Granulin-epithelin precursor is an oncofetal protein defining hepatic cancer stem cells

Abstract

Background and aims: Increasing evidence has suggested that hepatocellular carcinoma (HCC) might originate from a distinct subpopulation called cancer stem cells (CSCs), which are responsible for the limited efficacy of conventional therapies. We have previously demonstrated that granulin-epithelin precursor (GEP), a pluripotent growth factor, is upregulated in HCC but not in the adjacent non-tumor, and that GEP is a potential therapeutic target for HCC. Here, we characterized its expression pattern and stem cell properties in fetal and cancerous livers.

Methods: Protein expression of GEP in fetal and adult livers was examined in human and mouse models by immunohistochemical staining and flow cytometry. Liver cancer cell lines, isolated based on their GEP and/or ATP-dependent binding cassette (ABC) drug transporter ABCB5 expression, were evaluated for hepatic CSC properties in terms of colony formation, chemoresistance and tumorigenicity.

Results: We demonstrated that GEP was a hepatic oncofetal protein that expressed in the fetal livers, but not in the normal adult livers. Importantly, GEP+ fetal liver cells co-expressed the embryonic stem (ES) cell-related signaling molecules including β-catenin, Oct4, Nanog, Sox2 and DLK1, and also hepatic CSC-markers CD133, EpCAM and ABCB5. Phenotypic characterization in HCC clinical specimens and cell lines revealed that GEP+ cancer cells co-expressed these stem cell markers similarly as the GEP+ fetal liver cells. Furthermore, GEP was shown to regulate the expression of ES cell-related signaling molecules β-catenin, Oct4, Nanog, and Sox2. Isolated GEP(high) cancer cells showed enhanced colony formation ability and chemoresistance when compared with the GEP(low) counterparts. Co-expression of GEP and ABCB5 better defined the CSC populations with enhanced tumorigenic ability in immunocompromised mice.

Conclusions: Our findings demonstrate that GEP is a hepatic oncofetal protein regulating ES cell-related signaling molecules. Co-expression of GEP and ABCB5 further enriches a subpopulation with enhanced CSC properties. The current data provide new insight into the therapeutic strategy.

Figure 1. GEP expression in mouse and human livers.

(A) Immunohistochemical analysis showing GEP protein in fetal livers of mouse (embryonic day 17.5) and human (10 weeks 6 days), but not in adult livers (Magnification ×400). (B) Flow cytometric analyzes demonstrating the expression of GEP and hepatic stem cell markers in mouse embryonic hepatocytes. The co-expression of GEP and hepatic stem cell markers CD133, EpCAM, DLK1 and β-catenin were performed by quadruple-color flow cytometry, gating on the AFP+ and/or albumin+ hepatocytes of embryonic livers. Cells co-expressing the respective markers were shown in the upper right quadrant of dot plots. Data are expressed as mean percentage of cells+SD.

Figure 2. GEP-expressing cells isolated from liver cancer cell lines possess higher tumorigenic potential in vitro.

(A) GEP expression in unsorted Hep3B, HepG2 and Huh7 cells, and the freshly isolated GEP^high, and GEP_low subpopulations after cell sorting. Cells were sorted based on surface expression of GEP, but not on its intracellular expression, in order to keep the cells viable for subsequent functional assays. After cell isolation, 2 million of each sorted population was collected to assess the cell viability by trypan blue staining and the purity of the sorted subpopulations by flow cytometry using a different anti-GEP antibody (recognizing distinct epitopes compared to the antibodies used for cell sorting). Data are expressed as mean percentage of GEP+ cells ± SD. (B) Protein expression levels of stem cell markers β-catenin, Oct4, Nanog, Sox2 and ABCB5 in the sorted subpopulations were measured by intracellular staining and flow cytometric analysis. Data are expressed as mean percentage of positive cells ± SD. *P<0.05, **P<0.01, *** P<0.001 when compared with GEP_low cells. (C) Colony formation efficiencies of GEP^high subpopulations were higher than their respective GEP_low and unsorted Hep3B and HepG2 cells. (D, E) After exposure to doxorubicin (0.5 µg/ml for 24 h), GEP^high subpopulations retained significantly less doxorubicin and fewer cell apoptosis than GEP_low and unsorted Hep3B and HepG2 cells. *P<0.05, **P<0.01, *** P<0.001 when compared with unsorted controls.

Figure 3. Co-expression of GEP and ABCB5 further enriches a hepatic CSC subpopulation.

(A) GEP and ABCB5 expression in unsorted Hep3B cells, and the freshly isolated GEP^highABCB5+, GEP^highABCB5− and GEP_lowABCB5− subpopulations. Cells were sorted based on surface expression of GEP and ABCB5. After cell isolation, 2 million of each sorted subpopulation was collected to assess purity by flow cytometry using different anti-GEP and anti-ABCB5 antibodies that recognized epitopes distinct from those of the antibodies used for cell sorting. Note that most ABCB5+ cells were also GEP+, thus there was no GEP_lowABCB5+ cells (left panel). Mean percentage of cells ± SD is shown in each quadrant. (B) Colony formation efficiencies of GEP^highABCB5+ subpopulation were higher than GEP^highABCB5−, GEP_lowABCB5− and unsorted cells. (C, D) After exposure to doxorubicin (0.5 µg/ml for 24 h), GEP^highABCB5+ cells retained significantly less doxorubicin and fewer cell apoptosis than the other subpopulations. *P<0.05, **P<0.01 when compared with unsorted controls; # P<0.05, # # P<0.01, # # # P<0.001 between groups denoted by horizontal lines.

Figure 4. GEP^highABCB5+ Hep3B cells possess enhanced tumorigenic potential in vivo.

(A) Nude mice injected subcutaneously with GEP^highABCB5+ and GEP_lowABCB5− cells after 8 weeks. The right panel shows the subcutaneous tumors derived from 2.5×10⁵ GEP^highABCB5+ and 1×10⁶ GEP_lowABCB5− cells at week 16. (B) Tumorigenicity of GEP^highABCB5+ and GEP_lowABCB5− cells. (C) Serial transplantation of primary tumors generated from GEP^highABCB5+ and GEP_lowABCB5− cells.

Figure 5. Phenotypic characterization of GEP-expressing cells in HCC clinical specimens.

Liver tissues obtained from HCC patients were digested into single cell suspension, and assessed for the expression of albumin, GEP and stem cell markers. (A) Flow cytometric analyzes showing GEP expression in HCC, but not in adjacent non-tumor liver tissues. GEP expression analysis was performed by dual-color flow cytometry, gating on the albumin+ hepatocytes of liver specimens. (B) Co-expression of GEP and stem cell markers was performed by triple-color flow cytometry, gating on the albumin+ hepatocytes of HCC specimens. Protein expression of GEP, β-catenin, Oct4, Nanog, Sox2 and ABCB5 was measured by intracellular staining, while that of CD133 and EpCAM was assessed by surface staining. Cells co-expressing the respective markers were shown in the upper right quadrant of dot plots. Data were presented as mean percentage of cells ± SD.