Mammary gland tumor promotion by chronic administration of IGF1 and the insulin analogue AspB10 in the p53R270H/⁺WAPCre mouse model

Abstract

Introduction: Insulin analogues are structurally modified molecules with altered pharmaco-kinetic and -dynamic properties compared to regular human insulin used by diabetic patients. While these compounds are tested for undesired mitogenic effects, an epidemiological discussion is ongoing regarding an association between insulin analogue therapy and increased cancer incidence, including breast cancer. Standard in vivo rodent carcinogenesis assays do not pick up this possible increased carcinogenic potential.

Methods: Here we studied the role of insulin analogues in breast cancer development. For this we used the human relevant mammary gland specific p53R270H/⁺WAPCre mouse model. Animals received life long repeated treatment with four different insulin (-like) molecules: normal insulin, insulin glargine, insulin X10 (AspB10) or insulin-like growth factor 1 (IGF1).

Results: Insulin-like molecules with strong mitogenic signaling, insulin X10 and IGF1, significantly decreased the time for tumor development. Yet, insulin glargine and normal insulin, did not significantly decrease the latency time for (mammary gland) tumor development. The majority of tumors had an epithelial to mesenchymal transition phenotype (EMT), irrespective of treatment condition. Enhanced extracellular signaling related kinase (Erk) or serine/threonine kinase (Akt) mitogenic signaling was in particular present in tumors from the insulin X10 and IGF1 treatment groups.

Conclusions: These data indicate that insulin-like molecules with enhanced mitogenic signaling increase the risk of breast cancer development. Moreover, the use of a tissue specific cancer model, like the p53R270H/⁺WAPCre mouse model, is relevant to assess the intrinsic pro-carcinogenic potential of mitogenic and non-mitogenic biologicals such as insulin analogues.

Figure 1

Effect of insulin analogues on mammary gland tumor development in p53 ^R270H/+ WAPCre female mice. A) Upper graphs represent the average weight of the mice in the indicated groups during the first 35 weeks of the experiment. Statistical analysis was determined with an unpaired t-test. B) Effect of different compounds on mammary gland tumor free survival shown as in Kaplan Meier curves. Statistical analysis for the survival curves was determined with the log rank test.

Figure 2

Insulin analogues induce primarily EMT tumors in p53 ^R270H/+ WAPCre female mice. A) H & E images of representative EMT tumors with predominantly epithelial cells (left panel) and an EMT tumor with predominantly mesenchymal cells (right panel). B) Immunofluoresent images of CK5/8, area with normal mammary duct on the same slide that serves as control for CK5/CK8 staining, E-cadherin and smooth muscle actin. C) Tumor type distribution per treatment. D) Mammary gland tumor type distribution per treatment. E) Average number of mammary gland tumors. F) Percentage of epithelial cells in the EMT tumors over the different treatment groups. EMT, epithelial to mesenchymal transition.

Figure 3

Molecular profiling of IR and IGF1R signaling in insulin analogue derived mammary gland tumors. Tumor protein levels of critical mammary gland tumor related receptors (IR, IGF1R, ER, EGFR, Her2) and downstream signaling pathways (Erk, phospho-Erk, Akt, phospho-Akt) as well as epithelial differentiation markers (N-cadherin and E-cadherin) were determined by quantitative Western blotting of all primary mammary gland tumors (n = 148). A) A small subset of the Western blot data (n = 36) is shown which are representative for the quality of the blots. B) The quantitative IR, IGF1R, p-Akt and p-Erk levels of all primary EMT tumors are presented in dot-plots (n = 148). C) The IR versus IGF1R protein levels are plotted, five clusters are defined. D) The mean tumor latency time has been determined of each cluster. Error bars represent SEM, ns = P >0.05, * = P <0.05, ** = P <0.01, *** = P <0.001. EGFR, epidermal growth factor receptor; EMT, epithelial to mesenchymal transition; ER, estrogen receptor; IGF1R, insulin-like growth factor 1 receptor; IR, insulin receptor; SEM, standard error of the mean.

Figure 4

Hierarchical clustering of insulin (analogue) tumors based on IR and IGF1R signaling components. Quantitative expression levels of IGF1R-β, IR-β, p-Akt and p-Erk in mammary gland EMT tumors of all treatment groups were clustered using Euclidean distance and average linkage (A). Distinct clusters appeared in which the treatment groups are not equally distributed. In protein clusters 1, 2, 5 and 6, the highly mitogenic treatment groups (IGF1 and X10) are overrepresented. In graphs B and C the distribution of several parameters of the clusters are shown. B) Treatment group distribution. C) Latency time per cluster, in which it became apparent that the cluster with the highest p-Erk levels has the shortest tumor latency time. In the last two graphs the correlation between latency time and p-Erk (D) or latency time and p-Akt (E) is presented. Interestingly, high p-Akt levels are positively correlated with latency time, but only in the compound treatment groups and not in the vehicle treated animals. EMT, epithelial to mesenchymal transition; IGF1R, insulin-like growth factor 1 receptor; IR, insulin receptor.