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Defective processing of the transforming growth factor‐β1 in azoxymethane‐induced mouse colon tumors
Author(s) -
Guda Kishore,
Claffey Kevin P.,
Dong Mei,
Nambiar Prashant R.,
Rosenberg Daniel W.
Publication year - 2003
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/mc.10120
Subject(s) - biology , azoxymethane , plasmin , transforming growth factor , plasminogen activator , endocrinology , activator (genetics) , extracellular , in vivo , medicine , extracellular matrix , cancer research , microbiology and biotechnology , carcinogenesis , enzyme , biochemistry , receptor , gene
High levels of the cell growth inhibitor transforming growth factor‐β1 (TGF‐β1) are often found in a variety of human cancers. However, the physiological significance of this overexpression depends on the availability of the biologically active form of TGF‐β1 within the extracellular matrix of the tumor microenvironment. To determine the expression and activation status of TGF‐β1 in chemically induced tumors, 6‐wk‐old A/J mice were injected intraperitoneally with either azoxymethane (AOM) (10 mg/kg body weight, once a week for 6 wk) or normal saline solution, and colon tumors were isolated 24 wk following the last injection. An enzyme‐linked immunosorbent assay for TGF‐β1 revealed a significant increase (1.7‐fold, P < 0.05) in total TGF‐β1 protein in tumors. Interestingly, while 80% of the total TGF‐β1 in the control colon tissues was in the active form, only 50% was found to be active in tumors. Together with our earlier observations that TGF‐β1 mRNA levels are unchanged in A/J tumors, these data further support a mechanism whereby elevated TGF‐β1 levels result from a defective activation and turnover of this protein. Because plasmin is known to be a major activator of TGF‐β1 in vivo, we hypothesized that reduced plasmin activity may be responsible for the observed dysregulation of TGF‐β1 processing in these behaviorally benign tumors. With a fluorogenic peptide substrate for serine proteases, a deficiency in plasmin activity was found in the tumors. Furthermore, semiquantitative reverse transcription (RT)‐polymerase chain reaction (PCR) analysis of a panel of genes involved in the plasminogen activation system, including plasminogen activator inhibitor‐1 ( PAI‐1 ), urokinase‐plasminogen activator ( u‐PA ), and urokinase‐receptor ( u‐PAR‐1 ), demonstrated a significant upregulation (approximately fourfold to sixfold, P < 0.05) in the expression of each of these genes in the tumor tissue. In addition, no significant changes were observed in the expression levels of thrombospondin‐1 ( TSP‐1 ) and insulin‐like growth factor type II receptor ( IGF‐IIR ), which also mediate the activation of latent TGF‐β1. To gain further insight into the functionality of the TGF‐β1 pathway, cDNA microarrays were performed and the expression levels of a panel of 21 TGF‐β1–specific target genes were determined in AOM‐induced tumors that overexpress the ligand. A significant dysregulation in the expression of each of these targets was observed, providing evidence of aberrant TGF‐β1 signaling in tumors. Overall, the present study demonstrates a very low plasmin activity in A/J colon tumors, possibly as a result of the potent inhibitory effect of PAI‐1 on the plasminogen activation cascade. The observed deficiency in plasmin activity may not be sufficiently compensated for by other mechanisms of latent TGF‐β1 activation, including TSP‐1 and IGF‐IIR , thereby resulting in a decreased fraction of the biologically active form of TGF‐β1 and subsequent aberration in TGF‐β1‐specific gene regulation in A/J tumors. © 2003 Wiley‐Liss, Inc.