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-1R) 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 ( approximately 200 kDa) cleavage into IGF-1R (beta-subunit, approximately 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 [(3)H]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.
Khatib
A M
AM
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada.
Siegfried
G
G
Prat
A
A
Luis
J
J
Chrétien
M
M
Metrakos
P
P
Seidah
N G
NG
D000818:Animals; D017209:Apoptosis; D003110:Colonic Neoplasms / Q000098:blood supply / Q000188:drug therapy* / Q000473:pathology; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D008297:Male; D051379:Mice; D009361:Neoplasm Invasiveness; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011498:Protein Precursors / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D011498:Protein Precursors; D000515:alpha 1-Antitrypsin; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1