IGFBP4 inhibits IGF1 driven tumour cell proliferation and VEGF expression

Abstract

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Insulin like growth factors (IGFs) play key roles in growth, development, cellular transformation and survival of malignant cells. Bioavailability of IGFs is regulated by the IGF binding proteins (IGFBPs), which bind IGFs. IGF release is dependent on cleavage of the IGFBPs by their proteases. There is conflicting data on the significance of IGF and IGFBP expression in tumour growth. We examined the expression of IGFBP4 and its recently identified protease pregnancy associated plasma protein A (PAPP-A), in normal and tumour mouse tissues and 4T1.2 mammary adenocarcinoma cells by Western blotting. The effects of wild type IGF1 and recombinant mutant IGF1, which is resistant to IGFBP binding (IGF1(RE)), were examined on proliferation of 4T1.2 cells. 4T1.2 cells over express IGFBP4 but not PAPP-A. IGF1 at of 50, 100, 500, 1000, and 2000ng/ml did not stimulate proliferation of 4T1.2 cells whereas IGF1 (RE) at 50, 100, 500, 1000, and 2000ng/ml, increased proliferation of these cells (p< 0.05 ANOVA with LSD post hoc). In normal tissues, intact IGFBP4 expression was highest in brain tissue, which did not express PAPP-A. In normal spleen, liver, lung, heart, skin and kidney high levels of PAPP-A were expressed and IGFBP4 was either present at very low levels or completely absent. In 4T1.2 mammary fat pad tumours, lung and bone metastases, the pattern of intact IGFBP4 was the inverse of PAPP-A expression. These data show that IGFBP4-resistant IGF1 stimulates tumour cell proliferation whereas IGF1 bound by IGFBP4 does not. Furthermore, intact IGFBP4 is only present in tissues where PAPP-A is absent. Our results suggest that PAPP-A regulates IGF1 bioactivity via cleavage of IGFBP4. It has been shown in tumour cells that IGF1 increases the expression of VEGF. IGF1 had no effect on VEGF expression of 4T1.2 cells (≥ 50 ng/ml) increased VEGF expression. Our data suggests that IGFBP4 inhibits IGF1 driven tumour cell proliferation and VEGF production. However in vivo IGFBP4 cleavage by PAPP-A, releases bioactive IGF1. Therefore development of a protease-resistant IGFBP4 or inhibiting production of PAPP-A, may be of therapeutic value in blocking the activity of IGF1 in breast cancer Funded by the Irish Cancer Society/ Cancer Research Ireland