Insulin-mediated acceleration of breast cancer development and progression in a nonobese model of type 2 diabetes

Abstract

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

Figure 1

Metabolic abnormalities in MKR^+/+ female mice. (A) Left panel: Plasma insulin levels in 8 wk old WT (close bars) and MKR^+/+ (open bars) mice (n=7). Right panel: Insulin tolerance test was performed on fasted 12 wk old WT (circles) and MKR^+/+ (triangles) mice (n=5) after intraperitoneal injection of insulin (0.75 U/kg). Blood samples were obtained from the tail vein, and glucose concentrations were determined at the indicated time points. (B) Left panel: Blood glucose in 12 wk old WT (close bars) and MKR^+/+ (open bars) mice (n=15). Right panel: Glucose tolerance test was performed on fasted 10 wk old WT (circles) and MKR^+/+ (triangles) mice (n=5) after intraperitoneal injection of glucose (2 g/kg). Blood samples were obtained from the tail vein, and glucose concentrations were determined at the indicated time points. (C) Total body weight and total body adiposity in 10-12 wk old WT (close bars) and MKR^+/+ (open bars) mice (n=6). All results are expressed as a mean ± SEM. Statistically significant difference between WT and MKR groups is indicated (*), P < 0.05 (Student's t-test).

Figure 2

Effect of type 2 diabetes on normal mammary gland development. Whole-mount analysis of (A) prepubertal (3 wk) and(B) late virgin (15 wk) mammary glands obtained from WT and MKR^+/+ female mice. Arrows indicate the terminal end buds. Original magnification: x16. Representative image of 7 mice (C) Total IR levels in mammary epithelial cells (MECs) isolated from 15 wk old WT and MKR^+/+ virgin female mice (n=3) were measured by ELISA. Relative levels of total IR receptor levels are expressed as fold change compared to WT. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test). (D) Proteins (50 ug) extracted from mammary tissue of virgin 7 week old WT and MKR^+/+ mice were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRβ^Y1150/51/IGF-IRβ^Y1135/36, anti-phospho-Akt^S473 and anti-phospho-p42/p44^T202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total IR(, Akt and p42/p44. At least four animals per group were analyzed, the representative blots are included. (E) The results of densitometric analyses of IR/IGF-IR, Akt and ERK1/2 phosphorylation are presented as a fold change compared to WT mammary tissue. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test).

Figure 3

Effect of type 2 diabetes on PyVmT-induced mammary hyperplasia. (A) Whole-mount and (B) histological analysis of mammary glands obtained from virgin 6 wk old PyVmT^+/− and PyVmT^+/−/MKR^+/+ female mice. Arrows indicate secondary hyperplastic foci (A) and ductal hyperplasia (B). At least seven animals per group were analyzed, the representative images are included. LN, lymph node; PF, primary tumor focus. Original magnification: x4 (A), x100 (B). (C) Proteins (50 ug) extracted from mammary tissue of virgin 7 week old PyVmT^+/− and PyVmT^+/−/MKR^+/+ mice were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRβ^Y1150/51/IGF-IRβ^Y1135/36, anti-phospho-Akt^S473 and anti-phospho-p42/p44^T202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total IRβ, Akt and p42/p44. At least seven animals per group were analyzed, the representative blots are included. The results of densitometric analysis of IR/IGF-IR, Akt and ERK1/2 phosphorylation in PyVmT^+/− and PyVmT^+/−/MKR^+/+ mammary tissue and Met-1 orthograft tumor tissue are presented as a fold change compared to PyVmT^+/− mammary tissue. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test).

Figure 4

(A) Growth and (B) terminal weight of Met-1 tumors obtained from WT and MKR^+/+ female mice (7 animals per group; 4 weeks after orthotopic inoculation of 500,000 Met-1 cells). This study was replicated three times; the representative graphs are presented. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test). (C) Proteins (50 ug) extracted from Met-1 tumor tissues were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRβ^Y1150/51/IGF-IRβ^Y1135/36, anti-phospho-Akt^S473 and anti-phospho-p42/p44^T202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total IRβ, Akt and p42/p44. Five animals per group were analyzed, the representative blots are included. (D) The results of densitometric analysis of IR/IGF-IR, Akt and ERK1/2 phosphorylation in Met-1 tumor tissue are presented as a fold change compared to WT tumor tissue. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test).

Figure 5

(A) Growth and (B) terminal weight of MCNeuA tumors obtained from WT and MKR^+/+ female mice (4-7 animals per group; 4 weeks after orthotopic inoculation of 1,000,000 MCNeuA cells). This study was replicated three times; the representative graphs are presented. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test). (C) Proteins (50 ug) extracted from MCNeuA tumor tissues were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRβ^Y1150/51/IGF-IRβ^Y1135/36, anti-phospho-Akt^S473 and anti-phospho-p42/p44^T202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total IRβ, Akt and p42/p44. 3-5 animals per group were analyzed, the representative blots are included. (D) The results of densitometric analysis of IR/IGF-IR, Akt and ERK1/2 phosphorylation in MCNeuA orthograft tumor tissue are presented as a fold change compared to WT tumor tissue. Statistically significant difference is indicated (*), P < 0.05 (Student's t-test).

Figure 6

Effect of pharmacological IR/IGF-IR blockade on Met-1 tumor growth and carbohydrate metabolism in MKR^+/+ female mice. Eight week-old WT and MKR^+/+ female mice (n=5) were subjected to orthotopic inoculation of 500,000 Met-1 cells. Three weeks after tumor inoculation, small-molecule IR/IGF-IR tyrosine kinase inhibitor BMS-536924 (100 mg/kg/day orally by gavage) or the respective vehicle (80% polyethylene glycol, 20% water) was applied for the next 14 days. (A) Blood glucose and (B) plasma insulin levelsin vehicle- or BMS-536924-treated WT and MKR^+/+ female mice at the end of the study. (C) Growth and (D) terminal weight of Met-1 tumors obtained from WT and MKR^+/+ female mice treated with vehicle or BMS-536924. Statistically significant difference (P < 0.05, two-factor ANOVA followed by Holm-Sidak post-hoc test) is indicated: (a) WT-vehicle versus MKR-vehicle, (b) MKR-vehicle versus MKR-BMS-536924, (c) WT-BMS-536924 versus MKR-BMS-536924.