SHP-1 Acts as a Tumor Suppressor in Hepatocarcinogenesis and HCC Progression

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer mortality worldwide (1). Although significant progress has been achieved over the past decades, the outcomes of patients with late-stage HCC are still unsatisfactory. Therefore, the molecular pathogenesis of HCC remains to be defined, and novel diagnostic and therapeutic techniques need to be developed.

Protein tyrosine phosphorylation is critical for signal transduction in eukaryotic cells, which is reversibly and coordinately controlled by protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP; refs. 2, 3). The disturbed PTK–PTP balance often induces aberrant protein tyrosine phosphorylation in cancers and promotes tumorigenesis, including that of HCC (3–6). PTKs are mainly associated with oncogenic and tumorigenic activities, whereas PTPs play tumor-suppressor roles (3, 6–9). Although the cancer-related PTKs have been well accepted as therapeutic targets of human cancers in recent years, PTPs are considered as next-generation drug targets (3, 9).

The nonreceptor PTPs, Src homology region 2 (SH2) domain–containing phosphatase 1 (SHP-1, also known as PTPN6), and SHP-2 (also known as PTPN11) are important regulators of fundamental cellular processes, including proliferation, differentiation, inflammation, and intermediary metabolism (10). SHP-2 is a ubiquitously expressed modulator of inflammatory signaling and involved in hepatocarcinogenesis and HCC progression (11, 12). SHP-1 is predominantly expressed in hematopoietic and epithelial cells, and widely accepted as a negative regulator of inflammation (13). Recent studies reported that multikinase inhibitors, including sorafenib (14) and dovitinib (15), and Mcl-1 inhibitor SC-2001 (16, 17) exert their antitumor effects through enhancing the phosphatase activity of SHP-1. It was also reported that SHP-1 overexpression abolishes TGFβ1-induced STAT3^Tyr705 phosphorylation and the epithelial-to-mesenchymal transition as well as the migration and invasion of HCC cells (18). However, the association of SHP-1 expression and its influence on the prognosis of patients with HCC and the direct effects of SHP-1 on hepatocarcinogenesis are largely unknown.

Here, we report that SHP-1 expression was markedly decreased in HCC tissues compared with the surrounding noncancerous tissues, and reduced SHP-1 expression predicted poor prognosis of patients with HBV-associated HCC. Moreover, using hepatocyte-specific Ptpn6-knockout mice (Ptpn6^HKO), we demonstrate that SHP-1 plays a critical role in the development and progression of HCC through regulating the activation of STAT3, NF-κB, and AKT signaling.

Liver samples were obtained from patients with HCC undergoing surgical resection at the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). Written-informed consent was obtained from all patients. HCC tissues with typical macroscopic features were collected from tumor nodules, which were examined using hematoxylin and eosin (H&E) staining to confirm the diagnosis. Paired adjacent noncancerous tissues without histopathologically identified tumor cells were collected from ≥5 cm from the tumor border. A tissue microarray block containing 271 HCCs and paired noncancerous surrounding tissues was constructed using a tissue microarrayer (Outdo Biotech). Tissue microarray blocks containing 271 HCCs along with case-matched noncancerous tissues were constructed using a tissue microarrayer. IHC of tissue microarray slides was performed using an anti–SHP-1 antibody (CST). SHP-1 expression was assessed using a 4-point scale (negative, 1; weak positive, 2; positive, 3; strong positive, 4), according to the percentage of stained cells using Image-scope software (Aperio Technologies; ref. 19). Overall survival (OS) was defined as the interval between the date of surgery and death. All human experiments were conducted according to the CIOMS ethical guidelines and approved by the Ethics Committee of the Second Military Medical University (Shanghai, China).

Publicly available data were collected from The Cancer Genome Atlas (TCGA) database LIHC project (https://portal.gdc.cancer.gov/projects/TCGA-LIHC). A total of 867 HCC tissues from 865 patients with HCC were used for analyzing the genetic alterations by cBioportal (http://www.cbioportal.org; refs. 20, 21). All of the 310 patients with expression data, methylation data, and survival information in TCGA database were used to analyze the correlation between mRNA levels of SHP-1 and DNA methylation of PTPN6 locus. For survival analysis, gene expression data of a cohort mainly composed of patients with HBV-related HCC were downloaded from NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE14520 (22).

Total RNA was isolated from cells or tissues following the standard TRIzol (Takara) protocol. First-strand cDNA was synthesized using total RNA with a PrimeScript RT Master Mix (Takara). Transcript levels were detected using SYBR Green–based real-time PCR performed using an ABI StepOne Real-time PCR Detection System (Life Technologies). mRNA levels were normalized to those of β-actin mRNA. At least three independent experiments were performed using each condition. Primer sequences are shown in Supplementary Table S1.

Proteins were extracted using RIPA buffer (P0013B, Beyotime) supplemented with protease inhibitor cocktail (Roche), separated using SDS-PAGE, and then electrophoretically transferred to a nitrocellulose membrane (HAHY00010; Millipore). The membrane was blocked in PBS-T containing 5% milk/BSA for 2 hours before overnight incubation with a primary antibody at 4°C. After a 2-hour incubation with a secondary antibody (donkey-anti-mouse or donkey-anti-rabbit, IRDye 700 or IRDye 800, respectively; LI-COR), signals were quantitated using an Odyssey infrared imaging system (LI-COR) at 700 or 800 nm. The primary antibodies are listed in Supplementary Table S2.

Formalin-fixed paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase was blocked by 3% H₂O₂ followed by antigen retrieval. Slides were blocked in 10% goat serum for 2 hours at room temperature, incubated with primary antibodies overnight at 4°C, and incubated with secondary antibody at room temperature for 30 minutes. The staining was developed using an EnVision Detection Rabbit/Mouse Kit (GK500710; GeneTech).

The HCC cell line Huh7 was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The HCC cell line PLC and human embryonic kidney cell lines 293 and 293T were purchased from the American Type Culture Collection. Cell lines were routinely tested for mycoplasma contamination using a Mycoalert detection kit (Lonza) and authenticated by short tandem repeat analysis every 6 months. The cells were cultured in DMEM containing 10% heat-inactivated FCS.

The recombinant adenoviruses AdSHP-1 and AdGFP were previously established in our lab (23). SiRNA for SHP-1 (5′-CGCAGUACAAGUUCAUCUAtt-3′) and the negative controls (NC) siRNA (5′-UUCUCCGAACGUGUCACGUtt-3′) were purchased from GenePharma (Shanghai GenePharma Co., Ltd.).

HCC cells were infected or transfected for 8 to 12 hours and then plated onto 96-well plates (3,000 cells per well). Cell proliferation was measured using Cell Counting Kit-8 (Dojinodo) according to the manufacturer's instructions. At least three independent experiments were performed for each condition.

In vitro migration and invasion assays were performed using Transwell chambers (BD Bioscience), without or with Matrigel, according to the manufacturer's instructions. HCC cells infected or transfected for 8 to 12 hours were seeded in serum-free medium in the upper chamber. Medium supplemented with 10% FBS was added to the lower chamber. After incubation for 24 to 48 hours at 37°C, cells remaining on the upper membrane were removed with a cotton swab. Cells on the lower surface of the membrane were fixed and stained with 0.1% crystal violet and 20% methanol. Five fields of cells on the lower membrane were photographed and counted to estimate cell density. Image analysis software (Image-Pro Plus 6.0; Media Cybernetics) was used to measure the stained area.

Male NOD/SCID mice (aged 5–6 weeks) were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. To detect the effect of SHP-1 on the tumorigenicity of HCC cells, 2 × 10⁶ Huh-7 cells infected with AdSHP-1 or control virus were subcutaneously injected into the flanks of BALB/c nude mice. Tumor formation was estimated as previously described (24).

To detect the therapeutic effect of SHP-1 in vivo, Huh-7 cells were labeled with luciferase gene by lentivirus infection. Huh-7 cells stably expressing luciferase were injected subcutaneously into the flanks of NOD/SCID mice to generate tumor xenografts. The tumor nodules from the subcutaneous xenograft model were cut into 1 mm³ pieces and implanted into the left lobe of the livers of NOD/SCID mice (male, 5-week-old) to mimic primary HCC. AdSHP-1 or AdGFP was then injected via the tail vein twice each week for 3 weeks. The mice were monitored using an IVIS 200 imaging system once each week and killed 4 weeks after the transplantation of tumor fragments.

Ptpn6^f/f mice were obtained from The Jackson Laboratory. The Alb-Cre strain is described elsewhere (25, 26). To induce HCC, male Ptpn6^HKO mice and Ptpn6^f/f littermates were i.p. injected with DEN (25 mg/kg, Sigma-Aldrich) on postnatal day 15 (11). Liver tissues were collected 2, 4, 6, 8, 10, and 11 months after birth. No difference in the liver weight or liver weight to body weight ratio between the Ptpn6^HKO mice and Ptpn6^f/f mice was observed. To investigate the antitumor effect of SHP-1 in vivo, AdSHP-1 or AdGFP was injected via the tail veins of 10-month-old DEN-treated Ptpn6^HKO mice twice each week for 3 weeks. The livers were collected 1 week after the final injection of virus. Tumor nodules in the livers and lungs were counted and histopathologically analyzed using H&E staining.

Mice were housed in a temperature- and light-controlled (12-hour light/dark cycle) specific pathogen-free animal facility. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Second Military Medical University.

Primary mouse hepatocytes were isolated from adult male mice by using a modified version of a two-step collagenase perfusion protocol, as previously described (13). In brief, the flushed livers were perfused with DMEM plus collagenase IV (1 mg/mL; Sigma) following D-Hank's Balanced Salt Solution including EDTA (0.5 mmol/L). After perfusion, the digested hepatocytes were dispersed in DMEM and filtered through 80 and 200 mesh sieves to remove the undigested debris. The filtrates were centrifuged at 300 rpm for 5 minutes at 4°C. The hepatocytes in the precipitate were washed with DMEM 3 times and harvested for subsequent analysis.

To isolate the primary mouse HSC and Kupffer cells, the flushed livers were perfused with DMEM-free containing collagenase IV (1 mg/mL) and pronase (2 mg/mL; Roche) following D-Hank's Balanced Salt Solution including EDTA (0.5 mmol/L). The digested hepatic cells were dispersed in DMEM. DNA enzymes were added to prevent filamentous gelatinous material, and the undigested debris was removed through a filter. The filtrates were centrifuged at 300 rpm in a centrifuge tube for 5 minutes at 4°C. To isolate primary HSCs, the supernatant was collected following gradient centrifugation with 25% Nycodenz (Sigma). To isolate primary Kupffer cells, the supernatant was collected following gradient centrifugation with double Percoll gradient (20% and 50%, Sangon; ref. 10).

Statistical analyses were performed using SPSS software (18.0 version), and P < 0.05 was considered statistically significant. The Student t test was used to analyze the data of experiments involving two groups. The Wilcoxon signed-rank test was used for comparison of the expression levels of SHP-1 in human HCC tissues and their adjacent noncancerous tissues. The Mann–Whitney U test was used for comparison of tumor weight and volume of mice. The χ² test was used to compare two sample rates. The survival curves were assessed using the Kaplan–Meier method, and statistical differences between two groups were evaluated using a log-rank test.

To assess the clinical significance of SHP-1 expression, real-time PCR was performed to determine the expression of SHP-1 mRNA in human HCC tissues (T) and their noncancerous tissues (NT) from 84 patients. The expression of SHP-1 was decreased in HCC tissues (Fig. 1A). Moreover, significant downregulation of SHP-1 (T/NT ≤ 0.5) was observed in 45.23% (38/84) of the HCC tissues compared with their paired noncancerous tissues (Fig. 1B). Interestingly, the expression levels of SHP-1 inversely correlated with the diameter of tumors (r = −0.4130, P = 0.0002; Fig. 1C) and the serum α-fetoprotein (AFP) levels in patients (P = 0.047; Fig. 1D). Moreover, we also found that lower levels of SHP-1 expression were associated with a more aggressive HCC phenotype, characterized by larger tumor size (P = 0.010), younger age of onset (P = 0.038), and more advanced tumor stage (P = 0.019; Supplementary Table S3).

IHC was performed to detect SHP-1 protein levels in an HCC tissue microarray prepared from 271 other patients. Consistently, decreased SHP-1 expression levels were detected in HCC tissues compared with the paired surrounding noncancerous tissues (Supplementary Fig. S1A). The Kaplan–Meier analysis revealed that patients with the low SHP-1 expression levels experienced shorter OS compared with those with the high SHP-1 expression levels (median OS, 18.93 and 37 months, respectively; difference >18 months, P = 0.002; Fig. 1E). Moreover, the correlation of SHP-1 expression level and patient survival was analyzed using the data of GSE14520 from GEO database, in which most of the patients (96.31%) had a history of HBV infection like Chinese patients in our cohort (22). The results showed that the patients with low SHP-1 levels experienced shorter OS compared with the patients with high SHP-1 levels in HBV-associated HCCs (P = 0.0287; Supplementary Fig. S1B).

We next analyzed the potential mechanism of the downregulation of SHP-1 in patient HCCs. Only a few genetic alterations of PTPN6 gene were detected in 865 patients with HCC using cBioPortal (http://www.cbioportal.org; refs. 20, 21), including 3 amplifications, 4 missense mutations, and 1 truncating mutation. The previous studies suggested that the DNA methylation of PTPN6 locus affected the expression of SHP-1 in leukemia cells, colon cancer, and endometrial carcinoma cells (27–29). Calvisi and colleagues reported the hypermethylation of SHP-1 promoter in patient HCC tissues (30). In this study, we observed the correlation between DNA hypermethylation of PTPN6 locus and the reduction of SHP-1 expression in 310 HCC samples from TCGA database (r = −0.5006, P < 0.0001; Fig. 1F). In addition, treatment of DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine significantly increased the mRNA level of SHP-1 in HCC cells (Supplementary Fig. S1C). These data suggested that DNA methylation of PTPN6 locus could be involved in the decreased SHP-1 expression in HCC.

SHP-1 is a tumor suppressor in hematopoietic cancers (31, 32). However, the role of SHP-1 in hepatocarcinogenesis and HCC progression awaits further studies. To evaluate the effect of SHP-1 on the malignant phenotype of HCC cells, SHP-1 expression was upregulated in Huh7 and PLC cells using a recombinant adenovirus expressing SHP-1 (AdSHP-1; Fig. 2A). The CCK8 assay indicated that SHP-1 overexpression inhibited the proliferation of Huh7 and PLC cells (Fig. 2B). In contrast, downregulation of SHP-1 using siSHP-1 promoted the growth of HCC cells (Fig. 2C and D).

We next performed transwell assays to evaluate the metastatic potential of HCC cells. The results show that overexpression of SHP-1 suppressed the migration and invasion of both Huh7 and PLC cells (Fig. 2E and F), whereas siSHP-1 treatment exacerbated their metastatic potential (Fig. 2G and H).

We next assessed the effect of SHP-1 on the tumorigenicity of HCC cells in vivo. Huh7 cells infected with AdSHP-1 or the control adenovirus (AdGFP) were injected subcutaneously into the flanks of NOD/SCID mice. In the AdGFP group, xenografts were detected in 37.5% (3/8) of mice as early as day 19 after inoculation, and all mice developed tumor nodules by day 25. In contrast, xenografts were not observed until day 25 in the AdSHP-1 group, and only small nodules were detected in 62.5% (5/8) of the mice by day 34 (P < 0.001; Fig. 3A).

Moreover, xenografts of the AdSHP-1 group were significantly smaller compared with those of the control group at each time (P < 0.01; Fig. 3B). Similarly, xenograft weight was significantly reduced in the AdSHP-1 group (P < 0.001; Fig. 3C). IHC showed that AdSHP-1 treatment induced SHP-1 overexpression, which was accompanied by a significant decrease of Ki67 expression (Supplementary Fig. S2A).

We next utilized an orthotopic model of HCC to investigate the antitumor effect of SHP-1 in vivo. Luciferase-expressing Huh7 cells were injected into nude mice to establish subcutaneous tumors. Subsequently, the tumors were removed and implanted into NOD/SCID mouse liver to establish an orthotopic model of HCC. The luciferase activity of the xenografts in mice between AdGFP and AdSHP-1 group was not significantly different before adenovirus delivery (Fig. 3D). The mice were next injected with AdSHP-1 or the control virus (AdGFP) through the tail vein. After 3 weeks, AdSHP-1–injected mice emitted significantly reduced bioluminescence compared with mice injected with the control virus (Fig. 3D). Tumors from AdSHP-1–treated mice were significantly smaller compared with those of the AdGFP group in which 2 mice died because of an excess tumor burden (Fig. 3E; Supplementary Fig. S2B). Real-time PCR and IHC revealed that SHP-1 expression was significantly elevated in AdSHP-1–treated tumors, accompanied by repression of Ki67 compared with the AdGFP control (Fig. 3F and G).

To further investigate the effect of SHP-1 on hepatocarcinogenesis, hepatocyte-specific Ptpn6 knockout mice (Ptpn6^HKO) were established by crossing Ptpn6^f/f mice with Alb-Cre mice (26). SHP-1 expression was significantly reduced in the liver tissues of Ptpn6^HKO mice. Western blotting analysis indicated the deletion of SHP-1 in the hepatocytes without affecting SHP-1 expression in hepatic stellate and Kupffer cells in Ptpn6^HKO mice (Supplementary Fig. S3A–S3D).

Ptpn6^f/f and Ptpn6^HKO mice were injected with a single dose of DEN on postnatal day 15. We regularly sacrificed one cohort of mice after DEN injection every 2 months to monitor the development of HCC. We found that the incidence of liver tumors was higher in Ptpn6^HKO mice compared with that of Ptpn6^f/f mice at all times (Fig. 4A and B; Table 1). On 11 months after DEN treatment, liver tumors were detected in 100% (8/8) of the Ptpn6^HKO mice, whereas macroscopic and microscopic observations revealed that only 37.5% (3/8) and 50% (4/8) of the control Ptpn6^f/f mice developed liver tumors, respectively (Table 1). Moreover, the numbers and sizes of liver tumors in Ptpn6^HKO mice were significantly increased compared with those of controls (Fig. 4C). Notably, microscopic examination indicated that 62.5% (5/8) of Ptpn6^HKO mice developed lung metastases, whereas only 1 mouse in the control group had a lung metastasis (Fig. 4D–F). IHC validated the depletion of SHP-1 from the hepatocytes of Ptpn6^HKO mice (Fig. 4G), and Ki67 staining indicated the active proliferation of tumor cells in Ptpn6^HKO mice (Fig. 4G). The DEN-induced tumors in Ptpn6^HKO mice were diagnosed as HCC by experimental pathologists in our hospital, which displayed typical HCC features, including enlargement of hepatocytic plates, absence of portal tracts, and focal expression of AFP and osteopontin (Supplementary Fig. S4).

We next evaluated the therapeutic effects of SHP-1 on liver tumors in 40-week-old DEN-treated Ptpn6^HKO mice via systematic delivery of AdSHP-1 (Fig. 5A). SHP-1 expression was significantly increased in the livers of Ptpn6^HKO mice treated with AdSHP-1 compared with those of the AdGFP-treated group (Fig. 5B). IHC verified the restoration of SHP-1 expression in hepatocytes (Fig. 5C). As expected, restoration of SHP-1 expression in the mice significantly reduced the numbers and sizes of liver tumors in Ptpn6^HKO mice. In particularly, liver tumors were not detected in 2 mice treated with AdSHP-1 (Fig. 5D and E). Moreover, the numbers of lung metastases in AdSHP-1–treated mice were significantly fewer compared with those of their counterparts (Fig. 5F–H). Together, these results demonstrate that SHP-1 acts as a tumor suppressor to prevent the initiation and progression of HCC in mice.

SHP-1 modulates the cellular signals that involve in PI3K/AKT, JAK/STAT, MAPKs, and NF-κB (33–36). The activities of these signaling pathways are closely associated with the hepatocarcinogenesis and progression of HCC (37–41). Previous studies have indicated that SHP-1 affects the progression of HCC through targeting STAT3 phosphorylation (34). Here, we further investigated the effect of SHP-1 on these signaling pathways during the development and progression of HCC. As shown in Fig. 6A, depletion of SHP-1 led to the activation of JAK/STAT3, NF-κB, and PI3K/AKT signaling in the primary hepatocytes from 2-month-old DEN-treated Ptpn6^HKO mice. Moreover, the phosphorylation of STAT3, p65, and AKT was also markedly increased in the liver tissues and the tumor tissues of Ptpn6^HKO mice compared with that of Ptpn6^f/f mice (Fig. 6B and C). Nevertheless, the level of p-p38 and p-ERK did not significantly increase in hepatocytes and the livers of Ptpn6^HKO mice (Supplementary Fig. S5A–S5C). The JAK/STAT3, PI3K/AKT, and NF-κB signaling pathways are closely related to inflammation of the liver (19, 42). Therefore, we examined the expression levels of proinflammatory factors. The mRNA levels of IL6, TGFβ1, and TNFα were significantly increased in the hepatocytes of 2-month-old DEN-treated Ptpn6^HKO mice (Fig. 6D). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were also elevated in DEN-treated Ptpn6^HKO mice (Supplementary Fig. S5D). These data suggest that the inhibitory effect of SHP-1 on hepatocarcinogenesis may be achieved by inhibiting liver inflammation.

Consistently, the activation of STAT3, p65, and AKT as well as the expression of IL6, TGFβ1, and TNFα were significantly reduced by overexpression of SHP-1 in HCC cells (Fig. 6E and F). In contrast, enhanced SHP-1 expression did not affect the phosphorylation of p38 and ERK (Supplementary Fig. S5E). Western blotting analysis also showed that phosphorylation of STAT3 and AKT was decreased in orthotopic xenograft from the mice treated with AdSHP-1 (Fig. 6G). Taken together, these results implied that SHP-1 might suppress hepatocarcinogenesis and the malignant phenotype of HCC via inhibiting the activation of STAT3, NF-κB, and AKT signaling pathway.

SHP-1 has been demonstrated as a tumor suppressor in hematopoietic cancers (32). However, its potential function in epithelium-derived tumors is contradictory, and the effect of SHP-1 in oncogenesis is poorly understood. It has been reported that the expression of SHP-1 is increased in clear-cell renal carcinoma cells, but decreased in ER-negative breast cancer and prostate cancer tissues (43, 44). Calvisi and colleagues showed the protein levels of SHP-1 are decreased in HCC versus normal tissues (30). Here, we found that SHP-1 mRNA and protein levels were downregulated in HCC tissues. The lower levels of SHP-1 expression in HCCs were associated with more aggressive pathologic features, implying that SHP-1 reduction could be involved in the progression of HCC. Moreover, protein levels of SHP-1 in 271 HCCs from Chinese patients significantly correlated with the OS of patients. We also observed the correlation of SHP-1 expression and patient survival in a cohort from GEO database, in which most of patients (96.31%) had a history of HBV infection. Therefore, we proposed that SHP-1 might serve as a prognostic biomarker in patients with HBV-associated HCC. The prognostic value of SHP-1 in patients with other etiologies of HCC is worthy of further investigation.

SHP-1 plays a crucial role in glucose homeostasis and lipid metabolism in the liver (25, 26, 45). Certain target drugs such as sorafenib, dovitinib, and SC-2001 induce apoptosis and autophagy and inhibit the growth of HCC cells through enhancing the activity of SHP-1 tyrosine phosphatase (14, 15, 17, 46). In the present study, we further demonstrate that SHP-1 reversed the malignant properties of HCC in vitro and in vivo. Using a mouse model with a hepatocyte-specific deletion of Ptpn6, we show that SHP-1 plays a crucial role in the development and metastasis of HCC. Moreover, the upregulation of SHP-1 markedly abrogated the progression of HCC in mice. These findings suggest that SHP-1 may be a potential target for HCC therapy.

SHP-1 and SHP-2 are cytoplasmic PTPs that share similar signature sequences, comprising two Src homology 2 (SH2) NH₂-terminal domains and a C-terminal protein-tyrosine phosphatase domain (14, 23). Both of SHP-1 and SHP-2 govern a host of cellular functions with similar or parallel signal pathways (10). Previous studies reported that SHP-2 suppresses tumorigenesis, but promotes the progression of HCC, suggesting that SHP-2 plays bidirectional roles in HCC (47). However, our present data demonstrate that SHP-1 acted as a suppressor in initiation and progression of HCC in mice. The different roles of SHP-1 and SHP-2 in HCC may be attributed to their distinct effects on downstream signaling pathways. SHP-2 suppresses the initiation of HCC by dephosphorylating p-STAT3, which inhibits signaling through the JAK/STAT pathway, but promotes the progression of HCC by coordinately activating the Ras/Raf/Erk and PI3K/Akt/mTOR signaling pathways (11, 12). Here, we showed that SHP-1 suppressed the oncogenesis and progression of HCC by inhibiting the activation of the JAK/STAT, NF-κB, and PI3K/AKT signaling pathways, but not that of the MAPK signaling pathway.

Liver inflammation is a primary oncogenic factor associated with HCC (38, 41, 48). The inflammatory cytokines induced by liver injury stimulate the activation of inflammatory signaling pathways such as the JAK/STAT and NF-κB pathways and, in turn, increase the expression of IL6, TGFβ, and TNFα (40, 41, 49, 50). Our previous study demonstrated that SHP-1 acts as a downstream effector of HNF1α to inhibit liver inflammation during hepatic fibrogenesis (23). Here, we found that the levels of IL6, TGFβ1, and TNFα were significantly increased in the hepatocytes of DEN-treated Ptpn6^HKO mice. Moreover, the serum levels of ALT and AST were elevated in these mice, suggesting that depletion of SHP-1 from hepatocytes enhanced liver inflammation. Moreover, overexpression of SHP-1 inhibited the activation of STAT3 and p65 as well as the expression of IL6, TGFβ1, and TNFα in HCC cells. Therefore, we propose that SHP-1 suppressed hepatocarcinogenesis and HCC progression at least partly through impeding hepatic inflammation.

In conclusion, the present work is the first to report the prognostic value of SHP-1 for patients with HBV-associated HCC and demonstrates that SHP-1 suppressed tumorigenesis and the progression of HCC. These data further broaden our understanding of the biological function of SHP-1, which may serve as a novel target for therapy of HCC.

No potential conflicts of interest were disclosed.

Conception and design: X. Zhang, W.-F. Xie

Development of methodology: L.-Z. Wen, K. Ding, Z.-R. Wang, S.-J. Lei, J.-P. Liu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.-Z. Wen, K. Ding, Z.-R. Wang, S.-J. Lei, J.-P. Liu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.-Z. Wen, K. Ding, Z.-R. Wang, J. Ding, X. Zhang

Writing, review, and/or revision of the manuscript: L.-Z. Wen, C. Yin, P.-F. Hu, J. Ding, X. Zhang, W.-F. Xie

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.-H. Ding, C. Yin, P.-F. Hu, W.-S. Chen

Study supervision: W.-F. Xie

This work was supported by the National Natural Science Foundation of China (81230011 and 81530019 to W.-F. Xie; 81572377 and 81772523 to X. Zhang; and 81300305 to P.-F. Hu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.