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Suppression of proHB-EGF Carboxy-Terminal Fragment Nuclear Translocation: A New Molecular Target Therapy for Gastric Cancer

Authors' Affiliation: Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Requests for reprints: Hiromi Kataoka, Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho, Nagoya 467-8601, Japan. Phone: 81-52-853-8211; Fax: 81-52-852-0952; E-mail: hkataoka{at}med.nagoya-cu.ac.jp.

Purpose: Inactivation of epidermal growth factor (EGF) receptor (EGFR) represents a promising strategy for the development of selective therapies against epithelial cancers and has been extensively studied as a molecular target for cancer therapy. However, little attention has been paid to remnant cell-associated domains created by cleavage of EGFR ligands. The present study focused on recent findings that cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), an EGFR ligand, induces translocation of the carboxyl-terminal fragment (CTF) of HB-EGF from the plasma membrane to the nucleus and regulates cell cycle.

Experimental Design: Two gastric cancer cell lines, MKN28 and NUGC4, were used. KB-R7785, an inhibitor of proHB-EGF shedding, was used to suppress HB-EGF-CTF nuclear translocation with cetuximab, which inhibits EGFR phosphorylation. Cell growth was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, apoptosis was evaluated by assay of caspase-3 and caspase-7, and cell cycle was investigated by flow cytometry.

Results: Immunofluorescence study confirmed that KB-R7785 inhibited HB-EGF-CTF nuclear translocation under conditions of proHB-EGF shedding induction by 12-O-tetradecanoylphorbol-13-acetate in gastric cancer cells. KB-R7785 inhibited cell growth in a dose-dependent manner and high-dose KB-R7785 induced apoptosis. Moreover, KB-R7785 induced cell cycle arrest and increased sub-G₁ DNA content. KB-R7785 suppressed cyclin A and c-Myc expression. All effects of KB-R7785 were reinforced by combination with cetuximab.

Conclusions: These results suggest that both inhibition of EGFR phosphorylation and inhibition of HB-EGF-CTF nuclear translocation play crucial roles in inhibitory regulation of cancer cell growth. Suppression of HB-EGF-CTF nuclear translocation might offer a new strategy for treating gastric cancer.