PKC agonists stimulate mitogenesis of SNU-C4 colon cancer cells by augmenting autocrine stimulation of epidermal growth factor receptor

Abstract

PKC agonists induce rapid autophosphorylation of epidermal growth factor receptor (EGFR) family members and also induce the release and posttranslational processing of ligands for EGFR. However, the importance of these phenomena in cell growth is unknown. We therefore examined the role of EGFR and its ligands in the proliferative effects of PKC agonists in a human colon cancer cell line SNU-C4. The PKC agonists, PMA, thymeleatoxin, dPPA and ingenol each increased viable cell mass and 3Hthymidine uptake of SNU-C4 ceils. Proliferation stimulated by 1 nM PMA treatment was dose-dependently inhibited by PD 153035 (a selective EGFR tyrosine kinase inhibitor), MAh 225 (a competitive EGFR antagonist) and by GF 109203X (a selective PKC inhibitor). Immunoreactive TGF-ct levels in the medium of SNU-C4 cells increased approximately 100-fold after 24h treatment with proliferative concentrations of PKC agonists, an effect that was 90% reversible by co-incubation with 1 ~aM GF 109203X. However, immunoneutralizing anti-TGF-ct antibody did not suppress PMA-induced growth. To determine the potential involvement by other ligands, Northern analysis was performed. PMA (1 nM) strongly induced transcription of amphiregulin at 24 h but only slightly induced TGF-a and HB-EGF transcripts. GF 109203X suppressed induction of amphiregulin but had little effect on induced transcript levels of TGF-ct or HB-EGF. In summary, the proliferative effect of PKC agonists in SNU-C4 cells is dependent on EGFR ligation, EGFR tyrosine kinase activity, and PKC-like activity, and is associated with induction of amphiregulin and release of TGF-ct. The failure of immunoneutralizing TGF-ct antibody to inhibit PKC agonist-induced growth suggests that other EGFR ligands, either alone or in combination, mediate this autocrine proliferative response. This research was funded by NCI R29 CA71974, and the Mayo Foundation. Irene Kline was funded by S.U.R.F.