Uterine fibroids and growth factors

Uterine leiomyomas are benign (noncancerous) tumors originating from the smooth muscle of the uterus (myometrium) and are the most common indication for hysterectomy in the world . They affect about 77% of women of reproductive age, but approximately only 25% bear clinically apparent tumors . Heavy or abnormal uterine bleeding, pelvic pain or pressure, infertility, and recurrent pregnancy loss are associated with leiomyoma. Despite the high prevalence, significant health problems, and huge economical impact on the health-care system, relatively little is understood about the pathogenesis of uterine leiomyoma. Consequently, medical treatments are still limited. Growth of leiomyoma is believed to be dependent on ovarian hormone activity through intermediate elements such as growth factors .

Growth factors are polypeptides or proteins that are secreted by a number of cell 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 transduction pathways, including Smad 2/3, ERK 1/2, PI3K, and β-catenin by binding to their receptors ( Fig. 1 ). A number of studies reported that multiple growth factors, including activin-A , acidic fibroblast growth factor (aFGF) , basic fibroblast growth factor (bFGF) , epidermal growth factor (EGF) , heparin-binding EGF (HB-EGF) , insulin-like growth factor (IGF) , myostatin , platelet-derived growth factor (PDGF) , transforming growth factor-β (TGF-β) , transforming growth factor- α (TGF-α) , and vascular endothelial growth factor (VEGF) , are differentially expressed in myometrium and leiomyoma.

Growth factors activate several signaling pathways following enhancement of cell proliferation, extracellular matrix deposition, and angiogenesis which are essential for leiomyoma growth. The therapeutic effects of current and future medical treatments on uterine leiomyoma (indicated by blue rectangles) are mediated by regulation of cell proliferation, extracellular matrix, and angiogenesis through downregulation of growth factors and their signaling pathways.

Activin-A

Activin-A is a pleiotropic growth factor belonging to the TGF-β superfamily. It was originally isolated based on its activity in regulating follicle-stimulating hormone released from the anterior pituitary . Later, its functions has been extended in cell proliferation, differentiation, apoptosis, immune response, wound repair, and fibrosis Activin-A exerts multiple effects through the activation of Smad 2/3-dependent signaling pathway by binding to type II (ActRIIA or ActRIIB) and type I receptors (ActRIB, also known as activin receptor-like kinase 4 or ALK4). Activin A/Smad 2/3 signaling can be regulated by binding protein follistatin , follistatin-related gene (FLRG) , and Cripto at extracellular and plasma membrane levels. In addition, Smad7 is known as an inhibitory downstream modulator of activin/TGF-β-like signaling, and its expression is induced by activin-A .

The role of activin-A in leiomyoma and myometrial cell functions has been studied . The mRNA expression levels of activin-A and FLRG were found to be highly expressed in leiomyoma compared to myometrial tissues, whereas the receptors (ALK4, ActRIIA, and ActRIIB), follistatin, and Smad7 mRNAs remained unchanged . Activin-A has been reported to downregulate primary myometrial cell proliferation but not leiomyoma cells . Interestingly, activin-A was reported to increase mRNA expressions of extracellular matrix (ECM) components (collagen1A1, fibronectin, and versican) in primary leiomyoma cells compared to untreated cells . Furthermore, activin-A significantly increases phosphorylation of Smad signaling components, Smad2 and Smad3, in both leiomyoma and myometrial cells compared to untreated cells , suggesting that the fibrotic role of activin-A is mediated, at least in part, by the activation of Smad 2/3 signaling pathway.

Activin-A

Activin-A is a pleiotropic growth factor belonging to the TGF-β superfamily. It was originally isolated based on its activity in regulating follicle-stimulating hormone released from the anterior pituitary . Later, its functions has been extended in cell proliferation, differentiation, apoptosis, immune response, wound repair, and fibrosis Activin-A exerts multiple effects through the activation of Smad 2/3-dependent signaling pathway by binding to type II (ActRIIA or ActRIIB) and type I receptors (ActRIB, also known as activin receptor-like kinase 4 or ALK4). Activin A/Smad 2/3 signaling can be regulated by binding protein follistatin , follistatin-related gene (FLRG) , and Cripto at extracellular and plasma membrane levels. In addition, Smad7 is known as an inhibitory downstream modulator of activin/TGF-β-like signaling, and its expression is induced by activin-A .

The role of activin-A in leiomyoma and myometrial cell functions has been studied . The mRNA expression levels of activin-A and FLRG were found to be highly expressed in leiomyoma compared to myometrial tissues, whereas the receptors (ALK4, ActRIIA, and ActRIIB), follistatin, and Smad7 mRNAs remained unchanged . Activin-A has been reported to downregulate primary myometrial cell proliferation but not leiomyoma cells . Interestingly, activin-A was reported to increase mRNA expressions of extracellular matrix (ECM) components (collagen1A1, fibronectin, and versican) in primary leiomyoma cells compared to untreated cells . Furthermore, activin-A significantly increases phosphorylation of Smad signaling components, Smad2 and Smad3, in both leiomyoma and myometrial cells compared to untreated cells , suggesting that the fibrotic role of activin-A is mediated, at least in part, by the activation of Smad 2/3 signaling pathway.

Acidic fibroblast growth factor

aFGF (also known as FGF-1) is a member of the FGF family of growth factors and plays an important role in proliferation and angiogenesis. Immunohistochemical localization of aFGF was detected in human uterine leiomyomas with expression primarily localized to the smooth muscle cells . Wolanska and Bankowski reported that leiomyomas contained several times more aFGFs compared to myometrium .

Basic fibroblast growth factor

bFGF, a potent inducer of angiogenesis, belongs to the FGF family of growth factors. Leiomyomas show an increased protein expression of bFGF compared to the normal myometrium . Of note, bFGF was found primarily bound to the ECM of myometrium and fibroids . This observation suggests that the enhanced growth of leiomyomas may be due, in part, to the presence of large amounts of bFGF that are stored 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 . Rauk and colleagues reported that bFGF is mitogenic for both human uterine myometrial and leiomyoma cells, but leiomyoma cells are less responsive .

Epidermal growth factor

EGF, a member of the EGF family of growth factors, 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 . Dixon and coworkers reported that protein expression of EGF and EGF-R was observed in the cytoplasm of smooth muscle cells of leiomyomas and the matched myometrium . However, the intensity and percentage of positive staining of EGF were significantly decreased in leiomyoma tissue (the proliferative phase) compared to matched myometrium . EGF has been reported to be mitogenic for both cultured myometrium and leiomyoma cells . Mesquita et al. reported that the stimulation of primary leiomyoma cells with EGF caused a marked increase in nicotinamide adenine dinucleotide phosphate (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 . Furthermore, it has been shown that EGF treatment induced transient phosphorylation activation of EGFR and protein kinase B (Akt) in leiomyomal smooth muscle cells, followed by cell proliferation .

Heparin-binding EGF

HB-EGF is a member of the EGF family of growth factors. HB-EGF 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 . However, HB-EGF showed a decreased mRNA and protein expression in leiomyomas compared to normal myometrium . The receptor HER1 protein was detected in leiomyoma and myometrial cells with elevated expression in myometrial cells compared to leiomyoma . Treatment with HB-EGF upregulated HER1 expression in leiomyoma and myometrial cells . Wang and coworkers reported that HB-EGF showed a stimulatory effect on cultured leiomyoma cells and myometrial cells and inhibited apoptosis, at least in part, by augmentation of HER1 expression .

Insulin-like growth factor

IGFs are multifunctional peptides with high sequence similarity to insulin and play an important role in regulating cell proliferation, differentiation, apoptosis, and transformation. IGFs have two ligands, including IGF-I and IGF-II. They exert their effects 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 . Leiomyomas showed higher IGF-I and IGF-IR protein levels compared to myometrium . IGF-I was reported to increase the proliferation of uterine leiomyoma cells , partly by upregulation of expression of proliferating cell nuclear antigen (PCNA) and downregulation of apoptosis . Furthermore, IGF-I increased phosphorylation of IGF-IRβ, Shc, and MAPKp44/42 (ERK1/2) in uterine leiomyoma cells . Peng and colleagues reported that IGF-Akt signaling was positively associated with large and actively growing fibroids . It was shown that larger fibroids had higher levels of IGF-I and p-Akt activity when compared with the small ones .

Myostatin

Myostatin, or growth differentiation factor-8 (GDF-8), a secreted protein, was first cloned 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 via activation of the Smad 2/3 signaling pathway by binding to type II (ActRIIB) and type I receptors [(ALK4 or ActRIB) or (ALK5 or TGF-βRI)]. Myostatin/Smad 2/3 signaling can be interrupted by binding to protein follistatin and FLRG , Cripto and Smad7 .

The role of myostatin in leiomyoma and myometrial cell functions has been studied . We found that myostatin and FLRG mRNA expression levels were higher in leiomyoma compared to myometrial tissues, and the receptors (ALK4, ALK5, and ActRIIB), follistatin, and Smad7 mRNAs were unchanged . Cripto mRNA was found to be expressed only in human uterine leiomyoma explants . Myostatin significantly reduced cell proliferation in primary myometrial cells but not in leiomyoma cells ; interestingly, myostatin mRNA expression correlated significantly and directly with the intensity of dysmenorrhea . Furthermore, we demonstrated that myostatin can induce phosphorylation of Smad2 and 3 but does not affect pERK or p38 MAPK in both myometrial and leiomyoma cells .

Platelet-derived growth factor

PDGF is a dimeric glycoprotein consisting 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, matrix synthesis, antiapoptosis, and vascularization by binding to two different receptors, PDGF-α and PDGF-β. PDGF and PDGF-R expressions have been documented in both normal myometrium and leiomyoma . The elevated protein expressions levels of PDGF-AA and PDGF-BB and of their receptors were found in leiomyoma compared to myometrial tissues . In addition, high expression of PDGF-CC was reported to be in leiomyoma compared to adjacent myometrial tissues and smooth muscle cells . The profibrotic role of PDGF has been documented in myometrium and leiomyoma cells as it increased collagen alpha1 (I) expression in those cells . PDGF was reported to stimulate both DNA and protein synthesis , and cell proliferation of myometrial and leiomyoma cells , at least in part, by regulation of VEGF and ERK 1/2 signaling pathway . Furthermore, it has been suggested that PDGF can interact with other growth factors (such as TGF-β and EGF) to enhance cell proliferation .

Prolactin-releasing peptide

Prolactin-releasing peptide (PrRP) binds to GPR10 and phosphorylates PI3K at the cell membrane through G protein, α, β, and γ. A recent study shows that the activation of GPR10 by its cognate ligand PrRP promotes PI3K-AKT-mTOR pathways and proliferation of cultured primary leiomyoma cells .

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 EGF-R to exert its effects on cell proliferation, differentiation, and development . The role of TGF-α in leiomyoma pathogenesis has not been studied extensively. Dixon and coworkers reported the immunolocalization of TGF-α and its receptor (EGF-R) in the cytoplasm of smooth muscle cells of leiomyomas and matched myometrium . By contrast, Moore and colleagues reported that TGF-α and EGF-R were highly expressed in uterine leiomyosarcomas but were absent in leiomyomas and also in control myometrium in B6C3F1 mice (an outbred strain originated from crossing C57BL/6 with C3H mice) .

Transforming growth factor-β

TGF-β, a 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 ECM remodeling via activation of Smad 2/3-dependent and Smad 2/3-independent signaling pathways by binding to transmembrane receptors, TGFβRII and TGFβRI (Alk5) .

It has been shown that myometrial and leiomyoma smooth muscle cells expressed TGF-β1-3, TGFβRII, and TGFβRI mRNAs and proteins . TGFβRII and TGFβRI mRNAs and proteins were reported to be higher in leiomyoma compared to myometrium . The elevated expression levels of Smad3, Smad4, and pSmad3 were also found in leiomyoma compared to myometrium . Of note, TGF-β1 was reported to increase the rate of Smad and pSmad3, and pERK1/2 induction in both leiomyoma and myometrial smooth muscle cells . TGF-β1, at low concentrations (0.01 ng/mL), induced an increase in cell proliferation for both myometrial and leiomyoma cells , at least in part, by the upregulation of PDGF secretion . TGF-β1 also increased mRNA expressions of inflammatory and profibrotic mediators such as interleukin-11 (IL-11) , connective tissue growth factor (CTGF) , c-fos, c-jun, and plasminogen activator inhibitor 1 (PAI-1) , and fibromodulin (a collagen-binding protein) 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 response element binding protein-binding protein/p300-interacting transactivator with ED-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 .

Similar to TGF-β1, TGF-β3 at low concentrations (0.01–1 ng/mL) stimulated the proliferation of omyoma cells . TGF-β3 also increased mRNA expression of ECM components such as collagen 1A1, CTGF , fibronectin , and versican V0 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.

Vascular endothelial growth factor

VEGF, a key regulator of angiogenesis, was originally described as an endothelial cell-specific mitogen . VEGF stimulates cellular responses by binding to tyrosine kinase receptors, VEGFR-1 (fms-like tyrosine kinase: flt-1), and VEGFR-2 (kinase domain-containing receptor: KDR/flk-1). The role of VEGF in leiomyoma growth has been studied . VEGF mRNA and protein have been detected in both myometrium and leiomyoma with elevated expression of VEGF-A antigen in leiomyomas compared to the adjacent myometrium (Gentry et al., 2001). The VEGF receptors, VEGFR-1 and VEGFR-2, were found to be expressed in myometrial smooth muscle cells and leiomyoma tissue . Hassan and coworkers reported that the pretreatment of leiomyoma xenografts with VEGF was required for the continuous growth of leiomyoma tissue in vivo .

Growth factors and their signaling pathways as common target of current and future medical treatments

GnRHa has been used as a preoperative therapy for a long time, and 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. The abovementioned compounds exert their therapeutic effects on leiomyoma growth, at least in part, by downregulating growth factors and their signaling pathways ( Fig. 1 ).

Gonadotropin-releasing hormone agonist (GnRHa) is a US Food and Drug Administration (FDA)-approved short-term medical therapy for uterine fibroids. This treatment is concomitantly used with iron therapy for the preoperative hematologic improvement of patients with anemia caused by uterine fibroids. GnRHa has the ability to reduce leiomyoma and uterine volume and alleviates bleeding and other leiomyoma-related symptoms, but this treatment is restricted to a 3–6-month interval, because of the risk of irreversible bone loss and osteoporosis . 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 VEGF), and signaling pathways [Smad 2/3, ERK 1/2, PI3K-PKB/AKT] .

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 has been approved in Europe and Canada for preoperative fibroid treatment . Several recent studies reported that ulipristal acetate has the ability to reduce leiomyoma and uterine volume and positively improves leiomyoma-related symptoms and quality of life without serious complications . The therapeutic effect of ulipristal acetate on leiomyoma growth was associated with downregulation of growth factors and growth factor-mediated events, including proliferation, angiogenesis, and fibrotic process . Ulipristal acetate was reported to inhibit proliferation and induce apoptosis in cultured human uterine leiomyoma cells . 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 . 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 . Furthermore, ulipristal acetate was reported to increase ECM metalloproteinase inducer, matrix metalloproteinase (MMP)-1, MMP-8 contents and decrease tissue inhibitors of MMP (TIMP)-1, TIMP-2 as well as type I and type III collagen contents in cultured leiomyoma cells, without comparable effects on cultured normal myometrial cells .

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 incidence and size of spontaneously occurring leiomyoma of the oviduct in the Japanese quail . This phytoestrogen has been reported to exert an antiproliferative effect and inhibit the TGF-β/Smad signaling pathway genes in leiomyoma cells . Genistein at high concentration (≥ 10 μg/mL) inhibited leiomyoma as well as myometrial cell proliferation . In addition, genistein downregulated the TGF-β signaling pathway genes, activin A, activin B, Smad3, and TGF-β2, in human uterine leiomyoma cells at a concentration of 50 μg/mL .

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 . Curcumin inhibited leiomyoma cell proliferation as well as fibronectin, ERK 1, ERK 2, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in leiomyoma cells .

Tranilast (N-[3,4-Dimethoxycinnamoyl]-anthranilic acid) is an orally administered synthetic drug of low toxicity for the treatment of allergic disorders commonly used in Japan and South Korea. The antiproliferative and antifibrotic effects of tranilast have been documented in myometrial and leiomyoma cells . Tranilast inhibited the proliferation of uterine leiomyoma cells in vitro through G1 arrest associated with the induction of p21 (waf1) and p53 . The antifibrotic effect of tranilast in myometrial and leiomyoma cells was mediated by the downregulation of profibrotic growth factor, activin-A, and mRNA expression of ECM components such as collagen1A1, fibronectin, and versican .

Vitamin D is a fat-soluble vitamin found in natural foods such as fish-liver oils, fatty fishes, mushrooms, egg yolks, and liver. The two major physiologically 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 . The antiproliferative effect of vitamin D3 has been reported in both myometrial and leiomyoma cells . Vitamin D3 inhibited leiomyoma cell proliferation through the downregulation of PCNA, CDK1, and BCL-2 and suppressed catechol-O-methyltransferase expression . It has been reported that vitamin D3 consistently reduced TGF-β3 effects involved in the process of fibrosis in human leiomyoma cells . Vitamin D3 reduced TGF-β3-induced fibronectin, collagen type 1, and PAI-1 protein expression in human uterine leiomyoma cells . Vitamin D3 also reduced TGF-β3-induced phosphorylation of Smad2 as well as nuclear translocation of Smad2 and Smad3 in human uterine leiomyoma cells .

AG1478 and TKS050 are selective EGF-R blockers with therapeutic potential in a variety of cells. Since EGF has been shown to regulate leiomyoma growth , AG1478 and TKS050 were evaluated as potential targets for fibroids . AG-1478 was found to inhibit the growth of leiomyoma and myometrial cell cultures with IC-50 values of 5.6 and 5.7 μM, respectively . Another EGF-R blocker TKS050 effectively inhibited the growth of leiomyoma and myometrial cell cultures in a dose- and time-dependent manner with IC-50 values of 0.7 μmol/L for leiomyoma and 1.1 μmol/L for myometrial cell cultures, respectively . The antiproliferative effect of TKS050 was associated with the induction of cell cycle arrest and apoptosis, and inhibition of EGF-R autophosphorylation and phosphorylated signal transducer and activator of transcription 3 (STAT3) .

SB-525334 is a potent inhibitor of TGFβRI (IC-50 = 14.36 nM). Since TGF-β has been shown to regulate leiomyoma growth , SB 525334 was evaluated as a potential target for fibroids . Using the Eker rat model, Laping and coworkers demonstrated that SB-525334 significantly decreased tumor incidence and multiplicity and reduced the size of these mesenchymal tumors (leiomyoma) .

MK-2206 is a highly selective inhibitor of Akt1, Akt2, and Akt3 with IC-50 values of 8, 12, and 65 nM, respectively. Since Akt promotes uterine leiomyoma cell survival , Sefton and coworkers evaluated MK-2206 as a viable target for inhibiting uterine leiomyoma growth . In a mouse xenograft model, it was found that MK-2206 is able to inhibit phosphorylation of Akt and reduce uterine leiomyoma cell viability and uterine leiomyoma tumor volumes . As mTOR signaling contributes to the growth of uterine leiomyoma , mTOR inhibitor WAY-129327 was evaluated as a potential therapeutic target for fibroids . By performing 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 size .