Differential expression of receptor tyrosine kinases (RTKs) and IGF-I pathway activation in human uterine leiomyomas

Abstract

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 with 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 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-IRbeta and phosphorylated IGF-IRbeta. Additionally, we showed 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. 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-IRbeta, 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-IRbeta 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.

Figure 1

Differential expression of phosphorylated growth factor RTKs in leiomyoma and myometrial tissues. (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 with 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 ten leiomyoma tissue lysates and ten patient-matched myometrial tissue lysates from ten patients, and the dot and triangle symbols represent the dot blot (n = 4) intensity values for pooled myometrial and leiomyoma samples, respectively.

Figure 2

Immunolocalization of IGF-I, IGF-IRβ, phospho-MAP kinase, and phospho-AKT in leiomyoma and myometrial tissues. The immunohistochemical staining was performed on leiomyoma and matched myometrial tissue samples from eight 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 tissues. (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 with 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 eight patients with comparable results. The band images shown represent four of eight patient samples. The phospho/total ratio data was expressed as mean ± SE in eight patient samples.

Figure 4

Immunoprecipitation and western blot analysis of IGF-IR pathway protein expression in leiomyoma and myometrial tissues. (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 with myometrial (M) tissue samples. Levels of Grb2 associated with Shc were also significantly higher (P < 0.001) in leiomyomas compared with myometrial samples. However, there was no difference in levels of phosphorylated IRS-I associated with 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 eight patients with comparable results. The band intensity of the blots was expressed as means ± SE in eight 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 (SS) media plus IGF-I (P < 0.0001) compared with UtLM cells cultured in media without FBS (SF), 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 leiomyoma cells. (A,B) Phosphorylated IGF-IRβ was significantly higher 5 min after IGF-I (100ng/mL) treatment and remained increased until 60 min (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 (P < 0.05) were significantly increased at 5 min and peaked at 10 min when the cells were treated with IGF-I, then fell back to basal levels at 60 min. (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 then is 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 also may 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.