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

Author Manuscript Published OnlineFirst on May 18, 2018; DOI: 10.1158/0008-5472.CAN-17-3896 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

Liang-Zhi Wen1,5,#, Kai Ding1,#, Ze-Rui Wang1,#, Chen-Hong Ding1, Shu-Juan Lei1, Jin-Pei

Liu1, Chuan Yin1, Ping-Fang Hu1, Jin Ding2, Wan-Sheng Chen3, Xin Zhang1,3*, and Wei-Fen

Xie1,4*

1Department of Gastroenterology, Changzheng Hospital, Second Military Medical University,

Shanghai, 200003, China

2International Cooperation Laboratory on Signal Transduction of Eastern Hepatobiliary

Surgery Institute, Second Military Medical University, Shanghai, China

3Department of Pharmacy, Changzheng Hospital, Second Military Medical University, 415

Fengyang Road, Shanghai 200003, China

4Department of Gastroenterology, Shanghai East Hospital, Tongji University School of

Medicine, Shanghai, 200120, China

5Present address: Department of Gastroenterology, Institute of Surgery Research, Daping

Hospital, Third Military Medical University, Chongqing 400042, China

#These authors contributed equally.

Running title: SHP-1 suppresses hepatocarcinogenesis and HCC progression.

*Correspondence to:

Wei-Fen Xie, Department of Gastroenterology, Changzheng Hospital, Second Military

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 18, 2018; DOI: 10.1158/0008-5472.CAN-17-3896 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Medical University, 415 Fengyang Road, Shanghai 200003, China. Tel.: (86-21) 8188-5341;

Fax: (86-21) 8188-9624; E-mail: [email protected]

Xin Zhang, Department of Pharmacy, Changzheng Hospital, Second Military Medical

University, 415 Fengyang Road, Shanghai 200003, China. E-mail: [email protected]

Conflict of interest: The authors declare no conflicts of interest.

Abstract

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

alpha-fetoprotein(AFP) levels (P = 0.047). Reduced SHP-1 expression was associated with

shorter overall survival of HCC patients 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.

Introduction

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 (PTKs) and

protein tyrosine phosphatases (PTPs) (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). While the cancer-related PTK 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), 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 transforming growth factor-β1 (TGF-β1)-induce

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 HBV-associated HCC patients. 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.

Materials and Methods

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 (HE) 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, Shanghai, China). Tissue microarray blocks containing

271 HCCs along with case-matched noncancerous tissues were constructed using a tissue

microarrayer. Immunohistochemistry (IHC) of tissue microarray slides was performed using

an anti-SHP-1 antibody (CST, Boston, MA, USA). SHP-1 expression was assessed using a

four-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) (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 TCGA database LIHC project

(https://portal.gdc.cancer.gov/projects/TCGA-LIHC). 867 HCC tissues from 865 HCC

patients were used for analyzing the genetic alterations by cBioportal

(http://www.cbioportal.org) (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 HBV-related HCC patients 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 Table S1.

Western blotting analysis

Proteins were extracted using RIPA buffer (P0013B, Beyotime, Suzhou, China)

supplemented with protease inhibitor cocktail (Roche), separated using sodium dodecylsulfate

polyacrylamide gel electrophoresis (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 h before overnight incubation with a primary antibody at 4°C.

After a 2 h 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 nm or 800 nm. The primary antibodies are listed in

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 h at room temperature, incubated with

primary antibodies overnight at 4°C and incubated with secondary antibody at room

temperature for 30 min. The staining was developed using an EnVision Detection

Rabbit/Mouse Kit (GK500710, GeneTech, Shanghai, China).

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 Mycoalert detection kit

(Lonza) and authenticated by short tandem repeat analysis every 6 months. The cells were

cultured in Dulbecco’s Modified Eagle’s Medium containing 10% heat-inactivated fetal calf

serum.

Virus and siRNA

The recombinant adenoviruses AdSHP-1 and AdGFP were previously established in our

lab (23). Small interfering RNA for SHP-1 (5′-CGCAGUACAAGUUCAUCUAtt-3′) and the

negative controls (NC) siRNA (5′-UUCUCCGAACGUGUCACGUtt-3′) were purchased

from GenePharma (Shanghai GenePharma Co., Ltd, Shanghai, China).

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

HCC cells were infected or transfected for 8–12 h and then plated onto 96-well plates (3,000

cells per well). Cell proliferation was measured using Cell Counting Kit-8 (Dojinodo, Tokyo,

Japan) 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 8–12 h were seeded in serum-free medium in the upper chamber.

Medium supplemented with 10% fetal bovine serum was added to the lower chamber. After

incubation for 24–48 h at 37ºC, 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, 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 Jackson Laboratory. The Alb-Cre strain is described

elsewhere (25,26). To induce HCC, male Ptpn6HKO mice and Ptpn6f/f littermates were

intraperitoneally 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 one week after the final injection of virus. Tumor nodules in

the livers and lungs were counted and histopathologically analyzed using HE staining.

Mice were housed in a temperature- and light-controlled (12-h 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 mM). 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 min 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 mM). 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 min 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) (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.

Results

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 down-regulation 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 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) (Table S3).

Immunohistochemistry 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

(Fig. S1A). 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 months 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; Fig. S1B).

We next analyzed the potential mechanism of the down-regulation of SHP-1 in patient

HCCs. Only a few genetic alterations of PTPN6 gene was detected in 865 HCC patients using

cBioPortal (http://www.cbioportal.org) (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 et al 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-2A-deoxycytidine (5-Aza-CdR)

significantly increased the mRNA level of SHP-1 in HCC cells (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 up-regulated 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, down-regulation of SHP-1 using siSHP-1 promoted the

growth of HCC cells (Fig. 2C, 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, F), whereas siSHP-1 treatment exacerbated their metastatic

potential (Fig. 2G, 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 post 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 (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 two mice died because of an excess tumor burden (Fig. 3E and 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, 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 (Fig. S3A-D).

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 two 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, B; Table 1).On 11 months after

DEN treatment, liver tumors were detected in 100% (8/8) of the Ptpn6HKO mice, while

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 one 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 α-fetoprotein (AFP) and osteopontin (OPN) (Fig. S4).

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 two mice treated with AdSHP-1(Fig. 5D, 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 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 Figure 6A, depletion of SHP-1 led to the

activation of JAK/STAT3, NF-кB 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 6C). Nevertheless, the level of p-p38 and

p-ERK did not significantly increase in hepatocytes and the livers of Ptpn6HKO mice (Fig.

S5A-C). The JAK/STAT3, PI3K/AKT, and NF-кB signaling pathways are closely related to

inflammation of the liver (19,42). Therefore, we examined the expression levels of

pro-inflammatory factors. The mRNA levels of IL-6, TGFβ1, and TNFα were significantly

increased in the hepatocytes of 2-month-old DEN-treated Ptpn6HKO mice (Fig. 6D). ALT and

AST levels were also elevated in DEN-treated Ptpn6HKO mice (Figure 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 IL-6,

TGFβ1, and TNFα were significantly reduced by overexpression of SHP-1 in HCC cells (Fig.

6E and 6F). In contrast, enhanced SHP-1 expression did not affect the phosphorylation of p38

and ERK (Fig. S5E). Western blotting analysis also showed that phosphorylation of STAT3

and AKT was decreased in orthotopicxenograft 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-кB, and

AKT signaling pathway.

Discussion

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 et al showed the protein levels of SHP-1 are

decreased in HCC vs normal tissues (30). Here we found that SHP-1 mRNA and protein

levels were down-regulated in HCC tissues. The lower levels of SHP-1 expression in HCCs

was associated with more aggressive pathological 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 overall survival 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 HBV-associated HCC patients.

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 up-regulation 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 protein tyrosine phosphatases 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

PI3-K/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/STAT3 and NF-κB pathways, and in turn, increase the

expression of IL-6, 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 IL-6, 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 IL-6, 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.

Acknowledgments

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)

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Table 1. Incidence of DEN-induced HCC in mice.

Age Ptpn6f/f (n = 28) Ptpn6HKO (n = 30) Months Macroscopic Microscopic Macroscopic Microscopic 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)

Figure Legend

Fig. 1.Reduced SHP-1 expression is associated with aggressive clinicopathological

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) Down-regulation 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. Down-regulation 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 overall survival of 271 patients with HCC. The median level of

SHP-1 of the 271 HCC samples was chosen as the cut-off. The overall survival rates of 271

HCC patients 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).

Fig. 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.

(D)Knockdown of SHP-1 promoted the proliferation of HCC cells. (E-F) Overexpression of

SHP-1 inhibited the migration (E) and invasion (F) of HCC cells. (G-H) Knockdown of

SHP-1 enhanced the migration (G) and invasion (H) of HCC cells. Data represent the mean ±

standard deviation (SD) of triplicate experiments. *P < 0.05, **P <0.01, ***P <0.001.

Fig. 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.

Fig. 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 HE 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–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) HE 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.

Fig. 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-G) Representative

images of the lung metastasis (F) and HE 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.

Fig. 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 IL-6, 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 IL-6, 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.

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

Liang-Zhi Wen, Kai Ding, Ze-Rui Wang, et al.

Cancer Res Published OnlineFirst May 18, 2018.

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