Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation

Abstract

Background: To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC.

Methods: Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens.

Results: Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage.

Conclusions: Coronin-1C could be a candidate biomarker to predict HCC invasive behavior.

Figure 1

Coronin-1C was identified as differentially expressed protein between HCCLM9 and MHCC97L cells. (A) Membrane proteins were extracted from MHCC97L and HCCLM9 cells and compared by SDS-PAGE analyses. (B) The differential and interesting protein bands were excised and analyzed by ESI-MS/MS. One of MS/MS maps for Coronin-1C identification and the sequence of precursor were analyzed by MS/MS to be R.AIFLADGNVFTTGFSR.M.

Figure 2

Coronin-1C expression from membrane proteins of HCCLM9 cell rose significantly as compared with MHCC97L. (A) Confirmation of coronin-1C expression by western blot analysis between HCCLM9 and MHCC97L cells. ITGA3, a typical membrane protein, was used as a control. (B) Densiometric scan of immunoblots shown in A.

Figure 3

Coronin-1C expression in HCCLM9- and MHCC97L- nude mice model of HCC. Elevated coronin-1C expression was observed in liver cancer tissues of HCCLM9-nude mice. (A) Coronin-1C expression in tumor tissues of MHCC97L nude mice model of HCC by IHC. ×400; (B) Coronin-1C expression in tumor tissues of HCCLM9 nude mice model of HCC by IHC. ×400; (C) Spontaneous lung metastases occurred in HCCLM9- nude mice.

Figure 4

Tissues coronin-1C level and development of spontaneous pulmonary metastasis in nude mice model of HCC. Tumor tissues coronin-1C level rose remarkably at the end of the fifth wk. (A) Coronin-1C expression at the end of the fourth wk by IHC, ×400; (B) Coronin-1C expression at the end of the fifth wk by IHC, ×400;

Figure 5

The expression of coronin-1C human HCC specimens. Significant differences in coronin-1C expression were observed among HCC specimens of different clinical stages. (A) Score-, ×400; (B) Score +, ×400; (C) Score ++, ×400.