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


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.
Department of Surgery, The University of Hong Kong, Hong Kong.
Ching Yan
CY
Lam
Chi Wai
CW
Yip
Terence C W
TC
Poon
Christine K C
CK
Cheng
Eddy W Y
EW
Ng
Nicholas C L
NC
Wong
Phyllis F Y
PF
Cheung
Paul B S
PB
Lai
Irene O L
IO
Ng
Sheung Tat
ST
Fan
Siu Tim
ST
Cheung
D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D018572:Disease-Free Survival; D015870:Gene Expression; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D053208:Kaplan-Meier Estimate; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D009364:Neoplasm Recurrence, Local; D000077153:Progranulins; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011498:Protein Precursors; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018709:Statistics, Nonparametric; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins; D011498:Protein Precursors; D012333:RNA, Messenger; C498783:TPM3 protein, human; D014335:Tropomyosin