Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. There is an urgent need to develop novel biomarkers for early diagnosis, as well as to identify new drug targets for therapeutic interventions.

Patients and methods: 54 paired HCC samples and 21 normal liver tissues were obtained from West China Hospital of Sichuan University. Informed consent was obtained from all the patients or their relatives prior to analysis, and the project was approved by the Institutional Ethics Committee of Sichuan University. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics was employed to profile the differentially expressed proteins between a HepG2 human hepatoma cell line and an immortal hepatic cell line L02. Validation of PGAM1 expression was performed by semi-quantitative RT-PCR, immunoblot and immunohistochemistry using clinical samples. shRNA expressing plasmids specifically targeting PGAM1 were designed and constructed by GenePharma Corporation (Shanghai, China), and were utilized to silence expression of PGAM1 in vitro and in vivo. Cell proliferation was measured by a combination of colony formation assay and Ki67 staining. Apoptosis was examined by flow cytometry and TUNEL assay.

Results: A total of 63 dysregulated proteins were identified, including 51 up-regulated proteins, and 12 down-regulated proteins (over 2-fold, p < 0.01). Phosphoglycerate mutase 1 (PGAM1) was found markedly upregulated. Clinico-pathological analysis indicated that overexpression of PGAM1 was associated with 66.7% HCC, and strongly correlated with poor differentiation and decreased survival rates (p < 0.01). shRNAs-mediated repression of PGAM1 expression resulted in significant inhibition in liver cancer cell growth both in vitro and in vivo.

Conclusion: Our studies suggested that PGAM1 plays an important role in hepatocarcinogenesis, and should be a potential diagnostic biomarker, as well as an attractive therapeutic target for hepatocellular carcinoma.

Figure 1

Identification and quantification of proteins by LC-MS/MS Based on SILAC method. (A) Clustering analysis of the identified proteins from L02 cells and HepG2 cells. Protein cluster map generated by Cluster software. Expression of proteins in the normal group was constant at 0, proteins up-regulated in cancer tissue were in red, and the down-regulated proteins were in green. The intensity of the color green or red corresponds to the degree of alteration, respectively, according to the color strip at the bottom of the figure. These data were derived from three independent analyses. (B) A total of 63 dysregulated proteins were classified into 11 groups with diverse functions including metabolism (34.9%), signal transduction (12.7%), structural component (7.9%), and other functions (44.5%). (C), (D), and (E) showed output of the LC MS/MS database using the MASCOT program. LC MS/MS analysis revealed 8 matched peptides with 38% sequence coverage and a MOWSE score of 172. The matched peptides were shown in bold red. (F), left, MS spectrum showed a SILAC peptide matching PGAM1, with an up-regulation up to 6-fold. The m/z presents a difference of 3 mass units between labeled and unlabeled peptide pair, resulting in a 2⁺ change state; right, control SILAC peptide from β-actin.

Figure 2

Overexpression of PGAM1 in HCCs. (A), expression level of PGAM1 mRNA was semi-quantified by RT-PCR. Pairs of the total mRNA were normalized by GAPDH. (B), representative results of Western blot analysis of HCCs and the adjacent noncancerous samples with β--actin as an internal control. (C), box chart, quantitative Western blot results of HCCs vs. the corresponding normal tissues. To ensure reproducibility, three independent experiments were performed. *, p < 0.01, Student's t test. N, normal liver tissue. C, cancer tissue.

Figure 3

Immunohistochemical analysis indicated that overexpression of PGAM1 in HCCs was closely associated with the degree of differentiation and the lower survival rates. (A), staining against PGAM1 confirmed significant differences between normal liver tissues and hepatocellular carcinoma samples more likely to present poor differentiation (p < 0.05). a, normal liver tissue; b, well differentiated tissue; c, moderate differentiation; d, poor differentiation. a-d, representative HE staining; a'-d', expression of PGAM1 was examined by immunohistochemistry. a"-d", representative insets of a'-d'. Original magnification, × 200. (B), Kaplan-Meier survival curves showed the correlation between higher levels of PGAM1 expression and lower survival rates (p < 0.05).

Figure 4

Suppression of PGAM1 by PGAM1-shRNA-a resulted in cell growth arrest and induced apparent apoptosis in vitro. (A) Evaluation of the antiproliferation effects using MTT assay. Inhibition of cell growth by siRNA against PGAM1 was in a duration-dependent manner and the proliferation rate was decreased to 48.6% at 72 h posttransfection. (B) Survival rates of hepatoma cells were examined by colony formation assay. 24 h after transfection, cells were allowed to grow at a density of 300 cells/dish for additional two weeks before staining with Crystal Violet. (C) Flow cytometry analysis was performed and the subdiploid peak increased after 72 h of transfection (p < 0.05). (D) Cell apoptosis was assessed by TUNEL assay which showed a remarkably greater percentage of TUMEL positive nuclei of the PGAM1-shRNA-a group vs. the control group. a, untreated; b, Lipofectamine 2000; c, shNC; d, PGAM1-shRNA-a. Results represent the average of three independent experiments and data were shown as mean ± S.D.

Figure 5

Treatment with PGAM1-shRNA-a inhibited xenograft tumor growth and induced apoptosis in vivo. (A), tumor volume growth curve after tail intravenous injection for 20 days. PGAM1-shRNA-a treatment resulted in significantly decreased tumor growth compared with other control groups (p < 0.05). (B) & (C), a significant decrease of PGAM1 expression in tumor-bearing mice examined by immunohistochemical analysis (upper lane). Cell proliferation was obviously inhibited and apparent apoptosis was induced (p < 0.05). a, PBS; b, Lipofectamine 2000; c, shNC; d, PGAM1-shRNA-a. Data were shown as mean ± S.D.

Figure 6

A model of glycolysis illustrating the possible involvement in cellular energy signaling pathways. Arrows represent activation, whereas bars represent inhibition. Dotted arrows indicate the increased ratio of AMP/ATP. Small circles represent ATP. AMPK, AMP-activated protein kinase; TSC, tuberous sclerosis complex; Rheb, Ras homologue enriched in brain; mTOR, mammalian target of rapamycin; S6K, ribosomal protein S6 kinases; 4E-BP1, eIF4E-binding protein; ACC, acetyl-CoA carboxylase; FASN, fatty-acid synthase; CDKs, cyclin dependent kinases.