Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis

Abstract

The human gene Ptpn11, which encodes the tyrosine phosphatase Shp2, may act as a proto-oncogene because dominantly activating mutations have been detected in several types of leukemia. Herein we report a tumor-suppressor function of Shp2. Hepatocyte-specific deletion of Shp2 promotes inflammatory signaling through the Stat3 pathway and hepatic inflammation/necrosis, resulting in regenerative hyperplasia and development of tumors in aged mice. Furthermore, Shp2 ablation dramatically enhanced diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) development, which was abolished by concurrent deletion of Shp2 and Stat3 in hepatocytes. Decreased Shp2 expression was detected in a subfraction of human HCC specimens. Thus, in contrast to the leukemogenic effect of dominant-active mutants, Ptpn11/Shp2 has a tumor-suppressor function in liver.

Figure 1. Hepatic damage and inflammation in Shp2^hep−/− mice

(A) Circulating blood levels of AST and ALT were quantified in control or Shp2^hep−/− mice at indicated ages; n = 4–8, * p<0.05; ** p<0.01 for WT versus Shp2^hep−/−. (B) Gross appearance of the right lobe of a Shp2^hep−/− liver showing pale acellular regions. (C) H&E staining of a Shp2^hep−/− liver section showed inflammation and macroscopic necrosis. (D) H&E staining showed a small necrotic area surrounded and infiltrated by inflammatory cells in Shp2^hep−/− liver section. In D–H, Arrows indicate portal triads and diamond arrow shows central vein. (E) A necrosis area was surrounded by inflammatory infiltrates in H&E stained Shp2^hep−/− liver section. (F) H&E staining showed severe inflammatory infiltrates in Shp2^hep−/− liver. (G–H) Trichrome staining showed low and high levels of collagen secret surrounding the portal triad of control (G) and Shp2^hep−/− (H) liver sections, respectively. (I) Circulating blood levels of inflammatory cytokines were quantified at 2–3 months of age (n = 7–18, * p<0.05). (J) Hepatic gene expression of IL-6, TNF, Bcl-xl and HGF was assessed by qRT-PCR of total mRNAs isolated from 2–3-month-old mouse livers. The results were average of 3 mice, and absolute mRNA values were determined and normalized to cyclophilin. See also Figure S1.

Figure 2. Hypersensitivity to LPS in Shp2^hep−/− animals

(A) Circulating blood levels of ALT were quantified in WT and Shp2^hep−/− mice at indicated time points after LPS injection (n = 3–4). (B) Spleen weight was measured and normalized to body weight at several time points after LPS challenge (n = 5). (C) Graphic representation of hepatic inflammation levels. Values from 0 to 4 were given to each H&E slide according to the level of infiltration by inflammatory cells. An average was calculated from 5–10 mice of each genotype. (D) Circulating serum levels of TNFα and IL-6 were determined at indicated time points after LPS injection (n = 3–7, * p<0.05 for WT versus Shp2^hep−/−). (E) Serum levels of IFNγ, RANTES, IL-12, IL-1β and IL-10 were quantified at indicated time points after LPS administration (n = 3–7).

Figure 3. Altered hepatic LPS and IL-6 signaling events in vivo and in vitro

(A) Mice were injected with LPS, and pY-Stat1, pY-Stat3, p-Jnk1/2, p-Erk1/2, and p-p38 MAPK were assessed by immnoblotting with their protein levels as controls. IκBα degradation was assessed, using Erk2 protein as control. (B) The phospho-signals were quantified and normalized against total liver protein amounts. IκBα protein amounts were normalized against Erk2 The relative signal levels were determined by setting the value of the control at 1 hr as 1 unit. (n = 3–6, * p<0.05, ** p<0.01 for WT versus Shp2^hep−/−). (C) Immunoblotting of liver lysates was performed 5 min after injection of IL-6 (5 µg) into portal vein. pY-Stat3 and pS-Stat3, and p-Erk levels were assessed. (D, E) pY-Stat3 and pS-Stat3, p-Jnk1/2, p-Akt (Ser473), p-Erk, p-and p38 MAPK were assessed by immunoblot analysis with protein levels as control. The IκBα degradation was compared to Erk2 protein level. (F–G) Amounts of inflammatory cytokines were quantified in supernatants from WT and Shp2^hep−/− hepatocytes (n = 3).

Figure 4. Spontaneous development of hepatocellular adenoma in aged Shp2^hep−/− mice

(A) Immunoblot analysis of Shp2 protein levels in the liver and muscle isolated from young or old WT and Shp2^hep−/− mice. (B) Macroscopic view of hepatic adenomas that bulged from the surface of Shp2^hep−/− liver and appeared pale compared to the hepatic parenchyma. (C) Reticulin staining of an adenoma. (D) Hepatocyte proliferation in Shp2^hep−/− (left panel) and WT (right panel) aged littermate mice. BrdU-positive cells were shown green, Dapi stained nuclei were red. (E) H&E staining of an adenoma. Arrowhead points to a focal granuloma. (F) H&E staining showed nodular regenerative hyperplasia. (G) H&E staining showed hepatocyte degeneration, associated with vacuoles containing eosinophilic material at the periphery of adenoma. (H) TUNEL assay revealed an area of focal apoptosis (dark brown nuclei) in the liver parenchyma. Slides were counterstained with methylgreen. (I) pY-Stat3 levels in WT, tumoral Shp2^hep−/− parenchyma (T) and non-tumoral (N) parenchyma of 18-month-old mice, as assessed by immunoblotting of tissue extracts. (J) Human HCC specimens and corresponding surrounding tissue (ST) from the same patient were immunostained for Shp2. Shown here are 3 representative samples with Shp2 contents lower in HCC than in ST. See also Figure S2.

Figure 5. Enhanced HCC development in Shp2^hep−/− livers is compromised by additional deletion of Stat3

(A) Gross appearances of representative livers with tumors in control (WT), Shp2^hep−/−, Shp2/Stat3^hep−/− (DKO), and Stat3^hep−/− male mice. (B) Liver tumor numbers were compared between WT, Shp2^hep−/−, Stat3^hep−/− and DKO mice (male, n = 5–13). (C) Average maximal diameters of tumors were measured and compared between WT, Shp2^hep−/−, Stat3^hep−/− and DKO livers (male, n = 5–13). (D) The liver/body weight ratios were determined and compared between the four groups of mice (male, n = 5–13). (E) Tumor incidences were determined in WT, Shp2^hep−/−, Stat3^hep−/−, and DKO female mice (n = 6–13). (F) Immunoblotting was performed to evaluate pY-Stat3 levels in tumors (T) and surrounding hepatic tissues (N). (G) Deletion of Shp2 and/or Stat3 in Shp2^hep−/−, Stat3^hep−/− or DKO livers was evaluated by PCR analysis of genomic DNA extracted from tumor or surrounding liver tissues. (H) Immunoblotting was performed for protein extracts from tumor (T) and surrounding liver tissues (N), to determine Shp2 and/or Stat3 removal.

Figure 6. Increased inflammation in Shp2^hep−/− livers is reduced by combined deletion of Shp2 and Stat3

(A–D) Representative H&E staining of tumor samples showed typical trabecular structures in control and Stat3^hep−/− livers, but not in Shp2^hep−/− and DKO livers, and reduced size of eosinophilic vacuoles of degeneration in DKO liver. (E–H) Representative H&E staining of liver samples showed infiltrate of inflammatory cells in portal triads of Shp2^hep−/− and DKO livers, and fat droplets in Stat3^hep−/− liver. (I) Serum ALT levels were measured in the four groups of mice. (J–K) IL-6 and TNFα levels in liver extracts were measured by ELISA. (L–M) qRT-PCR was performed to determine IL-6 and TNFα mRNA levels. (I–M: n = 5–11; * p<0.05; ** p<0.01; *** p<0.001).