Src homology region 2 (SH2) domain–containing phosphatase 1 (SHP-1, also known as PTPN6) is a nonreceptor protein tyrosine phosphatase that acts as a negative regulator of inflammation. Emerging evidence indicates that SHP-1 plays a role in inhibiting the progression of hepatocellular carcinoma (HCC). However, the role of SHP-1 in hepatocarcinogenesis remains unknown. Here, we find that levels of SHP-1 are significantly downregulated in human HCC tissues compared with those in noncancerous tissues (P < 0.001) and inversely correlate with tumor diameters (r = −0.4130, P = 0.0002) and serum α-fetoprotein levels (P = 0.047). Reduced SHP-1 expression was associated with shorter overall survival of patients with HCC with HBV infection. Overexpression of SHP-1 suppressed proliferation, migration, invasion, and tumorigenicity of HCC cells, whereas knockdown of SHP-1 enhanced the malignant phenotype. Moreover, knockout of Ptpn6 in hepatocytes (Ptpn6HKO) enhanced hepatocarcinogenesis induced by diethylnitrosamine (DEN) as well as metastasis of primary liver cancer in mice. Furthermore, systemic delivery of SHP-1 by an adenovirus expression vector exerted a therapeutic effect in an orthotopic model of HCC in NOD/SCID mice and DEN-induced primary liver cancers in Ptpn6HKO mice. In addition, SHP-1 inhibited the activation of JAK/STAT, NF-κB, and AKT signaling pathways, but not the MAPK pathway in primary hepatocytes from DEN-treated mice and human HCC cells. Together, our data implicate SHP-1 as a tumor suppressor of hepatocarcinogenesis and HCC progression and propose it as a novel prognostic biomarker and therapeutic target of HCC.

Significance: The nonreceptor protein tyrosine phosphatase SHP-1 acts as a tumor suppressor in hepatocellular carcinoma. Cancer Res; 78(16); 4680–91. ©2018 AACR.

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 STAT3Tyr705 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 (Ptpn6HKO), 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.

Human tissues and microarray analysis

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).

Real-time PCR

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.

Western blotting analysis

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.

Immunohistochemical staining

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).

Cell culture

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.

Virus and siRNA

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.).

Assays of cell proliferation, in vitro migration, and in vitro invasion

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.

Animal models

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.

Ptpn6f/f mice were obtained from The Jackson Laboratory. The Alb-Cre strain is described elsewhere (25, 26). To induce HCC, male Ptpn6HKO mice and Ptpn6f/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 Ptpn6HKO mice and Ptpn6f/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 Ptpn6HKO 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.

Isolation of primary hepatocytes, HSCs, and Kupffer cells

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

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.

Reduced SHP-1 expression in HCC predicts aggressive tumor behavior and poor prognosis of patients

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).

Figure 1.

Reduced SHP-1 expression is associated with aggressive clinicopathologic features and poor prognosis of human HCC. A, The mRNA levels of SHP-1 in 84 HCC tissues (T) and their adjacent noncancerous tissues (NT) were detected using real-time PCR. The expression of SHP-1 in HCC tissues was markedly lower compared with that of noncancerous tissues (***, P < 0.001, Wilcoxon signed-rank test). B, Downregulation SHP-1 was detected in 45.23% (38/84) of primary HCC tissues. Data are presented as the log2 ratio of the SHP-1 mRNA levels in HCC tissues compared with their paired surrounding noncancerous tissues. Downregulation was defined as log2 (T/NT) ≤ 1. C, The negative correlation between mRNA levels of SHP-1 and tumor diameter of HCCs (r = −0.4130, P = 0.0002, n = 78). D, Reduced SHP-1 mRNA expression was more frequent in HCC samples from patients (n = 48) with high AFP serum levels (>20 ng/mL) compared with those (n = 36) with low AFP serum levels (AFP ≤ 20 ng/mL). E, Kaplan–Meier analysis of the OS of 271 patients with HCC. The median level of SHP-1 of the 271 HCC samples was chosen as the cutoff. The OS rates of 271 patients with HCC were compared between the low- and high-SHP-1 groups (P = 0.002, log-rank test). F, The expression levels of SHP-1 were negatively correlated with the methylation status of PTPN6 locus (r = −0.5006, P < 0.0001, n = 310).

Figure 1.

Reduced SHP-1 expression is associated with aggressive clinicopathologic features and poor prognosis of human HCC. A, The mRNA levels of SHP-1 in 84 HCC tissues (T) and their adjacent noncancerous tissues (NT) were detected using real-time PCR. The expression of SHP-1 in HCC tissues was markedly lower compared with that of noncancerous tissues (***, P < 0.001, Wilcoxon signed-rank test). B, Downregulation SHP-1 was detected in 45.23% (38/84) of primary HCC tissues. Data are presented as the log2 ratio of the SHP-1 mRNA levels in HCC tissues compared with their paired surrounding noncancerous tissues. Downregulation was defined as log2 (T/NT) ≤ 1. C, The negative correlation between mRNA levels of SHP-1 and tumor diameter of HCCs (r = −0.4130, P = 0.0002, n = 78). D, Reduced SHP-1 mRNA expression was more frequent in HCC samples from patients (n = 48) with high AFP serum levels (>20 ng/mL) compared with those (n = 36) with low AFP serum levels (AFP ≤ 20 ng/mL). E, Kaplan–Meier analysis of the OS of 271 patients with HCC. The median level of SHP-1 of the 271 HCC samples was chosen as the cutoff. The OS rates of 271 patients with HCC were compared between the low- and high-SHP-1 groups (P = 0.002, log-rank test). F, The expression levels of SHP-1 were negatively correlated with the methylation status of PTPN6 locus (r = −0.5006, P < 0.0001, n = 310).

Close modal

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 inhibits the malignant phenotype of HCC cells in vitro

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).

Figure 2.

SHP-1 suppresses the malignant phenotypes of HCC cells in vitro. A, Western blotting analysis of SHP-1 expression in HCC cells infected with AdSHP-1 or the control virus. B, Enforced expression of SHP-1 suppressed the proliferation of HCC cells. C, Expression levels of SHP-1 in Huh-7 and PLC cells transfected with siSHP-1 or siNC. D, Knockdown of SHP-1 promoted the proliferation of HCC cells. E and F, Overexpression of SHP-1 inhibited the migration (E) and invasion (F) of HCC cells. G and H, Knockdown of SHP-1 enhanced the migration (G) and invasion (H) of HCC cells. Data represent the mean ± SD of triplicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

SHP-1 suppresses the malignant phenotypes of HCC cells in vitro. A, Western blotting analysis of SHP-1 expression in HCC cells infected with AdSHP-1 or the control virus. B, Enforced expression of SHP-1 suppressed the proliferation of HCC cells. C, Expression levels of SHP-1 in Huh-7 and PLC cells transfected with siSHP-1 or siNC. D, Knockdown of SHP-1 promoted the proliferation of HCC cells. E and F, Overexpression of SHP-1 inhibited the migration (E) and invasion (F) of HCC cells. G and H, Knockdown of SHP-1 enhanced the migration (G) and invasion (H) of HCC cells. Data represent the mean ± SD of triplicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

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).

SHP-1 suppresses the tumorigenicity and growth of HCC cells in vivo

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).

Figure 3.

SHP-1 represses the tumorigenicity of Huh-7 cells and growth of orthotopic HCC. A, HCC-free survival of NOD/SCID mice transplanted with Huh-7 cells infected with AdGFP or AdSHP-1 was analyzed using the Kaplan–Meier method. B, Growth curves of tumors in mice injected with Huh-7 cells infected with AdGFP or AdSHP-1 (n = 8 for each group). C, Images (top) and weights (bottom) of tumor nodules from subcutaneous mouse xenograft model. D, Images (left) and statistical analysis (right) of luciferase activity of NOD/SCID mice transplanted with Huh7 cells stably expressing luciferase. E, Images (top) and weights (bottom) of tumors from the model mice. F, SHP-1 mRNA levels of the tumor nodules. G, Immunohistochemical analysis of SHP-1 and Ki67 expression in tumors. Scale bars, 100 μm. **, P < 0.01; ***, P < 0.001.

Figure 3.

SHP-1 represses the tumorigenicity of Huh-7 cells and growth of orthotopic HCC. A, HCC-free survival of NOD/SCID mice transplanted with Huh-7 cells infected with AdGFP or AdSHP-1 was analyzed using the Kaplan–Meier method. B, Growth curves of tumors in mice injected with Huh-7 cells infected with AdGFP or AdSHP-1 (n = 8 for each group). C, Images (top) and weights (bottom) of tumor nodules from subcutaneous mouse xenograft model. D, Images (left) and statistical analysis (right) of luciferase activity of NOD/SCID mice transplanted with Huh7 cells stably expressing luciferase. E, Images (top) and weights (bottom) of tumors from the model mice. F, SHP-1 mRNA levels of the tumor nodules. G, Immunohistochemical analysis of SHP-1 and Ki67 expression in tumors. Scale bars, 100 μm. **, P < 0.01; ***, P < 0.001.

Close modal

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).

SHP-1 acts as a tumor suppressor of HCC in mice

To further investigate the effect of SHP-1 on hepatocarcinogenesis, hepatocyte-specific Ptpn6 knockout mice (Ptpn6HKO) were established by crossing Ptpn6f/f mice with Alb-Cre mice (26). SHP-1 expression was significantly reduced in the liver tissues of Ptpn6HKO 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 Ptpn6HKO mice (Supplementary Fig. S3A–S3D).

Ptpn6f/f and Ptpn6HKO 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 Ptpn6HKO mice compared with that of Ptpn6f/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 Ptpn6HKO mice, whereas macroscopic and microscopic observations revealed that only 37.5% (3/8) and 50% (4/8) of the control Ptpn6f/f mice developed liver tumors, respectively (Table 1). Moreover, the numbers and sizes of liver tumors in Ptpn6HKO mice were significantly increased compared with those of controls (Fig. 4C). Notably, microscopic examination indicated that 62.5% (5/8) of Ptpn6HKO 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 Ptpn6HKO mice (Fig. 4G), and Ki67 staining indicated the active proliferation of tumor cells in Ptpn6HKO mice (Fig. 4G). The DEN-induced tumors in Ptpn6HKO 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).

Figure 4.

Ptpn6 ablation enhances DEN-induced hepatocarcinogenesis in mice. A, Representative images of livers from 11-month-old DEN-treated Ptpn6f/f and Ptpn6HKO mice. B, Representative images of H&E staining of liver tissues from Ptpn6f/f and Ptpn6HKO mice treated with DEN. C, Tumor numbers (left) and tumor sizes (right) in the livers of 11-month-old mice. Horizontal lines indicate the median values. *, P < 0.05. D and E, Lung metastasis in the mice treated with DEN. D, Representative images of the lungs from 11-month-old Ptpn6f/f and Ptpn6HKO mice treated with DEN. E, H&E staining of lung tissues. F, Lung metastasis tumor numbers in DEN-treated mice (n = 8 for each group). G, Immunohistochemical analysis of SHP-1 and Ki67 expression in Ptpn6f/fandPtpn6HKO mice. Scale bars, 100 μm. *, P < 0.05.

Figure 4.

Ptpn6 ablation enhances DEN-induced hepatocarcinogenesis in mice. A, Representative images of livers from 11-month-old DEN-treated Ptpn6f/f and Ptpn6HKO mice. B, Representative images of H&E staining of liver tissues from Ptpn6f/f and Ptpn6HKO mice treated with DEN. C, Tumor numbers (left) and tumor sizes (right) in the livers of 11-month-old mice. Horizontal lines indicate the median values. *, P < 0.05. D and E, Lung metastasis in the mice treated with DEN. D, Representative images of the lungs from 11-month-old Ptpn6f/f and Ptpn6HKO mice treated with DEN. E, H&E staining of lung tissues. F, Lung metastasis tumor numbers in DEN-treated mice (n = 8 for each group). G, Immunohistochemical analysis of SHP-1 and Ki67 expression in Ptpn6f/fandPtpn6HKO mice. Scale bars, 100 μm. *, P < 0.05.

Close modal
Table 1.

Incidence of DEN-induced HCC in mice

AgePtpn6f/f (n = 26)Ptpn6HKO (n = 30)
MonthsMacroscopicMicroscopicMacroscopicMicroscopic
2 0 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 
4 0 (0/3) 0 (0/3) 0 (0/3) 0 (0/3) 
6 0 (0/2) 0 (0/2) 0 (0/3) 33% (1/3) 
8 0 (0/3) 33% (1/3) 0 (0/5) 80% (4/5) 
10 20% (1/5) 40% (2/5) 67% (4/6) 83% (5/6) 
11 37.5% (3/8) 50% (4/8) 100% (8/8) 100% (8/8) 
AgePtpn6f/f (n = 26)Ptpn6HKO (n = 30)
MonthsMacroscopicMicroscopicMacroscopicMicroscopic
2 0 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 
4 0 (0/3) 0 (0/3) 0 (0/3) 0 (0/3) 
6 0 (0/2) 0 (0/2) 0 (0/3) 33% (1/3) 
8 0 (0/3) 33% (1/3) 0 (0/5) 80% (4/5) 
10 20% (1/5) 40% (2/5) 67% (4/6) 83% (5/6) 
11 37.5% (3/8) 50% (4/8) 100% (8/8) 100% (8/8) 

We next evaluated the therapeutic effects of SHP-1 on liver tumors in 40-week-old DEN-treated Ptpn6HKO mice via systematic delivery of AdSHP-1 (Fig. 5A). SHP-1 expression was significantly increased in the livers of Ptpn6HKO 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 Ptpn6HKO 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.

Figure 5.

The therapeutic effect of SHP-1 on DEN-induced primary liver cancers in Ptpn6HKO mice. A, Schematic representation of adenovirus delivery to DEN-treated Ptpn6HKO mice. B, Western blotting analysis of the expression of SHP-1 in the livers of mice treated with AdGFP and AdSHP-1. C, IHC analysis of SHP-1 expression in hepatocytes of the mice treated with AdSHP-1. D, Representative images of the mouse livers from Ptpn6HKO mice injected with AdGFP and AdSHP-1. E, Tumor numbers (left) and tumor sizes (right) in DEN-treated Ptpn6HKO mice injected with AdGFP and AdSHP-1. F and G, Representative images of the lung metastasis (F) and H&E staining of lung tissues (G). Scale bars, 100 μm. H, Lung metastasis tumor numbers in AdGFP- and AdSHP-1–treated Ptpn6HKO mice (n = 7 for each group). *, P < 0.05.

Figure 5.

The therapeutic effect of SHP-1 on DEN-induced primary liver cancers in Ptpn6HKO mice. A, Schematic representation of adenovirus delivery to DEN-treated Ptpn6HKO mice. B, Western blotting analysis of the expression of SHP-1 in the livers of mice treated with AdGFP and AdSHP-1. C, IHC analysis of SHP-1 expression in hepatocytes of the mice treated with AdSHP-1. D, Representative images of the mouse livers from Ptpn6HKO mice injected with AdGFP and AdSHP-1. E, Tumor numbers (left) and tumor sizes (right) in DEN-treated Ptpn6HKO mice injected with AdGFP and AdSHP-1. F and G, Representative images of the lung metastasis (F) and H&E staining of lung tissues (G). Scale bars, 100 μm. H, Lung metastasis tumor numbers in AdGFP- and AdSHP-1–treated Ptpn6HKO mice (n = 7 for each group). *, P < 0.05.

Close modal

SHP-1 inhibits the activation of STAT3, NF-κB, and AKT signaling pathways in HCC

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 Ptpn6HKO mice. Moreover, the phosphorylation of STAT3, p65, and AKT was also markedly increased in the liver tissues and the tumor tissues of Ptpn6HKO mice compared with that of Ptpn6f/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 Ptpn6HKO 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 Ptpn6HKO mice (Fig. 6D). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were also elevated in DEN-treated Ptpn6HKO mice (Supplementary Fig. S5D). These data suggest that the inhibitory effect of SHP-1 on hepatocarcinogenesis may be achieved by inhibiting liver inflammation.

Figure 6.

SHP-1 inhibits the activation of STAT3, NF-κB, and AKT signaling pathways during hepatocarcinogenesis and HCC progression. A, Western blotting analysis of SHP-1 and the phosphorylation of STAT3, p65, and AKT in hepatocytes isolated from 2-month-old DEN-treated mice. B, The phosphorylation status of STAT3, p65, and AKT in livers of 8-month-old mice exposed to DEN. C,Ptpn6 ablation increased the phosphorylation of STAT3, p65, and AKT in tumor nodules of 11-month-old Ptpn6HKO mice. D, mRNA levels of IL6, TGFβ1, and TNFα in primary hepatocytes from 2-month-old DEN-treated mice (n = 3 for each group). E, SHP-1 overexpression decreased the phosphorylation of STAT3, p65, and AKT in Huh-7 cells. F, RT-PCR showed reduced expression of IL6, TGFβ1, and TNFα in Huh-7 cells infected with AdSHP-1. G, Western blotting analysis of p-STAT3, STAT3, p-AKT, AKT, and SHP-1 expression in orthotopic HCC model mice systemically injected with AdGFP or AdSHP-1. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

SHP-1 inhibits the activation of STAT3, NF-κB, and AKT signaling pathways during hepatocarcinogenesis and HCC progression. A, Western blotting analysis of SHP-1 and the phosphorylation of STAT3, p65, and AKT in hepatocytes isolated from 2-month-old DEN-treated mice. B, The phosphorylation status of STAT3, p65, and AKT in livers of 8-month-old mice exposed to DEN. C,Ptpn6 ablation increased the phosphorylation of STAT3, p65, and AKT in tumor nodules of 11-month-old Ptpn6HKO mice. D, mRNA levels of IL6, TGFβ1, and TNFα in primary hepatocytes from 2-month-old DEN-treated mice (n = 3 for each group). E, SHP-1 overexpression decreased the phosphorylation of STAT3, p65, and AKT in Huh-7 cells. F, RT-PCR showed reduced expression of IL6, TGFβ1, and TNFα in Huh-7 cells infected with AdSHP-1. G, Western blotting analysis of p-STAT3, STAT3, p-AKT, AKT, and SHP-1 expression in orthotopic HCC model mice systemically injected with AdGFP or AdSHP-1. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

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 Ptpn6HKO 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.

1.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics, 2017
.
CA Cancer J Clin
2017
;
67
:
7
30
.
2.
Hunter
T
. 
Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signaling
.
Cell
1995
;
80
:
225
36
.
3.
He
RJ
,
Yu
ZH
,
Zhang
RY
,
Zhang
ZY
. 
Protein tyrosine phosphatases as potential therapeutic targets
.
Acta Pharmacol Sin
2014
;
35
:
1227
46
.
4.
Scott
LM
,
Lawrence
HR
,
Sebti
SM
,
Lawrence
NJ
,
Wu
J
. 
Targeting protein tyrosine phosphatases for anticancer drug discovery
.
Curr Pharm Des
2010
;
16
:
1843
62
.
5.
Murakami
M
,
Kobayashi
S
,
Marubashi
S
,
Tomimaru
Y
,
Noda
T
,
Wada
H
, et al
Tyrosine kinase inhibitor PTK/ZK enhances the antitumor effects of interferon-alpha/5-fluorouracil therapy for hepatocellular carcinoma cells
.
Ann Surg Oncol
2011
;
18
:
589
96
.
6.
Wang
ZC
,
Gao
Q
,
Shi
JY
,
Guo
WJ
,
Yang
LX
,
Liu
XY
, et al
Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor-induced epithelial-mesenchymal transition
.
Hepatology
2015
;
62
:
1201
14
.
7.
Frankson
R
,
Yu
ZH
,
Bai
Y
,
Li
Q
,
Zhang
RY
,
Zhang
ZY
. 
Therapeutic targeting of oncogenic tyrosine phosphatases
.
Cancer Res
2017
;
77
:
5701
5
.
8.
Drake
JM
,
Lee
JK
,
Witte
ON
. 
Clinical targeting of mutated and wild-type protein tyrosine kinases in cancer
.
Mol Cell Biol
2014
;
34
:
1722
32
.
9.
Meeusen
B
,
Janssens
V
. 
Tumor suppressive protein phosphatases in human cancer: Emerging targets for therapeutic intervention and tumor stratification
.
Int J Biochem Cell Biol
2018
;96:98–134.
10.
Chong
ZZ
,
Maiese
K
. 
The Src homology 2 domain tyrosine phosphatases SHP-1 and SHP-2: diversified control of cell growth, inflammation, and injury
.
Histol Histopathol
2007
;
22
:
1251
67
.
11.
Bard-Chapeau
EA
,
Li
S
,
Ding
J
,
Zhang
SS
,
Zhu
HH
,
Princen
F
, et al
Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis
.
Cancer Cell
2011
;
19
:
629
39
.
12.
Han
T
,
Xiang
DM
,
Sun
W
,
Liu
N
,
Sun
HL
,
Wen
W
, et al
PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients
.
J Hepatol
2015
;
63
:
651
60
.
13.
An
H
,
Hou
J
,
Zhou
J
,
Zhao
W
,
Xu
H
,
Zheng
Y
, et al
Phosphatase SHP-1 promotes TLR- and RIG-I-activated production of type I interferon by inhibiting the kinase IRAK1
.
Nat Immunol
2008
;
9
:
542
50
.
14.
Tai
WT
,
Shiau
CW
,
Chen
PJ
,
Chu
PY
,
Huang
HP
,
Liu
CY
, et al
Discovery of novel Src homology region 2 domain-containing phosphatase 1 agonists from sorafenib for the treatment of hepatocellular carcinoma
.
Hepatology
2014
;
59
:
190
201
.
15.
Chen
KF
,
Tai
WT
,
Hsu
CY
,
Huang
JW
,
Liu
CY
,
Chen
PJ
, et al
Blockade of STAT3 activation by sorafenib derivatives through enhancing SHP-1 phosphatase activity
.
Eur J Med Chem
2012
;
55
:
220
7
.
16.
Su
JC
,
Tseng
PH
,
Wu
SH
,
Hsu
CY
,
Tai
WT
,
Li
YS
, et al
SC-2001 overcomes STAT3-mediated sorafenib resistance through RFX-1/SHP-1 activation in hepatocellular carcinoma
.
Neoplasia
2014
;
16
:
595
605
.
17.
Chen
KF
,
Su
JC
,
Liu
CY
,
Huang
JW
,
Chen
KC
,
Chen
WL
, et al
A novel obatoclax derivative, SC-2001, induces apoptosis in hepatocellular carcinoma cells through SHP-1-dependent STAT3 inactivation
.
Cancer Lett
2012
;
321
:
27
35
.
18.
Fan
LC
,
Shiau
CW
,
Tai
WT
,
Hung
MH
,
Chu
PY
,
Hsieh
FS
, et al
SHP-1 is a negative regulator of epithelial-mesenchymal transition in hepatocellular carcinoma
.
Oncogene
2015
;
34
:
5252
63
.
19.
Ning
BF
,
Ding
J
,
Liu
J
,
Yin
C
,
Xu
WP
,
Cong
WM
, et al
Hepatocyte nuclear factor 4alpha-nuclear factor-kappaB feedback circuit modulates liver cancer progression
.
Hepatology
2014
;
60
:
1607
19
.
20.
Gao
J
,
Aksoy
BA
,
Dogrusoz
U
,
Dresdner
G
,
Gross
B
,
Sumer
SO
, et al
Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal
.
Sci Signal
2013
;
6
:
pl1
.
21.
Cerami
E
,
Gao
J
,
Dogrusoz
U
,
Gross
BE
,
Sumer
SO
,
Aksoy
BA
, et al
The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data
.
Cancer Discov
2012
;
2
:
401
4
.
22.
Roessler
S
,
Long
EL
,
Budhu
A
,
Chen
Y
,
Zhao
X
,
Ji
J
, et al
Integrative genomic identification of genes on 8p associated with hepatocellular carcinoma progression and patient survival
.
Gastroenterology
2012
;
142
:
957
66
e12
.
23.
Qian
H
,
Deng
X
,
Huang
ZW
,
Wei
J
,
Ding
CH
,
Feng
RX
, et al
An HNF1alpha-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells
.
Cell Res
2015
;
25
:
930
45
.
24.
Yin
C
,
Wang
PQ
,
Xu
WP
,
Yang
Y
,
Zhang
Q
,
Ning
BF
, et al
Hepatocyte nuclear factor-4alpha reverses malignancy of hepatocellular carcinoma through regulating miR-134 in the DLK1-DIO3 region
.
Hepatology
2013
;
58
:
1964
76
.
25.
Xu
E
,
Charbonneau
A
,
Rolland
Y
,
Bellmann
K
,
Pao
L
,
Siminovitch
KA
, et al
Hepatocyte-specific Ptpn6 deletion protects from obesity-linked hepatic insulin resistance
.
Diabetes
2012
;
61
:
1949
58
.
26.
Xu
E
,
Forest
MP
,
Schwab
M
,
Avramoglu
RK
,
St-Amand
E
,
Caron
AZ
, et al
Hepatocyte-specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet-induced obesity: potential role of PPARgamma
.
Hepatology
2014
;
59
:
1803
15
.
27.
Witzig
TE
,
Hu
G
,
Offer
SM
,
Wellik
LE
,
Han
JJ
,
Stenson
MJ
, et al
Epigenetic mechanisms of protein tyrosine phosphatase 6 suppression in diffuse large B-cell lymphoma: implications for epigenetic therapy
.
Leukemia
2014
;
28
:
147
54
.
28.
Xu
SB
,
Liu
XH
,
Li
BH
,
Zhang
Y
,
Yuan
J
,
Yuan
Q
, et al
DNA methylation regulates constitutive expression of Stat6 regulatory genes SOCS-1 and SHP-1 in colon cancer cells
.
J Cancer Res Clin Oncol
2009
;
135
:
1791
8
.
29.
Sheng
Y
,
Wang
H
,
Liu
D
,
Zhang
C
,
Deng
Y
,
Yang
F
, et al
Methylation of tumor suppressor gene CDH13 and SHP1 promoters and their epigenetic regulation by the UHRF1/PRMT5 complex in endometrial carcinoma
.
Gynecol Oncol
2016
;
140
:
145
51
.
30.
Calvisi
DF
,
Ladu
S
,
Gorden
A
,
Farina
M
,
Conner
EA
,
Lee
JS
, et al
Ubiquitous activation of Ras and Jak/Stat pathways in human HCC
.
Gastroenterology
2006
;
130
:
1117
28
.
31.
Li
Y
,
Yang
L
,
Pan
Y
,
Yang
J
,
Shang
Y
,
Luo
J
. 
Methylation and decreased expression of SHP-1 are related to disease progression in chronic myelogenous leukemia
.
Oncol Rep
2014
;
31
:
2438
46
.
32.
Esposito
N
,
Colavita
I
,
Quintarelli
C
,
Sica
AR
,
Peluso
AL
,
Luciano
L
, et al
SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia
.
Blood
2011
;
118
:
3634
44
.
33.
Sharma
Y
,
Ahmad
A
,
Bashir
S
,
Elahi
A
,
Khan
F
. 
Implication of protein tyrosine phosphatase SHP-1 in cancer-related signaling pathways
.
Future Oncol
2016
;
12
:
1287
98
.
34.
Huang
TT
,
Su
JC
,
Liu
CY
,
Shiau
CW
,
Chen
KF
. 
Alteration of SHP-1/p-STAT3 signaling: a potential target for anticancer therapy
.
Int J Mol Sci
2017
;
18
.
35.
Wang
P
,
Xue
Y
,
Han
Y
,
Lin
L
,
Wu
C
,
Xu
S
, et al
The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation
.
Science
2014
;
344
:
310
3
.
36.
Khan
TH
,
Srivastava
N
,
Srivastava
A
,
Sareen
A
,
Mathur
RK
,
Chande
AG
, et al
SHP-1 plays a crucial role in CD40 signaling reciprocity
.
J Immunol
2014
;
193
:
3644
53
.
37.
Schneider
AT
,
Gautheron
J
,
Feoktistova
M
,
Roderburg
C
,
Loosen
SH
,
Roy
S
, et al
RIPK1 suppresses a TRAF2-dependent pathway to liver cancer
.
Cancer Cell
2017
;
31
:
94
109
.
38.
Zhang
J
,
Li
Z
,
Liu
L
,
Wang
Q
,
Li
S
,
Chen
D
, et al
Long noncoding RNA TSLNC8 is a tumor suppressor that inactivates the interleukin-6/STAT3 signaling pathway
.
Hepatology
2018
;
67
:
171
87
.
39.
Zhang
Y
,
Liu
Y
,
Duan
J
,
Yan
H
,
Zhang
J
,
Zhang
H
, et al
Hippocalcin-like 1 suppresses hepatocellular carcinoma progression by promoting p21(Waf/Cip1) stabilization by activating the ERK1/2-MAPK pathway
.
Hepatology
2016
;
63
:
880
97
.
40.
Yuan
JH
,
Yang
F
,
Wang
F
,
Ma
JZ
,
Guo
YJ
,
Tao
QF
, et al
A long noncoding RNA activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular carcinoma
.
Cancer Cell
2014
;
25
:
666
81
.
41.
He
G
,
Karin
M
. 
NF-kappaB and STAT3 - key players in liver inflammation and cancer
.
Cell Res
2011
;
21
:
159
68
.
42.
Wang
Q
,
Yu
WN
,
Chen
X
,
Peng
XD
,
Jeon
SM
,
Birnbaum
MJ
, et al
Spontaneous hepatocellular carcinoma after the combined deletion of Akt isoforms
.
Cancer Cell
2016
;
29
:
523
35
.
43.
Tao
T
,
Yang
X
,
Zheng
J
,
Feng
D
,
Qin
Q
,
Shi
X
, et al
PDZK1 inhibits the development and progression of renal cell carcinoma by suppression of SHP-1 phosphorylation
.
Oncogene
2017
;
36
:
6119
31
.
44.
Lopez-Ruiz
P
,
Rodriguez-Ubreva
J
,
Cariaga
AE
,
Cortes
MA
,
Colas
B
. 
SHP-1 in cell-cycle regulation
.
Anticancer Agents Med Chem
2011
;
11
:
89
98
.
45.
Dubois
MJ
,
Bergeron
S
,
Kim
HJ
,
Dombrowski
L
,
Perreault
M
,
Fournes
B
, et al
The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis
.
Nat Med
2006
;
12
:
549
56
.
46.
Su
JC
,
Tseng
PH
,
Hsu
CY
,
Tai
WT
,
Huang
JW
,
Ko
CH
, et al
RFX1-dependent activation of SHP-1 induces autophagy by a novel obatoclax derivative in hepatocellular carcinoma cells
.
Oncotarget
2014
;
5
:
4909
19
.
47.
Luo
X
,
Liao
R
,
Hanley
KL
,
Zhu
HH
,
Malo
KN
,
Hernandez
C
, et al
Dual Shp2 and Pten deficiencies promote non-alcoholic steatohepatitis and genesis of liver tumor-initiating cells
.
Cell Rep
2016
;
17
:
2979
93
.
48.
Duran
A
,
Hernandez
ED
,
Reina-Campos
M
,
Castilla
EA
,
Subramaniam
S
,
Raghunandan
S
, et al
p62/SQSTM1 by binding to vitamin D receptor inhibits hepatic stellate cell activity, fibrosis, and liver cancer
.
Cancer Cell
2016
;
30
:
595
609
.
49.
Park
EJ
,
Lee
JH
,
Yu
GY
,
He
G
,
Ali
SR
,
Holzer
RG
, et al
Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression
.
Cell
2010
;
140
:
197
208
.
50.
Li
N
,
Zhou
ZS
,
Shen
Y
,
Xu
J
,
Miao
HH
,
Xiong
Y
, et al
Inhibition of the sterol regulatory element-binding protein pathway suppresses hepatocellular carcinoma by repressing inflammation in mice
.
Hepatology
2017
;
65
:
1936
47
.