Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase II in Hepatocellular Carcinoma

Abstract

Early detection and intervention are key strategies to reduce mortality, increase long-term survival, and improve the therapeutic effects of hepatocellular carcinoma (HCC) patients. Herein, the isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues, surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal noncancerous tissues. Among these proteins, carbonic anhydrase II (CA2) was identified to be significantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identified a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.

Keywords: Carbonic anhydrase II; Early diagnosis/prognosis; Epithelial mesenchymal transition; Hepatocellular carcinoma; Tissue secretome.

Figure 1

Strict quality control of the secretory proteins from the cultured HCC-related tissues A. The quality control workflow for the cultured HCC-related tissues and the secretory proteins in the supernatant. B. H&E staining of cultured HCC-related tissues. Scale bar, 1 mm. C. TUNEL staining of cultured HCC-related tissues. We examined the densities of DAPI (blue) and FITC (green) for each tissue. Scale bar, 150 µm. D. The molecular weight distribution of secretory proteins determined by SDS-PAGE. E. Western blot demonstrating the effective prevention of intracellular protein contamination in extracted secretory proteins. Actin was used as a control. HCC, hepatocellular carcinoma; C, cancerous tissue; SN, surrounding noncancerous tissue; DN, distal noncancerous tissue; CM, conditioned medium; H&E, haematoxylin and eosin; TUNEL, TdT-mediated dUTP nick-end labeling.

Figure 2

Experimental workflow for thesecretomeanalysis of HCC-relatedtissues The three types of tissues (C, SN, and DN) from HCC patients were cultured in vitro, and the CM was collected to extract secretory proteins. Secretory proteins were digested with trypsin, directly labeled using iTRAQ-8plex, and analyzed through 2D LC–MS/MS. The target proteins screened by bioinformatics were then verified in vitro to find potential biomarkers of HCC and to investigate the molecular mechanisms of HCC recurrence. iTRAQ, isobaric tag for relative and absolute quantitation; 2D LC–MS/MS, two-dimensional liquid chromatography–tandem mass spectrometry; ELISA, enzyme linked immunosorbent assay; PRM, parallel reaction monitoring.

Figure 3

The overall characteristics and GO enrichment analysesfor the secretoryproteins identified fromHCCtissue secretomes A. and B. The distribution of molecular weight (A) and isoelectric point (B) of the identified secretory proteins. C.–E. GO enrichment analyses of the cell components (C), biological processes (D), and molecular functions (E) of the secretory proteins. F. Venn diagrams showing the numbers of differentially abundant secretory proteins in the two comparison groups (the C/DN group and the SN/DN group). G. and H. The signaling pathway networks in which the differentially abundant secretory proteins of the C/DN group (G) and the SN/DN group (H) involved. GO, Genetic Ontology.

Figure 4

Clinical values of serum CA2 in HCC diagnosis and prognosis A. Distribution (upper panel) and ROC curve (lower panel) of serum CA2 levels in HCC patients and healthy volunteers from the training cohort through PRM targeted proteomics. B. Distribution (upper panel) and ROC curve (lower panel) of serum CA2 levels in HCC patients, with and without recurrence, from the validation cohort assessed by ELISA. C. Comparison of cumulative recurrence rate and overall survival rate between the high- and low-CA2 groups in the validation cohort by Kaplan-Meier analysis. D. Distribution of serum CA2 levels in AFP-negative HCC patients. E. Comparison of cumulative recurrence rate between the high- and low-CA2 groups in AFP-negative HCC patients by Kaplan-Meier analysis. F. AUC of the CA2/AFP combination in HCC patients. G. Prognostic value of the CA2/AFP combination in HCC patients. Two-tailed unpaired Student’s t-test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). CA2, carbonic anhydrase II; ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval; AFP, alpha fetoprotein.

Figure 5

Extracellular CA2 promotes cell migration and invasion through activating EMT A. Representative images and quantification results of migration and invasion of MHCC97L cells treated with exogenous recombinant CA2. B. Cellular morphology of MHCC97L cells treated with exogenous recombinant CA2. Scale bar, 50 μm. C. Western blot showing the expression levels of E-cadherin, N-cadherin, and Zeb1 in MHCC97L cells treated with exogenous recombinant CA2. Two-tailed unpaired Student’s t-test (*, P < 0.05; **, P < 0.01).

Figure 6

Expressionpatternof intracellular CA2 is opposite to its extracellular form A. Western blot showing the intracellular CA2 expression in C tissues and their paired SN tissues in HCC patients. B. Quantification of the intracellular CA2 expression shown in (A). C. Statistical analysis of the intracellular CA2 expression in C tissues and their paired SN tissues on in-house TMAs determined by IHC. D. Representative images of CA2 IHC in a C tissue (score 1) and its paired SN tissue (score 3). Two-tailed paired Student’s t-test (****, P < 0.0001). TMA, tissue microarray; IHC, immunohistochemistry.