Identification and characterization of tropomyosin 3 associated with granulin-epithelin precursor in human hepatocellular carcinoma

Abstract

Background and aim: Granulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry.

Methods and results: Specific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining.

Conclusions: TPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.

Figure 1. Identification of TPM3 as the predominant binding partner of GEP.

(A) SDS-PAGE analysis of co-immunoprecipitation reactions using total protein lysate from liver cancer cells Hep3B and monoclonal anti-GEP antibody. M: Protein marker (Fermentus prestained protein marker); Lane 1: Immunoprecipitation product of protein lysate and anti-GEP antibody; Lane 2: Protein lysate alone; Lane 3: Anti-GEP antibody alone. The protein band indicated by the arrow was excised from the gel and the protein identity was examined by MS. (B) MALDI-TOF/TOF-MS analysis of the tryptic peptides. The peptides matched to TPM3 were asterisked. (C) Protein sequence of TPM3 was shown, and matched peptides were underlined. (D) An annotated representative tandem MS mass spectrum of a tryptic peptide (M/Z 1284.8) with the amino acid sequence identified as KLVIIEGDLER.

Figure 2. Association of TPM3 and GEP.

(A–B) Protein-protein interaction of TPM3 and GEP was validated by immunoprecipitation using anti-TPM3 antibody, followed with immunoblot detection using anti-GEP antibody in different cell lines (A) Hep3B and (B) HepG2 cells. (C) Positive correlation of GEP and TPM3 on protein level. AS: Cells suppressed for GEP by transfection with anti-sense GEP fragment; FL: Cells overexpressed for GEP by transfection with full-length (FL) GEP cDNA construct. Cells with overexpression of GEP showed elevated TPM3 levels, while suppression of GEP showed decreased TPM3.

Figure 3. Over-expression of TPM3 and GEP mRNA levels in liver cancer.

(A) Elevated expressions of TPM3 were observed in liver cancer (hepatocellular carcinoma, HCC) compared with the paralleled adjacent non-tumor liver tissues (P  = 0.001, respectively). (B) Transcript levels of GEP and TPM3 were significantly correlated (Spearman’s ρ correlation coefficient  = 0.658, P<0.001). (C) Kaplan-Meier recurrence-free survival plot according to TPM3 levels (log-rank test, P  = 0.0496). Patients in the TPM3-high group showed poor recurrence-free survival compared to TPM3-low group (median recurrence-free survivals 8.0 and 52.0 months, respectively).

Figure 4. Immunohistochemistry analysis of TPM3 and GEP on clinical specimens.

(A–D) Elevated TPM3 protein in liver cancer. TPM3 was observed in the cytoplasm of liver cancer cells (A and B) but rarely in the adjacent non-tumor liver tissue (C and D). (E–H) GEP and TPM3 co-localization in liver cancer. Similar pattern of protein expressions were observed in GEP protein (E and F) and TPM3 protein (G and H). The protein signal was visualized as brown. Magnification×100 in A, C, E and G; ×400 in B, D, F and H.