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.

The Her2 oncogene is expressed in approximately 25% of human breast cancers and is associated with metastatic progression and poor outcome. Epidemiological studies report that breast cancer incidence and mortality rates are higher in women with type 2 diabetes. Here we use a mouse model of Her2-mediated breast cancer on a background of hyperinsulinemia to determine how elevated circulating insulin levels affect Her2-mediated primary tumor growth and lung metastasis. Hyperinsulinemic (MKR+/+) mice were crossed with doxycycline-inducible NeuNT (MTB/TAN) mice to produce the MTB/TAN/MKR+/+ mouse model. Both MTB/TAN and MTB/TAN/MKR+/+ mice were administered doxycycline in drinking water to induce NeuNT mammary tumor formation. In tumor tissues removed at two, four and six weeks of Neu-NT overexpression, we observed increased tumor mass and higher phosphorylation of the insulin receptor (IR)/insulin-like growth factor receptor 1 (IGF-1R), suggesting that activation of these receptors in conditions of hyperinsulinemia could contribute to the increased growth of mammary tumors. After 12 weeks on doxycycline, although no significant further increase in tumor weight was observed in MTB/TAN/MKR+/+ compared to MTB/TAN mice, the number of lung metastases was significantly higher in MTB/TAN/MKR+/+ mice compared to controls (MTB/TAN/MKR+/+ 16.41 ± 4.18 vs. MTB/TAN 5.36 ± 2.72). In tumors at the six week time-point, we observed an increase in vimentin, a cytoskeletal protein and marker of mesenchymal cells, associated with epithelial-to-mesenchymal transition and cancer associated fibroblasts. We conclude that hyperinsulinemia in MTB/TAN/MKR+/+ mice resulted in larger primary tumors, with more mesenchymal cells and therefore, more aggressive tumors with more numerous pulmonary metastases.

Increased breast cancer risk and mortality has been associated with obesity and Type 2 diabetes (T2D). Hyperinsulinemia, a key factor in obesity, pre-diabetes and T2D, has been associated with decreased breast cancer survival. In the current study, a mouse model of pre-diabetes (MKR mouse) was used to investigate the mechanisms through which endogenous hyperinsulinemia promotes mammary tumor metastases. The MKR mice developed larger primary tumors and greater number of pulmonary metastases compared to wild type (WT) mice after injection with c-Myc/Vegf overexpressing MVT-1 cells. Analysis of the primary tumors showed significant increase in Vimentin protein expression in the MKR mice compared to WT. We hypothesized that Vimentin was an important mediator in the effect of hyperinsulinemia on breast cancer metastasis. Lentiviral shRNA knockdown of Vimentin led to a significant decrease in invasion of the MVT-1 cells and abrogated the increase in cell invasion in response to insulin. In the pre-diabetic MKR mouse, Vimentin knockdown led to a decrease in pulmonary metastases. In vitro, we found that insulin increased pAKT, prevented Caspase 3 activation, and increased Vimentin. Inhibiting the PI3K/AKT pathway, using NVP-BKM120, increased active Caspase 3 and decreased Vimentin levels. This study is the first to show that Vimentin plays an important role in tumor metastasis in vivo in the setting of pre-diabetes and endogenous hyperinsulinemia. Vimentin targeting may be an important therapeutic strategy to reduce metastases in patients with obesity, pre-diabetes or T2D.

Background Individuals with type 2 diabetes (T2D) are at greater risk of bone fractures than those without diabetes. Certain oral diabetic medications may further increase the risk of fracture. Dipeptidyl peptidase-IV (DPP-IV) inhibitors are incretin-based therapies that are being increasingly used for the management of T2D. It has been hypothesized that these agents may reduce fracture risk in those with T2D. In this study, we used a mouse model of T2D to examine the effects of the DPP-IV inhibitor, MK-0626, on bone. Methods Male wild type (WT) and diabetic muscle-lysine-arginine (MKR) mice were treated with MK-0626, pioglitazone, alendronate or vehicle. The effects of treatment with MK-0626 on bone microarchitecture and turnover were compared with treatment with pioglitazone, alendronate and vehicle. Osteoblast differentiation was determined by alkaline phosphatase staining of bone marrow cells from WT and MKR mice after treatment with pioglitazone, MK-0626 or phosphate buffered saline. Results We found that MK-0626 had neutral effects on cortical and trabecular bone in diabetic mice. Pioglitazone had detrimental effects on the trabecular bone of WT but not of diabetic mice. Alendronate caused improvements in cortical and trabecular bone architecture in diabetic and WT mice. MK-0626 did not alter osteoblast differentiation, but pioglitazone impaired osteoblast differentiation in vitro. Conclusions Overall, the DPP-IV inhibitor, MK-0626, had no adverse effects on bone in an animal model of T2D or directly on osteoblasts in culture. These findings are reassuring as DPP-IV inhibitors are being widely used to treat patients with T2D who are already at an increased risk of fractures.

Aims/hypothesis Previous epidemiological studies have reported a potential link between insulin analogues and breast cancer; however, a prospective randomised controlled trial showed neutral effects of insulin glargine on cancer risk. Insulin glargine is metabolised in vivo to an M1 metabolite. A question remains whether a subset of individuals with slower rates of glargine metabolism or who are on high doses could, theoretically, have an increased risk of cancer progression if a tumour is already present. In this study, we aimed to determine whether a non-metabolisable form of insulin glargine induced murine breast cancer growth. Methods A mouse model of type 2 diabetes (MKR) was used for these studies. MKR mice were injected with two murine mammary cancer cell lines: Mvt-1 cells (derived from MMTV-c-Myc/Vegf tumours) and Met1 cells (derived from MMTV-polyoma virus middle T antigen tumours). Mice were treated with 25 U/kg per day of the long-acting insulin analogues, insulin glargine, insulin detemir, insulin degludec or non-metabolisable glargine, or vehicle. Results No difference in tumour growth was seen in terms of tumour size after insulin glargine, detemir, degludec or vehicle injections. Non-metabolisable glargine did not increase tumour growth compared with insulin glargine or vehicle. Insulin glargine and non-metabolisable glargine led to insulin receptor phosphorylation in vivo rather than IGF-1 receptor phosphorylation. Conclusions/interpretation These results demonstrate that in a mouse model of type 2 diabetes, at high concentrations, basal insulin analogues and a non-metabolisable glargine analogue do not promote the progression of breast tumours.