Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis

Abstract

Liver cancer is one of the most common solid tumors, with poor prognosis and high mortality. Mutation or deletion of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is strongly correlated with human liver cancer. Glucose-regulated protein 94 (GRP94) is a major endoplasmic reticulum (ER) chaperone protein, but its in vivo function is still emerging. To study the role of GRP94 in maintaining liver homeostasis and tumor development, we created two liver-specific knockout mouse models with the deletion of Grp94 alone, or in combination with Pten, using the albumin-cre system. We demonstrated that while deletion of GRP94 in the liver led to hyperproliferation of liver progenitor cells, deletion of both GRP94 and PTEN accelerated development of liver tumors, including both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), suggestive of progenitor cell origin. Furthermore, at the premalignant stage we observed disturbance of cell adhesion proteins and minor liver injury. When GRP94 was deleted in PTEN-null livers, ERK was selectively activated.

Conclusion: GRP94 is a novel regulator of cell adhesion, liver homeostasis, and tumorigenesis.

Fig. 1

Hyperproliferation of cells adjacent to portal veins in cGrp94^f/f livers. (A) Liver PCR genotyping and (B) Western blot of liver lysates at 2 months. (C) Liver appearance and weight at indicated ages. (D) Liver H&E staining and Ki67 staining of proliferative cells (black arrows) at 2 months and 9 months. Insets show 2X magnification. PV: portal vein. (E) Immunofluorescence staining with LPC marker A6 (green) and Ki67 (red) in cGrp94^f/f frozen liver sections. White arrows denote double-positive cells. Nuclei were stained with DAPI (blue). (F) Quantitation of Ki67⁺ cells (left) (student’s t test) and A6, Ki67 double-positive cells (right) (χ² test). Scale bar: 25 μm. All data are presented as mean ± s.e. (*P<0.05, **P<0.01, and ***P<0.001).

Fig. 2

Increased LPC pool in cGrp94^f/f mice. (A) Immunofluorescence staining with LPC markers A6 (green) and panCK (red) in frozen liver sections at 2 months. Nuclei were stained with DAPI (blue). (B) Quantitation of PVs bearing over 50 A6⁺ cells (**P<0.01 and ***P<0.001; χ² test). (C) Quantitation and examples of free A6⁺ cells (white arrows) in cGrp94^f/f livers. (D) Plasma ALT, total bilirubin, and ALP measurements (*P<0.05 and **P<0.01). (E) TUNEL staining (red) of liver sections for apoptotic cells (white arrows) at 2 months. (F) Necrosis marker HMGB1 staining. PV: portal vein. Scale bar: 50 μm. All data are presented as mean ± s.e.

Fig. 3

Disruption of cell adhesion molecules in cGrp94^f/f livers at 2 months. (A and B) Immunofluorescence staining of Cx26 (A) and Cx32 (B) in frozen liver sections. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (C) Schematic drawing of E-cadherin zonal expression pattern (red) in WT and diffuse expression (pink) in cGrp94^f/f livers. Immunofluorescence co-staining of integrin β1 (green) and E-cadherin (red) of the boxed areas (U1,2 and L1,2) is shown below. White arrows denote clusters of liver progenitor/bile duct cells expressing high levels of E-cadherin near PVs. (D) Immunofluorescence staining of E-cadherin (green) and panCK (red). Double-positive cells are indicated by white arrows. PV: portal vein; CV: central vein. Scale bar (C, D): 50 μm.

Fig. 4

Biallelic deletion of Pten and Grp94 in the liver accelerates liver tumorigenesis. (A) Western blot of liver lysates at 2 months. (B) Liver H&E and Ki67 staining at 2 months showing proliferative cells (black arrows). Arrowheads denote bile ducts. Scale bar: 25 μm. (C) Liver appearance and weight at 8–9 months presented as mean ± s.e. (**P<0.01 and ***P<0.001). (D) Same as B but at 8–9 months.

Fig. 5

HCC and CC formation in cPten^f/fGrp94^f/f mice. (A) Liver tumor spectrum. Each circle represents one mouse. The solid and open circles represent mice with and without tumors, respectively. (B) H&E staining of liver tumors in cP^f/f94^f/f mice (8 months) showed compact trabecular growth structures of HCC and altered tubule structures protruding to the duct lumen characteristic of CC. Right panels represent 2X magnification of the boxed regions. (C) Immunofluorescence staining with HepPar1 (green) and panCK (red) identified HCC and CC, respectively in cP^f/f94^f/f livers (9 months). Nuclei were stained with DAPI (blue). PV: portal vein. Scale bar: 50 μm.

Fig. 6

Expansion of LPCs in cPten^f/fGrp94^f/f mice. (A) Quantitative PCR analysis of LPC markers EpCAM, AFP, and CK19. (B) Immunofluorescence staining of cP^f/f94^f/f livers at 9 months with HepPar1 (green) and panCK (red) identifies bi-lineage LPCs (white arrows). Nuclei were stained with DAPI (blue). PV: portal vein. Scale bar: 25 μm. (C) Plasma ALT measurements. (D) Quantitation of liver TUNEL staining at 2 months. All data are presented as mean ± s.e. (*P<0.05 and **P<0.01).

Fig. 7

Disturbance of cell junctions in cPten^f/fGrp94^f/f livers at 2 months. Immunofluorescence staining of Cx26 (A) and Cx32 (B) in frozen liver section. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (B) Schematic drawings of liver E-cadherin expression patterns. PV: portal vein; CV: central vein. Double staining of integrin β1 (green) and E-cadherin (red) of the boxed areas (U1,2,3 and L1,2,3) is shown below. White arrows indicate clusters of liver progenitor/bile duct cells with strong E-cadherin. Scale bar: 50 μm.

Fig. 8

ERK activation in premalignant cPten^f/fGrp94^f/f livers at 2 months. (A) Representative Western blots of liver lysates for the indicated proteins. (B) Same as (A) except ERK was analyzed. (C) Staining of p-ERK in the liver. The boxed area was enlarged with black arrows denoting positive cells morphologically similar to LPCs. Scale bar: 50 μm. (D) Summary model on how hepatic GRP94 depletion promotes PTEN-null induced tumorigenesis. PV: portal vein.