RANKL/RANK Pathway and Its Inhibitor RANK-Fc in Uterine Leiomyoma Growth

Abstract

Context: Uterine leiomyomas are the most common type of gynecologic tumor in women.

Objective: To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth.

Design: Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo.

Setting: Research laboratory.

Patients: Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy.

Interventions: None.

Main outcome measure: Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc.

Results: RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4).

Conclusions: Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

Figure 1.

(A) RANKL mRNA fold-change in leiomyoma compared with myometrium; RANKL expression is five times higher in leiomyoma than in myometrium. (B) RANK mRNA fold change in leiomyoma compared with myometrium; RANK expression is 50% lower in leiomyoma than myometrium. n = 12 independent tissue samples. The error bars are the standard error of the mean.

Figure 2.

(A) RANKL expression is significantly higher in LICs and (B) RANK expression is significantly higher in LSCs on qRT-PCR. (C) Western blot analysis confirmed the highest RANKL and RANK protein levels in LICs and LSCs, respectively. n = 3 (Western blot) to n = 5 (qRT-PCR) independent tissue samples. The error bars are the standard error of the mean.

Figure 3.

The PR agonist R5020 induces RANKL but not RANK gene expression. (A and B) Total RNA was extracted from leiomyoma explants treated with R5020 for 24 hours (A) or 48 hours (B). RANKL mRNA was quantified by using qRT-PCR; n = 3 independent tissue samples. (C) RANK mRNA was quantified in explants treated with R5020 for 48 hours; n = 3. (D) R5020 significantly induced RANKL expression in LICs. LSCs, LICs, and LDCs were sorted from leiomyoma explants treated with R5020 for 24 hours, and then RANKL mRNA level was quantified in each cell population. Data show a representative experiment performed in triplicate from three independent tissue samples. The error bars are the standard error of the mean. NS, not significant.

Figure 4.

Treatment of the total leiomyoma cells with RANKL increased cyclin D1 expression that was suppressed with the RANKL/RANK pathway inhibitor RANK-Fc. Upper panel shows immunoblot densities quantified with ImageJ software. Lower panel shows images of representative immunoblots. Experiments were repeated with cells from five patient samples. The error bars are the standard error of the mean. RFc, RANK-Fc; RL, RANKL.

Figure 5.

(A) Xenografted leiomyoma tumors from mice treated with RANK-Fc showed decreased tumor volume compared with mice treated with DPBS. (B) Representative images of regenerated tumors (red arrows) 4 weeks after engrafting. (C) A significantly lower number of cells expressing Ki67 (brown-stained) was seen in xenografted tumors from mice treated with RANK-Fc; n = 4. (D) Representative images of immunohistochemistry staining for Ki67, ×20. The inset in D (yellow rectangle) is the region in the red rectangle at a high magnification, ×2.7. The error bars are the standard error of the mean. E, estradiol; HPF, high-power field; P, progesterone.