Background and Objective: Granulin-epithelin precursor (GEP), a novel growth factor, has been identified as a potential therapeutic target from our earlier genome-wide expression profile study on liver cancer. GEP over-expressed in liver cancer, and regulated proliferation, invasion and tumorigenesis. We demonstrated the therapeutic approach of GEP targeted therapy in human cancer using the home-made GEP monoclonal antibody on animal model, and GEP antibody showed dose-dependent inhibitory effect on tumor growth. However, the exact mechanism on how GEP executes its biological function, and in particular its interacting protein partners, is largely unknown. The present study aims to identify and characterize the GEP interacting protein in liver cancer.

Methods: Coimmunoprecipitation (co-IP) was employed to investigate the protein-protein interacting partners of GEP. Total protein was extracted from liver cancer cells Hep3B, and specific anti-GEP monoclonal antibody was used to precipitate its interacting protein partner. The precipitated proteins were analyzed by SDS-PAGE. The extra band (compared with control 1: lysate alone, and control 2: antibody alone) was excised and analysed for protein identification using mass-spectrometry. The interaction of the GEP partner was validated in additional liver cancer cells Hep3B and HepG2 with swap co-IP and western blot using specific antibodies. The expression of GEP and the interacting partner in liver cancer cells and clinical samples were further examined by immunohistochemistry, western blot and real-time quantitative RT-PCR.

Results: The GEP co-IP analysis showed an extra band at 30kDa, and the protein was identified as tropomyosin 3 (TPM3). The interaction of GEP and TPM3 was demonstrated in additional liver cancer cells Hep3B and HepG2, with co-IP using specific TPM3 antibody and detection by western blot using GEP monoclonal antibody. Elevated TPM3, both on mRNA and protein levels, were observed in the liver cancer tissues compared to cirrhosis, hepatitis and normal livers. Overexpression of TPM3 was demonstrated in 74% of liver cancer tissues compared to their paralleled adjacent non-tumor livers. Immunohistochemistry demonstrated cytoplasmic TPM3 protein, and co-localized with GEP protein. High TPM3 level was associated with advanced tumor stage.

Summary: TPM3 locates at 1q21.2, a region with recurrent chromosomal gain in liver cancer. The TPM3-ALK fusion gene has been reported to associate with transformation, proliferation, invasion and metastasis. The current study demonstrated that TPM3 is an interacting partner of GEP in liver cancer, and strong TPM3 was associated with advanced stage. Thus, TPM3/GEP signaling should play important role in liver cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3130.