Inhibition of STAT3 activity delays obesity-induced thyroid carcinogenesis in a mouse model

Abstract

Compelling epidemiologic studies indicate that obesity is a risk factor for many human cancers, including thyroid cancer. In recent decades, the incidence of thyroid cancer has dramatically increased along with a marked rise in obesity prevalence. We previously demonstrated that a high fat diet (HFD) effectively induced the obese phenotype in a mouse model of thyroid cancer (Thrb(PV/PV)Pten(+/-) mice). Moreover, HFD activates the STAT3 signal pathway to promote more aggressive tumor phenotypes. The aim of the present study was to evaluate the effect of S3I-201, a specific inhibitor of STAT3 activity, on HFD-induced aggressive cancer progression in the mouse model of thyroid cancer. WT and Thrb(PV/PV)Pten(+/-) mice were treated with HFD together with S3I-201 or vehicle-only as controls. We assessed the effects of S3I-201 on HFD-induced thyroid cancer progression, the leptin-JAK2-STAT3 signaling pathway, and key regulators of epithelial-mesenchymal transition (EMT). S3I-201 effectively inhibited HFD-induced aberrant activation of STAT3 and its downstream targets to markedly inhibit thyroid tumor growth and to prolong survival. Decreased protein levels of cyclins D1 and B1, cyclin dependent kinase 4 (CDK4), CDK6, and phosphorylated retinoblastoma protein led to the inhibition of tumor cell proliferation in S3I-201-treated Thrb(PV/PV)Pten(+/-) mice. Reduced occurrence of vascular invasion and blocking of anaplasia and lung metastasis in thyroid tumors of S3I-201-treated Thrb(PV/PV)Pten(+/-) mice were mediated via decreased expression of vimentin and matrix metalloproteinases, two key effectors of EMT. The present findings suggest that inhibition of the STAT3 activity would be a novel treatment strategy for obesity-induced thyroid cancer.

Keywords: JAK2-STAT3 signaling; Pten-deficiency; STAT3 inhibitor; obesity; preclinical mouse model; thyroid cancer; thyroid hormone receptor β mutant.

Figure 1. The effects of S3I-201 on thyroid tumor growth and survival of HFD-wild type and HFD-Thrb^PV/PVPten^+/− mice

(A) Survival curves for HFD- Thrb^PV/PVPten^+/− mice treated with S3I-201 (5 mg/kg body weight, n=12) or vehicle (0.05% DMSO, n=12)) I.P. injection 3 times a week from 8 weeks of age until they had to be euthanized because of sickness. Data are presented by Kaplan-Meir methods and analyzed by log-rank test. The p values are indicated. (B) Thyroid weights of vehicle-treated or S3I-201-treated wild type (n=10–11) or Thrb^PV/PVPten^+/− mice (n=10–13). (C-I). Comparison between the two groups of representative microphotographs of immunohistochemistry (IHC) one using anti-Ki67 antibody on thyroid sections from wild type mice with/without S3I-201 (panels a, b, c and d) and one without the treatment with primary antibodies are shown in the corresponding panels (a and c). The thyroid sections from Thrb^PV/PVPten^+/− mice (e, f, g, and h) and the negative without the treatment with primary antibodies are shown in the corresponding panels (e and g). (C-II) The Ki67-positive cells were counted and the data expressed as percentage of Ki67-positive cells versus total cells. The data are expressed as mean ± SE (n=2 slides). The p values are shown.

Figure 2. The effects of S3I-201 on the protein levels of key regulators of the cell cycle in thyroid tumors from HFD-wild type and HFD-Thrb^PV/PVPten^+/− mice

(A) Western blot analysis of cyclin D1, cyclin B1, cdk4, cdk6, phosphorylated retinoblastoma (p-Rb; S780), and total Rb in mice treated with vehicle (lanes 1, 2, 5, & 6) or with S3I-201 (lanes 3, 4, 7, & 8). Two mice were used in each group. GAPDH was used as a loading control (panel g). (B) The band intensities of the protein detected in A were quantified and compared. The filled bars were from mice treated with the inhibitor, S3I-201, and the open bars are from mice treated with vehicle. The data, shown as mean ± SE, were analyzed by Student’s t test.

Figure 3. The effects of S3I-201 on serum leptin levels and size of adipocytes in the HFD-wild type and HFD- Thrb^PV/PVPten^+/− mice

(A) The serum leptin concentrations in wild type or Thrb^PV/PVPten^+/− mice were determined as described in Materials and Methods. The serum leptin levels from wild type mice treated with vehicle (n=8) and S3I-201 (n=8) were determined, as were the serum leptin levels from Thrb^PV/PVPten^+/− mice treated with vehicle (n=23) and S3I-201 (n=13). The p values are indicated. NS, Not significant. (B) Representative histological features of inguinal fat of HFD-wild type and HFD- Thrb^PV/PVPten^+/− mice with or without S3I-201 (panel a, b, c and d). (C) Fat cell size was measured for each genotype with vehicle or S3I-201 shown in B. Data are expressed as mean ± SE (n=4). The p values are shown.

Figure 4. The effects of S3I-201 on protein levels of regulators in the STAT3 pathway in thyroids of HFD-wild type and HFD- Thrb^PV/PVPten^+/− mice

(A) Western blot analysis of protein abundance of leptin receptor, phosphorylated -JAK2 (Y1007/1008), total JAK-2, phosphorylated STAT3 (Y705), total-STAT3, and GAPDH as a loading control after treatment with vehicle or S3I-201 in wild type (n=2) and Thrb^PV/PVPten^+/− mice (n=2). (B) The band intensities of the protein detected in (A) were quantified and compared. The data, shown as mean ± SE, were analyzed by Student’s t test.

Figure 5. The effect of S3I-201 on STAT3 protein abundance in the thyroid of HFD-wild type and HFD- Thrb^PV/PVPten^+/− mice

(A-I) Comparison between the two groups of representative microphotographs of immunohistochemical analyses using anti-p-STAT3 antibody on thyroid sections from vehicle-treated or S3I-201-treated wild type (panels b and d) and Thrb^PV/PVPten^+/− mice (panels f and h). The negative controls from no primary antibodies are shown in the corresponding panels (wild type; a and c, Thrb^PV/PVPten^+/− mice e and g). (A-II) The p-STAT3-positively stained cells were counted and the data are expressed as percentage of p-STAT3-positive cells versus total cells. The data are expressed as mean±SE (n= 3 slides). The p values are shown. (B) The relative mRNA expression of Ccnd1, Myc, Mcl1, Bcl2, and Socs3 in thyroid tumors from vehicle-treated (n=4) and S3I-201-treated (n=4) groups. The data, presented as mean ± SE, were analyzed by Student’s t test.

Figure 6. The effect of S3I-201 on pathological progression during thyroid carcinogenesis of HFD- Thrb^PV/PVPten^+/− mice

(A-I) Pathologic analysis in the vehicle-treated (n=4) and S3I-201-treated (n=4) groups of Thrb^PV/PVPten^+/− mice. The prevelence of each pathological feature in thyroid carcinogenesis, according to the vehicle or S3I-201 treatment, is shown a percentage. (A-II) Representative examples of hematoxylin and eosin (H&E)-stained thyroid sections from wild type mice treated with vehicle (panel a) and S3I-201 (panel b) and lung sections treated with vehicle (panel a’) and S3I-201 (panel b’) and thyroid tumor sections from Thrb^PV/PVPten^+/− mice treated with vehicle (panel c) and S3I-201 (panel d) and lung sections treated with vehicle (panel c’) and S3I-201 (panel d’). The arrows indicate vascular invasion (panel d) and anaplasia (panel c). (B-I) Western blot analysis of vimentin, active MMP2 and GAPDH as a loading control after treatment with vehicle and S3I-201 in wild type and Thrb^PV/PVPten^+/− mice. (B-II) The band intensities of the protein detected in B-I were quantified and compared. The data, shown as mean ± SE, were analyzed by Student’s t test.