMP-1,
    resulting in positive feedback regulation of TGF- activity. Of note, deregulation of TGF- /BMP
    activity can lead to developmental defects and pathological disease, including cancer. Fibulins are
    reported to play an important role in the modulation of TGF- activation. They are a family of
    seven secreted glycoproteins with homologous epidermal growth factor-like domains and a unique
    C-terminal structure. Fibulins interact with ECM proteins forming anchoring structures that can
    regulate cell proliferation and migration [101]. Interestingly, similar to TGF- , the fibulins possess
    context-specific pro-tumorigenic and tumor-suppressive properties [102]. Of the different fibulins
    interacting with TGF- 1 (see also stromal inhibitors) fibulin-2 enhances the release of TGF- 1 from
    its latent complex in the ECM (Figure 3A) [103]. The stimulatory role of fibulin-2 on TGF- has been
    further highlighted in a fibulin-2 null mouse model, where depletion of the protein is associated with
    a decrease in canonical TGF- signaling activity [104]. Another line of research based on genomic
    microarray analysis of neuronal stem cells, has reported that inhibition of fibulin-2 leads to the
    blockade of TGF- 1 mediated pro-neurogenic effects. Of note, TGF- can also stimulate the expression
    of fibulin-2, thus establishing a positive feedback loop [105].
    Latent TGF- can also be activated by other stromal-derived proteinases, such as plasmin and
    thrombin, with numerous studies demonstrating that these enzymes are involved in its indirect
    activation via LTBP1 cleavage [106,107]. Cleavage of LTBP1 results in the release of matrix-bound
    J. Clin. Med. 2017, 6, 7
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    TGF- from the ECM, where it is subsequently activated by MMPs (Figure 3A). Accordingly, a study
    examining the relevance of human fibroblast and fibrosarcoma-derived pericellular structures in TGFregulation has revealed that these matrices act as a storage compartment for latent TGF- . The addition
    of plasmin and thrombin to the cellular structures results in the rapid release of LTBP1 [65]. The latter
    observations are consistent with a number of other studies that demonstrate that the latent form of
    TGF- can also be activated by plasmin-mediated proteolysis in co-cultures of endothelial and smooth
    muscle cells [106,108,109].
    4. Stromal Inhibitors of TGF- in Cancer
    One of the most significant consequences of TGF- release into the tumor microenvironment is a
    robust fibrotic response, known as a desmoplastic reaction. The desmoplastic stroma is characterized by
    the overproduction of ECM proteins and enhanced proliferation of cells associated with a myofibroblast
    phenotype [110]. Many solid tumors display a dense and fibrotic stroma, where CAFs, in response
    to the cytokines released by cancer cells, contribute to the overproduction of ECM components.
    This process inevitably leads to an increase in matrix rigidity, myofibroblast contractility [111] and,
    as previously described in this review, the subsequent release of mature TGF- from the LLC. Thus,
    TGF- activation, myofibroblast contraction and ECM remodeling collectively participate in a positive
    feedback loop that drives the initiation and progression of tumors. In the present section, we discuss
    stromal proteins known to suppress TGF- activity.
    4.1. Proteoglycans
    Proteoglycans are a family of highly glycosylated proteins mainly involved in tissue organization
    and in the regulation of collagen fibrillogenesis [112]. Proteoglycans and in particular small
    leucine-rich proteoglycan (SLRP) family members, such as decorin, biglycan, asporin, lumican and
    fibromodulin, exert an inhibitory influence on the TGF- signaling pathways. The mechanism, however,
    leading to TGF- signaling inhibition is variable, largely consisting of SLRP protein binding to:
    (i) soluble TGF- ; (ii) TGF- type I and/or type II receptors; or (iii) latent TGF- , consequently
    impairing ligand-receptor interactions.
    Decorin, a well-known member of the SLRP family, inhibits TGF- signaling activity by binding
    with high affinity to all TGF- isoforms (Figure 4A). Data also reveal that decorin can act as an
    endogenous tumor suppressor through sustained inhibition of tumor growth and angiogenesis [113].
    More importantly, the loss of stromal decorin predicts poor prognosis in metastatic breast cancer
    and is associated with a higher incidence of progressive disease [114]. As demonstrated in a murine
    model of liver fibrosis, decorin null mice display increased expression of both TGF- 1 and its early
    inducible response gene, TIEG [115]. This loss of decorin expression leads to enhanced accumulation
    of ↵-SMA-positive cells and high levels of ERK1/2 and SMAD3 activity [115]. In human mesangial
    cells, other investigations have demonstrated that decorin disrupts TGF- /SMAD signaling events
    (Figure 4C), through a mechanism involving the mobilization of Ca2+ /calmodium-dependent kinase
    II and the phosphorylation of SMAD2 at serine-240, a negative regulatory site [116]. In addition to
    these observations, decorin has also been reported to induce the formation and nuclear translocation
    of SMAD2/SMAD4 hetero-oligomeric complexes [116]. Therefore, an important aspect controlling
    decorin-mediated regulation of TGF- canonical signaling is the nuclear sequestration of cytoplasmic
    co-SMAD4, rendering it unavailable for TGF- receptor-initiated SMAD signaling. In malignant
    glioma cells, decorin contributes to reduced TGF- pathway activity by preventing the synthesis and
    release of TGF- 1 and TGF- 2 [117,118]. These events promote tumor regression and prolong survival
    in experimental murine models [117,118]. Additionally, decorin-mediated inhibition of TGF- 1 activity
    has been shown to regulate the inflammatory profile of immune cells, such as macrophages [119].
    In this cellular subset, suppression of TGF- 1 activity by decorin is associated with reduced levels
    of miR-21, a post-transcriptional repressor of PDCD4, a pro-inflammatory molecule. The increase
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    10 of 25
    in
    PDCD4 leads to a lower production of the anti-inflammatory cytokine, IL-10, and the ensuing
    J. Clin. Med. 2017, 6, 7 10 of 24 establishment of a pro-inflammatory environment [119].
    Biglycan, a small interstitial proteoglycan structurally‐related to decorin, is considered a potent Biglycan,
    a small interstitial proteoglycan structurally-related to decorin, is considered a potent
    modulator of
    of cytokine
    cytokine function
    function due
    due to
    to its
    its ability
    ability to
    to bind
    bind TGFTGF‐β (Figure
    (Figure 4A)
    4A) [120],
    [120], TNF↵,
    TNFα, BMP2,
    BMP2, modulator
    BMP4, BMP6 and WISP1 [121]. In vitro studies conducted in the context of lung fibrosis, have shown BMP4,
    BMP6 and WISP1 [121]. In vitro studies conducted in the context of lung fibrosis, have shown
    that biglycan and decorin can suppress TGF‐β activity in a dose dependent manner [122]. However, that
    biglycan and decorin can suppress TGF- activity in a dose dependent manner [122]. However,
    while the overexpression of decorin is sufficient in reducing TGF‐β‐induced fibrosis, biglycan does while
    the overexpression of decorin is sufficient in reducing TGF- -induced fibrosis, biglycan does not
    not exert a similar effect. A plausible hypothesis could be that these differences are associated with exert
    a similar effect. A plausible hypothesis could be that these differences are associated with the
    the distinct distribution of the two SRLPs within tissue compartments [122]. distinct
    distribution of the two SRLPs within tissue compartments [122].
    Figure 4.4. Stromal
    Stromal inhibitors of TGF‐β the tumor microenvironment. The decorin,
    SLRPs, biglycan,
    decorin, Figure
    inhibitors
    of TGFin thein tumor
    microenvironment.
    (A) The(A) SLRPs,
    biglycan, lumican and asporin, bind with high affinity to TGF‐β, preventing the biologically‐active lumican
    and asporin, bind with high affinity to TGF- , preventing the biologically-active protein from
    protein from binding to its cognate receptor. (B) Fibulin‐3 and ‐4indirectly inhibit TGF‐β activity by binding
    to its cognate receptor. (B) Fibulin-3 and -4indirectly inhibit TGF- activity by interacting
    interacting TGF‐β toRI, leading in
    to TGF
    a decrease complex formation. with TGF- with RI, leading
    a decrease
    RI/TGF in RIITGFβRI/TGFβRII complex formation.
    (C) Decorin
    can (C) Decorin can indirectly disrupt TGF‐β activity by negative regulation of SMAD2 phosphorylation. indirectly disrupt TGF- activity by negative regulation of SMAD2 phosphorylation. (D) Nephrocan
    (D) Nephrocan indirectly regulates activity by inhibiting canonical SMAD3 signaling. and
    (E) indirectly
    regulates
    TGF- activity
    byTGF‐β inhibiting
    canonical
    SMAD3 signaling.
    (E) Fibromodulin
    Fibromodulin and LLC,
    ‐2 bind to the TGF‐β LLC, preventing its release from the ECM. fibrilin-1
    and -2and bindfibrilin‐1 to the TGFpreventing
    its release
    from
    the ECM.
    Fibronectin
    mediates
    the
    Fibronectin mediates of LTBP1 the ECM, thereby suppressing regulation
    of LTBP1
    in thethe ECM,regulation thereby suppressing
    TGF-in 1 bioavailability.
    Solid arrows
    indicate TGF‐β1 bioavailability. Solid arrows indicate activation and/or enzymatic activity while blunted activation and/or enzymatic activity while blunted arrows indicate inhibitory activity (solid—direct;
    arrows indicate inhibitory activity (solid—direct; interrupted—indirect activity). interrupted—indirect
    activity).
    Fibromodulin, another SLRP that shares extensive sequence homology with decorin and Fibromodulin, another SLRP that shares extensive sequence homology with decorin and biglycan,
    biglycan, is known for its role in collagen fibril structural organization [123]. Interestingly, several is known for its role in collagen fibril structural organization [123]. Interestingly, several studies
    studies have reported that the expression of fibromodulin is significantly reduced in metastatic have reported that the expression of fibromodulin is significantly reduced in metastatic cancers
    cancers compared to primary cancers [124–126]. In human fibroblasts, fibromodulin expression compared to primary cancers [124–126]. In human fibroblasts, fibromodulin expression suppresses
    suppresses nuclear factor‐kappa b (NF‐κb) signaling through a series of events that results in their nuclear factor-kappa b (NF-b) signaling through a series of events that results in their diminished
    diminished survival [127]. Moreover, fibromodulin‐mediated regulation of TGF‐β occurs through its survival [127]. Moreover, fibromodulin-mediated regulation of TGF- occurs through its interaction
    interaction with latent TGF‐β (Figure 4E). Experimental evidence has demonstrated that with latent TGF- (Figure 4E). Experimental evidence has demonstrated that fibromodulin binds to
    fibromodulin binds to LLC of TGF‐β, preventing its release from the ECM [120,128]. Importantly, the LLC of TGF- , preventing its release from the ECM [120,128]. Importantly, the direct influence of
    direct influence of fibromodulin on TGF‐β bioavailability has recently been uncovered in a fibromodulin on TGF- bioavailability has recently been uncovered in a fibromodulin-null mouse
    fibromodulin‐null mouse model of skin wound repair [129]. The absence of fibromodulin in this model of skin wound repair [129]. The absence of fibromodulin in this model was associated with
    model was associated with an increase in TGF‐β3 bioavailability in cells of stromal origin, as well as an increase in TGF- 3 bioavailability in cells of stromal origin, as well as a corresponding increase
    a corresponding increase in levels of its cognate receptors, TGFβRI and TGFβRII. However, a recent in levels of its cognate receptors, TGF RI and TGF RII. However, a recent study by Adini et al. has
    study by Adini et al. has demonstrated that fibromodulin exerts a stimulatory influence on TGF‐β1 expression and secretion in endothelial cells in angiogenesis‐dependent disease such as macular degeneration [130]. In their study, endothelial cells incubated with fibromodulin displayed enhanced levels of phosphorylated SMAD1/5, in addition to an increase in migratory and angiogenic capacity. The authors further highlight that fibromodulin‐induced secretion of TGF‐β1 can J. Clin. Med. 2017, 6, 7
    11 of 25
    demonstrated that fibromodulin exerts a stimulatory influence on TGF- 1 expression and secretion in
    endothelial cells in angiogenesis-dependent disease such as macular degeneration [130]. In their study,
    endothelial cells incubated with fibromodulin displayed enhanced levels of phosphorylated SMAD1/5,
    in addition to an increase in migratory and angiogenic capacity. The authors further highlight that
    fibromodulin-induced secretion of TGF- 1 can co-activate TGF RII, stimulating canonical SMAD
    transcriptional complexes [130].Given that emerging evidence has demonstrated the dual function of
    fibromodulin in TGF- modulation, further investigation is required to fully determine its pertinent
    function in different cellular subsets.
    Another SLRP known to modulate TGF- 1 activity is asporin. Asporin structurally differs from
    other SLRP family members due to the presence of an aspartic acid repeat in its N-terminal region
    and the absence of carbohydrates moieties [131]. Aberrant expression of stromal-derived asporin has
    been reported to contribute to tumor promoting effects in several human cancers [131,132]. However,
    our group has been the first to demonstrate a tumor suppressive role for asporin in breast cancer [131].
    In our study, we have shown that CAF-secreted asporin leads to the inhibition of TGF- -induced
    SMAD2 phosphorylation and the reversal of an EMT phenotype in breast cancer cells. Collectively,
    our results suggest that asporin directly binds TGF- 1 (Figure 4A), rather than acting as competitive
    inhibitor of its cognate receptor, TGF RII. In support of our conclusions, a previous report has
    established that the interaction between asporin and TGF- 1 occurs between residue His159 and Asn205
    of the asporin amino acid sequence [133]. Interestingly, the expression of asporin positively correlates
    with active levels of TGF- 1, indicating the existence of a negative feedback loop that can lead to better
    regulation of TGF- 1 activity [131]. This loop can be disrupted in triple negative breast cancer, where
    overexpression of IL-1 results in the effective inhibition of CAF-induced asporin expression [131].
    Lumican is a TGF- modulator known to be overexpressed in several solid tumors, however
    its exact role in tumorigenesis remains poorly understood. Clinical evidence suggests that lumican
    expression in the stromal compartment of pancreatic and breast malignancies is associated with lower
    patient survival and a higher incidence of metastatic disease [134,135]. On the other hand, pre-clinical
    evidence has demonstrated that lumican reduces the proliferative capacity and adhesive properties of
    osteosarcoma cells in the ECM [136]. Mechanistically this inhibitory activity occurs via high affinity
    binding of lumican to TGF- 2 (Figure 4A) and the negative regulation of downstream targets such as
    SMAD2, integrin 1 and FAK [136,137]. Interestingly, down-regulation of lumican mRNA levels can
    abrogate the latter effects, further indicating that lumican influences the bioavailability of TGF- 2 [136].
    Nephrocan, a member of a new class of SLRPs, contains the canonical cysteine cluster at the
    N-terminal region and the 17 leucine-rich repeat (LRR) motif. However, nephrocan differs from
    other SLRP members, due to the presence of four cysteine residues in the C-terminal flanking
    domain, five potential N-glycosylation sites, and a polyacidic amino acid tail at the C- terminus.
    Following the general trend of the other SLRPs, nephrocan has also been reported to function as an
    endogenous inhibitor of canonical TGF- signaling, exerting its tumor suppressive function through
    negative regulation of phosphorylated SMAD3 (Figure 4D) [138]. The entire sequence of events behind
    nephrocan-mediated inhibition of TGF- biological activity is, however, very poorly understood and
    thus requires further exploration.
    4.2. Fibrillins
    The fibrillins are evolutionally considered as the main structural proteins of microfibrils [139].
    These proteins have been described to regulate the bioavailability of local TGF- during tissue
    formation and remodeling. The mechanism by which fibrillin-1 inhibits the activation of TGFis not well elucidated, but it is thought that it may involve the association of the LLC with fibrillin-1
    rich microfibrils (Figure 4E) [140]. Accordingly, fibrillin-1 deficiency, causes the release of elevated
    amounts of active TGF- from the ECM, a condition that in Marfan syndrome patients is associated
    with severe cardiovascular disease, developmental emphysema and skeletal abnormalities [101].
    Fibrillin-2 knock-out mice, display reduced bone formation. This phenotype is dependent on the
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    aberrant activation of latent TGF- via its interaction with the LLC (Figure 4E), and the consequent
    blunting of OSTERIX expression, the transcriptional regulator of osteoblast maturation, and collagen
    I, the structural template for bone mineralization [102]. In addition, fibrillin-1 and -2 can directly
    bind the propeptides of other TGF- superfamily members, such as BMP2, BMP4, BMP7, BMP10,
    and GDF8 [104]. The fibrillin-1 promoter has been reported to be hypermethylated in colorectal cancer
    cell lines [141], colorectal cancer patient samples [142] and endothelial tumor cells [143]. Consequently,
    the FBN1 gene can be considered as a potential tumor suppressor and its down-regulation could play
    a role in tumor angiogenesis.
    4.3. Fibulins
    Fibulin-3, expressed and secreted by both cancer cells and fibroblasts, has recently been reported
    to be down-regulated in several tumor types [144,145]. Fibulin-3 inhibits the TGF- canonical
    signaling pathway essentially by interacting with TGF- RI (Figure 4B), thus leading to a decrease in
    TGF RI/TGF RII complex formation [146]. As demonstrated in an in vitro model of breast cancer,
    fibulin-3 overexpression limits TGF- -induced EMT, cell migration, invasion and endothelial cells
    permeability [146].In addition, aberrant promoter methylation and the resultant loss of fibulin-3 gene
    expression, is associated with a higher risk of metastasis and tumor progression in breast cancer [146],
    CRC [147], lung cancer [148] and hepatocellular carcinoma [149]. Similar to Fibulin-3, Fibulin-4
    has also been reported to suppress canonical TGF- signaling (Figure 4B). Experimental evidence
    has demonstrated that patients with a cardiovascular pathology harboring a recessive Fibulin-4
    mutation, display increased phosphorylation of SMAD2/3 and enhanced CTGF expression in their
    tissue samples [150]. Additional data from the same study also establishes that an increased level of
    phosphorylated SMAD2 in patient-derived skin fibroblasts is associated with the mutant fibulin-4
    cohort [150]. These observations are in agreement with another pre-clinical investigation demonstrating
    conditional knockout of fibulin-4 enhances TGF- signaling activity [151]. However, following the
    observation that fibulin-4 deficiency results in marked upregulation of ERK1/2 phosphorylation,
    the investigators have suggested that this protein may also impair non-canonical TGF- signaling
    activity [151]. While the aforementioned findings may have a relevance in a cancerous setting, the body
    of evidence linking the fibulins to tumor development is yet to be fully elucidated.
    4.4. Fibronectin
    Fibronectin is a glycoprotein involved in tissue repair and ECM regulation [152,153]. Similar to
    many members of the integrin receptor family, fibronectin also plays a critical role in LTBP1 deposition
    in the ECM of osteoblasts and fibroblasts [152]. Moreover, Dallas et al. have demonstrated that
    fibronectin is incorporated into the ECM prior to LTBP1 deposition, thus creating a temporary
    scaffold template that gradually depletes during the maturation of fibrillar networks (Figure 4E) [154].
    As previously described in this review, LTBP1 is a critical part of the TGF- 1 latent complex, therefore
    fibronectin-mediated regulation of LTBP1 in the ECM directly diminishes TGF- 1 bioavailability.
    However, other data suggests that TGF- 1 can also induce the expression of fibronectin [155] and its
    receptor, integrin ↵5 1 [156] in endothelial cells, creating a key positive-feedback loop that regulates
    ECM deposition and turnover.
    5. Clinical Outlook: Targeting Stromal Modulators of TGF- in Cancer
    Considering its multi-modal role in tumor progression, targeting the TGF- signaling axis
    represents a promising therapeutic prospect in cancer therapy. Several classes of TGF- pathway
    inhibitors have been evaluated in the preclinical setting including TGF- neutralizing monoclonal
    antibodies, anti-TGF receptor monoclonal antibodies, anti-sense oligonucleotides and small molecule
    kinase inhibitors. Importantly, some of these agents are currently under various clinical phases of
    development, demonstrating a good safety profile but modest efficacy in a wide variety of human
    cancers. The anti-tumor activity and clinical impact of these pharmacological agents has been
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    extensively reviewed by a number of investigators [157–160]. The lack of significant therapeutic
    outcomes indicates that we still need to understand more about the complex and intricate mechanisms
    involved in TGF- activity. As described previously, the bioavailability of active TGF- depends
    significantly on the activity of distinct TGF- modulators located in the ECM. The regulation of stromal
    proteins that directly or indirectly alter TGF- signal transduction cascades might be exploited as
    a potential therapeutic strategy in targeted cancer treatment. For example, adenovirus mediated
    decorin gene transfer (Ad-DCN), has been reported to significantly reduce growth in tumor xenografts.
    (Ad)-mediated transfer and expression of human decorin cDNA induced apoptosis in vivo via
    overexpression of p21, a potent inhibitor of cyclin-dependent kinases, and Caspase-8. Moreover,
    data has revealed that the effect of decorin is specific for tumor cells, as neither apoptosis nor growth
    inhibition was observed in non-cancerous cells [161]. Accordingly, a clinical overview of various
    anti-cancer agents designed to either suppress or stimulate some stromal activators and inhibitors of
    TGF- , is shown in Table 1.
    Table 1. Clinical development of agents targeting TGF- modulators.
    Name
    Class
    Target
    Developmental Stage
    Peptidomimetic
    Peptidomimetic
    Small molecule
    Small molecule
    Small molecule
    Peptidomimetic
    Tetracycline derivative
    Tetracycline derivative
    Broad spectrum
    Broad spectrum
    MMP-2, 8, 9
    MMP-2, 3, 9, 14
    MMP-2, 3, 9, 13
    MMP-1, 2, 9
    Broad spectrum
    N/A
    Cancelled in phase III
    Cancelled in phase III
    Cancelled in phase I
    Cancelled in phase III
    Cancelled in phase III
    Cancelled in phase III
    Phase I/II
    Approved/ongoing
    Monoclonal antibody
    Monoclonal antibody
    Cyclic RGD peptide
    Small molecule
    Small molecule
    Small molecule
    Small molecule
    7
    ↵v 3, ↵v 5
    ↵v 3, ↵v 5
    FAK
    FAK
    N/A
    FAK, PYK2
    Cancelled in phase II
    Phase I/II
    Cancelled in phase III
    Phase I
    Phase I
    Phase II
    Phase II
    Monoclonal antibody
    ED-B domain of fibronectin
    Phase II
    Matrix Metalloproteinases
    Batimastat [162]
    Marimastat (BB-2516) [163]
    CGS-27023A (MMI270) [164]
    Prinomastat (AG3340) [165]
    Tanomastat (BAY-129566) [166]
    BMS-275291 (D2163) [167]
    Metastat (COL-3) [168]
    Doxycycline [169]
    Integrins
    Vitaxin/etaracizumab [170]
    Intetumumab (CTNO 95) [171]
    Cilengitide [172]
    PF-00562271 (VS-6062) [173]
    GSK2256098 (NCT00996671)
    VS-4718 (PND-1186) [174]
    PF-04554878 (VS-6063) (NCT01951690)
    Fibronectin
    L-19 (NCT02076633)
    5.1. MMPs
    One of the most extensively studied drug targets are the MMPs. TGF- and MMPs are
    mutually regulated in normal and cancer tissues. As previously reported in this review, MMPs
    activate latent TGF- which in turn, up-regulates MMPs production in both cancer and stromal
    cells. TGF- and MMPs, both contribute to the progression and metastatic potential of tumors. Thus,
    the inhibition of the amplification loop operated between the TGF- and MMP system in tumor cells
    could impair cancer dissemination, proliferation and survival. In addition, the validity of MMPs as
    therapeutic targets has been investigated in several cancer types. MMP inhibitors such as tanomastat,
    have demonstrated considerable preclinical efficacy and are well tolerated in cancer patients [166].
    However, phase III assessment of these inhibitors has demonstrated a lack of survival benefitcompared
    to standard chemotherapy [175]. A more promising example is doxycycline, an FDA-approved
    antibiotic, also known to inhibit MMP secretion [176]. Treatment with doxycycline is associated
    with reduced tumor growth and metastastic spreading in experimental models of breast cancer [169]
    and pancreatic cancer [177]. Moreover, doxycycline is non-toxic to normal cells, has anti-inflammatory
    properties and is known to cross the blood-brain barrier, pointing to its therapeutic value in treating
    brain malignancies [178]. The anti-tumor efficacy of doxycycline is currently being tested in primary
    cancers (NCT02874430) and metastatic breast cancer (NCT01847976). A key future step is seen in the
    development of combined therapy that specifically targets the TGF- -MMP axis.
    J. Clin. Med. 2017, 6, 7
    14 of 25
    5.2. Integrins
    The integrins, are another class of TGF- modulators that have been identified as attractive
    drug targets [179]. The active cross-talk between TGF- and integrins is triggered in tumors in
    response to several stimuli including hypoxia, oxidative stress or therapy, and it promotes tumor
    survival. In a pre-clinical model of non-small cell lung cancer (NSCLC),the concomitant inhibition of
    TGF- 1 and integrin 3 silencing, resulted in decreased lymph node metastasis. The data provided
    in the study support the hypothesis that combined targeted therapy toward TGF- and 3 integrin
    could be a promising approach to attenuate metastatic lung cancer [180]. Preclinical and clinical
    studies with various integrin antagonists have demonstrated their effectiveness in blocking tumor
    progression.Integrin antagonists, currently in clinical trials, include monoclonal antibodies and
    RGD peptide mimetics. Etaracizumab, a monoclonal antibody targeting integrin ↵v 3, showed
    a good efficacy in preclinical studies and was one of the first drug targeting integrins to enter into
    clinical trials [179]. However, while etaracizumab demonstrated a favourable toxicity profile in
    initial clinical studies, it was discontinued after phase II evaluation following negative outcome data.
    Likewise, intetumumab, a dual-specific monoclonal antibody targeting integrin ↵v 3 and ↵v 5 has
    also shown disappointing clinical results. Clinical investigation in melanoma patients demonstrated
    that intetumumabmonotherapy or its combination withstandard therapy did not improve overall
    survival compared to placebo [181]. Interestingly, however, cilengitide, a cyclic pentapeptide designed
    to selectively inhibit integrin ↵v 3 and ↵v 5, has demonstrated in phase II clinical evaluation, robust
    anti-tumor activity when combined with systemic therapy in the treatment of recurrent glioma [182].
    The clinical activity of cilengitide was unexpectedly less promising in phase III evaluation. In this study,
    the cilengitide combined with systemic therapy did not improve patient outcome, indicating that it
    will not undergo further clinical development [172]. Alternatively, the integrin signalling pathway
    can be suppressed with the use of Focal Adhesion Kinases (FAK) small molecule inhibitors such as
    VS-6062 and GSK2256098. The efficacy of these inhibitors has been evaluated in phase I clinical trials
    initiated for several solid tumors, including head and neck cancer, prostate cancer, pancreatic cancer
    and ovarian cancer [183].
    5.3. Fibronectin
    Fibronectin has also been examined as a candidate target in the treatment of malignant disease.
    However, unlike the previously discussed targets, fibronectin has been developed primarily for
    antibody-based drug conjugates. Indeed, the abundance, accessibility and stability of certain isoforms
    of fibronectin suggests it bears excellent properties for the intratumoral delivery of therapeutic
    antibodies [184]. In cancer and other angiogenesis-related disease, the majority of clinical studies have
    been performed using the ScFv (L19), an antibody against a fragment of fibronectin. To date, more than
    30 L19-based biopharmaceuticals have been tested in preclinical trials. For example, Santimaria et al.
    have shown that radiolabelled L19 is accumulated in tumor lesions in aggressive types of lung cancer
    and colorectal cancer [185]. Furthermore, other investigators have demonstrated decreased tumor
    growth in mice grafted with subcutaneous tumors after intravenous injection of L19 [186]. Interestingly,
    the L19 antibody has also been fused with a large number of chemical derivatives and fusion proteins
    such as cytokines and chemokines. In preclinical studies, the fusion of L19 with IL-2 has shown potent
    tumor-targeting effects and therapeutic efficacy in orthotopic models of hepatocellular carcinoma and
    pancreatic cancer [187]. The delivery of TNF↵ to tumor blood vessels has also been investigated by
    using a fusion protein composed of mouse TNF-↵ and a high-affinity antibody fragment targeted to the
    extradomain B of fibronectin (L19mTNF-↵) [188]. Radiolabeled L19mTNF↵ selectively targeted tumor
    neovasculature and demonstrated significant anti-tumoractivity. Interestingly, the safety and efficacy
    of L19-TNF↵ combined with L19-IL2 has been investigated in a phase II clinical study for patients with
    metastatic melanoma [189]. A single intratumoral administration of the L19-TNF↵ /L19-IL2 mixture
    was sufficient to eliminate the tumor, while neither of these agents alone was effective [189]. Finally,
    the recombinant human fibronectin fragment (FN-CH296, RetroNectin), designedto enhance T-cell
    J. Clin. Med. 2017, 6, 7
    15 of 25
    therapy in patients with advanced cancers has shown a good safety profile and a high level of efficacy
    in phase I clinical evaluation [190].
    6. Conclusions
    The importance of TGF- as a regulator of host-tumor interactions during the initiation and
    progression of human cancers is well documented. Indeed, the tumor stroma is an important element
    in cancer development and a large proportion of the cells that mediate stromal-epithelial interactions
    are of fibroblastic origin. It cannot be excluded, however, that TGF- - induced bi-directional signaling
    between epithelial cells and other cell types within the stromal microenvironment, such as endothelial
    cells and inflammatory cells, may also play an important role in tumor formation. This, however,
    has been far less investigated.
    The present review has explored the role of stromal modulators of TGF- in cancer. These stromal
    modulators, however, need not only be limited to cancer, but can also play a role in a host of other
    pathologies and degenerative disease. The MMPs, integrins, ROS, and other stromal-derived molecules
    activate TGF- by assessing the latent cytokine, generating the mature protein and thereby regulating
    its bioavailability. In addition, under normal physiological conditions a homeostatic balance exists
    between active levels of TGF- and its sequestered latent form. However, in pathological conditions
    such as cancer and inflammation, the integrity of the ECM is lost thus enabling stromal cells such
    as fibroblasts and CAFs to assess the defective ECM and induce increased expression of activators
    of latent TGF- , as part of the process of repair [29]. This shift in balance in the physiological and
    pathological setting, broadly impacts the bioavailability of TGF- ; its apparent paradoxical effects on
    malignant cells and their biological processes. On the other hand, the mechanism underlying negative
    control of TGF- by inhibitors such as the proteoglycans, fibrillins, fibulins and fibronectin, is more
    diverse compared to its positive control and activation. The interaction of TGF- inhibitors and the
    cytokine are not only limited to their interaction with its sequestered latent form, but also with their
    inhibition of the activity of downstream signaling protein such as SMAD2 and SMAD3. Moreover,
    the mature active form of TGF- can also induce the expression of some of these stromal inhibitors such
    as fibronectin [155] and asporin [131], further highlighting the paradox of TGF- and the complexity
    of its regulation in the tumor microenvironment. Taken together, additional work is needed to better
    classify these stromal modulators of TGF- and their impact on malignant disease. Further elucidation
    will thus depend on further identification of the underlying molecular mechanisms associated with
    these modulators of TGF- , through profiling and OMICS approaches. This will ultimately enable
    greater understanding of the tumor microenvironment and its fundamental process, paving the way
    for a new era of personalized medicine.
    Acknowledgments: The authors are thankful to University of Liège, the National Fund for Scientific Research
    (FNRS) as well as Televie (FNRS) for financial support.
    Author Contributions: B.C., I.A.U., J.B., V.C. and A.T. drafted the paper. A.T. supervised and directed the work.
    J.B., B.C., I.A.U. and A.T. created the figures and table. All authors participated in proofreading.
    Conflicts of Interest: Authors declare no conflict of interest.
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