Inhibition of STAT3 signaling blocks obesity-induced mammary hyperplasia in a mouse model

Abstract

Compelling epidemiologic evidence indicates that obesity is a risk factor for human cancers, including breast. However, molecular mechanisms by which obesity could contribute to the development of breast cancer remain unclear. To understand the impact of obesity on breast cancer development, we used a mutant mouse that expresses a mutated thyroid hormone receptor β (denoted as PV) with haplodeficiency of the Pten gene (Thrb^PV/PVPten^+/- mice). We previously showed that adult nulliparous female Thrb^PV/PVPten^+/- mice developed extensive mammary hyperplasia and breast tumors. In this study, we induced obesity in Thrb^PV/PVPten^+/- mice by feeding them a high fat diet (HFD). We found HFD exacerbated the extent of mammary hyperplasia in Thrb^PV/PVPten^+/- mice. HFD elevated serum leptin levels but had no effect on the levels of serum thyroid stimulating hormone, thyroid hormones, and estrogens. Molecular analysis showed that the obesity-induced hyperplasia was mediated by the leptin/leptin receptor-JAK1-STAT3 pathway to increase key cell cycle regulators to stimulate mammary epithelial cell proliferation. Activated STAT3 signaling led to altered expression in the key regulators of epithelial-mesenchymal-transition (EMT) to augment invasiveness and migration of mammary proliferating epithelial cells. Moreover, treatment of HFD-Thrb^PV/PVPten^+/- mice with a STAT3 inhibitor, S3I-201, markedly reversed the obesity-induced mammary hyperplasia and reduced EMT signals to lessen cell invasiveness and migration. Our studies not only elucidated how obesity could contribute to mammary hyperplasia at the molecular level, but also, importantly, demonstrated that inhibition of the STAT3 activity could be a novel treatment strategy for obesity-induced breast cancer progression.

Keywords: JAK2-STAT3 signaling; Mammary carcinogenesis; STAT3 inhibitor; obesity; preclinical mouse model; thyroid hormone receptor β mutant.

Figure 1

Effects of obesity and S3I-201 on lobulo-alveolar development and morphology of the mammary glands of wild type (WT) and Thrb^PV/PVPten^+/- mice. (A) Histopathology of mammary glands of WT and Thrb^PV/PVPten^+/- mice fed with low fat diet (LFD) or high fat diet (HFD), and treated with vehicle or S3I-201. The histopathological changes were given a score based on the rating scale in (a). The distribution of scores is shown in (b). The genotypes of mice are as marked. (B) Effects of S3I-201 on obesity-induced aberrant lobulo-alveolar development in HFD-Thrb^PV/PVPten^+/- mice. Hematoxylin and eosin-stained sections of mouse mammary tissue from LFD-WT with/without S3I-201 (a, b) and HFD-WT with or without S3I-201 (e and f), and a LFD-Thrb^PV/PVPten^+/- with/without S3I-201 (c and d) and HFD-Thrb^PV/PVPten^+/- with/without S3I-201 (g and h) mice.

Figure 2

Effects of S3I-201 on cell proliferation in mammary tissue sections from HFD-WT and HFD-Thrb^PV/PVPten^+/- mice. (A) Representative microphotographs of immunohistochemical analysis of Ki67 on mammary tissue sections of WT mice treated with vehicle (a, b) or S3I-201 (c and d) or Thrb^PV/PVPten^+/- mice treated with vehicle (e and f) or treated with S3I-201 (g and h). (a, c, e, and g) were the negative controls from using anti-IgG antibodies and (b, d, f, and h) were from using anti-Ki67 antibodies. (B) The Ki67-positively stained cells were counted and the data expressed as percentage of Ki67-positive cells versus total cells. The data are expressed as mean ± SE (n = 3 slides). The p values are shown.

Figure 3

Effects of HFD and S3I-201 on protein levels of key regulators of the STAT3 signaling pathway in mammary glands from WT and Thrb^PV/PVPten^+/- mice. (A) Western blot analysis of p-STAT3 (Y705), total STAT3, cyclin D1, phosphorylated retinoblastoma (p-Rb; S780), total Rb, the leptin receptor, p-JAK1, and total JAK1 in mammary glands from WT mice and Thrb^PV/PVPten^+/- mice fed with LFD (lanes 1, 2, 5, 6, and 7) or with HFD (lanes 3, 4, 8, 9, and 10). Two or 3 mice were used for WT mice and Thrb^PV/PVPten^+/- mice, respectively. GAPDH was used as a loading control (g and J). (B) The band intensities of the proteins detected in (A) were quantified and graphed. The open bars represent mice fed with LFD and filled bars represent mice fed with HFD. The data, shown as mean ± SE, were analyzed by Student’s t test. (C) Western blot analysis of p-STAT3 (Y705), t-STAT3, cyclin D1, p-Rb (S780), total Rb, the leptin receptor, p-JAK1 (Y1022/1023), and total JAK1 in mammary glands from WT or Thrb^PV/PVPten^+/- mice treated with vehicle (lanes 1, 2, 5, 6, and 7) or with S3I-201 (lanes 3, 4, 8, 9, and 10). Two or 3 mice were used from WT and Thrb^PV/PVPten^+/- mice, respectively. GAPDH was used as a loading control (g and J). (D) The band intensities of the proteins detected in (C) were quantified and graphed. The open bars represent mice treated with vehicle and filled bars represent mice treated S3I-201. The data, shown as mean ± SE, were analyzed by Student’s t test. NS = not significant.

Figure 4

Effects of HFD and S3I-201 on the serum levels of TSH, thyroid hormones, and estrogens in WT and Thrb^PV/PVPten^+/- mice. (A) Thyroid function tests of WT and Thrb^PV/PVPten^+/- mice fed with LFD or HFD and treated with vehicle or with S3I-201. Levels of TSH (Aa, n = 7-11), total T4 (Ab, n = 7-18), total T3 (Ac, n = 7-12) were determined as described in Methods and Materials. The genotypes are marked. (B) E2 was determined as described in Methods and Materials (n = 6-12).

Figure 5

Effects of HFD and S3I-201 on the protein levels of EMT regulators in mammary glands from of WT and Thrb^PV/PVPten^+/- mice. (A) Western blot analysis of E-cadherin, vimentin, and pro- and active-MMP2 in mammary tissues fed with LFD (lanes 1, 2, 5, 6, and 7) or HFD (lanes 3, 4, 8, 9, and 10) from WT or Thrb^PV/PVPten^+/- mice. Two WT and Thrb^PV/PVPten^+/- mice were used. GAPDH was used as a loading control (d). (B) The band intensities of the protein detected in (A) were quantified and graphed. The open bars represent mice treated with LFD and filled bars represent those treated with HFD. The data, shown as mean ± SE, were analyzed by Student’s t test. (C) Western blot analysis of E-cadherin, vimentin, pro- and active-MMP2 in mammary tissues treated with vehicle (lanes 1, 2, 5, 6, and 7) or with S3I-201 (lanes 3, 4, 8, 9, and 10) from WT or Thrb^PV/PVPten^+/- mice. Two WT and Thrb^PV/PVPten^+/- mice were used. GAPDH was used as a loading control (d). (D) The band intensities of the protein detected in (C) were quantified and graphed. The open bars represent mice treated with vehicle and filled bars represent those treated with S3I-201. The data, shown as mean ± SE, were analyzed by Student’s t test.

Figure 6

Schematic representation of the effect of S3I-201 in obesity-induced extensive mammary hyperplasia. Obesity-induced aberrant activation of the STAT3 pathway contributes to the mammary hyperplasia via increased cell proliferation and EMT signals in Thrb^PV/PVPten^+/- mice. HFD induces elevated leptin to activate STAT3 signaling, resulting in increased cyclin D1, p-Rb, vimentin, and active MMP2 and decreased E-cadherin. All of these STAT3 downstream effectors are reversed by S3I-201 to attenuate STAT3 signaling, leading to reduced cell proliferation and decreased EMT signals.