Differential Expression of Receptor Tyrosine Kinases (RTKs) and IGF-I Pathway
Activation in Human Uterine Leiomyomas
Running head: IGF-I Pathway Activation in Fibroids
Linda Yu, 1 Katrin Saile, 1 Carol D. Swartz, 2 Hong He, 1 Xiaolin Zheng, 1 Grace E.
Kissling, 3 Xudong Di, 1 Shantelle Lucas, 1 Stanley J. Robboy, 4,5 and Darlene Dixon1*
1Laboratory of Experimental Pathology and the 3Biostatistics Branch, National Institute
of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH),
Department of Health and Human Services (DHHS), Research Triangle Park, North
Carolina 27709, 2Environmental Carcinogenesis Division, US Environmental Protection
Agency, Research Triangle Park, North Carolina 27709, 4Department of Pathology, and
the 5Department of Obstetrics and Gynecology, Duke University Medical Center,
Durham, North Carolina 27710
* To whom correspondence should be addressed:
Dr. Darlene Dixon
Laboratory of Experimental Pathology
P.O. Box 12233, MDC2-09
111 Alexander Drive, Bldg. 101, rm. C254A
Research Triangle Park, North Carolina 27709 USA
Telephone: (919) 541-3814
Fax Number: (919) 541-0637
Key words: Fibroids, RTKs array, MAPK pathway, phosphorylation, and proliferation
Uterine leiomyomas (fibroids) are benign tumors that are prevalent in women of
reproductive age. Research suggests that activated receptor tyrosine kinases (RTKs) play
an important role in the enhanced proliferation observed in fibroids. In this study, a
phospho-RTK array technique was used to detect RTK activity in leiomyomas compared
to myometrial tissue. We found that fifteen out of seventeen RTKs evaluated in this study
were highly expressed (p<0.02-0.03) in the leiomyomas, and included the IGF-I/IGF-IR,
EGF/EGFR, FGF/FGF-R, HGF/HGF-R and PDGF/PDGF-R gene families. Due to the
higher protein levels of IGF-IR observed in leiomyomas by us in earlier studies, we
decided to focus on the activation of the IGF-IR, its downstream effectors, and
MAPKp44/42 to confirm our earlier findings, and to validate the significance of the
increased IGF-IR phosphorylation observed by RTK array analysis in this study. We used
immunolocalization, western blot or immunoprecipitation studies and confirmed that
leiomyomas overexpressed IGF-IRβ and phosphorylated IGF-IRβ, in addition to showing
that the downstream effectors, Shc, Grb2 and MAPKp44/42 (p < 0.02-0.001) were also
overexpressed and involved in IGF-IR signaling in these tumors, while IRS-I, PI3K and
AKT were not. Additionally, in vitro studies showed that IGF-I (100 ng/ml) increased
the proliferation of uterine leiomyoma cells (UtLM) (p <0.0001), and that phosphorylated
IGF-IRβ, Shc and MAPKp44/42 were also overexpressed in IGF-I-treated UtLM cells
(p<0.05), similar to the tissue findings. A neutralizing antibody against the IGF-IRβ
blocked these effects. These data indicate that overexpression of RTKs and in particular,
activation of the IGF-IR signaling pathway through Shc/Grb2/MAPK are important in
mediating uterine leiomyoma growth. These data may provide new anti-tumor targets for
noninvasive treatment of fibroids.
Uterine leiomyomas (fibroids) are benign neoplasms of the myometrium that are
prevalent in reproductive-aged women in the United States (1). Some fibroids are
asymptomatic; however, many can cause pelvic pain, menstrual bleeding and infertility.
In the United States, fibroids represent a tremendous public health burden for women and
a great economic cost to society (2). Treatment options for leiomyomas are currently very
limited; surgery remains the main form of treatment (3).
Receptor tyrosine kinases (RTKs) are the main mediators of the signaling network
that transmit extracellular signals into the cell, and control cellular differentiation and
proliferation (4). Recent and rapid advances in the understanding of cellular signaling by
RTKs in normal and tumor cells have brought to light the potential of RTKs as selective
anti-tumor targets. Their activity is normally tightly controlled and regulated; however,
overexpression of RTK proteins or abnormal stimulation by autocrine growth factor
loops contribute to constitutive RTK signaling, resulting in dysregulated cell growth and
tumor formation (4). Uterine leiomyomas express many types of growth factors (5, 6).
Those factors may foster leiomyoma growth through local paracrine and/or autocrine
mechanisms (5, 7). However, the association between upregulation of various growth
factor RTKs and leiomyoma development is not fully understood. In this study, we
obtained uterine leiomyomas and patient-matched myometrial tissue from premenopausal
women to investigate the differential expression of growth factor RTKs involved in cell
mitogenesis by using a RTK array technique.
Previous studies in our laboratory (5, 8) and by others (7, 9-11) have indicated
that the IGF-I pathway plays an important role in uterine leiomyoma development and
growth. IGF-I is a single-chain polypeptide whose structure is highly similar to that of
pro-insulin. It can bind either to its specific cell surface receptor IGF-IR or to the closely
related insulin receptor (IR) although it has a much higher affinity for its own receptor
(12). IGF-IR is one of the major receptor tyrosine kinase proteins, and its central role in
the IGF-I family in the regulation of both physiological and pathological growth
processes has been firmly established (13). The IGF-IR utilizes IRS-I/Shc as immediate
downstream adaptors which can ultimately lead to the activation of the IRS/PI3K/AKT
cell survival pathway and/or the Shc/Ras/Grb2/MAP kinase cell proliferation pathway
(14). Based on our previous studies, and to confirm the overexpression level of
phosphorylated IGF-IR observed in the RTKs array in this study, we chose to further
evaluate the IGF-IR and its pathway activation in leiomyoma and myometrial tissues, and
to assess IGF-IR signaling in uterine leiomyoma cells in culture.
MATERIALS AND METHODS
Uterine leiomyoma and patient-matched myometrial tissue samples were
collected from 8-10 women ranging from 41 – 49 years of age who underwent
hysterectomy for symptomatic leiomyomas. All subjects had taken no hormonal
medication within at least 3 months prior to hysterectomy. Informed consent was
obtained, and the Institutional Review Board (IRB) of the NIEHS, NIH approved the
study. All leiomyomas and unaffected myometrial samples were confirmed by
histological evaluation. The endometrium from each uterus was evaluated to determine
the menstrual cycle phase, with all tumors taken from women in the proliferative phase of
the menstrual cycle.
Phosphorylation of Receptor Tyrosine Kinases (RTKs) Array
Expression of phosphorylated growth factor receptor tyrosine kinases (RTKs) was
detected using the Proteome ProfilerTM Array Kit (R&D Systems, Minneapolis, MN
USA). Samples of 50-100 mg of frozen leiomyoma and myometrial tissue from each of
ten patients were collected in cold solubilization buffer (1% Triton X-100, 1 mM sodium
vanadate, 1 mM sodium fluoride, 0.05 mM sodium molybdate, 20μg/ml aprotinin,
20μg/ml leupeptin, 4μg/ml (4-amidinophenyl) methane sulfonyl fluoride, 150 mM
sodium chloride in 50 mM Tris-HCl, pH 7.4). The samples were minced and then
homogenized on ice using a 30-second burst of a homogenizer at its highest setting
followed by centrifugation at 14,000 rpm for 5 min at 4°C. The supernatants were
collected and stored at -80°C. Three-hundred μg of pooled total protein from ten
leiomyomas and patient-matched myometrial tissue (30 μg from each patient sample)
were incubated with RTK array membrane spotted with various anti-phospho-RTK
antibodies. The procedures were performed according to the manufacturer’s protocol.
Histology and Immunohistochemistry
The tissues from 8 of the 10 women from the RTK studies were fixed overnight in
10% neutral buffered formalin, embedded in paraffin, sectioned at 6 μm and mounted
onto charged glass slides (ProbeOn TM Plus, Fisher Scientific, Pittsburgh, PA) for
immunohistochemical staining. Tissues were deparaffinized with xylene and rehydrated
with ethanol. Endogenous peroxidase activity was blocked with 0.3% H2O2 [0.1 ml 30%
hydrogen peroxide (Fisher Scientific, Fair Lawn, NJ)] for 30 minutes. Tissues were
pressurized and depressurized by a decloaker and blocked for 30 minutes with normal
serum (Vector Laboratories, Inc. Burlingame, CA). Tissues were incubated overnight at
4°C with respective diluted antibodies: 1:25 for IGF-I and 1:75 for IGF-IRβ (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), 1:50 for IR (Upstate Biotechnology, Lake Placid,
NY), and 1:50 for phospho-MAPKp44/42 and phospho-AKT (Cell Signaling
Technology, Beverly, MA). The same concentrations of non-immune rabbit or goat
serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used as negative
controls. Secondary antibodies were applied for 60 min (1 drop of biotinylated anti-rabbit
or goat IgG, respectively, with 3 drops normal goat or rabbit serum, respectively).
Tissues were labeled for 60 min with 2 drops of Avidin + 2 drops biotinylated Enzyme
(Vector Laboratories). DAKO® Liquid DAB (3,3’-diaminobenzidine tetrahydrachloride)
large volume substrate-chromogen system (DAKO Corporation, Carpinteria, CA) was
applied for 6 min in the dark. Tissues were counterstained for 45 seconds with Mayer’s
hematoxylin (Polyscientific, Bay Shore, NY), dehydrated with ethanol and xylene and
mounted with Permount, then coverslipped.
Cell Culture Studies and MTT Assay
Human uterine leiomyoma (UtLM) cells (GM10964) were purchased from Coriell
Institute for Medical Research (Camden, NJ), and grown in UtLM media containing
Minimum Essential Medium (MEM; Gibco Life Technologies), 1X vitamins, 1X non-
essential amino acids, 1X essential amino acids, 2X L- glutamine (Gibco Life
Technologies) and 20% Fetal Bovine Serum (FBS; Sigma, St. Louis, MO). UtLM cells
were seeded into 96-well plates at 5,000 cells/well in growth medium. The media were
switched to phenol red-free Dulbecco’s Modified Eagel’s Medium (DMEM) with 10%
FBS, or 10% charcoal/dextran-treated FBS after 24 hours, and switched to phenol red-
free DMEM only after 48 hours. Twenty-four hours later, the cells were treated with
media containing serum (FBS) + DMEM, or serum free DMEM with or without LongR-
IGF-I (GroPep Limited, The Barton SA, Australia), or charcoal/dextran treated serum +
DMEM with or without IGF-I peptide. The IGF-I treatment media consisted of 100ng/ml
IGF-I and 0.1% bovine serum albumin. The cells were treated with IGF-I every 3 days
and were counted on each of days 0, 3, 9, 12, and 15 using a commercially available
MTT assay kit (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega,
Madison, WI) according to the manufacturer’s protocol. Plates were consistently read
after 1.5 hours of incubation with the assay reagent. The study was repeated at least three
times and statistical analysis was used to test for differences between IGF-I treatments
and their respective non-treated controls, between serum (FBS) and serum free medium
conditions, and between IGF-I treatments before and after anti-IGF-IR blocking.
Readings from the columns with no cells were subtracted as background.
In order to assess early activation of a specific pathway, in particular the IGF-IR
pathway, cells must be tested in a serum free environment to eliminate the possibility of
activation by cytokines in the serum. UtLM cells were grown in UtLM media until 70%
confluent, and maintained in charcoal/dextran treated FBS for 3 days and serum free for 1
day. The cells were treated with IGF-I peptide (100 ng/ml) for 0, 5, 10, 30, and 60
minutes separately and cell lysates were harvested and stored at -80oC prior to western
blotting. For IGF-IR blocking, the cells were blocked with anti- goat-hIGF-IR (2μg/ml)
(R&D systems) for 2 hours, then cells were induced with LongR-IGF-I peptide (100
ng/ml) for 0, 5, 10, 30 and 60 minutes. The cell lysates were harvested and stored at -
80oC prior to western blotting.
Western Blotting of Tissue Homogenates and Cells
A total of 50-100 mg of frozen leiomyoma and myometrial tissues from each of
eight patients same as those in RTKs and Immunohistochemistry studies were collected
in cold solubilization buffer (the same as described in the RTKs array procedure). The
supernatants containing protein lysate were collected and stored at -80°C for western blot
For the in vitro studies, UtLM cells were harvested in RIPA buffer (phosphate-
buffered saline, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing
protease inhibitors aprotinin (10μg/ml), leupeptin (10ug/ml) and phenymethanesulfonyl
fluoride (1mM), after treatment with IGF-I at different time points. The cell lysates were
centrifuged and stored at -80°C prior to western blot analysis as previously described
(15). The western blots were incubated overnight with a specific diluted primary
antibody: 1: 1000 for total and phosphorylated IGF-IRβ, IRβ, MAPKp44/42, IRS-I, Shc
and AKT, and PI3K (Cell Signaling Technology Incorporated, Beverly, MA), and 1: 300
for Grb2 (Santa Cruz Biotechology, Santa Cruz, CA). A densitometer (Fluor
ChemTM8900, Alpha Innotech, San Leandro, CA) was used for quantitation of band
A total of 300 μg of total protein from tissue homogenates (as described in the
RTKs array procedure) in 500ml solubilization buffer was precleared by incubation with
50 μl of Protein A- sepharose CL-4B Beads (Amersham Pharmacia Biotech, Piscataway,
NJ) at 4oC for 30 min. Precleared samples were incubated for 2 hours with rabbit
polyclonal IGF-IRβ or phosphorylated Shc at 4oC. The antigen-antibody complex was
captured by incubation with 50μl of protein A-sepharose CL 4B overnight at 4oC. The
beads were washed 3-4 times in solubilization buffer. Immune complexes were eluted
from the beads using 2X laemmli sample buffer (BIO-RAD, Hercules CA, with 5% 2-
mercaptoethanol added to the buffer before use).
A Seize Primary Immunoprecipitation Kit (Pierce Biotechnology, Rockford, IL)
was used to keep the antigen free from antibody contamination. The kit was applied
because IgG of anti-IGF-IRβ, which was used to pull down Shc, has the same molecular
weight as the target protein Shc and this kit allows the immunoglobulin to remain
adherent to aminolink Plus Gel following elution. Amounts of 200μg of antibody IGF-
IRβ were coupled to 400μl of 50% of aminolink Plus Gel Slurry in coupling buffer
overnight at 4oC. The coupled gel and antibody complex were divided into 8 tubes, and
each tube incubated with 300μg of the total protein in binding buffer over night at 4oC.
The gel was washed 3 times with washing buffer (manufacturer’s protocol). Only the
antigen in the antigen-antibody complexes was eluted by the elution buffer (all buffers
supplied in the Kit).
Mann-Whitney tests (16) were used to determine statistically significant
differences between leiomyoma and myometrial tissue for each RTK receptor dot
intensity value. Mann-Whitney tests were also used to compare the western blot intensity
value of leiomyoma and myometrial tissue with respect to total and phospho-IGF-IRβ,
IRS-I, Shc, AKT, and MAPKp44/42, and their ratios; and to compare myometrial tissue
with leiomyoma tissue for intensity values of PI3K, IR and immuno-precipitated values
of Shc, IRS-I and Grb2. For in vitro studies, the Wilcoxon signed rank test was used to
test for differences between IGF-I treatments and their respective non-treatment controls,
between serum (FBS) and serum free medium conditions, and between with and without
anti-IGF-IR blocking with IGF-I treatments. Also, Mann-Whitney tests were used to
compare the western intensity values of the IGF-I treatment and non-treatment groups,
and the IGF-IR blocked and non-blocked groups. The analysis of RTKs was based on
four replicates of data. The western blot densitometry procedure from tissue was based
on data from eight patients. For the in vitro IGF-I treatment MTT assay, 8 or 16 wells in
96 wells plates were treated as replicates for each treatment condition, and the assay was
repeated at least three times. The western blotting analysis of IGF-I treated UtLM cells
was based on three replicates of data.
Differential Expression of Growth Factor RTKs in Leiomyoma and Myometrial
To determine the expression profile of growth factor RTKs in leiomyoma and
patient-matched myometrial tissues, we performed phospho-receptor tyrosine kinase
arrays. The degree of differential expression of growth factor RTK proteins between
leiomyoma and myometrial tissue was quantitated by dot blot (17 phosphorylated growth
factor RTK antibodies evaluated on the membrane). Ten leiomyomas and patient-
matched myometrial samples were collected, and pooled tumor and myometrial protein
samples were incubated on separate RTK membranes followed by densitometric
measurements of dot blots.
The various growth factor receptor tyrosine kinases were differentially expressed
in leiomyoma and myometrial tissues (Figure 1A). Of seventeen RTKs evaluated on the
array membrane for this study, significantly greater expression levels were observed with
fifteen of the phosphorylated RTKs in the leiomyomas compared to myometrial samples
(p < 0.02 - 0.03) (Figure 1B). The significant mean (n=4 dots) fold changes of expression
of RTKs in leiomyoma tissue over that of myometrial tissue were EGFR (1.9), ErbB4
(3.2), FGFR1 (3.0), FGFR2α (3.3), FGFR3 (2.9) FGFR4 (3.8), InsulinR (2.4), IGF-IR
(2.7), HGF-R (3.5), MSP-R (2.7), PDGF-Rα (3.8), PDGF-Rβ (4.8), SCF-R (7.9), Fit3
(6.2), and M-CSF-R (6.5). These receptors basically belong to the EGF, Insulin, IGFI,
FGF, HGF, and PDGF growth factor gene families, in addition to the hematopoietic
growth factors, which are all involved in cell proliferation and differentiation.
Immunohistochemical localization of IGF-I, IGF-IRβ, IRβ, and MAP Kinase
(MAPK)p44/42 in Leiomyoma and Myometrial Tissue
Based on our previous studies, and to confirm the overexpression level of
phosphorylated IGF-IR observed in the RTKs array in this study, we chose to further
evaluate the IGF-IR and its pathway activation in leiomyoma and myometrial tissue.
Immunohistochemical localization of IGF-I, IGF-IRβ, phospho-MAPKp44/42 and
phospho-AKT in leiomyoma and myometrial tissue was assessed. To check the
specificity of the IGF-Rβ antibody, IRβ was also assessed (data not shown).
Immunoexpression of the IGF-I peptide was much more pronounced in
leiomyomas than in myometrial tissue. Positive staining for IGF-I peptide in the
myometrium was mostly perivascular and minimal (Figure 2A). In the leiomyomas, the
cytoplasm of smooth muscle tumor cells and the fibroblasts in the extracellular matrix of
the tumor tissue stained moderately to intensely positive for IGF-I; whereas, the smooth
muscle cells and interstitial connective tissue of myometrium did not (Figures 2A and
2B). Both tumor and myometrial samples stained positively for IGF-IRβ. However,
staining was more intense in the tumor samples and was found mostly in the cytoplasm
and cytoplasmic membranes of both the smooth muscle cells in the myometrium and the
leiomyoma cells (Figure 2C and 2D). Phospho-MAPKp44/42 staining was nuclear, and
was minimal in the myometrium (Figure 2E). However, the nuclei of fibroblasts in the
extracellular matrix regions and smooth muscle tumor cells in the leiomyoma tissue
showed intense positive nuclear staining for phospho-MAPKp44/42 (Figure 2F).
Interestingly, there was no difference in staining between leiomyoma and myometrial
tissues for phospho-AKT in most of the samples; however, 2 out of 8 (25%) of the tumor
samples showed slightly more intense intranuclear and cytoplasmic phospho-AKT
staining compared to patient-matched myometrial tissue (Figures 2G and 2H). Using
immunohistochemistry, both leiomyoma and myometrial samples were only minimally
positive for the insulin receptor (data not shown).
Western Blot Analysis of Phosphorylated IGF-IR and Associated RTK Pathway
Proteins in Leiomyoma and Myometrial Tissue
To further investigate the activation of the IGF-IR pathway and to delineate which
specific proteins are phosphorylated in leiomyoma tissue compared to myometrium, we
performed western blot analysis to assess the status of IGF-IR, IRS-I, Shc, PI3K, Grb2,
AKT and MAPKp44/42 expression in eight leiomyomas and patient-matched myometrial
tissue samples. Both total and phosphorylated IGF-IRβ, Shc and MAPKp44/42 were
identified by western blotting in leiomyoma and myometrial tissues (Figure 3A).
However, greater phosphorylation of IGF-IRβ, Shc and MAPKp44/42 was observed in
tumor samples, as indicated by the increased intensity of the bands in tumors compared to
the bands from myometrial tissue (Figure 3A). Furthermore, the ratios of the mean
intensities of bands of phosphorylated over total protein for IGF-IRβ (p < 0.04), Shc (p <
0.05), and MAPKp44/42 (p < 0.02) were significantly higher in tumors compared to
patient-matched myometrial samples (Figure 3B). Lower levels of expression of PI3K
were observed in both myometrial and leiomyoma tissues (Figure 3A). Although there
was expression of IRS-I and AKT in both tissue types (Figure 3A), the ratios of the mean
intensities of phosphorylated IRS-I and AKT over total protein levels were not
significantly different between leiomyoma and myometrial tissues (Figure 3B).
Immunoprecipitation of Phospho-Shc, Phospho-IRS-1 and Grb2
Immunoprecipitation studies using leiomyoma and patient-matched myometrial
tissue lysates were performed to determine which proteins downstream of IGF-IR were
activated and associated with the IGF-IR. We found that more phospho-Shc was
precipitated to the IGF-IRβ in leiomyoma samples compared to myometrial samples (p
<0.02); however, there was no difference in IRS-I protein associated with the IGF-IRβ in
tumor versus myometrial tissue (Figure 4A and 4B). Grb2, a protein immediately
downstream of Shc in the MAPK signaling pathway, showed increased precipitation to
Shc (p < 0.001) in the leiomyoma samples compared to myometrial tissues (Figure 4A
and 4B). Our data indicate that phospho-IGF-IRβ and the downstream adaptor and
effector proteins phospho-Shc, Grb2 and the MAP kinase pathway were activated in
uterine leiomyomas compared to myometrial samples, and more so than the PI3K/AKT
Functional Effects of IGF-I on UtLM Cell Proliferation
To examine the functional properties of IGF-I on proliferation of uterine
leiomyoma (UtLM) cells in vitro, UtLM cells were cultured in DMEM containing IGF-I
(100ng/ml) in the presence or absence of 10% charcoal/dextran-treated (stripped serum)
FBS, and in the presence or absence of 10% normal FBS. There were significant
differences in proliferation under the different culture conditions as evidenced by using a
MTT assay (Figure 5). The cell numbers were significantly higher in the presence of
10% FBS, or presence of IGF-I (100 ng/ml) with 10% charcoal/dextran treated FBS
(stripped serum, SS) compared to cells receiving no FBS (serum free, SF) and no IGF-I at
days 3, 9, 12 and 15 (p<0.0002, p<0.0001). Surprisingly, UtLM cell growth in SS plus
IGF-I medium equaled the growth of UtLM cells cultured in complete FBS medium at 9
days after treatment. In serum free medium, there was insufficient IGF-I and other
growth factors and cytokines needed to maintain UtLM cell survival and promote cell
growth. The proliferative effect of IGF-I on UtLM cells was inhibited by a neutralizing
antibody against IGF-IRβ (p<0.0002, Figure 5).
Effects of IGF-I on Activation of IGF-IR Pathway in UtLM cells
To determine whether the IGF-I ligand could activate the IGF-IR and its
associated downstream proteins in vivo, UtLM cells were exposed to 100 ng/ml of IGF-I
in serum-free medium for 0, 5, 10, 30 and 60 minutes. Western immunoblots of the cell
lysates at different time points were probed with anti-phospho and anti-total IGF-IRβ,
IRS-I, Shc, AKT and MAPKp44/42. UtLM cells exposed to IGF-I were found to have
increased protein tyrosine kinase phosphorylation in a time-dependent manner. When
UtLM cells were cultured in media containing IGF-I peptide the phosphorylated IGF-IR
and IRS-I protein expression levels were increased within 5 minutes, reaching maximum
levels at 10 minutes and plateauing by 60 minutes. However, phosphorylated Shc,
MAPKp44/42 and AKT expression levels were increased in 5 minutes, reached
maximum levels at 10 minutes, thereafter declining toward baseline by 60 minutes
(Figure 6A). The ratio of phosphorylated over total of IGF-IRβ, IRS-I, Shc and
MAPKp44/42 followed the same pattern, and was significantly increased in UtLM cells
treated with IGF-I compared to untreated UtLM cells (p<0.05; Figure 6B).
Two hours of blocking with anti-IGF-IRβ resulted in a significant reduction in
phosphorylated IGF-IRβ, Shc, and MAPKp44/42 (p< 0.05) compared to UtLM cells in
IGF-I containing medium without the neutralization antibody (Figure 6A). Densitometric
scanning showed that IGF-IRβ level in anti-IGF-IRβ treated UtLM cells was
approximately 20% of that seen in UtLM cells without the antibody treatment. There
was also a reduction of approximately 40% in phosphorylated Shc and 80% in
phosphorylated MAP kinase levels respectively (Figure 6C). However, the inhibitory
effect on expression of phosphorylated IRS-I was not statistically significant at the 10-,
30- and 60-minute time points. In addition, the expression of phosphorylated AKT was
not significantly altered at any time point (Figure 6C), which might be due to IGF-IR
activation being partially blocked by the neutralization antibody, or the activation of IRS-
1 and AKT occurring through activation of the IR in UtLM cells after treatment with
IGF-I. These data indicate that the MAP kinase pathway was activated through
phosphorylation of IGF-IRβ and its adaptor protein Shc when stimulated with IGF-I at
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, 6-8, 18). 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
In this study, leiomyoma and patient-matched myometrial samples from 10
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 to myometrial samples, and many of these receptors
belonged to the 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, 21, 22). 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
partially due 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, 27). 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, 29 30), 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 (32-34), 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
to myometrial tissues in our RTK array studies. These receptors might be worthwhile
studying in the future.
The up-regulation 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 further regulated
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
Among these highly expressed RTKs in leiomyoma tissues, we observed that the
IGF-IR exhibited increased phosphorylation in leiomyomas compared to myometrial
samples in this study, and in earlier studies we have found the receptor protein to be
overexpressed in leiomyomas compared to myometrium (5). IGF-IR affects cell
mitogenesis and survival by binding of its ligand IGF-I, and 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, 18). 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, 36). 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, 36). In
keeping with this concept and based on increased immunolocalization of IGF-IR in
leiomyoma compared to myometrial tissue observed in our earlier studies (5, 18), 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 (37-39). 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 upregulation 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 to
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, 42). 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 of tumor samples compared to 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, which 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,
44), 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) .
In vitro, IGF-I is mitogenic for a variety of cells including fibroblasts, smooth
muscle cells and leiomyoma cells (11, 45, 46). In this study, we found that IGF-I
treatment resulted in significantly enhanced proliferation of UtLM cells treated with IGF-
I peptide compared to 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, 36). 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 significantly blocked 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 to myometrial tissue. These data indicate that
activation of the IGF-IR/MAPK pathway in fibroids is important in uterine leiomyoma
growth as proposed in Figure 7. 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 (Figure 7). In this model, IGF-I peptide
binds to the IGF-IR to induce tyrosine autophosphorylation of the beta 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-IR• 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
recruits Grb2, but the Shc-Grb2 pathway seems to be 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, 48).
We conclude that upregulation of multiple RTKs and activation of the IGF-I/IGF-
IR pathway plays an important role in uterine leiomyoma growth. The results from this
study may potentially provide non-invasive therapeutic intervention for clinical cases of
The authors would like to thank Dr. Gregg Richards for his extensive review of the
original version of this manuscript. This research was supported, in part, by the
Intramural Research Program of the NIH, National Institute of Environmental Health
1. Payson M, Leppert P, Segars J. (2006) Epidemiology of myomas. Obstet Gynecol
Clin North Am 33: 1-11.
2. Mauskopf J, Flynn M, Thieda P, Spalding J, Duchane J. (2005) The economic
impact of uterine fibroids in the United States: a summary of published estimates.
J Womens Health (Larchmt) 14: 692-703.
3. Wallach EE, Vlahos NF. (2004) Uterine myomas: an overview of development,
clinical features, and management. Obstet Gynecol 104: 393-406.
4. Bennasroune A, Gardin A, Aunis D, Cremel G, Hubert P. (2004) Tyrosine kinase
receptors as attractive targets of cancer therapy. Crit Rev Oncol Hematol 50: 23-
5. Dixon D, He H, Haseman JK. (2000) Immunohistochemical localization of
growth factors and their receptors in uterine leiomyomas and matched
myometrium. Environ Health Perspect 108 Suppl 5: 795-802.
6. Brahma PK, Martel KM, Christman GM. (2006) Future directions in myoma
research. Obstet Gynecol Clin North Am 33: 199-224, xiii.
7. Walker CL, Stewart EA. (2005) Uterine fibroids: the elephant in the room.
Science 308: 1589-1592.
8. Flake GP, Andersen J, Dixon D. (2003) Etiology and pathogenesis of uterine
leiomyomas: a review. Environ Health Perspect 111: 1037-1054.
9. Burroughs KD, Howe SR, Okubo Y, Fuchs-Young R, LeRoith D, Walker CL.
(2002) Dysregulation of IGF-I signaling in uterine leiomyoma. J Endocrinol 172:
10. Wei J, Chiriboga L, Mizuguchi M, Yee H, Mittal K. (2005) Expression profile of
tuberin and some potential tumorigenic factors in 60 patients with uterine
leiomyomata. Mod Pathol 18: 179-188.
11. Giudice LC, Irwin JC, Dsupin BA, et al. (1993) Insulin-like growth factor (IGF),
IGF binding protein (IGFBP), and IGF receptor gene expression and IGFBP
synthesis in human uterine leiomyomata. Hum Reprod 8: 1796-1806.
12. Rubin R, Baserga R. (1995) Insulin-like growth factor-I receptor. Its role in cell
proliferation, apoptosis, and tumorigenicity. Lab Invest 73: 311-331.
13. Werner H, Roberts CT, Jr. (2003) The IGFI receptor gene: a molecular target for
disrupted transcription factors. Genes Chromosomes Cancer 36: 113-120.
14. Mauro L, Surmacz E. (2004) IGF-I receptor, cell-cell adhesion, tumour
development and progression. J Mol Histol 35: 247-253.
15. Swartz CD, Afshari CA, Yu L, Hall KE, Dixon D. (2005) Estrogen-induced
changes in IGF-I, Myb family and MAP kinase pathway genes in human uterine
leiomyoma and normal uterine smooth muscle cell lines. Mol Hum Reprod 11:
16. Conover WJ, Iman RL. (1982) Analysis of covariance using the rank
transformation. Biometrics 38: 715-724.
17. Arslan AA, Gold LI, Mittal K, et al. (2005) Gene expression studies provide clues
to the pathogenesis of uterine leiomyoma: new evidence and a systematic review.
Hum Reprod 20: 852-863.
18. Dixon D, Flake GP, Moore AB, et al. (2002) Cell proliferation and apoptosis in
human uterine leiomyomas and myometria. Virchows Arch 441: 53-62.
19. Wang J, Ohara N, Wang Z, et al. (2006) A novel selective progesterone receptor
modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I,
TGFbeta3 and their receptors in cultured uterine leiomyoma cells. Hum Reprod
20. Harrison-Woolrych ML, Charnock-Jones DS, Smith SK. (1994) Quantification of
messenger ribonucleic acid for epidermal growth factor in human myometrium
and leiomyomata using reverse transcriptase polymerase chain reaction. J Clin
Endocrinol Metab 78: 1179-1184.
21. Rein MS, Nowak RA. (1992) Biology of uterine myomas and myometrium in
vitro. Semin Reprod Endocrinol 10: 310-319.
22. Shimomura Y, Matsuo H, Samoto T, Maruo T. (1998) Up-regulation by
progesterone of proliferating cell nuclear antigen and epidermal growth factor
expression in human uterine leiomyoma. J Clin Endocrinol Metab 83: 2192-2198.
23. Stewart EA, Nowak RA. (1996) Leiomyoma-related bleeding: a classic
hypothesis updated for the molecular era. Hum Reprod Update 2: 295-306.
24. Mangrulkar RS, Ono M, Ishikawa M, Takashima S, Klagsbrun M, Nowak RA.
(1995) Isolation and characterization of heparin-binding growth factors in human
leiomyomas and normal myometrium. Biol Reprod 53: 636-646.
25. Wolanska M, Bankowski E. (2006) Fibroblast growth factors (FGF) in human
myometrium and uterine leiomyomas in various stages of tumour growth.
Biochimie 88: 141-146.
26. Fayed YM, Tsibris JC, Langenberg PW, Robertson AL, Jr. (1989) Human uterine
leiomyoma cells: binding and growth responses to epidermal growth factor,
platelet-derived growth factor, and insulin. Lab Invest 60: 30-37.
27. Liang M, Wang H, Zhang Y, Lu S, Wang Z. (2006) Expression and functional
analysis of platelet-derived growth factor in uterine leiomyomata. Cancer Biol
Ther 5: 28-33.
28. Wolanska M, Bankowski E. (2007) Transforming growth factor beta and platelet-
derived growth factor in human myometrium and in uterine leiomyomas at
various stages of tumour growth. Eur J Obstet Gynecol Reprod Biol 130: 238-
29. Boehm KD, Daimon M, Gorodeski IG, Sheean LA, Utian WH, Ilan J. (1990)
Expression of the insulin-like and platelet-derived growth factor genes in human
uterine tissues. Mol Reprod Dev 27: 93-101.
30. Hoppener JW, Mosselman S, Roholl PJ, et al. (1988) Expression of insulin-like
growth factor-I and -II genes in human smooth muscle tumours. Embo J 7: 1379-
31. Toscani GK, Chaves EM, Cervi FL, et al. (2004) Gene expression and tyrosine
kinase activity of insulin receptor in uterine leiomyoma and matched
myometrium. Arch Gynecol Obstet 270: 170-173.
32. Tommola P, Pekonen F, Rutanen EM. (1989) Binding of epidermal growth factor
and insulin-like growth factor I in human myometrium and leiomyomata. Obstet
Gynecol 74: 658-662.
33. Chandrasekhar Y, Heiner J, Osuamkpe C, Nagamani M. (1992) Insulin-like
growth factor I and II binding in human myometrium and leiomyomas. Am J
Obstet Gynecol 166: 64-69.
34. Van der Ven LT, Roholl PJ, Gloudemans T, et al. (1997) 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 75: 1631-1640.
35. Schlessinger J. (2000) Cell signaling by receptor tyrosine kinases. Cell 103: 211-
36. Baserga R. (1998) The IGF-I Receptor in Normal and Abnormal Growth. In:
Hormones and Growth Factors in Development and Neoplasia. Dickson RB,
Salomon DS (ed.) Wiley-Liss Inc, Wilmington, DE, pp. 269-287.
37. van der Ven LT, Gloudemans T, Roholl PJ, et al. (1994) Growth advantage of
human leiomyoma cells compared to normal smooth-muscle cells due to
enhanced sensitivity toward insulin-like growth factor I. Int J Cancer 59: 427-
38. Englund K, Lindblom B, Carlstrom K, Gustavsson I, Sjoblom P, Blanck A.
(2000) Gene expression and tissue concentrations of IGF-I in human myometrium
and fibroids under different hormonal conditions. Mol Hum Reprod 6: 915-920.
39. Wolanska M, Bankowski E. (2004) An accumulation of insulin-like growth factor
I (IGF-I) in human myometrium and uterine leiomyomas in various stages of
tumour growth. Eur Cytokine Netw 15: 359-363.
40. O'Connor R. (2003) Regulation of IGF-I receptor signaling in tumor cells. Horm
Metab Res 35: 771-777.
41. Butler AA, Yakar S, Gewolb IH, Karas M, Okubo Y, LeRoith D. (1998) Insulin-
like growth factor-I receptor signal transduction: at the interface between
physiology and cell biology. Comp Biochem Physiol B Biochem Mol Biol 121:
42. Surmacz E. (2003) Growth factor receptors as therapeutic targets: strategies to
inhibit the insulin-like growth factor I receptor. Oncogene 22: 6589-6597.
43. Kovacs KA, Lengyel F, Kornyei JL, et al. (2003) Differential expression of
Akt/protein kinase B, Bcl-2 and Bax proteins in human leiomyoma and
myometrium. J Steroid Biochem Mol Biol 87: 233-240.
44. Kovacs KA, Lengyel F, Vertes Z, et al. (2007) Phosphorylation of PTEN
(phosphatase and tensin homologue deleted on chromosome ten) protein is
enhanced in human fibromyomatous uteri. J Steroid Biochem Mol Biol 103: 196-
45. Zumstein P, Stiles CD. (1987) Molecular cloning of gene sequences that are
regulated by insulin-like growth factor I. J Biol Chem 262: 11252-11260.
46. Strawn EY, Jr., Novy MJ, Burry KA, Bethea CL. (1995) Insulin-like growth
factor I promotes leiomyoma cell growth in vitro. Am J Obstet Gynecol 172:
1837-1843; discussion 1843-1844.
47. Tao Y, Pinzi V, Bourhis J, Deutsch E. (2007) Mechanisms of disease: signaling of
the insulin-like growth factor 1 receptor pathway--therapeutic perspectives in
cancer. Nat Clin Pract Oncol 4: 591-602.
48. Clemmons DR. (2007) Modifying IGF1 activity: an approach to treat endocrine
disorders, atherosclerosis and cancer. Nat Rev Drug Discov 6: 821-833.
Figure 1. Differential expression of phosphorylated growth factor RTKs in
Leiomyoma and myometrial tissue
(A, B) Out of 17 RTKs analyzed in the Phosphorylation of Receptor Tyrosine
Kinases Array, 15 growth factor RTKs were highly expressed (p<0.02-0.03) in
leiomyoma (L) tissue compared to myometrial (M) tissue. These RTKs belong to the
EGF, FGF, IGF-I, HGF, and PDGF growth factor receptor gene families. The array was
done on pooled samples of 10 leiomyoma tissue lysates and 10 patient-matched
myometrial tissue lysates from 10 patients, and the bar represents mean+ SE of the dot
blot (n=4) intensity values.
Figure 2. Immunolocalization of IGF-I, IGF-IRβ β, phospho-MAP kinase and
phospho-AKT in leiomyoma and myometrial tissue
The Immunohistochemical staining was performed on leiomyoma and matched
myometrial tissue samples from 8 patients with comparable results. Both myometrial
(M) and leiomyoma (L) tissues stained positive for IGF-I, IGF-IRβ, MAPK and AKT;
however, IGF-I (A, B) was localized to the cytoplasm of smooth muscle cells and within
the fibroblasts in the extracellular matrix in the tumors, whereas, positive staining in
myometrial tissue was mostly perivascular and minimal ; (C, D) IGF-IRβ staining was
observed in the cytoplasm and cytoplasmic membranes, and was more intense in tumor
smooth muscle cells . (E, F) Phospho-MAPKp44/42 was strongly positive in nuclei of
smooth muscle cells in tumor, but minimal in myometrial tissue. (G, H) There were some
staining for phospho-AKT, but no staining difference between myometrial and tumor
tissues was observed.
Figure 3. Western blot analysis of IGFI/IGF-IR pathway activation in leiomyoma
and myometrial tissue
(A,B) Phosphorylated IGF-IRβ (P<0.04), Shc (p<0.05), and MAPKp44/42
(p<0.02) expression levels were significantly higher in leiomyomas (L) compared to
myometrial (M) tissue samples. There was some expression of phosphorylated and total
IRS-I and AKT, but no significant differences were found between tumor and myometrial
tissue samples. There was very minimal expression of PI3K. Western blot analysis was
done at least three times in three independent leiomyoma and matched myometrial tissue
preparations from 8 patients with comparable results. The band images shown represent
four of eight patient samples. The phospho/total ratio data was expressed as means+SE in
eight patient samples.
Figure 4. Immunoprecipitation and western blot analysis of IGF-IR pathway
protein expression in leiomyoma and myometrial tissue
(A) Immunoprecipitation and Immunoblots for phospho-Shc or IRS-I, and Grb2
were done to look for an association of phospho-Shc or IRS-I to the IGF-IRβ, and an
association of Grb2 to phospho-Shc . (B) Concentrations of phosphorylated Shc
associated with IGF-IRβ were significantly higher (p<0.02) in leiomyoma (L) tissue
samples compared to myometrial (M) tissue samples. Levels of Grb2 associated with Shc
were also significantly higher (p<0.001) in leiomyomas compared to myometrial
samples. However, there was no difference in levels of phosphorylated IRS-I
precipitated with anti-IGF-IRβ in myometrial and leiomyoma samples. The procedure
was performed at least three times in three independent leiomyoma and matched
myometrial tissue preparations from 8 patients with comparable results. The band
intensity of the blots was expressed as means+SE in 8 patient samples.
Figure 5. Proliferative effects of IGF-I on uterine leiomyoma (UtLM) cells
IGF-I (100 ng/ml) increased UtLM cell growth in a time-dependent manner. The UtLM
cells had significantly higher growth rates when cultured in media with FBS (P<0.0001)
or with charcoal/dextran stripped media plus IGF-I (p<0.0001) compared to UtLM cells
cultured in media without FBS, or with charcoal/dextran stripped media without IGF-I, or
with IGF-I in the absence of SS. Note that UtLM cell growth in SS plus IGF-I medium
equaled UtLM cell growth in full FBS medium at 9 days after treatment. The
proliferative effect of IGF-I was inhibited by a neutralizing antibody to IGF-IRβ
(p<0.0002). The MTT cell proliferation assay was repeated at least three times with
comparable results. The absorbance of MTT was expressed as means+SE of 8 or 16 wells
in 96 wells plates for each treatment condition.
Figure 6. Activation Effects of IGF-I on IGF-IR pathway in human uterine
(A,B) Phosphorylated IGF-IRβ was significantly higher 5 minutes after IGF-I (100ng/ml)
treatment and remained increased until 60 minutes (p<0.05). Downstream adapter
protein IRS-1 followed the same pattern (p<0.05). Phosphorylation of another adapter
protein Shc (p<0.05) and downstream effector protein AKT (p<0.05) and MAPKp44/42
Linda Yu Page 38
(p<0.05) were significantly increased at 5 minutes and peaked at 10 minutes when the
cells were treated with IGF-I, then fell back to basal levels at 60 minutes. (C) The
activation of IGF-IRβ, Shc and MAPKp44/42 induced by IGF-I was neutralized partially
(Shc: about 40% and MAPKp44/42: about 80%, P<0.05) when the cells were incubated
with anti-IGF-IRB antibody before IGF-I treatment. However, the induction of
phosphorylated IRS-I at 10’, 30’ and 60’, and phosphorylated AKT at 5’, 10’, 30’ and
60’, were not significantly decreased. The western blot analysis was done at least three
times in three independent in vitro experiments with comparable results. The
phospho/total ratio data was expressed as means+SE of three replicates.
Figure 7. Proposed Pathway
In this model, IGF-I peptide binds to IGF-IR to induce tyrosine
autophosphorylation and phosphorylation of its adaptor protein Shc. Phosphorylated Shc
is then associated with the Grb2-mSOS complex to activate p21/ Ras, which leads to
proliferation by activation of the Ras/Raf/MAPK pathway. The IGF-I and IGF-IR
complex also autophosphorylates its docking protein IRS-I, which in turn activates the
survival PI3K/AKT pathway. However, on the basis of our studies this pathway appears
not to be playing a major role in leiomyoma growth. IRS-I may also recruit Grb2, but it
appears that the Shc-Grb2 pathway is the predominant activator of p21/Ras in IGF-IR
signaling in UtLM cells and uterine leiomyoma tissue.
Molecular Medicine www.molmed.org
Molecular Medicine www.molmed.org
Molecular Medicine www.molmed.org
Molecular Medicine www.molmed.org
Molecular Medicine www.molmed.org
Molecular Medicine www.molmed.org
UNCORRECTED PROOF Download full-text
Molecular Medicine www.molmed.org