Validation of HB-EGF and amphiregulin as targets for human cancer therapy

https://doi.org/10.1016/j.bbrc.2007.11.015Get rights and content

Abstract

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.

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Materials and methods

Reagents. Diphtheria toxin was prepared as described previously [9]. All the siRNAs for EGFR ligands and EGFR were purchased from TaKaRa Bio Inc. (Shiga, Japan). Polyclonal rabbit anti-EGFR (sc03) and anti-ERK1/2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibodies against phospho-tyrosine clone 4G10 and phospho-MAPK were acquired from Upstate Biotechnology Inc. (Lake Placid, NY).

Cells and cell culture. The following cell lines were obtained

mRNA expression of EGFR ligands, EGFR, and ErbB2 as well as secretion of EGFR ligand proteins in cancer cell lines

HB-EGF or amphiregulin was identified as the primarily predominant EGFR ligand in most of cancer cells, whereas the expression levels of the other EGFR ligands appeared to vary (Fig. 1A and B, and Supplemental Fig. 1A–L). In ovarian, gastric, bladder, and breast cancers, malignant melanoma and glioblastoma cells with high expression of HB-EGF mRNA, and abundant secretion of soluble HB-EGF were found (Fig. 2A and Supplemental Fig. 2A). In colon, pancreatic, and prostate cancer, renal cell

Discussion

In the present study, we have confirmed the validity of HB-EGF and amphiregulin as targets for cancer therapy (Supplemental Table 1). HB-EGF and amphiregulin possess a heparin-binding motif, whereas the other EGFR ligands do not [7]. Accordingly, HB-EGF and amphiregulin have quite an advantage toward accumulating on the cell surface and activating the EGFR pathway through binding to heparin sulfate proteoglycans (HSPGs) compared to other EGFR ligands without the motif, although a difference

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 17014057) and Research Promotion for Innovative Therapies against Cancers from the Ministry of Education, Culture, Sports, Science and Technology to E.M.

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