FGFR2/STAT3 Signaling Pathway Involves in the Development of MMTV-Related Spontaneous Breast Cancer in TA2 Mice

Abstract

The Tientsin Albino 2 (TA2) mouse has a high incidence of spontaneous breast cancer (SBC) in the absence of external inducers or carcinogens. The initiation of SBC is related to mouse mammary tumor virus (MMTV) infection and pregnancy. Pathologic analysis showed that breast cancer cells in TA2 mice are triple negative. Our previous study confirmed that fibroblast growth factor receptor 2 (FGFR2) expression increased in SBC tissue compared to that in their corresponding normal breast tissues of TA2 mice. The present study focused on the function of the FGFR2/STAT3 signaling pathway in the initiation of SBC. In this study, the expression of FGF3, FGFR2, STAT3, p-STAT3^Tyr705, and p-STAT3^Ser727 was detected in serum and normal mammary gland tissues of TA2 mice with different number of pregnancies and SBC. The proliferation, invasiveness, and migration abilities of MA-891 cells from TA2 SBC were compared before and after cryptotanshinone and Stattic treatment. Transient siRNA transfection was used to detect the invasiveness, and migration abilities to avoid the off-targets effects. Downstream protein expression of STAT3 was also detected in MA-891 cells and TA2 xenografts from MA-891 inoculation. In addition, STAT3 expression was analyzed in 139 cases of human breast cancer including 117 cases of non-triple negative breast cancer (non-TNBC) (group I) and 22 cases of triple-negative breast cancer (TNBC) (group II). Results of our study confirmed that MMTV-LTR amplification, and FGFR2, p-STAT3^Tyr705, p-STAT3^Ser727 expression increased with the number of pregnancies in the breast tissue of TA2 mice and were the highest in SBC. Serum FGF3 expression of SBC was higher than it of TA2 mice with different number of pregnancies. After STAT3 was inhibited, the abilities of proliferation, invasiveness, and migration in MA-891 decreased and the expression levels of STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1, and c-myc in MA-891 and animal xenografts were also down-regulated. In human breast cancer, STAT3 expression was significantly higher in TNBC than that in non-TNBC. Our results showed that the FGFR2/STAT3 signaling pathway may be related to SBC initiation in TA2 mice. Inhibition of STAT3 can decrease proliferation, invasiveness, and migration in MA-891 cells and the growth of TA2 xenografts.

Keywords: FGFR2/STAT3; MMTV; spontaneous breast cancer; tientsin albino 2; triple-negative breast cancer.

Figure 1

Expression of mouse mammary tumor virus-long terminal repeat (MMTV-LTR) and related proteins in the serum and breast tissue of Tientsin Albino 2 (TA2) mice with different numbers of pregnancies and spontaneous breast cancer (SBC). (A) (a–e) Morphological changes in the breast tissues in TA2 mice with zero, two, four, and six pregnancies and SBC, respectively (H&E,×40). (B) Results of reverse transcription-polymerase chain reaction (RT-PCR) analysis of MMTV-LTR from TA2 mice with no, two, four, and six pregnancies and SBC, respectively. (C) Serum FGF3 concentrations in TA2 mice with different numbers of pregnancies and SBC by enzyme-linked immunosorbent assay (ELISA). (D) Expression levels of fibroblast growth factor receptor 2 (FGFR2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3)^Tyr705, p-STAT3^Ser727, and STAT3 by western blot analysis in TA2 mice with different numbers of pregnancies and SBC. (E) Histograms showing quantitative differences in MMTV-LTR and related protein expression in the serum and breast tissue of TA2 mice with different number of pregnancies and SBC. Each bar represents the mean ± standard deviation (SD) of three independent experiments. (F) Immunohistochemical staining of FGFR2 and p-STAT3^Ser727 (IHC, ×40). (a–e). FGFR2 staining in breast tissues of TA2 mice with 0, 2, 4, and 6 pregnancies and with SBC, respectively. (f–j). p-STAT3^Ser727 staining in breast tissues of TA2 mice with no, two, four, and six pregnancies and SBC, respectively. Statistically differences are indicated: **P < 0.001; *P < 0.05.

Figure 2

Protein expression in control and cryptotanshinone (CTS) and Stattic-treated MA-891 cells. (A) Western blot analysis showing STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1, and c-myc expression in control, CTS-treated, and Stattic-treated cells. (B) Histograms of quantitative differences in protein expression among control, CTS and Stattic-treated MA-891 cells. Each bar represents the mean ± standard deviation (SD) of three independent experiments. (C) Immunocytochemical (ICC) staining of STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1, and c-myc. (a–f). STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1 and c-myc staining in control MA-891 cells, respectively. (g–l). STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1, and c-myc staining in the CTS-treated group, respectively. (m–r). STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1 and c-myc in staining in the Stattic-treated group, respectively. Statistically differences are indicated: **P < 0.001; *P < 0.05.

Figure 3

Comparisons of proliferation, migration, and invasive ability in control, cryptotanshinone (CTS)-treated, and Stattic-treated MA-891 cells. (A) Cell counting kit-8 (CCK8) shows that CTS and Stattic inhibit viability in a time- and dose-dependent manner. (a) Comparisons of viability in control cells and cells treated with different concentrations of CTS for 24 h. (b) Comparisons of viability in control cells and cells treated with 60 μM of CTS for different times. (c) Comparisons of viability in control cells and cells treated with different Stattic concentrations for 24 h. (d) Comparisons of viability in control cells and cells treated with 2 μM Stattic for different time. (B) Cell proliferation ability based on clone formation. (a–c) Proliferation ability of 2,000, 1,000, and 500 control cells, respectively. (d–f) Proliferation ability of 2,000, 1,000, and 500 cells after CTS treatment, respectively. (g–i) Proliferation ability of 2,000, 1,000, and 500 cells after Stattic treatment, respectively. (C) Wound-healing assay in MA-891 cells at 0, 12, and 24 h, respectively, after different treatments. (a–c) Representative images in control cells. (d–f) Representative images after CTS treatment. (g–i) Representative images after Stattic treatment. (D) Western blot showed STAT3 and GAPDH expression in MA-891 cells with siRNA STAT3-2315, 1415, 1107, positive control, and negative control. (E) Western blot showed STAT3, cyclin D1, c-myc and Bcl2 expression in MA-891 cells with siRNA STAT3-1415, positive control, and negative control. (F) Transwell migration and invasion assay in MA-891 cells before and after treatment. (a–d) Migration ability in control, CTS-treated, Stattic-treated cells, and cells after STAT knockdown. (e–h) Invasion assay of control, CTS-treated, Stattic-treated cells and cells after STAT knockdown. (G) Histograms showing the quantitative results of the proliferation, migration, and invasive ability of MA-891 cells before and after treatment. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Statistically differences are indicated: **P < 0.001; *P < 0.05. 1415: siRNA STAT3-1415. PC, positive control; NC, negative control.

Figure 4

Fibroblast growth factor receptor 2/signal transducer and activator of transcription 3 (FGFR2/STAT3) signaling pathway-related proteins associated with the development of animal xenografts from MA-891 and human breast cancer. (A) Gross pictures of tumor masses (each tumor was from one mouse) after the 18th day. (a–d) Tumor tissue of TA2 xenografts from control MA-891 cell injection (a), control MA-891 cell injection and cryptotanshinone (CTS) administration (b), control MA-891 cell injection (c), and control MA-891 cell injection and Stattic administration, respectively (d). (e–f) Growth curves of animal xenografts from control and CTS administration (e), control and Stattic administration (f), respectively. (B) Western blot analysis of the expression of proteins including STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1, and c-myc in xenografts with control and CTS and Stattic treatments. (C) Histograms of quantitative differences in protein expression. Each bar represents the mean ± standard deviation (SD) of three independent experiments. (D) Immunohistochemical (IHC) staining of human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), progesterone receptor (PR), and STAT3 in human breast cancer tissues (IHC × 100). (a–c) HER-2-, ER-, and PR-positive breast cancer. (d) STAT3 expression in non-TNBC. (e) FGFR2 expression in non-TNBC. (f) HER-2-negative breast cancer. (g) ER-negative breast cancer. (h) PR-negative breast cancer. (i) STAT3 expression in TNBC. (j) FGFR2 expression in TNBC. Statistically differences are indicated: **P < 0.001; *P < 0.05.