Targeting granulin-epithelin precursor in breast cancer

Abstract

Modulation of hormone action by tamoxifen is an important therapeutic strategy for breast cancer. Despite initial benefit, resistance is common and the main obstacle to complete cure. Constitutive overexpression of autocrine growth factor by tumor cells has been proposed as a possible mechanism for tamoxifen resistance.1 Granulin-epithelin precursor (GEP) is an important growth factor up-regulated in breast cancer.2 In breast cancer cell lines, GEP overexpression has been demonstrated to confer resistance to Herceptin and tamoxifen.3,4 We hypothesize that GEP is a potential target for treatment of breast cancer and utilized a newly established monoclonal antibody A23, previously demonstrated to specifically target GEP.5 GEP expression in clinical samples and breast cancer cell lines were investigated by immunohistochemistry and Western blot analysis respectively. Tissue array containing 112 breast cancers and 31 non-tumor tissue from Department of Pathology, Queen Mary Hospital of Hong Kong were used. GEP in breast cancer cells was neutralized using A23 monoclonal antibody and its effect on cell proliferation investigated in-vitro and in-vivo. Underlying signaling pathways were examined by Western blot. Immunohistochemistry showed GEP was positive in 93.7% of cancers, with 59.8% showing high expression, while >90% non-tumor samples were GEP-negative. GEP was expressed in both estrogen receptor (ER)-positive and -negative cancer tissue. Importantly, 94.1% of ER-negative cases were GEP-positive. Western blot analysis also showed that GEP was expressed in most of the breast cancer cell lines independent of ER status or tamoxifen sensitivity. Neutralizing GEP by the antibody suppressed breast cancer cell growth in ER-positive (MCF7, T47D and ZR-75-1), ER-negative (MDA-MB-468) and tamoxifen-resistant (AK47 and R27) breast cancer cell lines. In a mouse model by orthotopic implantation of MCF7 into mammary fat pad of nude mice, intraperitoneal administration of the monoclonal antibody could suppress tumor growth effectively without observable side effect. The antibody was found to reduce tumor cell growth via inhibition of MAPK and Akt pathway. Phosporylated Foxo3A, the downstream target of Akt, was also inhibited. In conclusion, GEP appears to be a useful target particularly for ER-negative breast cancer. Targeting GEP is effective in inhibiting breast cancer growth and provides a potential strategy to circumvent tamoxifen resistance especially in ER-negative cells. It thus provides a novel and essential platform for advancement of targeted therapy in breast cancer. Further investigation in the mechanism of tamoxifen resistance by alteration of GEP expression in breast cancer cell lines is currently underway. References: 1. J Steroid Biochem Mol Biol, 76:71-84, 2001 2. Carcinogenesis, 25:1587-82, 2004 3. Can Res, 64:1737-43, 2004 4. Clin Can Res, 12:4192-9, 2006 5. Hepatology, 47:1524-32, 2008