Insulin receptor phosphorylation by endogenous insulin or the insulin analog AspB10 promotes mammary tumor growth independent of the IGF-I receptor

Abstract

Endogenous hyperinsulinemia and insulin receptor (IR)/IGF-I receptor (IGF-IR) phosphorylation in tumors are associated with a worse prognosis in women with breast cancer. In vitro, insulin stimulation of the IR increases proliferation of breast cancer cells. However, in vivo studies demonstrating that IR activation increases tumor growth, independently of IGF-IR activation, are lacking. We hypothesized that endogenous hyperinsulinemia increases mammary tumor growth by directly activating the IR rather than the IGF-IR or hybrid receptors. We aimed to determine whether stimulating the IR with the insulin analog AspB10 could increase tumor growth independently of IGF-IR signaling. We induced orthotopic mammary tumors in control FVB/n and hyperinsulinemic MKR mice, and treated them with the insulin analog AspB10, recombinant human IGF-I, or vehicle. Tumors from mice with endogenous hyperinsulinemia were larger and had greater IR phosphorylation, but not IGF-IR phosphorylation, than those from control mice. Chronic AspB10 administration also increased tumor growth and IR (but not IGF-IR) phosphorylation in tumors. IGF-I led to activation of both the IGF-IR and IR and probably hybrid receptors. Our results demonstrate that IR phosphorylation increases tumor growth, independently of IGF-IR/hybrid receptor phosphorylation, and warrant consideration when developing therapeutics targeting the IGF-IR, but not the IR.

FIG. 1.

Insulin and AspB10 led to IR phosphorylation but not IGF-IR phosphorylation in vitro. MVT1 and Met1 cells were stimulated with 10 nmol/L insulin (Ins), the insulin analog AspB10 (AspB10), rhIGF-I (IGF-I), or PBS. Western blot (WB) analysis of protein lysates (from MVT1 cells, A; from Met1 cells, B) and densitometry (from MVT1 cells, C; and from Met1 cells, D) demonstrated that insulin and AspB10 led to phosphorylation of the IRβ^{(Tyr 1150/1151)} at 95 kDa, whereas IGF-I stimulation led to phosphorylation of the IGF-IRβ^{(Tyr 1135/1136)} at 97 kDa and the IRβ^{(Tyr 1150/1151)} at 95 kDa, most likely through hybrid receptor phosphorylation (A and B). Immunoprecipitation (IP) of the IR in Met1 cell lysates after in vitro stimulation with PBS, 10 nmol/L Ins, IGF-I, or AspB10 confirmed that insulin and AspB10 caused phosphorylation of the IRβ^{(Tyr 1150/1151)}, whereas IGF-I led to IGF-IRβ^{(Tyr 1135/1136)} and IRβ^{(Tyr 1150/1151)} receptor phosphorylation, the latter likely through IR and hybrid receptor (E and F) with densitometry (G and H). Representative blots from experiments are shown. All experiments were performed two to three times. Bar graphs display means and SEM. *PBS group significantly lower than all other groups, P < 0.05; #IGF-I group significantly higher than all other groups, P < 0.05; &AspB10 group significantly higher than human insulin group, P < 0.05; ΦIGF-I group significantly higher than human insulin group, P < 0.05; Ŧhuman insulin group significantly greater than PBS, P < 0.05; ¶AspB10 significantly greater than PBS, P < 0.05; ‡IGF-I group greater than PBS, P = 0.05. p, phosphorylated.

FIG. 2.

IGF-I increased orthotopic MVT1 and Met1 tumor growth in the hyperinsulinemic MKR mice by increasing IGF-IR phosphorylation. WT and MKR mice were injected with tumor cells on Day 0. MKR mice developed larger MVT1 and Met1 tumors than WT mice (A and B). MVT1 and Met1 tumor cells were orthotopically injected into MKR mice, mice were divided into two groups with equal mean tumor size, and mice were administered either rhIGF-I or vehicle (vertical arrow indicates time when treatment began). Administration of rhIGF-I led to a further stimulation in tumor growth, over endogenous hyperinsulinemia (D and E). Serum IGF-I concentration in the rhIGF-I treatment group was 2.6 times that of the control group (C). The greater mean number of pulmonary macrometastases in the mice treated with rhIGF-I did not reach statistical significance, compared with vehicle-treated MKR mice (F). Western blot analysis of tumor lysates demonstrated that MKR mice with endogenous hyperinsulinemia (MKR V) show IRβ phosphorylation at 95 kDa, and rhIGF-I treatment led to increased IGF-IRβ and IRβ or hybrid receptor phosphorylation in MVT1 and Met1 tumors (G–J). Representative images from three repeated experiments of tumor volume and Western blots are displayed. The graphs represent the average for each group; error bars indicate SEM (A–F, H, and I). Statistical analysis was performed using a two-tailed t test; *indicates statistically significant differences (P < 0.05) between the groups. n = 8–11 mice per group. p, phosphorylated.

FIG. 3.

Chronic activation of the IR by the insulin analog AspB10 increased orthotopic Met1 and MVT1 tumor growth. MKR mice were injected with MVT1 or Met1 tumor cells on Day 0. Treatment was started with AspB10 (12.5 IU/kg, twice daily s.c.) or vehicle, indicated by vertical arrow (A and B). AspB10 led to increased growth of both MVT1 and Met1 tumors (A and B). The number of pulmonary macrometastases showed a nonsignificant increase in the AspB10-treated group (C). An insulin tolerance test was performed with AspB10 (12.5 IU/kg s.c.), regular human insulin (12.5 IU/kg s.c.), and PBS (vehicle). Blood glucose was measured at 0.5, 1, 2, 4, 7, and 10 h after injection (D). Statistical analysis was performed using a one-way ANOVA for comparing more than two groups: #P < 0.05 between PBS and AspB10 and human insulin groups; &P < 0.05 between PBS- and AspB10-treated groups; n = 4 per group. #PBS group was significantly greater than other groups, P < 0.05; &PBS group was significantly higher than AspB10 group, P < 0.05. No change in body weight (E) or difference in relative lean or fat mass (F and G) was observed after 2 weeks of AspB10 administration. Graphs are representative of two studies. All graphs show the mean for each group, and error bars represent the SEM. Statistical analysis was performed using two-tailed t test. *P < 0.05 between groups. n = 9–11 mice per group. MKR V, MKR mice with endogenous hyperinsulinemia.

FIG. 4.

Endogenous hyperinsulinemia and AspB10 treatment led to increased IR phosphorylation in MVT1 and Met1 tumors. Western blot (WB) analysis (representative blots, A and B) revealed that chronic AspB10 treatment led to increased IRβ phosphorylation at 95 kDa (C and D). Immunoprecipitation (IP) of the IRβ (representative blot, E) and IGF-IRβ (representative blot, F) was performed on MVT1 and Met1 tumor protein lysates. They were immunoblotted for the phosphorylated IGF-IRβ/IRβ. Endogenous hyperinsulinemia (MKR V) and chronic AspB10 administration (MKR AspB10) led to increased IR phosphorylation (E). rhIGF-I administration led to increased IGF-IRβ and IRβ phosphorylation (E and F). Graphs are representative of two studies. All graphs show the mean for each group, and error bars represent the SEM. Statistical analysis was performed using two-tailed t test. *P value < 0.05 between groups. n = 9–11 mice per group. p, phosphorylated; MKR V, MKR mice with endogenous hyperinsulinemia.

FIG. 5.

Chronic administration of IGF-I and AspB10 led to increased phosphorylation of Akt, but no change in Erk1/2 phosphorylation. Western blot analysis of MVT1 tumor lysates at the end of the treatment periods with rhIGF-I and AspB10 revealed that rhIGF-I and AspB10 treatment led to increased Akt phosphorylation and no change in Erk1/2 phosphorylation (A–D). Graphs are representative of two studies for AspB10 treatment and three studies for rhIGF-I treatment. All graphs show the mean for each group, and error bars represent the SEM. Statistical analysis was performed using two-tailed t test. *P < 0.05 between groups. n = 9–11 mice per group. p, phosphorylated; MKR V, MKR mice with endogenous hyperinsulinemia.