Lapatinib‑induced inhibition of ovarian function is counteracted by the STAT3 pathway both in vivo and in vitro

Abstract

The present study was designed to ascertain whether lapatinib, a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), affects ovarian reserve and fertility potential in a mouse model. Female C57BL/6 mice were treated with either vehicle or lapatinib (100 or 200 mg/kg/day orally) for 4 weeks, after which body weight, vaginal smears, follicle numbers, serum anti‑Müllerian hormone (AMH) levels and mating outcomes were analyzed to assess the ovarian reserve and reproductive function. Slices from the ovaries of 4‑week‑old mice were cultured with lapatinib (0, 5 or 10 µM) for 24 and 48 h, and protein expression levels were assessed to validate the changes in signaling pathways. The results indicated that mice treated with 200 mg/kg lapatinib showed a slight decrease in body weight compared to those treated with vehicle or 100 mg/kg lapatinib. There was no statistical difference in estrous cyclicity among the three groups. No significant difference was observed in follicle numbers, AMH levels, histological morphologies of the ovaries or mating outcomes in the three groups of mice. Western blotting and immunohistochemical staining of the EGF receptor and its main downstream signaling pathways showed decreased phosphorylation of EGFR and mitogen‑activated protein kinase (MAPK)3/1 and increased phosphorylation of signal transducers and activators of transcription (STAT)3 in the lapatinib‑treated groups compared to the control group. Our study suggests that lapatinib has little effect on ovarian reserve and reproductive function in a mouse model. This lack of effect of lapatinib on ovarian function may be due to the activation of the STAT3 signaling pathway that counteracts the inhibitory effects of lapatinib on EGF receptors.

Figure 1.

Mouse body weight gain and cyclicity monitored after treatment with lapatinib. (A) Body weight gain of mice was calculated based on the initial and final body weights. *P<0.05, statistical significance. (B) Representative vaginal smears with regular estrous cyclicity under a positive fluorescence microscope (magnification, ×100). (C) Percentage of mice with regular and irregular cyclicity after a 4-week treatment with lapatinib. Percentage of mice with regular cyclicity was 63.6% in the control group, 72.7% in the 100 mg/kg group and 43.8% in the 200 mg/kg group.

Figure 2.

Mouse ovarian reserve and fertility. (A) Representative ovarian histological sections from control mice and lapatinib-treated mice (magnification, ×200). (B) Follicle numbers per section of different stages after control, 100 mg/kg lapatinib and 200 mg/kg lapatinib treatment. (C) Serum AMH levels in control and lapatinib-treated mice. (D) Pregnancy rate and (E) average pup number after control, 100 mg/kg lapatinib and 200 mg/kg lapatinib treatment. AMH, anti-Müllerian hormone.

Figure 3.

Western blot analysis and immunohistochemical staining of the EGFR signaling pathway in vivo. Western blotting of ovaries treated with control and lapatinib in vivo showed (A) decreased phosphorylation of EGFR (p-EGFR) and (C) MAPK3/1 in the lapatinib-treated groups, and (D) increased phosphorylation of STAT3 in the lapatinib-treated groups. (B) No obvious difference was noted in the phosphorylation of the PI3K/AKT pathway. (E) Immunohistochemical staining confirmed these results (magnification, ×100). EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3 kinase; AKT, protein kinase B; PTEN, phosphatase and tensin homologue deleted on chromosome ten; STAT, signal transducers and activators of transcription.

Figure 4.

Histological assessment and western blot analysis of the ovarian slices in vitro. (A) Hematoxylin and eosin staining of ovarian slices cultured with or without lapatinib (5 and 10 µM) for 24 and 48 h (magnification, ×100). (B) Follicle numbers per area (mm²) of ovarian slices treated for 24 and 48 h. (C and D) Lapatinib (Lapa) inhibits the phosphorylation of EGFR (p-EGFR) and the PI3K/AKT and MAPK/ERK pathways; this inhibition was counteracted by activation of STAT3. EGFR, epidermal growth factor receptor; PI3K, phosphatidylinositol-3 kinase; AKT, protein kinase B; MAPK, mitogen-activated protein kinases; ERK, extracellular regulated kinase; STAT, signal transducers and activators of transcription; PTEN, phosphatase and tensin homologue deleted on chromosome ten.

Figure 5.

Schematic of STAT3 counteraction of EGFR inhibition by lapatinib. Lapatinib successfully inhibits EGFR activity and the downstream pathways in mouse ovaries, yet the inhibitory effects may be counteracted by the activation of STAT3. Thus, little effect of lapatinib treatment was conferred on oocyte maturation, ovarian reserve and reproductive function in mice. EGFR, epidermal growth factor receptor; STAT, signal transducers and activators of transcription; PI3K, phosphatidylinositol-3 kinase; AKT, protein kinase B; PTEN, phosphatase and tensin homologue deleted on chromosome ten; MEK, mitogen-activated extracellular signal-regulated kinase.