Growth factors and pathogenesis

Accepted Manuscript Growth factors and pathogenesis Md Soriful Islam, Stefania Greco, Milijana Janjusevic, Andrea Ciavattini, Stefano Raffaele Giannubilo, Assunta D’Adderio, Alessandra Biagini, Rosamaria Fiorini, Mario Castellucci, M.D., Ph.D., Pasquapina Ciarmela, Ph.D. PII:

S1521-6934(15)00164-9

DOI:

10.1016/j.bpobgyn.2015.08.018

Reference:

YBEOG 1545

To appear in:

Best Practice & Research Clinical Obstetrics & Gynaecology

Received Date: 13 August 2015 Accepted Date: 27 August 2015

Please cite this article as: Islam MS, Greco S, Janjusevic M, Ciavattini A, Giannubilo SR, D’Adderio A, Biagini A, Fiorini R, Castellucci M, Ciarmela P, Growth factors and pathogenesis, Best Practice & Research Clinical Obstetrics & Gynaecology (2015), doi: 10.1016/j.bpobgyn.2015.08.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Islam et al. 1

ACCEPTED MANUSCRIPT Full title: Growth factors and pathogenesis

Md Soriful Islam1,2, Stefania Greco1, Milijana Janjusevic1, Andrea Ciavattini3, Stefano Raffaele Giannubilo3, Assunta D’Adderio1, Alessandra Biagini3, Rosamaria Fiorini4, Mario

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Castellucci1*, Pasquapina Ciarmela1, 5*

Department of Experimental and Clinical Medicine, Faculty of Medicine, Polytechnic

Biotechnology and Microbiology Laboratory, Department of Botany, University of Rajshahi,

Rajshahi-6205, Bangladesh 3

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University of Marche, Ancona, Italy

Department of Clinical Science, Polytechnic University of Marche, Ancona, Italy

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Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy Department of Information Engineering, Polytechnic University of Marche, Ancona, Italy

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*CORRESPONDING AUTHOR’S:

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1) Pasquapina Ciarmela, Ph.D.

Department of Experimental and Clinical Medicine,

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Faculty of Medicine, Polytechnic University of Marche, via Tronto 10/a, 60020 Ancona, Italy Phone: +390712206270, Fax: +390712206087, E-mail: [email protected] 2) Mario Castellucci, M.D., Ph.D. Department of Experimental and Clinical Medicine, Faculty of Medicine, Polytechnic University of Marche, via Tronto 10/a, 60020 Ancona, Italy Phone: +390712206086, Fax: +390712206087, E-mail: [email protected]

Islam et al. 2

ACCEPTED MANUSCRIPT Abstract

Growth factors are relatively small and stable, secreted or membrane-bound polypeptide ligands, play important role in proliferation, differentiation, angiogenesis, survival, inflammation, and tissue repair, or fibrosis. They exert multiple effects through activating

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signal transduction pathways by binding to their receptors on the surface of target cells. A considerable amount of studies have demonstrated a central role of growth factors and their signaling pathways in the pathogenesis of uterine leiomyomas. A number of differentially

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expressed growth factors have been identified in leiomyoma and myometrium. Those growth factors can activate multiple signaling pathways (Smad 2/3, ERK 1/2, PI3K and β-catetin)

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and regulate major cellular processes, including inflammation, proliferation, angiogenesis and fibrosis which are linked to uterine leiomyoma development and growth. In this chapter, we will discuss role of growth factors and their signaling pathways in the pathogenesis of uterine

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leiomyomas.

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Keywords: uterine leiomyoma; uterine fibroid; growth factors; activin-A, TGF-β; VEGF

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UTERINE FIBROIDS AND GROWTH FACTORS Uterine leiomyomas are benign (non-cancerous) tumors originating from the smooth muscle of the uterus (myometrium), and are the most common indication for hysterectomy in the

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world (1). They affect about 77% of women of reproductive-age, but approximately only 25% of reproductive-age women bear clinically apparent tumors (2, 3). Heavy or abnormal uterine bleeding, pelvic pain or pressure, infertility, and recurrent pregnancy loss are

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associated with leiomyoma. Despite the high prevalence, significant health problems and huge economical impact on healthcare system, relatively little is understood about the

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pathogenesis of uterine leiomyoma. Consequently, medical treatments are still limited. Growth of leiomyoma is believed to be dependent on ovarian hormones activity through intermediate elements like growth factors (4, 5).

Growth factors are polypeptides or proteins that are secreted by a number of cell

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types. They are important for regulating a variety of cellular processes, such as proliferation, differentiation, angiogenesis, survival, inflammation, and tissue repair, or fibrosis. Growth factors exert their effects on leiomyoma growth through activating multiple signal

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transduction pathways, including Smad 2/3, ERK 1/2, PI3K and β-catenin by binding to their

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receptors (6-9) (Figure 1). A great deal of studies reported that multiple growth factors, including activin-A (10), acidic fibroblast growth factor (aFGF) (11), basic fibroblast growth factor (bFGF) (12), epidermal growth factor (EGF) (13), heparin-binding-EGF (12), insulinlike growth factor (IGF) (14), myostatin (10), platelet-derived growth factor (PDGF) (15), transforming growth factor-β (TGF-β) (16, 17), TGF-α (13) and vascular endothelial growth factor (VEGF) (18, 19) are differentially expressed in myometrium and leiomyoma.

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ACCEPTED MANUSCRIPT ACTIVIN-A

Activin-A is a pleiotropic growth factor belonging to the transforming growth factor-β (TGFβ) superfamily. It was originally isolated based on their activity in regulating folliclestimulating hormone released from the anterior pituitary (20). Later its functions has been

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extended in cell proliferation, differentiation, apoptosis, immune response, wound repair, and fibrosis (21, 22). Activin-A exerts multiple functions through the activation of Smad 2/3 dependent signaling pathway by binding to type II (ActRIIA or ActRIIB) and type I receptor

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(ActRIB, also known as activin receptor like kinase 4 or ALK4). Activin A/Smad 2/3 signaling can be regulated by binding protein follistatin (23), follistatin-related gene (FLRG)

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(24), and Cripto (25) at extracellular and plasma membrane level. In addition, Smad7 is known as an inhibitory downstream modulator of activin/TGF-β-like signaling, and its expression is induced by activin-A (26).

The role of activin-A in leiomyoma and myometrial cell functions has been studied

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(6, 10, 27). The mRNA expression levels of activin-A (10, 28) and FLRG (10) were found to be highly expressed in leiomyoma compared to myometrial tissues, whereas the receptors (ALK4, ActRIIA, and ActRIIB), follistatin, and Smad7 mRNAs were unchanged (10).

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Activin-A has been reported to downregulate primary myometrial cell proliferation (6) but

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not leiomyoma cells (6). Interestingly, activin-A was reported to increase mRNA expressions of extracellular matrix components (collagen1A1, fibronectin, and versican) in primary leiomyoma cells compared to untreated cells (6). Furthermore, activin-A significantly increases phosphorylation of Smad signaling components, Smad2 and Smad3 in both leiomyoma and myometrial cells compared to untreated cells (6), suggesting that the fibrotic role of activin-A is mediated, at least in part, by activation of Smad 2/3 signaling pathway.

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ACCEPTED MANUSCRIPT ACIDIC FIBROBLAST GROWTH FACTOR

Acidic fibroblast growth factor (aFGF) (also known as FGF-1) is a member of FGF family of growth

factors

and

plays

important

roles

in

proliferation

and

angiogenesis.

Immunohistochemical localization of aFGF was detected in human uterine leiomyomas with

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expression localized primarily to the smooth muscle cells (29). Wolanska and Bankowski reported that leiomyomas contained several times more aFGF compared to myometrium (11).

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BASIC FIBROBLAST GROWTH FACTOR

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis, belongs to the FGF

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family of growth factors. Leiomyomas show an increased protein expression of bFGF compared to normal myometrium (12). Of note, bFGF was found primarily bound to the ECM of myometrium and fibroids (12). This observation suggests that the enhanced growth of leiomyomas may be due, in part, to the presence of large quantities of bFGF that are stored

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in the ECM of these tumors. Furthermore, FGFR-1 and FGFR-2 expressions have also been demonstrated to be increased in leiomyoma compared with adjacent myometrium (11, 30). Rauk and colleagues reported that bFGF is mitogenic for both human uterine myometrial and

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leiomyoma cells, but leiomyoma cells are less responsive (31)

EPIDERMAL GROWTH FACTOR Epidermal growth factor (EGF), a member of the EGF growth factor family, regulates proliferation, survival, differentiation, tissue homeostasis, and tumorigenesis through activating multiple signaling pathways by binding to EGF receptor (also known as ErbB1 or HER1). The mRNA expressions of both EGF and ErbB1 have been identified in myometrial and leiomyoma cells (32). Dixon and co-workers reported that protein expression of EGF and EGF-R was observed in the cytoplasm of smooth-muscle cells of leiomyomas and matched

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myometrium (13). However, intensity and percentage of positive staining of EGF was significantly decreased in leiomyoma tissue from the proliferative phase compared to matched myometrium (13). EGF has been reported to be mitogenic for both cultured myometrium and leiomyoma cells (7, 33, 34). Mesquita et al. reported that stimulation of

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primary leiomyoma cells with EGF caused a marked increase in NADPH oxidase derived intracellular reactive oxygen species (ROS) production that activates mitogen-activated protein kinase (MAPK)3/MAPK1 (ERK 1/2) signaling leading to cell proliferation (7).

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Furthermore, it has been shown that EGF treatment induced transient phosphorylation

proliferation (35).

HEPARIN-BINDING EGF

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activation of EGFR and AKT in leiomyomal smooth muscle cells, followed by cell

Heparin-binding EGF (HB-EGF) is a member of the EGF family of growth factors. HB-EGF

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binds to an EGF-R following EGF-R (HER1) autophosphorylation, and exerts various biological activities such as cell proliferation and migration. The presence of HB-EGF protein expression has been detected in both normal myometrium and fibroid tumor tissues

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(36). However, HB-EGF showed a decreased mRNA and protein expression in leiomyomas

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compared to normal myometrium (12). The receptor HER1 protein was detected in leiomyoma and myometrial cells (13) with elevated expression in myometrial cells compared to leiomyoma cells (37). Treatment of leiomyoma and myometrial cells with HB-EGF upregulated HER1 expression in leiomyoma and myometrial cells (37). Wang and co-workers reported that HB-EGF showed stimulatory effect on cultured leiomyoma cells and myometrial cells and inhibited apoptosis, at least in part, by augmentation of HER1 expression (37).

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ACCEPTED MANUSCRIPT INSULIN-LIKE GROWTH FACTOR

Insulin-like growth factors (IGFs) are multifunctional peptides with high sequence similarity to insulin, plays an important role in regulating cell proliferation, differentiation, apoptosis, and transformation. IGFs have two ligands, included IGF-I and IGF-II. They exert their

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actions by interacting with a specific receptor on the cell membrane, namely, IGF-IR and IGF-IIR, and the interaction is regulated by a group of specific binding proteins (IGFBP 1-6). Both IGF-I and IGF-II mRNAs have been detected in leiomyoma and myometrium (38).

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Leiomyomas showed higher IGF-I and IGF-IR protein levels compared to myometrium (14). IGF-I was reported to increase the proliferation of uterine leiomyoma cells (30, 39), partly by

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upregulation of PCNA expression and downregulation of apoptosis (40). Furthermore, IGF-I increased phosphorylation of IGF-IRβ, Shc and MAPKp44/42 (ERK1/2) in uterine leiomyoma cells (30). Peng and colleagues reported that IGF-AKT signaling was positively associated with large and actively growing fibroids (41). It was shown that larger fibroids had

MYOSTATIN

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higher levels of IGF-I and p-AKT activity when compared with small ones (41).

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Myostatin, or growth differentiation factor-8 (GDF-8), a secreted protein that is first cloned

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in 1997 as a member of the TGF-β superfamily. It is well known for its ability to negatively regulate skeletal muscle growth. Myostatin exerts its actions in different cellular processes, such as cell proliferation, skeletal muscle fibrosis and adipogenesis by activating Smad 2/3 signaling pathway through binding to type II (ActRIIB) and type I receptor [(ALK4 or ActRIB) or (ALK5 or TGF-βRI)]. Myostatin/Smad 2/3 signaling can be interrupted by binding protein follistatin (23) and FLRG (24), Cripto (10) and Smad7 (42). The role of myostatin in leiomyoma and myometrial cell functions has been studied (6, 10, 43). We found that myostatin (10, 28) and FLRG (10) mRNA expression levels were

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higher in leiomyoma compared to myometrial tissues, and the receptors (ALK4, ALK5, and ActRIIB), follistatin, and Smad7 mRNAs were unchanged (10). Cripto mRNA was found to be expressed only in human uterine leiomyoma explants (10). Myostatin significantly reduced cell proliferation in primary myometrial cells but not in leiomyoma cells (6),

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interestingly, myostatin mRNA expression correlated significantly and directly with the intensity of dysmenorrhea (28). Furthermore, we demonstrated that myostatin can induce

and leiomyoma cells (6).

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PLATELET-DERIVED GROWTH FACTOR

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phosphorylation of Smad2 and 3 but does not affect pERK or p38 MAPK in both myometrial

Platelet-derived growth factor (PDGF) is a dimeric glycoprotein consists of four homodimers PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD, and one heterodimer, PDGF-AB. This multifunctional growth factor exerts biological effects on cellular chemotaxis, proliferation,

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matrix synthesis, antiapoptosis, and vascularization through binding to two different receptors, PDGF-α and PDGF-β. PDGF and PDGF-R expressions have been documented in both normal myometrium and leiomyoma (12, 44). The elevated protein expressions levels of

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PDGF-AA and PDGF-BB and of their receptors were found in leiomyoma compared to

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myometrial tissues (15, 30). In addition, PDGF-CC was reported to be higher expressed in leiomyoma compared to adjacent myometrial tissues and smooth muscle cells (45, 46). The profibrotic role of PDGF has been documented in myometrium and leiomyoma cells as it increased collagen alpha1 (I) expression in those cells (15). PDGF was reported to stimulate DNA synthesis as well as protein synthesis (33), and cell proliferation of myometrium and leiomyoma cells (7, 15, 47), at least in part, by regulation of vascular endothelial growth factor (VEGF) (48) and ERK 1/2 signaling pathway (7). Furthermore, it has been suggested

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that PDGF can interacts with other growth factors (such as TGF-β and EGF) to enhance cell proliferation (33, 49).

PROLACTIN RELEASING PEPTIDE

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Prolactin releasing peptide (PrRP) binds to GPR10 and phosphorylate PI3K at the cell membrane through G protein, α, β and γ. A recent study shows that the activation of GPR10

cultured primary leiomyoma cells (8).

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TRANSFORMING GROWTH FACTOR-α

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by its cognate ligand PrRP, promotes PI3K-AKT-mTOR pathways and proliferation of

Transforming growth factor-α (TGF-α) is a member of the EGF family. This growth factor is structurally and biologically similar to EGF, and interacts with the EGF-R to exert effects on cell proliferation, differentiation and development (50). The role of TGF-α in leiomyoma

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pathogenesis is limitedly studied. Dixon and co-workers reported the immunolocalization of TGF-α and its receptor (EGF-R) in the cytoplasm of smooth-muscle cells of leiomyomas and matched myometrium (13). In contrast, Moore and colleagues reported that TGF-α and EGF-

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R were intensely expressed in uterine leiomyosarcomas, but their expressions were absent in

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leiomyomas and also in control myometrium in B6C3F1 mice (an outbred strain originated from crossing C57BL/6 with C3H mice) (51).

TRANSFORMING GROWTH FACTOR-β Transforming growth factor βs are member of TGF-β superfamily, consists of three homodimeric isoforms called TGF-β1, TGF-β2 and TGF-β3. These multifunctional peptides control various biological processes, including cell growth, proliferation and differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling by activating Smad 2/3

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dependent and Smad 2/3 independent signaling pathways through binding to transmembrane receptors, TGF-βR-II and TGF-βR-I (Alk5) (52, 53). It has been shown that myometrial and leiomyoma smooth muscle cells expressed TGF-β1-3, TGF-βR-II and TGF-βR-I mRNAs and proteins (16, 17). TGF-βR-II and TGF-

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βR-I mRNAs and proteins were reported to be higher in leiomyoma compared to myometrium (54). The elevated expression levels of Smad3, Smad4 and pSmad3 were also found in leiomyoma compared to myometrium (54). Of note, TGF-β1 was reported to

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increase the rate of Smad and pSmad3, and pERK1/2 induction in both leiomyoma and myometrial smooth muscle cells (55, 56). TGF-β1, only at low concentrations (0.01 ng/mL),

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induced an increase in cell proliferation for both myometrial and leiomyoma cells (47), at least in part, by the upregulation of PDGF secretion (47). TGF-β1 also increased mRNA expressions of inflammatory and profibrotic mediators, such as IL-11 (57), connective tissue growth factor (CTGF) (58), c-fos, c-jun and plasminogen activator inhibitor 1 (PAI-1) (56),

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fibromodulin (a collagen-binding protein) (59) in myometrial and leiomyoma smooth muscle cells. Furthermore, TGF-β1 was reported to modulate mRNA expressions of TGF-β-induced factor, TGF-β-inducible early gene response, early growth response 3, CITED2 (cAMP

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response element binding protein-binding protein/p300-interacting transactivator with ED-

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rich tail), Nur77, Runx1, Runx2, p27, p57, growth arrest-specific 1, and G protein-coupled receptor kinase 5 in myometrial and leiomyoma smooth muscle cells (57). Similar to TGF-β1, TGF-β3 at low concentrations (0.01-1 ng/mL) stimulated the

proliferation of leiomyoma cells (60, 61). TGF-β3 also increased mRNA expression of ECM components such as collagen 1A1, CTGF (62), fibronectin (60) and versican V0 (63) in myometrial and leiomyoma cells. The above results suggest that TGF-β1 and TGF-β3 may influence leiomyoma growth by regulating inflammatory response, cell growth, apoptosis, and tissue remodeling through activating Smad 2/3 and ERK 2/3 signaling pathways.

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VASCULAR ENDOTHELIAL GROWTH FACTOR Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, was originally described as an endothelial cell-specific mitogen (64). VEGF stimulates cellular responses by

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binding to tyrosine kinase receptors, VEGFR-1 (fms-like tyrosine kinase: flt-1) and VEGFR2 (kinase domain-containing receptor: KDR/flk-1). The role of VEGF in leiomyoma growth has been studied (19, 65, 66). VEGF mRNA and protein have been detected in both

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myometrium and leiomyoma (18, 19) with elevated expression of VEGF-A antigen in leiomyomas compared to the adjacent myometrium (Gentry et al., 2001).

The VEGF

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receptors, VEGFR-1 and VEGFR-2 were found to be expressed in myometrial smooth muscle cells (67) and leiomyoma tissue (66). Hassan and co-workers reported that pretreatment of leiomyoma xenografts with VEGF was required for the continuous growth of

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leiomyoma tissue in vivo (65).

GROWTH FACTORS AND THEIR SIGNALING PATHWAYS AS COMMON TARGET OF CURRENT AND FUTURE MEDICAL TREATMENTS

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As current medical treatment, GnRHa is used as preoperative therapy for a long time, and

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recently ulipristal acetate has been added for the management of uterine fibroids. In addition, several other promising compounds such as, genistein, curcumin, tranilast, vitamin D, AG1478, TKS050, SB-525334, MK-2206 and WAY-129327 have been investigated for their efficacy against uterine fibroids. Those above compounds exert their therapeutic effects on leiomyoma growth, at least in part, by downregulating growth factors and their signaling pathways (Figure 1). Gonadotropin-releasing hormone agonist (GnRHa) is US Food and Drug Administration (FDA)-approved short-term medical therapy for uterine fibroids. This

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treatment is concomitantly used with iron therapy for the preoperative hematologic improvement of patients with anemia caused by uterine fibroids. GnRHa is able to reduce leiomyoma and uterine volume (68) and alleviates bleeding and other leiomyoma-related symptoms, but this treatment is restricted to a 3 to 6 month interval, because of the risk of

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irreversible bone loss and osteoporosis (69, 70). A considerable amount of studies have demonstrated that the therapeutic effect of GnRHa on leiomyoma growth was mediated, at least in part, by downregulation of multiple growth factors [TGF-β, bFGF, PDGF and

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VEGF), and signaling pathways [Smad 2/3, ERK 1/2, PI3K/protein kinase B (PKB/AKT)] (54, 71-74).

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Ulipristal acetate (also known as CDB-2914) is a selective progesterone receptor modulator that binds to progesterone receptors A and B with high affinity. This treatment has been approved in Europe and Canada for preoperative fibroid treatment (75). Several recent studies reported that ulipristal acetate is able to reduce leiomyoma and uterine volume and

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positively improves leiomyoma-related symptoms and quality of life without serious complications (76-78). The therapeutic effect of ulipristal acetate on leiomyoma growth was associated with downregulation of growth factors and growth factors mediated events,

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including proliferation, angiogenesis and fibrotic process (79-82). Ulipristal acetate was

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reported to inhibit proliferation and induce apoptosis in cultured human uterine leiomyoma cells (82). Ulipristal acetate also decreased VEGF receptors (VEGFR-1 and VEGFR-2), and progesterone-induced VEGF-A and VEGF-B contents in cultured leiomyoma cells but not in normal myometrial cells (80). Recently, we found that ulipristal acetate is also able to block the activin-A-induced increase in fibronectin or VEGF-A mRNA expression in myometrial and in leiomyoma cultured cells (79). Furthermore, ulipristal acetate was reported to increase extracellular matrix metalloproteinase inducer, matrix metalloproteinase (MMP)-1, MMP-8 contents and decrease tissue inhibitors of MMP (TIMP)-1, TIMP-2 contents as well as type I

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and type III collagen contents in cultured leiomyoma cells, without comparable effects on cultured normal myometrial cells (81). Genistein is a phytoestrogen mostly found in soybeans [Glycine max (L.). Dietary supplementation of genistein (400 or 800 mg of genistein/kg) was reported to reduce the

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incidence and size of spontaneously occurring leiomyoma of the oviduct in the Japanese quail (83). This phytoestrogen has been reported to exert antiproliferative effect and inhibits TGFβ/Smad signaling pathway genes in leiomyoma cells (84). Genistein at high concentration (≥

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10 µg/mL) inhibited leiomyoma as well as myometrial cells proliferation (84). In addition, downregulation of TGF-β signaling pathway genes, activin A, activin B, Smad3 and TGF-β2

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in human uterine leiomyoma cells was reported by genistein at 50 µg/mL (85). Curcumin, a polyphenol found in the rhizome of turmeric (Curcuma longa L.), has been reported to exert antiproliferative and antifibrotic effects in human uterine leiomyoma cells (86). Curcumin inhibited leiomyoma cell proliferation (86), as well as inhibited

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fibronectin, ERK 1, ERK 2, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in leiomyoma cells (86).

Tranilast (N-[3,4-Dimethoxycinnamoyl]-anthranilic acid), a orally administered

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synthetic drug of low toxicity for the treatment of allergic disorders commonly used in Japan

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and South Korea. The antiproliferative and antifibrotic effect of tranilast has documented in myometrial and leiomyoma cells (87-89). Tranilast inhibited the proliferation of uterine leiomyoma cells in vitro through G1 arrest associated with the induction of p21 (waf1) and p53 (88). The antifibrotic effect of tranilast in myometrial and leiomyoma cells was mediated by downregulation of profibrotic growth factor, activin-A, and ECM components such as collagen1A1, fibronectin and versican expression (87). Vitamin D is a fat-soluble vitamin found naturally in only a few foods, such as fishliver oils, fatty fishes, mushrooms, egg yolks, and liver. The two major physiologically

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relevant forms of vitamin D are D2 (ergocalciferol) and D3 (cholecalciferol). Vitamin D3 [1,25(OH)2D3] has been reported to shrink uterine leiomyoma tumors in the Eker rat model (90). The antiproliferative effect of vitamin D3 has been reported in both myometrial and leiomyoma cells (91). Vitamin D3 inhibited leiomyoma cell proliferation through the down-

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regulation of PCNA, CDK1, and BCL-2 and suppresses catechol-O-methyltransferase expression (92). It has been reported that vitamin D3 consistently reduced TGF-β3 effects that are involved in the process of fibrosis in human leiomyoma cells (93). Vitamin D3

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reduced TGF-β3 induced fibronectin, collagen type 1, and PAI-1 protein expression in human uterine leiomyoma cells (93). Vitamin D3 also reduced TGF-β3 induced phosphorylation of

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Smad2 as well as nuclear translocation of Smad2 and Smad3 in human uterine leiomyoma cells (93).

AG1478 and TKS050 are selective EGF-R blocker with therapeutic potential in a variety of cells. Since EGF has been shown to regulate leiomyoma growth (7, 33, 34),

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AG1478 and TKS050 were evaluated as potential target for fibroids (94, 95). AG-1478 was found to inhibit the growth of leiomyoma and myometrium cell cultures with IC-50 values of 5.6 and 5.7 µM, respectively (94). Another EGF-R blocker TKS050 effectively inhibited the

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growth of leiomyoma and myometrial cell cultures in a dose- and time-dependent manner

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with IC-50 values of 0.7 µmol/L for leiomyoma and 1.1 µmol/L for myometrial cell cultures, respectively (95). The antiproliferative effect of TKS050 was associated with induction of cell cycle arrest and apoptosis, and inhibition of EGF-R autophosphorylation and of phosphorylated signal transducer and activator of transcription 3 (STAT3) (95). SB-525334 is a potent inhibitor of the TGF-βR-I (IC50 = 14.36 nM). Since TGF-β has been shown to regulate leiomyoma growth (47, 60, 61), SB 525334 was evaluated as a potential target for fibroids (96). Using the Eker rat model, Laping and co-workers

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demonstrated that SB-525334 significantly decreased tumor incidence and multiplicity and reduced the size of these mesenchymal tumors (leiomyoma) (96). MK-2206 is a highly selective inhibitor of Akt1, Akt2 and Akt3 with IC50 of 8, 12 and 65 nM, respectively. Since AKT promotes uterine leiomyoma cell survival (97), Sefton

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and co-workers evaluated MK-2206 as viable target for inhibiting uterine leiomyoma growth (98). They found that MK-2206 is able to reduce phosphorylation of AKT, uterine leiomyoma cell viability and uterine leiomyoma tumor volumes in mouse xenograft model

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(98). As mTOR signaling contributes to the growth of uterine leiomyoma (99), mTOR inhibitor WAY-129327 was evaluated as potential therapeutic target for fibroids (100). Using

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in vivo and in vitro experiments, Crabtree and colleagues found that treatment of Eker rats with the WAY-129327 inhibits phosphorylation of the downstream mTOR target S6K and its effector S6 and cell proliferation in tumors, and decreases tumor incidence, multiplicity and

SUMMARY

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size (100).

Growth factors are increasingly accepted as major contributors for uterine leiomyoma

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development and growth, and their actions are regulated by steroid hormones. They activate

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several signaling pathways, including Smad 2/3, ERK 1/2, PI3K and β-catetin, and regulate major cellular processes, including inflammation, proliferation, angiogenesis and fibrosis which are linked to uterine leiomyoma development and growth (Figure 1). The ability of growth factors to promote leiomyoma growth makes them an attractive therapeutic target for the treatment of fibroids. Consequently, several current medical treatments (such as GnRHa, ulipristal acetate) were found to exert therapeutic effects on leiomyoma growth through regulation of growth factors and signaling pathways (Figure 1). Future medical treatment can also be established targeting growth factors and signaling pathway specific components.

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PRACTICE POINTS •

Growth factors are involved in the pathogenesis of uterine leiomyoma and they are differentially expressed in leiomyoma compared to myometrium. Growth factors exert multiple actions on leiomyoma cells through activating multiple

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•

signal transduction pathways. •

The therapeutic actions of current and future medical treatments are partly mediated

RESEARCH AGENDA •

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by regulation of growth factors and their signaling pathways.

Presence and actions of growth factors are incompletely studied in myometrial and leiomyoma cells; therefore, more complete studies are needed to explore their role in leiomyoma growth.

Other signaling pathways can be studied.

•

Future medical treatments can be studied on the basis of growth factors and their

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•

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signaling pathways.

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ACKNOWLEDGEMENTS

This work was supported by a grant from the “Fondazione Cassa di Risparmio di Fabriano e Cupramontana” (to M.C. and P.C.) and by Italian Ministry of the University and Research (PRIN 2010-2011, No. 20102CHST5_007, to S.R.G.).

Conflicts of interest The authors have no conflicts of interest.

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ACCEPTED MANUSCRIPT REFERENCES 1.

Islam MS, Protic O, Toti P, Giannubilo SR, Tranquilli AL, Petraglia F, et al. Uterine

leiomyoma: available medical treatments and new possible therapeutic options. Journal of Clinical Endocrinology & Metabolism. 2013;98(3):921–34. Buttram Jr VC, Reiter RC. Uterine leiomyomata: etiology, symptomatology, and

management. Fertility and Sterility. 1981;36(4):433-45. 3.

Cramer SF, Patel A. The frequency of uterine leiomyomas. American Journal of Clinical

Pathology. 1990;94(4):435-8.

Islam MS, Protic O, Stortoni P, Grechi G, Lamanna P, Petraglia F, et al. Complex networks of

SC

4.

RI PT

2.

5.

M AN U

multiple factors in the pathogenesis of uterine leiomyoma. Fertility and Sterility. 2013;100(1):178-93. Ciarmela P, Islam MS, Reis FM, Gray PC, Bloise E, Petraglia F, et al. Growth factors and

myometrium: biological effects in uterine fibroid and possible clinical implications. Human Reproduction Update. 2011 Nov-Dec;17(6):772-90. 6.

Islam MS, Catherino WH, Protic O, Janjusevic M, Gray PC, Giannubilo SR, et al. Role of

TE D

activin-A and myostatin and their signaling pathway in human myometrial and leiomyoma cell function. The Journal of Clinical Endocrinology & Metabolism. 2014;99(5):E775-E85. 7.

Mesquita FS, Dyer SN, Heinrich DA, Bulun SE, Marsh EE, Nowak RA. Reactive oxygen

EP

species mediate mitogenic growth factor signaling pathways in human leiomyoma smooth muscle cells. Biol Reprod. 2010 Feb;82(2):341-51. Varghese BV, Koohestani F, McWilliams M, Colvin A, Gunewardena S, Kinsey WH, et al.

AC C

8.

Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(6):2187-92. 9.

Ono M, Yin P, Navarro A, Moravek MB, Coon JS, Druschitz SA, et al. Paracrine activation

of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(42):17053-8.

Islam et al. 18

ACCEPTED MANUSCRIPT 10.

Ciarmela P, Bloise E, Gray PC, Carrarelli P, Islam MS, De Pascalis F, et al. Activin-A and

myostatin response and steroid regulation in human myometrium: disruption of their signalling in uterine fibroid. Journal of Clinical Endocrinology and Metabolism. 2011 March;96(03):755-65. 11.

Wolanska M, Bankowski E. Fibroblast growth factors (FGF) in human myometrium and

12.

RI PT

uterine leiomyomas in various stages of tumour growth. Biochimie. 2006 Feb;88(2):141-6. Mangrulkar RS, Ono M, Ishikawa M, Takashima S, Klagsbrun M, Nowak RA. Isolation and

characterization of heparin-binding growth factors in human leiomyomas and normal myometrium. Biol Reprod. 1995 Sep;53(3):636-46.

Dixon D, He H, Haseman JK. Immunohistochemical localization of growth factors and their

SC

13.

receptors in uterine leiomyomas and matched myometrium. Environ Health Perspect. 2000 Oct;108

14.

M AN U

Suppl 5:795-802.

Van der Ven LT, Roholl PJ, Gloudemans T, Van Buul-Offers SC, Welters MJ, Bladergroen

BA, et al. Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues. Br J Cancer. 1997;75(11):1631-40. Liang M, Wang H, Zhang Y, Lu S, Wang Z. Expression and functional analysis of platelet-

TE D

15.

derived growth factor in uterine leiomyomata. Cancer Biol Ther. 2006 Jan;5(1):28-33. 16.

Tang XM, Dou Q, Zhao Y, McLean F, Davis J, Chegini N. The expression of transforming

EP

growth factor-beta s and TGF-beta receptor mRNA and protein and the effect of TGF-beta s on human myometrial smooth muscle cells in vitro. Molecular Human Reproduction. 1997

17.

AC C

Mar;3(3):233-40.

Dou Q, Zhao Y, Tarnuzzer RW, Rong H, Williams RS, Schultz GS, et al. Suppression of

transforming growth factor-beta (TGF beta) and TGF beta receptor messenger ribonucleic acid and protein expression in leiomyomata in women receiving gonadotropin-releasing hormone agonist therapy. Journal of Clinical Endocrinology & Metabolism. 1996 Sep;81(9):3222-30. 18.

Harrison-Woolrych ML, Sharkey AM, Charnock-Jones DS, Smith SK. Localization and

quantification of vascular endothelial growth factor messenger ribonucleic acid in human myometrium and leiomyomata. J Clin Endocrinol Metab. 1995 Jun;80(6):1853-8.

Islam et al. 19

ACCEPTED MANUSCRIPT 19.

Gentry CC, Okolo SO, Fong LF, Crow JC, Maclean AB, Perrett CW. Quantification of

vascular endothelial growth factor-A in leiomyomas and adjacent myometrium. Clin Sci (Lond). 2001 Dec;101(6):691-5. 20.

Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, et al. Purification and

1986;321(6072):776-9. 21.

Werner S, Alzheimer C. Roles of activin in tissue repair, fibrosis, and inflammatory disease.

Cytokine & Growth Factor Reviews. 2006;17(3):157-71.

Tsuchida K, Nakatani M, Hitachi K, Uezumi A, Sunada Y, Ageta H, et al. Activin signaling

SC

22.

RI PT

characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature.

as an emerging target for therapeutic interventions. Cell Communication and Signaling.

23.

M AN U

2009;7(15):10.1186.

Tsuchida K, Arai KY, Kuramoto Y, Yamakawa N, Hasegawa Y, Sugino H. Identification and

characterization of a novel follistatin-like protein as a binding protein for the TGF-β family. Journal of Biological Chemistry. 2000;275(52):40788-96.

Hill JJ, Davies MV, Pearson AA, Wang JH, Hewick RM, Wolfman NM, et al. The myostatin

TE D

24.

propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum. The Journal of Biological Chemistry. 2002 Oct 25;277(43):40735-41. Kelber JA, Shani G, Booker EC, Vale WW, Gray PC. Cripto is a noncompetitive activin

EP

25.

antagonist that forms analogous signaling complexes with activin and nodal. Journal of Biological

26.

AC C

Chemistry. 2008 Feb 22;283(8):4490-500. Bilezikjian LM, Corrigan AZ, Blount AL, Chen Y, Vale WW. Regulation and actions of

Smad7 in the modulation of activin, inhibin, and transforming growth factor-beta signaling in anterior pituitary cells. Endocrinology. 2001 Mar;142(3):1065-72. 27.

Ciarmela P, Wiater E, Vale W. Activin-A in myometrium: characterization of the actions on

myometrial cells. Endocrinology. 2008 May;149(5):2506-16. 28.

Tsigkou A, Reis FM, Ciarmela P, Lee MH, Jiang B, Tosti C, et al. Expression levels of

myostatin and matrix metalloproteinase 14 mRNAs in uterine leiomyoma are correlated with dysmenorrhea. Reprod Sci. 2015 Jul 1:[Epub ahead of print].

Islam et al. 20

ACCEPTED MANUSCRIPT 29.

Hague S, Zhang L, Oehler MK, Manek S, MacKenzie IZ, Bicknell R, et al. Expression of the

hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density. Clin Cancer Res. 2000 Jul;6(7):2808-14. 30.

Yu L, Saile K, Swartz CD, He H, Zheng X, Kissling GE, et al. Differential expression of

RI PT

receptor tyrosine kinases (RTKs) and IGF-I pathway activation in human uterine leiomyomas. Mol Med. 2008 May-Jun;14(5-6):264-75. 31.

Rauk PN, Surti U, Roberts JM, Michalopoulos G. Mitogenic effect of basic fibroblast growth

factor and estradiol on cultured human myometrial and leiomyoma cells. Am J Obstet Gynecol. 1995

32.

SC

Aug;173(2):571-7.

Yeh J, Rein M, Nowak R. Presence of messenger ribonucleic acid for epidermal growth factor

M AN U

(EGF) and EGF receptor demonstrable in monolayer cell cultures of myometria and leiomyomata. Fertility and Sterility. 1991 Nov;56(5):997-1000. 33.

Fayed YM, Tsibris JC, Langenberg PW, Robertson AL, Jr. Human uterine leiomyoma cells:

binding and growth responses to epidermal growth factor, platelet-derived growth factor, and insulin.

34.

TE D

Lab Invest. 1989 Jan;60(1):30-7.

Rossi MJ, Chegini N, Masterson BJ. Presence of epidermal growth factor, platelet-derived

growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in

35.

EP

smooth muscle cells in vitro. Endocrinology. 1992 Mar;130(3):1716-27. Ren Y, Yin H, Tian R, Cui L, Zhu Y, Lin W, et al. Different effects of epidermal growth

AC C

factor on smooth muscle cells derived from human myometrium and from leiomyoma. Fertility and Sterility. 2011;96(4):1015-20. 36.

Nowak RA. Novel therapeutic strategies for leiomyomas: targeting growth factors and their

receptors. Environ Health Perspect. 2000 Oct;108 Suppl 5:849-53. 37.

Wang J, Ohara N, Takekida S, Xu Q, Maruo T. Comparative effects of heparin-binding

epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells. Hum Reprod. 2005 Jun;20(6):1456-65.

Islam et al. 21

ACCEPTED MANUSCRIPT 38.

Gloudemans T, Prinsen I, Van Unnik JA, Lips CJ, Den Otter W, Sussenbach JS. Insulin-like

growth factor gene expression in human smooth muscle tumors. Cancer Res. 1990 Oct 15;50(20):6689-95. 39.

Strawn EY, Jr., Novy MJ, Burry KA, Bethea CL. Insulin-like growth factor I promotes

40.

RI PT

leiomyoma cell growth in vitro. Am J Obstet Gynecol. 1995 Jun;172(6):1837-43; discussion 43-4. Gao Z, Matsuo H, Wang Y, Nakago S, Maruo T. Up-regulation by IGF-I of proliferating cell

nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells. J Clin Endocrinol Metab. 2001 Nov;86(11):5593-9.

Peng L, Wen Y, Han Y, Wei A, Shi G, Mizuguchi M, et al. Expression of insulin-like growth

SC

41.

factors (IGFs) and IGF signaling: molecular complexity in uterine leiomyomas. Fertil Steril. 2009

42.

M AN U

Jun;91(6):2664-75.

Zhu X, Topouzis S, Liang LF, Stotish RL. Myostatin signaling through Smad2, Smad3 and

Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism. Cytokine. 2004 Jun 21;26(6):262-72.

Ciarmela P, Wiater E, Smith SM, Vale W. Presence, actions, and regulation of myostatin in

TE D

43.

rat uterus and myometrial cells. Endocrinology. 2009 Feb;150(2):906-14. 44.

Boehm KD, Daimon M, Gorodeski IG, Sheean LA, Utian WH, Ilan J. Expression of the

Oct;27(2):93-101.

Hwu YM, Li SH, Lee RK, Tsai YH, Yeh TS, Lin SY. Increased expression of platelet-derived

AC C

45.

EP

insulin-like and platelet-derived growth factor genes in human uterine tissues. Mol Reprod Dev. 1990

growth factor C messenger ribonucleic acid in uterine leiomyomata. Fertil Steril. 2008 Feb;89(2):46871. 46.

Suo G, Jiang Y, Cowan B, Wang JY. Platelet-derived growth factor C is upregulated in

human uterine fibroids and regulates uterine smooth muscle cell growth. Biol Reprod. 2009 Oct;81(4):749-58. 47.

Arici A, Sozen I. Expression, menstrual cycle-dependent activation, and bimodal mitogenic

effect of transforming growth factor-beta1 in human myometrium and leiomyoma. American Journal of Obstetrics and Gynecology. 2003 Jan;188(1):76-83.

Islam et al. 22

ACCEPTED MANUSCRIPT 48.

Taniguchi Y, Morita I, Kubota T, Murota S, Aso T. Human uterine myometrial smooth

muscle cell proliferation and vascular endothelial growth-factor production in response to plateletderived growth factor. J Endocrinol. 2001 Apr;169(1):79-86. 49.

Wolanska M, Bankowski E. Transforming growth factor beta and platelet-derived growth

RI PT

factor in human myometrium and in uterine leiomyomas at various stages of tumour growth. European Journal of Obstetrics, Gynecology, and Reproductive Biology. 2007 Feb;130(2):238-44. 50.

Massague J. Epidermal growth factor-like transforming growth factor. I. Isolation, chemical

rat cells. J Biol Chem. 1983 Nov 25;258(22):13606-13. 51.

SC

characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed

Moore AB, He H, Yoshida A, Rico PJ, Haseman JK, Dixon D. Transforming growth factor-

M AN U

alpha, epidermal growth factor receptor, and PCNA immunoexpression in uterine leiomyosarcomas and leiomyomas in B6C3F1 mice. Exp Toxicol Pathol. 2000 Jun;52(3):195-200. 52.

Massagué J. TGF-beta signal transduction. Annual Review of Biochemistry. 1998;67(1):753-

91.

Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family

TE D

53.

signalling. Nature. 2003;425(6958):577-84. 54.

Chegini N, Luo X, Ding L, Ripley D. The expression of Smads and transforming growth

EP

factor beta receptors in leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. Molecular and Cellular Endocrinology. 2003 Nov 14;209(1-2):9-16. Xu J, Luo X, Chegini N. Differential expression, regulation, and induction of Smads,

AC C

55.

transforming growth factor-beta signal transduction pathway in leiomyoma, and myometrial smooth muscle cells and alteration by gonadotropin-releasing hormone analog. Journal of Clinical Endocrinology & Metabolism. 2003 Mar;88(3):1350-61. 56.

Ding L, Xu J, Luo X, Chegini N. Gonadotropin releasing hormone and transforming growth

factor beta activate mitogen-activated protein kinase/extracellularly regulated kinase and differentially regulate fibronectin, type I collagen, and plasminogen activator inhibitor-1 expression in leiomyoma and myometrial smooth muscle cells. Journal of Clinical Endocrinology & Metabolism. 2004 Nov;89(11):5549-57.

Islam et al. 23

ACCEPTED MANUSCRIPT 57.

Luo X, Ding L, Xu J, Chegini N. Gene expression profiling of leiomyoma and myometrial

smooth muscle cells in response to transforming growth factor-beta. Endocrinology. 2005 Mar;146(3):1097-118. 58.

Luo X, Ding L, Chegini N. CCNs, fibulin-1C and S100A4 expression in leiomyoma and

RI PT

myometrium: inverse association with TGF-beta and regulation by TGF-beta in leiomyoma and myometrial smooth muscle cells. Molecular Human Reproduction. 2006 Apr;12(4):245-56. 59.

Levens E, Luo X, Ding L, Williams RS, Chegini N. Fibromodulin is expressed in leiomyoma

and myometrium and regulated by gonadotropin-releasing hormone analogue therapy and TGF-beta

SC

through Smad and MAPK-mediated signalling. Molecular Human Reproduction. 2005 Jul;11(7):48994.

Arici A, Sozen I. Transforming growth factor-beta3 is expressed at high levels in leiomyoma

M AN U

60.

where it stimulates fibronectin expression and cell proliferation. Fertility and Sterility. 2000 May;73(5):1006-11. 61.

Lee BS, Nowak RA. Human leiomyoma smooth muscle cells show increased expression of

TE D

transforming growth factor-beta 3 (TGF beta 3) and altered responses to the antiproliferative effects of TGF beta. Journal of Clinical Endocrinology & Metabolism. 2001 Feb;86(2):913-20. 62.

Joseph DS, Malik M, Nurudeen S, Catherino WH. Myometrial cells undergo fibrotic

EP

transformation under the influence of transforming growth factor beta-3. Fertility and Sterility. 2010 Mar 15;93(5):1500-8.

Norian JM, Malik M, Parker CY, Joseph D, Leppert PC, Segars JH, et al. Transforming

AC C

63.

growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas. Reproductive Sciences. 2009 Dec;16(12):1153-64. 64.

Ferrara N, Houck K, Jakeman LYN, Leung DW. Molecular and biological properties of the

vascular endothelial growth factor family of proteins. Endocrine reviews. 1992;13(1):18-32. 65.

Hassan MH, Eyzaguirre E, Arafa HMM, Hamada FMA, Salama SA, Al-Hendy A. Memy I: a

novel murine model for uterine leiomyoma using adenovirus-enhanced human fibroid explants in severe combined immune deficiency mice. American journal of obstetrics and gynecology. 2008;199(2):156. e1-. e8.

Islam et al. 24

ACCEPTED MANUSCRIPT 66.

Sanci M, Dikis C, Inan S, Turkoz E, Dicle N, Ispahi C. Immunolocalization of VEGF, VEGF

receptors, EGF-R and Ki-67 in leiomyoma, cellular leiomyoma and leiomyosarcoma. Acta Histochem. 2011 May;113(3):317-25. 67.

Brown LF, Detmar M, Tognazzi K, Abu-Jawdeh G, Iruela-Arispe ML. Uterine smooth

RI PT

muscle cells express functional receptors (flt-1 and KDR) for vascular permeability factor/vascular endothelial growth factor. Lab Invest. 1997 Feb;76(2):245-55. 68.

Stovall TG, Muneyyirci-Delale O, Summitt Jr RL, Scialli AR. GnRH agonist and iron versus

placebo and iron in the anemic patient before surgery for leiomyomas: A randomized controlled trial.

69.

SC

The Leuprolide Study Group. Obstetrics & Gynecology. 1995;86(1):65-71.

Friedman AJ, Hoffman DI, Comite F, Browneller RW, Miller JD. Treatment of leiomyomata

M AN U

uteri with leuprolide acetate depot: a double-blind, placebo-controlled, multicenter study. The Leuprolide Study Group. Obstetrics and Gynecology. 1991;77(5):720-5. 70.

Leather AT, Studd JWW, Watson NR, Holland EFN. The prevention of bone loss in young

women treated with GnRH analogues with" add-back" estrogen therapy. Obstetrics & Gynecology.

71.

TE D

1993;81(1):104-7.

Bifulco G, Miele C, Pellicano M, Trencia A, Ferraioli M, Paturzo F, et al. Molecular

mechanisms involved in GnRH analogue-related apoptosis for uterine leiomyomas. Molecular human

72.

EP

reproduction. 2004;10(1):43-8.

Chegini N, Kornberg L. Gonadotropin releasing hormone analogue therapy alters signal

AC C

transduction pathways involving mitogen-activated protein and focal adhesion kinases in leiomyoma. Journal of the Society for Gynecologic Investigation. 2003;10(1):21-6. 73.

Di Lieto A, De Falco M, Mansueto G, De Rosa G, Pollio F, Staibano S. Preoperative

administration of GnRH-a plus tibolone to premenopausal women with uterine fibroids: evaluation of the clinical response, the immunohistochemical expression of PDGF, bFGF and VEGF and the vascular pattern. Steroids. 2005;70(2):95-102. 74.

Di Lieto A, De Rosa G, De Falco M, Iannotti F, Staibano S, Pollio F, et al. Relationship

between platelet-derived growth factor expression in leiomyomas and uterine volume changes after gonadotropin-releasing hormone agonist treatment. Hum Pathol. 2002 Feb;33(2):220-4.

Islam et al. 25

ACCEPTED MANUSCRIPT 75.

Melis GB, Piras B, Marotto MF, Orru MM, Maricosu G, Pilloni M, et al. Pharmacokinetic

evaluation of ulipristal acetate for uterine leiomyoma treatment. Expert Opin Drug Metab Toxicol. 2012 Jul;8(7):901-8. 76.

Donnez J, Tomaszewski J, Vázquez F, Bouchard P, Lemieszczuk B, Baró F, et al. Ulipristal

RI PT

acetate versus .euprolide acetate for uterine fibroids. New England Journal of Medicine. 2012;366(5):421-32. 77.

Donnez J, Tatarchuk TF, Bouchard P, Puscasiu L, Zakharenko NF, Ivanova T, et al. Ulipristal

acetate versus placebo for fibroid treatment before surgery. New England Journal of Medicine.

78.

SC

2012;366(5):409-20.

Donnez J, Hudecek R, Donnez O, Matule D, Arhendt HJ, Zatik J, et al. Efficacy and safety of

79.

M AN U

repeated use of ulipristal acetate in uterine fibroids. Fertil Steril. 2015 Feb;103(2):519-27 e3. Ciarmela P, Carrarelli P, Islam MS, Janjusevic M, Zupi E, Tosti C, et al. Ulipristal acetate

modulates the expression and functions of activin A in leiomyoma cells. Reproductive Sciences. 2014;21(9):1120-5.

Xu Q, Ohara N, Chen W, Liu J, Sasaki H, Morikawa A, et al. Progesterone receptor

TE D

80.

modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells.

81.

EP

Human Reproduction. 2006;21(9):2408–16.

Xu Q, Ohara N, Liu J, Amano M, Sitruk-Ware R, Yoshida S, et al. Progesterone receptor

AC C

modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells. Molecular Human Reproduction. 2008;14(3):181-91. 82.

Xu Q, Takekida S, Ohara N, Chen W, Sitruk-Ware R, Johansson EDB, et al. Progesterone

receptor modulator CDB-2914 down-regulates proliferative cell nuclear antigen and Bcl-2 protein expression and up-regulates caspase-3 and poly (adenosine 5'-diphosphate-ribose) polymerase expression in cultured human uterine leiomyoma cells. Journal of Clinical Endocrinology & Metabolism. 2005;90(2):953–61. 83.

Sahin K, Akdemir F, Tuzcu M, Sahin N, Onderci M, Ozercan R, et al. Genistein suppresses

spontaneous oviduct tumorigenesis in Quail. Nutrition and cancer. 2009;61(6):799-806.

Islam et al. 26

ACCEPTED MANUSCRIPT 84.

Moore AB, Castro L, Yu L, Zheng X, Di X, Sifre MI, et al. Stimulatory and inhibitory effects

of genistein on human uterine leiomyoma cell proliferation are influenced by the concentration. Hum Reprod. 2007 Oct;22(10):2623-31. 85.

Di X, Andrews DMK, Tucker CJ, Yu L, Moore AB, Zheng X, et al. A high concentration of

RI PT

genistein down-regulates activin A, Smad3 and other TGF-β pathway genes in human uterine leiomyoma cells. Experimental & molecular medicine. 2012;44(4):281-92. 86.

Malik M, Mendoza M, Payson M, Catherino WH. Curcumin, a nutritional supplement with

Fertil Steril. 2009 May;91(5 Suppl):2177-84. 87.

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antineoplastic activity, enhances leiomyoma cell apoptosis and decreases fibronectin expression.

Islam MS, Protic O, Ciavattini A, Giannubilo SR, Tranquilli AL, Catherino WH, et al.

M AN U

Tranilast, an orally active antiallergic compound, inhibits extracellular matrix production in human uterine leiomyoma and myometrial cells. Fertility and sterility. 2014;102(2):597-606. 88.

Shime H, Kariya M, Orii A, Momma C, Kanamori T, Fukuhara K, et al. Tranilast inhibits the

proliferation of uterine leiomyoma cells in vitro through G1 arrest associated with the induction of

89.

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p21waf1 and p53. Journal of Clinical Endocrinology & Metabolism. 2002;87(12):5610-7. Islam MS, Protic O, Giannubilo S, Stortoni P, Ciavattini A, Lamanna P, et al.

Antiproliferative effect of tranilast on human myometrial and leiomyoma cells. Biological and

90.

EP

Biomedical Reports. 2012;2(5):321-7.

Halder SK, Sharan C, Al-Hendy A. 1,25-dihydroxyvitamin D3 treatment shrinks uterine

91.

AC C

leiomyoma tumors in the Eker rat model. Biology of Reproduction. 2012;86(4):116. Bläuer M, Rovio PH, Ylikomi T, Heinonen PK. Vitamin D inhibits myometrial and

leiomyoma cell proliferation in vitro. Fertility and sterility. 2009;91(5):1919-25. 92.

Sharan C, Halder SK, Thota C, Jaleel T, Nair S, Al-Hendy A. Vitamin D inhibits proliferation

of human uterine leiomyoma cells via catechol-O-methyltransferase. Fertility and sterility. 2011;95(1):247-53. 93.

Halder SK, Goodwin JS, Al-Hendy A. 1,25-Dihydroxyvitamin D reduces TGF-β3-induced

fibrosis-related gene expression in human uterine leiomyoma cells. Journal of Clinical Endocrinology and Metabolism. 2011;96(4):E754-E62.

Islam et al. 27

ACCEPTED MANUSCRIPT 94.

Shushan A, Rojansky N, Laufer N, Klein BY, Shlomai Z, Levitzki R, et al. The AG1478

tyrosine kinase inhibitor is an effective suppressor of leiomyoma cell growth. Hum Reprod. 2004 Sep;19(9):1957-67. 95.

Shushan A, Ben-Bassat H, Mishani E, Laufer N, Klein BY, Rojansky N. Inhibition of

RI PT

leiomyoma cell proliferation in vitro by genistein and the protein tyrosine kinase inhibitor TKS050. Fertil Steril. 2007 Jan;87(1):127-35. 96.

Laping NJ, Everitt JI, Frazier KS, Burgert M, Portis MJ, Cadacio C, et al. Tumor-specific

efficacy of transforming growth factor-beta RI inhibition in Eker rats. Clinical Cancer Research. 2007

97.

SC

May 15;13(10):3087-99.

Hoekstra AV, Sefton EC, Berry E, Lu Z, Hardt J, Marsh E, et al. Progestins activate the AKT

M AN U

pathway in leiomyoma cells and promote survival. Journal of Clinical Endocrinology & Metabolism. 2009;94(5):1768-74. 98.

Sefton EC, Qiang W, Serna V, Kurita T, Wei J-J, Chakravarti D, et al. MK-2206, an AKT

Inhibitor, promotes caspase-independent cell death and inhibits leiomyoma growth. Endocrinology.

99.

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2013;154(11):4046-57.

Yin XJ, Wang G, Khan-Dawood FS. Requirements of phosphatidylinositol-3 kinase and

mammalian target of rapamycin for estrogen-induced proliferation in uterine leiomyoma-and

100.

Crabtree JS, Jelinsky SA, Harris HA, Choe SE, Cotreau MM, Kimberland ML, et al.

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e5.

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myometrium-derived cell lines. American journal of obstetrics and gynecology. 2007;196(2):176. e1-.

Comparison of human and rat uterine leiomyomata: identification of a dysregulated mammalian target of rapamycin pathway. Cancer Res. 2009 Aug 1;69(15):6171-8.

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Figure 1: Growth factors activate several signaling pathways following enhancement of cell proliferation, extracellular matrix deposition and angiogenesis which are important for leiomyoma growth. The therapeutic effects of current and future medical treatments on

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uterine leiomyoma (indicated by blue rectangles) are mediated by regulation of cell proliferation, extracellular matrix and angiogenesis through downregulation of growth factors

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and their signaling pathways.

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Figure 1 Ulipristal acetate Genistein Tranilast

GnRHa Genistein

GnRHa Ulipristal acetate

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P

P

AKT

P

P

AC C

ERK1/2

Raf

TE D

Raf

P

Grb2

GTP

Smad 4

Raf

Shc

Ras

Ras GDP

Ras GTP

P

SOS

Grb2

SOS

Ras GDP

PIP2

PI3K

P

IGFIRβ

AG1478 TKS050

Smad 2/3

IGFIRβ

II

SC

I

PDGFRβ

I

PDGFRα

P

EGFR

II

P

EGFR

Extracellular

RI PT

TGF-β

Angiogenesis

GnRHa Ulipristal acetate

P

ACCEPTED MANUSCRIPT Highlights

Growth of leiomyoma is dependent on ovarian hormones activity through growth factors. Growth factors exert their effects activating multiple signal transduction pathways. Growth factors signaling pathways include Smad 2/3, ERK 1/2, PI3K and β-catenin. Therapeutic compounds downregulate growth factors and their signaling pathways.

AC C

EP

TE D

M AN U

SC

RI PT

Growth factors are attractive therapeutic target for the treatment of fibroids.