Modulation of cathepsin D by IGF-II in breast cancer cells

What changes in cellular function account for the progression of breast cancer? Onepromising research area that addresses this fundamental question is the study of the interactions of insulin-like growth factor-II (IGF-II) and catbepsin D with the IGF-II/mannose-6-phosphale (M6P) receptor. IGF-II is a mitogen for breast cancer and stimulates cell motility important for metastasis. Cathepsin D is an enzyme involved in metastasis. Both bind the IGF-II/M6P receptor at distinct binding sites. Nevertheless, a reciprocal inhibitory interaction is observed when IGF-II or catbepsin D bind the IGF-II/M6P receptor. Normally, cathepsin D is stored intracellularly in the lysosomes. However, a significant amount of this protease is secreted in breast cancer. We have developed an in vitro model consisting of MCF-7 breast cancer cells stably transfected with IGF-II. The expression of IGF-II in these transfected cells significantly increases the secretion of cathepsin D. Since higher levels of IGF-II and cathepsin D represent an increased risk of malignancy and metastasis, we hypothesize that inhibition of IGF-II expression will decrease cathepsin D, thus decreasing malignancy and metastatic potential. To address this question, MCF-7 breast cancer cells overexpressing IGF-II were transfected with an antisense IGF-II construct Stable transfectants were cloned by limiting dilution in 96 well plates. Clones with reduced or no IGF-II were identified by performing a dot blot assay on aliquots of serum-free conditioned media Western blotting analysis to characterize the IGF-II forms on antisense transfected clones demonstrated that these clones expressed only the 14 kD IGF-II form. In contrast, IGF-II secreting clones expressed several forms of IGF-II ranging from 14-45 kD. The total expression of IGF-II in antisense clones was reduced approximately by ten fold when compared to IGF-II secreting clones. Northern blotting analysis revealed that expression of the IGF-II mRNA was also significantly decreased by approximately five fold in antisense clones. Preliminary cathepsin D analysis by western blotting showed that antisense IGF-II clones secreted less cathepsin D. Since IGF-II and cathepsin D promote tumor growth and metastasis, inhibition of these proteins represents a desirable target for treatment. Thus, regulation of IGF-II may offer a unique therapeutic target for a molecular medicine approach to the treatment of breast cancer.