Inhibition of proprotein convertases is associated with loss of growth and tumorigenicity of HT-29 human colon carcinoma cells -: Importance of insulin-like growth factor-1 (IGF-1) receptor processing in IGF-1-mediated functions

Proprotein convertases (PCs) of the subtilisin/kexin family are responsible for the activation of prohormones, protrophic factors, and their receptors. We sought to determine whether loss of PC-mediated activities might affect the malignant phenotypes of cancer cells. Stable transfectants of alpha (1)-antitrypsin Portland (alpha (1)-PDX) cDNA, coding for a potent PC inhibitor, were analyzed in model HT-29 cells (HT-29/PDX) and in other cell lines. Expression of alpha (1)-PDX resulted in a proinsulin-like growth factor-1 receptor (pro-IGF-IR) processing blockade, hence inhibiting the ability of exogenous IGF-1 to induce tyrosine phosphorylation of its beta -subunit and insulin-related substrate-1. Coexpression of IGF-1R with four different PCs or the novel convertase SKI-1 in the furin-defective LoVo-C5 cells demonstrated that pro-IGF-1R (similar to 200 kDa) cleavage into IGF-1R (beta -subunit, similar to 105 kDa) can be achieved by furin and PC5A, but not by PACE4, PC7, or SKI-1. Expression of alpha (1)-PDX resulted in reduction of DNA synthesis and in anchorage-independent growth. Following serum deprivation, the alpha (1)-PDX transfectants exhibited an enhanced apoptotic phenotype and were insensitive to IGF-1-mediated [H-3]thymidine incorporation and protection against apoptosis. These cells showed reduced invasiveness that paralleled decreased mRNA levels of urokinase-type plasminogen activator and its receptor, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. Comparative subcutaneous inoculation of cells in nude mice revealed that animals injected with HT-29/PDX cells exhibited delayed and lower incidence of tumor development as well as reduced tumor size. Immunohistochemical analysis of CD31 antigen expression, a marker of endothelial cells, revealed reduced HT-29/PDX tumor vascularization. These findings indicate that PCs actively contribute to the growth and malignant phenotypes of HT-29 tumors, suggesting that PC inhibition strategies may be a useful adduct to the arsenal of colorectal anticancer gene therapies.