Despite the major impact on gynecological morbidity, the etiology of uterine leiomyomas remains poorly understood (17
). Uterine leiomyomas are characterized by changes in cell proliferation and differentiation, and it appears that multiple growth factors are probably important in the pathogenesis of these tumors (1
). Growth factors mediate diverse biologic responses through control of cellular proliferation, differentiation, migration, and metabolism by binding to and activating cell-surface receptors that have intrinsic protein kinase activity. To date, about 60 receptor tyrosine kinases (RTKs), which belong to about 16 different receptor gene families, have been identified.
In this study, leiomyoma and patient-matched myometrial samples from ten women were examined for expression of 17 activated growth factor receptor tyrosine kinases. We found that 15 out of the 17 activated RTK receptors evaluated were highly expressed in tumor compared with myometrial samples, and many of these receptors belonged to the EGF, IGF-I, FGF, HGF, and PDGF growth factor gene families, which are important in cell proliferation and differentiation. To our knowledge, this is the first study of growth factor RTK expression profiles in human uterine leiomyoma and matched myometrial tissues.
Several studies have shown that growth factors and their receptor-mediated signaling pathways are important in uterine leiomyoma growth. One such growth factor, EGF, is mitogenic (19
) and is expressed more in leiomyomas than in myometrial tissue during the lutereal phase (20
). The EGF receptor in leiomyomas is reported to be more sensitive to regulation by sex steroids than those in the myometrium (8
). The growth factor bFGF also induces proliferation of smooth muscle cells in both leiomyomas and myometrial tissue (23
). The enhanced growth of leiomyomas may be due partially to the presence of large quantities of bFGF stored in the extracellular matrix (ECM) of these tumors (24
). The expression of FGF receptor protein was also reported to be more intense in leiomyomas than in the myometrium (25
). Another potent mitogen for smooth muscle cells is PDGF, its mRNA is expressed in both leiomyomas and in myometrium, and its receptor sites per cell are seen more in leiomyomas than in the myometrium (26
). However, it appears that PDGF does not act alone, but acts in concert with other growth factors such as TGF- β, EGF, and the IGFs. For example, low amounts of TGF-β stimulate autocrine PDGF secretion and promotes the synthesis of PDGF receptors (28
). When myometrial cells are treated with both PDGF and EGF, there is a synergistic decrease in DNA synthesis, whereas treatment of leiomyoma cells with both factors results in an additive increase in DNA synthesis (26
). Insulin and PDGF also exert an additive effect upon DNA synthesis in leiomyoma and myometrial cells. The mRNA expression level of IGF-I was reported higher in leiomyomas than in the myometrium (26
), although insulin and its receptor are not highly expressed in leiomyoma tissue (31
). The levels of IGF-I receptors in leiomyomas have also been reported to exceed those of the myometrium (32
), which suggests that IGF-I and the IGF-IR signaling pathway may be of major significance in the growth of uterine leiomyomas. There are limited studies done on the role of the hematopoietic growth factors, M-CSF-R, Fit-3, SCF-R, and MSP-R on uterine leiomyoma growth and development, although these RTKs were highly expressed in the leiomyomas compared with myometrial tissues in our RTK array studies. These receptors might be worthwhile studying in the future.
The upregulation of different families of phosphorylated growth factor RTK proteins in the leiomyoma samples found in this study further indicates that there are multiple growth factors that might be important in the pathogenesis and growth of fibroids. Different growth factors could play a role at different stages of the disease. Many of the growth factors may interact, sometimes resulting in a synergistic effect (8
). The signal specificity may be defined partially by a combinatorial control. Every RTK recruits and activates a unique set of signaling proteins via its own tyrosine autophosphorylation sites and by means of tyrosine phosphorylation sites on closely associated docking or adaptor proteins. The combinatorial recruitment of a particular complement of signaling proteins from a common preexisting pool of signaling cascades is one mechanism for control of signal specificity (35
). This process is regulated further by differential recruitment of stimulatory and inhibitory proteins by the different receptors and downstream effector proteins leading to fine tuning of cellular responses. Signaling pathways activated by RTKs are interconnected with other signaling pathways via protein networks that are subjected to multiple positive and negative feedback mechanisms (35
Among these highly expressed RTKs in leiomyoma tissues, we observed that the IGF-IR exhibited increased phosphorylation in leiomyomas compared with myometrial samples in this study, and, in earlier studies, we have found the receptor protein to be overexpressed in leiomyomas compared with myometrium (5
). IGF-IR affects cell mitogenesis and survival by binding of its ligand, IGF-I, and by activation of downstream effector proteins. Upregulation of IGF-I and/or the IGF-IR could increase fibroid growth and/or survival through its mitogenic and/or anti-apoptotic effects (6
). IGF-IR is a major receptor tyrosine kinase protein, which appears to be pivotal to the adequate function of other growth factors. Some researchers have reported that overexpression of EGF, PDGF, and insulin receptor is not sufficient for ligand dependent growth unless a functional IGF-IR is present (12
). On the other hand, overexpression of the IGF-IR renders mouse embryo-derived fibroblasts capable of growing in the presence of IGF-I only, without activation of PDGF and EGF receptors. These findings would suggest a critical role of the IGF-IR in the mitogenic action of other growth factors (13
). In keeping with this concept and based on increased immunolocalization of IGF-IR in leiomyoma compared with myometrial tissue observed in our earlier studies (5
), and increased expression levels of phosphorylated IGF-IR in the leiomyoma samples indicated by the RTKs array, we focused on IGF-I and IGF-IR pathway expression and activation in fibroids in this study.
IGF-I, the product of an estrogen-regulated gene, mediates the biologic effectors of growth hormone in many tissues. It exerts its mitogenic action by increasing DNA synthesis, accelerating the progression of the cell cycle from G- to S-phase, and inhibiting apoptosis (6
). Several studies have indicated increased expression of IGF-I mRNA and higher tissue concentrations of IGF-I protein in leiomyomas versus corresponding myometrium (37
). Our previous study also found that IGF-I peptide immunolocalized to the leiomyoma cells and the fibroblasts in the bands of intervening connective tissue comprising the extracellular matrix in some leiomyomas; the latter was not seen in the myometrial samples. In addition, a significant increase in the levels of IGF-IRβ in leiomyomas was noted (5
). These data support a possible autocrine or local paracrine mechanism for IGF-I induced growth in leiomyomas. Other data in rats have shown that IGF-I acts as an autocrine growth factor in the regulation of normal growth in the myometrium, and dysregulation of IGF-I signaling could contribute to the neoplastic growth of uterine leiomyomas (9
). Our findings of up-regulation of the IGF-I/IGF-IR pathway confirm what has been found in previous studies and demonstrate that higher expression levels of IGF-I and IGF-IRβ are present in leiomyomas compared with myometrial tissue. In addition, our study has demonstrated significantly higher expression levels of phosphorylated IGF-IRβ and its downstream adaptor/effector proteins, Shc, Grb2, and MAPKp44/42. These data support the involvement of the IGF-I/IGF-IR pathway in fibroid growth and development.
The best characterized signaling pathways activated by the IGF-IR are the MAP kinase and the PI3 kinase pathways (40
). Tyrosine-phosphorylated IRS-1 and Shc bind different adaptor/effector proteins inducing multiple signaling cascades, among them several interconnecting pathways controlling cell survival and proliferation. Activation of the Shc/Ras/MAP kinase pathway leads to transcriptional responses associated with mitogenesis and cell proliferation. The critical survival pathway activated by IGF-I stems from IRS-1. IRS-1 recruits and stimulates PI3K, which then transmits signals to the serine/threonine kinase AKT. Activated AKT phosphorylates and blocks a variety of proapoptotic proteins. Furthermore, AKT induces the expression of the anti-apoptotic protein Bcl-2 (41
). In this study, there was minimal expression of PI3K, and phospho- and total IRS-1 in both tumor and myometrial tissues. There was slightly higher expression of phosphorylated AKT in a few tumor samples compared with matched myometrium by immunohistochemical analysis. However, there were no significant differences in total and phosphorylated AKT expression levels observed between tumor and myometrial tissue from confirmative western blotting. This is consistent with our previous findings that a higher rate of cell proliferation appears to play the predominant role in uterine leiomyoma growth, and that neither prolonged cell survival, nor loss of expression of apoptosis-inducing proteins, or increased apoptosis, were likely to be significant mechanisms of uterine leiomyoma cell growth (18
). However, another group found higher expression of phospho-AKT in leiomyomas than in myometrial tissue (43
), which may be due to differences in sampling and/or assays used to determine phosphorylated AKT or total AKT levels. We used immunohistochemistry, western blot analysis, and in vitro
studies to evaluate the expression levels of total and phosphorylated AKT in leiomyoma and myometrial tissue and in leiomyoma cells. In our studies, we compared the ratio of phosphorylated AKT over total AKT, which was not done in one study where a difference in expression of phosphorylated and total AKT was noted in leiomyoma versus myometrial tissues (43
, IGF-I is mitogenic for a variety of cells including fibroblasts, smooth muscle cells, and leiomyoma cells (11
). In this study, we found that IGF-I treatment resulted in significantly enhanced proliferation of UtLM cells treated with IGF-I peptide compared with non-treated UtLM cells. Interestingly, exogenous IGF-I could compensate for steroid hormones, and possibly growth factor peptide effects, which had been reduced or removed from serum after charcoal/dextran treatment by restoring UtLM cell growth to the level of UtLM cells under full serum culturing conditions. IGF-I treatment also increased phosphorylated IGF-IRβ expression in UtLM cells and facilitated activation of the IGF-IR signaling cascade and the downstream effector proteins Shc and MAPKp44/42, which correlated positively with UtLM cell maintenance and proliferation. Taken together, our results further indicate that activation of IGF-IR reduced the requirement for hormones and other growth factors, and was necessary for UtLM cells to obtain optimal growth in vitro
, which is consistent with other groups’ findings of a central role of the IGF-IR in the mitogenic action of the IGF-I peptide and other growth factors (13
). A neutralizing antibody against IGF-IRβ inhibited IGF-I-induced stimulation of UtLM cell proliferation and partially blocked IGF-I-induced activation of the IGF-IR pathway, which is consistent with some reports that cells in monolayer culture are only partially sensitive to the inhibition of IGF-IR when IGF-IR is blocked (42
). The in vitro
data further support the involvement of the IGF-IR and MAPK pathways in orchestrating uterine leiomyoma growth.
In contrast to our in vivo findings, the IGF-I peptide increased the phosphorylation of IRS-I and AKT in UtLM cells. These differential effects might be due to differences in the biological environments between tissue and cells grown in culture. The activation of IRS-I and AKT was not blocked significantly by IGF-IR neutralization, which might indicate that IRS-I phosphorylation was most likely induced by the IGF-I peptide binding to the insulin receptor.
In summary, we have analyzed the expression profiles of RTKs in leiomyoma and patient-matched myometrial tissue, and identified phosphorylation of IGF-IR and 14 other growth factor RTKs in leiomyoma tissue. We have also found an overexpression of IGF-I and IGF-IRβ, and downstream phosphorylated effector proteins of the IGF-IRβ signaling pathway in leiomyomas compared with myometrial tissue. These data indicate that activation of the IGF-IR/MAPK pathway in fibroids is important in uterine leiomyoma growth as proposed in . Exogenously added IGF-I had a mitogenic effect on UtLM cells, and an effect on activation of IGF-IR and its downstream effector proteins in vitro. A neutralizing antibody against IGF-IRβ inhibited IGF-I-induced stimulation of UtLM cell proliferation and the expression of IGF-IR and downstream proteins.
The differential expression of IGF-I and MAPK pathway proteins in patient-matched leiomyoma and myometrial tissues and in UtLM cells before and after stimulation with IGF-I observed in this study suggests a model for an IGF-I induced signaling cascade in leiomyoma development (). In this model, IGF-I peptide binds to the IGF-IR to induce tyrosine autophosphorylation of the β subunit of the receptor and phosphorylation of its adaptor protein Shc. Phosphorylated Shc then associates with Grb2-mSOS complex to activate p21/Ras, which leads to transcriptional activation of genes involved in proliferation through the Ras/Raf/MAPK pathway. IGF-I and IGF-IRβ complex also autophosphorylates its docking protein, IRS-I, which in turn activates the survival PI3K/AKT pathway. In our in vivo study, however, this pathway does not appear to play a major role in the pathogenesis of leiomyomas. IRS-I may also recruit Grb2, but the Shc-Grb2 pathway seems to be a predominant activator of p21/Ras in IGF-IR signaling in UtLM cells and in the pathogenesis of uterine leiomyomas.
The findings in this study may indicate new anti-tumor targets for noninvasive treatment of uterine leiomyomas. A variety of approaches have been used in preclinical studies to inhibit IGF-IR signaling, including dominant negative mutants, kinase-defective mutants, antisense oligonucleotides, IGFBPs, soluble IGFR antagonistic and/or neutralizing antibodies, and small-molecule kinase inhibitors. Antagonistic antibodies and TK inhibitors are probably the most clinically viable options to date (47
We conclude that upregulation of multiple RTKs and activation of the IGF-I/IGF-IR pathway play an important role in uterine leiomyoma growth. The results from this study potentially may provide non-invasive therapeutic interventions for clinical cases of uterine fibroids.