Hyperinsulinemia enhances c-Myc-mediated mammary tumor development and advances metastatic progression to the lung in a mouse model of type 2 diabetes

Abstract

Introduction: Hyperinsulinemia, which is common in early type 2 diabetes (T2D) as a result of the chronically insulin-resistant state, has now been identified as a specific factor which can worsen breast cancer prognosis. In breast cancer, a high rate of mortality persists due to the emergence of pulmonary metastases.

Methods: Using a hyperinsulinemic mouse model (MKR+/+) and the metastatic, c-Myc-transformed mammary carcinoma cell line Mvt1, we investigated how high systemic insulin levels would affect the progression of orthotopically inoculated primary mammary tumors to lung metastases.

Results: We found that orthotopically injected Mvt1 cells gave rise to larger mammary tumors and to a significantly higher mean number of pulmonary macrometastases in hyperinsulinemic mice over a period of six weeks (hyperinsulinemic, 19.4 ± 2.7 vs. control, 4.0 ± 1.3). When Mvt1-mediated mammary tumors were allowed to develop and metastasize for approximately two weeks and were then surgically removed, hyperinsulinemic mice demonstrated a significantly higher number of lung metastases after a four-week period (hyperinsulinemic, 25.1 ± 4.6 vs. control, 7.4 ± 0.42). Similarly, when Mvt1 cells were injected intravenously, hyperinsulinemic mice demonstrated a significantly higher metastatic burden in the lung than controls after a three-week period (hyperinsulinemic, 6.0 ± 1.63 vs. control, 1.5 ± 0.68). Analysis of Mvt1 cells both in vitro and in vivo revealed a significant up-regulation of the transcription factor c-Myc under hyperinsulinemic conditions, suggesting that hyperinsulinemia may promote c-Myc signaling in breast cancer. Furthermore, insulin-lowering therapy using the beta-adrenergic receptor agonist CL-316243 reduced metastatic burden in hyperinsulinemic mice to control levels.

Conclusions: Hyperinsulinemia in a mouse model promotes breast cancer metastasis to the lung. Therapies to reduce insulin levels in hyperinsulinemic patients suffering from breast cancer could lessen the likelihood of metastatic progression.

Figure 1

Mvt1 tumor growth and metastasis development are more rapid in MKR^+/+ mice compared to controls. A total 100,000 Mvt1 cells were injected into the left inguinal mammary fat pad of control and MKR^+/+ mice (8 to 10 per group). (A) Mammary tumor volume (mm³) measured by calipers. (B) Terminal weights of Mvt1-induced mammary tumors. (C) Numbers of pulmonary macrometastases in control and MKR^+/+ mice. (D) H & E staining of control and MKR^+/+ lungs, arrows indicate metastases. Error bars represent SEM *, P ≤ 0.05.

Figure 2

Independent of primary tumor size, metastases formation is accelerated in MKR^+/+ mice. A total of 100,000 Mvt1 cells were injected into the inguinal mammary fat pad of 8- to 10-week-old mice (8 to 10 mice per group). Developing tumors were removed surgically when they reached a specific size (30 to 40 mm³). (A) Volumes and (B) weights of tumors from MKR^+/+ and control mice at time of surgical removal. (C) Number of metastases present in the lungs of control and MKR^+/+ mice four weeks after tumor resection. (D) Number of metastases present in the lungs of control and MKR^+/+ mice three weeks after intravenous injection of 10,000 Mvt1 cells. Error bars represent SEM, **, P ≤ 0.05.

Figure 3

Hyperinsulinemia results in increased expression of c-Myc, MMP-9, IR-β, IGF-IR and VEGF in Mvt1 tumors. (A) Tumor tissue was extracted from control and MKR^+/+ mice and subjected to SDS PAGE and Western blotting with antibodies to c-Myc, MMP-9, IR-β, IGF-IR and VEGF. β-actin was included as a loading control. (B) Fold change in expression of c-Myc, MMP-9, IR-β, IGF-IR and VEGF in control and MKR^+/+ tumor tissue compared to β-actin control. *, P ≤ 0.05. (C) Semi-quantitative PCR showing relative mRNA expression levels of Igf1, Igf-2 and β-actin in tumor tissue from control and MKR^+/+ mice. (D) Densitometric quantification of DNA gel electrophoresis showing mRNA expression of Igf1 and Igf2 relative to β-actin.

Figure 4

In Mvt1 cells, insulin elevates c-Myc, MMP-9 and phosphorylated Akt, and results in increased proliferation. (A) Akt phosphorylation in Mvt1 cells after insulin stimulation. Mvt1 cells were serum-starved for four hours prior to stimulation with increasing concentrations of insulin. Cells were then harvested and subjected to SDS PAGE and Western blotting with antibodies to insulin receptor-β (IR-β), phosphor-Akt^Ser473 and Akt, phospho-p42/p44^{Thr202Tyr/204} and p42/p44. Β-actin was included as a loading control. (B) Mvt1 cells were seeded at a density of 1 × 10⁴ cells/ml in 24-well plates and stimulated with either 10 nM or 100 nM insulin in growth medium containing 0.5% FBS. After 72 hours, cells were trypsinized and counted using a haemocytometer and trypan blue exclusion. Cell counts are representative of three independent experiments. Error bars represent SEM, *, P ≤ 0.05. (C) Mvt1 cells were stimulated with 10 nM or 100 nM insulin for between 10 and 60 minutes. Previously, cells were grown in medium containing 0.5% FBS for 72 hours. After stimulation, cells were harvested and subjected to SDS PAGE and Western blotting with antibodies to c-Myc and MMP-9. β-actin was included as a loading control.

Figure 5

Chronic CL-316243 treatment reduces the hyperinsulinemia and metastases burden in MKR^+/+ mice. The authors treated 8- to 10-week-old MKR^+/+ and control mice with CL-316243 or vehicle for three weeks (6 to 8 mice per group). (A) Whole body fat (% of total body weight) was measured after 10 days of treatment. (B) Blood glucose was measured after seven days of treatment. (C) Body weight was measured weekly. (D) Serum insulin was measured after 21 days of treatment. Data are expressed as SEM. *, P ≤ 0.05 and for control vs. MKR. ^#, P ≤ 0.05 for CL-316243 vs. vehicle. (E) Mvt1 cells (10,000) were injected intravenously into 8- to 10-week-old control and MKR^+/+ mice (7 to 9 per group). Mice were then treated with CL-316243 or vehicle for three weeks. Mice were sacrificed and the numbers of lung macrometasatases were counted. Data are expressed as SEM. *, P ≤ 0.05 for control vs. MKR^+/+. ^#, P ≤ 0.05 for CL-316243 vs. vehicle.