Structural and functional studies of insulin receptors in human breast cancer

Summary

We characterized the structure and the function of insulin receptors isolated from 10 human breast cancer specimens. We observed that the insulin receptor content, as determined by a specific radioimmunoassay, was four fold increased in human breast cancer tissue when compared to normal breast tissues. In both cancer and normal breast tissues, insulin receptor mRNA consisted of two major species of approximately 11.0 and 8.5 kilobases. The size of the insulin receptor alpha subunit was determined by¹²⁵I-insulin cross-linking followed by immunoprecipitation and polyacrylamide gel electrophoresis; a value of 135 kDa was observed for receptors from both breast cancer and normal breast tissues. The functional binding ability of insulin receptors from cancer tissues was slightly lower as compared to normal tissue derived insulin receptor (% B/T= 2.22±0.50 per ng of insulin receptor as determined by radioimmunoassay vs. 2.96±0.49, mean±S.E.M.). The concentration of insulin that caused half maximal inhibition of¹²⁵I-insulin binding was very similar for both cancer and normal breast receptors (80pM).

The size of the insulin receptor beta subunit as determined by receptor autophosphorylation was 95kDa. Basal and maximal insulin (100nM) stimulated receptor tyrosine kinase activity, in terms of both receptor autophosphorylation and phosphorylation of an exogenous substrate, was similar in malignant and normal breast tissue derived insulin receptor. Also, a very similar insulin stimulated Km value for ATP was showed by the tyrosine kinase of insulin receptors from breast cancer and normal breast tissue (11.1 and 10.8µM ATP, respectively). However, in insulin receptors from breast cancer tissue the average tyrosine kinase sensitivity to insulin, as calculated on the exogenous substrate, was higher, although not significantly, with respect to normal breast tissue (ED₅₀ at 0.28±0.09 and 1.08±0.33 nM insulin, respectively). A similarly different sensitivity to insulin was observed also for receptor autophosphorylation.

In conclusion, this study demonstrates that breast cancer tissues have an increased number of structurally and functionally normal insulin receptors. In some breast cancer tissues, however, the sensitivity of the receptor tyrosine kinase activity to insulin is greatly increased. These data suggest that, in vivo, the mitogenic effect of insulin may play a role in the biology of certain breast cancers.