Protein phosphatase magnesium-dependent 1δ is a novel tumor marker and target in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a lethal liver malignancy worldwide. In this study, we reported that protein phosphatase magnesium-dependent 1δ (PPM1D) was highly expressed in the majority of HCC cases (approximately 59%) and significantly associated with high serum α-fetoprotein (AFP) level (P= 0.044). Kaplan-Meier and Cox regression data indicated that PPM1D overexpression was an independent predictor of HCC-specific overall survival (HR, 2.799; 95% CI, 1.346–5.818, P = 0.006). Overexpressing PPM1D promoted cell viability and invasion, whereas RNA interference-mediated knockdown of PPM1D inhibited proliferation, invasion, and migration of cultured HCC cells. In addition, PPM1D suppression by small interfering RNA decreased the tumorigenicity of HCC cells in vivo. Overall, results suggest that PPM1D is a potential prognostic marker and therapeutic target for HCC.

Keywords

PPM1D / hepatocellular carcinoma / prognosis / target therapy

Introduction

Hepatocellular carcinoma (HCC) is the sixth most common liver malignancy and the second leading cause of cancer-related deaths worldwide [ 1]. Despite the implementation of various treatment strategies, HCC remains a lethal disease with a high recurrence rate and poor survival [ 2]. Surgical resection, liver transplantation, and tumor ablation are potential curative measures for patients with early-stage HCC. However, these treatments are unsuitable for patients in the advanced stages of the disease. These patients are offered alternative treatments, e.g., transarterial chemoembolization and Sorafenib, which could only moderately improve their survival [ 3].

Protein phosphatase magnesium-dependent 1d (PPM1D), also called wild-type p53-induced phosphatase 1, is a member of type 2C phosphatases [ 4]. Numerous studies demonstrate the amplification and overexpression of PPM1D in solid tumors, including mammary gland tumors [ 5, 6], ovarian clear cell carcinoma [ 7], pancreatic adenocarcinoma [ 8], neuroblastoma [ 9], and medulloblastoma [ 10]. High expression of PPM1D at the mRNA or protein level indicates poor survival outcomes in breast adenocarcinoma [ 11], ovarian clear cell carcinoma [ 7], neuroblastoma [ 9], medulloblastoma [ 12], pancreatic adenocarcinoma [ 8], colorectal cancer [ 13], gastric cancer [ 14], and non-small cell lung cancer [ 15]. PPM1D mRNA expression is also associated with tumor size, tumor stage, and overall survival time (OS) of HCC patients [ 16]. Despite these significant clinical results, the tumorigenic functions of PPM1D and its prognostic value in HCC remain largely unknown. In the present study, we examined the tumorigenic roles of PPM1D in HCC and evaluated the feasibility of targeting this protein.

Materials and methods

Patients, cell lines, and antibodies

This study included a retrospective cohort of 81 patients with HCC who underwent hepatectomy at the Department of Surgery, Nantong Tumor Hospital (Nantong, Jiangsu, China) between 1998 and 2002, with a median follow-up time of 41.5 months. The demographic features and clinicopathological data of the patients are summarized in Table 1. The median age of the patients was 48.9 years (21–75 years), and the median tumor size was 5.4 cm (0.7–17 cm). All patients were diagnosed with primary HCC, and none received radiotherapy or chemotherapy before the surgery. Resected tumor specimens were fixed in buffered paraformaldehyde. All procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation (Nantong Tumor Hospital, China) and with the Helsinki Declaration of 1975 revised in 2000. Informed consent was obtained from patients for their inclusion in this study. Human HCC cell lines, namely, HepG2, Huh7, MHCC97L, and Hep3B, were used and cultured as described previously [ 17, 18]. The following antibodies were used: rabbit polyclonal antibody against human PPM1D (H-300; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse monoclonal antibody against human b-actin (Sigma-Aldrich Co., St Louis, MO, USA), and HRP-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA).

Immunohistochemistry

HCC tissue microarrays were obtained from the Department of Pathology, Nantong Tumor Hospital. These microarrays composed of 81 pieces of HCC tissue and core biopsies in duplicate (1 mm in diameter), which were prepared from fixed, paraffin-embedded tumors. Immunohistochemistry was performed with rabbit polyclonal antibody against human PPM1D by using previously reported methods [ 19]. The stained tissue microarrays were blindly examined and scored by a pathologist (Huang WB) according to a semi-quantitative scoring method [ 19]. The degree of cytosolic and nuclear staining was scored, and the percentage of immunoreactivity in tumor cells was graded as follows: 0 (<5%), 1 (5%–24%), 2 (25%–49%), 3 (50–74%), and 4(≥75%).

Overexpression and suppression of PPM1D in HCC cell lines

For PPM1D overexpression, HepG2 and Huh7 cells were transiently transfected with full-length PPM1D cDNA (NM_003620.3)-containing plasmid (pcDNA3.1-PPM1D) or pcDNA3.1 empty vector (control) by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). For PPM1D suppression, PPM1D Stealth RNAi-targeting human PPM1D gene (siPPM1D-1: 5′-CCACCACAAGAUGCCAUCUCAAUGU-3′; siPPM1D-2: 5′-GGUGGGAGUGUAAUGAACAAGUCUG-3′) and non-targeting siRNA (Stealth RNAi™ siRNA Negative Control, siCon) (Invitrogen, Carlsbad, CA, USA) were transfected at 100 pmol into semi-confluent Hep3B or MHCC97L HCC cells. The cells were subjected to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay, wound healing assay, and Matrigel chamber assay 24 h post-transfection. The cells were then harvested 72 h post-transfection and subjected to biochemical analysis of PPM1D expression through quantitative PCR and immunoblotting. Subsequently, MTT assay, quantitative PCR, western blot, wound healing assay, and Matrigel chamber assay analyses were routinely performed [ 17]. Details on research methods are described in the Supplementary Methods.

Tumor xenograft mouse model

Male BALB/c athymic mice (nu/nu, 4–6 weeks of age) were housed under specific pathogen-free conditions in the Animal Laboratory of the Nanjing Key Gene Biotech. Co., Ltd. Institutional and national guidelines for the care and use of laboratory animals were also followed. For the tumor xenograft growth assay, 5×10⁶ MHCC97L-siCon or MHCC97L-siPPM1D-1 were injected subcutaneously into the right flank of the mouse (six mice per group), and signs of tumor formation were observed daily [ 20]. Tumor volume (V) was obtained using the formula V= length × width²/2.

Statistical analysis

SPSS statistical package 16.0 (SPSS, Chicago, IL, USA) was used for data analysis. PPM1D protein expression (relative fold ratio) and the number of invaded cells in the transfected and control cells were compared by using paired Student’s t-test. Cumulative tumor growth was calculated by measuring the AUC of the tumor growth curve for each mouse through the trapezoidal method. Independent Student’s t-test was used to assess the effects of PPM1D on the HCC cell lines and cumulative tumor growth. Clinicopathological features were compared between patients with high and low PPM1D expression through Pearson chi-square tests for categorical variables and Student’s t-test for continuous data. Kaplan-Meier plots and log-rank tests were used for survival analysis. OS was calculated from the date of surgery to death or the last follow-up. Cox regression was used in the univariate survival analysis to determine the association of individual clinicopathological variables with OS. All variables with P<0.05 in the univariate analysis, as well as age and gender, were subjected to multivariate Cox regression analysis to determine the hazard ratios (HRs) and the independence of effects. P <0.05 was considered statistically significant.

Results

Clinical significance of PPM1D in HCC

To determine the prevalence and clinical significance of PPM1D in HCC, we examined the protein expression of PPM1D by immunohistochemistry in a retrospective cohort of 81 resected tumor samples from patients with HCC. PPM1D immunoreactivity was graded according to low (score 0–2) and high (score 3–4) expression (Fig. 1A and Table 1). High PPM1D expression was significantly associated with increased serum AFP levels (>400 ng/ml, P<0.044) but not with other examined parameters. The prognostic value of PPM1D expression for patients with HCC was analyzed by Kaplan-Meier analysis. As shown in Fig. 1B, high PPM1D expression (grades 3–4) in HCC was significantly associated with short OS (log-rank= 6.643, P = 0.01). The mean OS for patients with high PPM1D expression was 66 months, and the median OS for those with low PPM1D expression was 32 months. Univariate Cox regression analysis further revealed that tumor size (P<0.001), tumor differentiation (P = 0.029), and tumor stage (AJCC stage III; P = 0.002) were significantly associated with OS (Table 2). Moreover, multivariate analysis results indicated that PPM1D expression was an independent prognostic marker for OS of HCC patients [HR, 2.799; 95% confidence interval (CI), 1.346–5.818, P = 0.006; Table 2].

Considering that high PPM1D expression was associated with poor OS in patients with HCC, we investigated the tumorigenic and metastatic potentials of PPM1D-overexpressing HCC cells. Western blot analysis showed that PPM1D was successfully overexpressed by transient transfection of PPM1D plasmid in HepG2 and Huh7 cells (Fig. 2A and Fig. S1). MTT assay was then employed to assess cell viability and proliferation in these transfectants. After transient transfection, HepG2-PPM1D cells exhibited faster growth rates than the empty vector control cells (HepG2-Vec) (Fig. 2B). Similarly, cell viability increased in Huh7 cells after PPM1D overexpression (Huh7-PPM1D) (Fig. 2B). To test the metastatic potential of PPM1D-transfected cells, we investigated cell invasion ability by using Matrigel chamber assay. More Huh7-PPM1D cells penetrated the Matrigel compared with the Huh7-Vec control cells after 36 h (Fig. 2C).

Targeting PPM1D alleviated tumor phenotypes of HCC cells

We investigated alterations in the tumor phenotypes of HCC cells after transfection with PPM1D siRNA. Both siRNAs targeting PPM1D reduced the endogenous mRNA and protein expression of PPM1D in Hep3B and MHCC97L cells (Fig. 3A). Cell viability was assessed by MTT assays 24 h after the transient transfection of siPPM1D. As shown in Fig. 3B, both HCC cell lines with suppressed PPM1D expression showed significantly lower cell viability rates than scrambled siRNA-transfected controls (Hep3B-siCon, MHCC97L-siCon). To determine the effects of suppressing PPM1D on the metastatic potential of HCC cells, we examined the cell motility and invasion ability of Hep3B-siPPM1D and MHCC97L-siPPM1D cells through wound healing assay (Fig. 3C) and Matrigel chamber assay (Fig. 3D). Compared with those of the scrambled siRNA-transfected controls, wound closure was slower in siPPM1D-transfected cells at 48 h and fewer cells penetrated the Matrigel at 36 h. In addition, tumorigenicity was significantly suppressed in nude mice injected with MHCC97L cells pretreated with siPPM1D-1 (Fig. 4). Furthermore, tumors were significantly larger in the control groups than those in the experimental group on day 23 after tumor cell inoculation. Hence, PPM1D was associated with tumorigenic properties.

Discussion

A growing body of evidence suggests that PPM1D is a potential tumor-related molecule and a promising prognostic marker in various solid tumors. In the present study, we demonstrated the tumorigenic potentials of PPM1D in HCC in vitro and in vivo. Our results showed that targeting PPM1D by using siRNA can inhibit the proliferation and invasion and reduce the tumorigenicity of HCC cells in a tumor xenograft mouse model. We also demonstrated the association between PPM1D and OS of patients with HCC; hence, PPM1D can be used as a prognostic marker in HCC.

PPM1D is a gene induced in p53-dependent manner in response to ionizing radiation in Burkitt lymphoma cells [ 4]. After exposure to radiation or other environmental stresses, PPM1D dephosphorylates stress-related signaling proteins, such as p53, p38 MAPK, and ataxia-telangiectasia mutated kinase; PPM1D is also involved in cell cycle arrest, apoptosis, and DNA repair [ 21, 22]. More than 10% of primary breast cancers exhibit PPM1D gene amplification at the DNA level [ 5, 6]. In vitro and in vivo experiments demonstrate that silencing PPM1D can inhibit the proliferation and invasion of colorectal cancer, lung cancer, bladder cancer, breast cancer, and human glioma cancer cells [ 23– 27].

PPM1D mRNA expression is significantly higher in HCC than that in the paired non-cancerous liver tissue. High PPM1D mRNA levels are associated with increased AFP level, large tumor size, advanced TNM stage, increased rate of tumor recurrence, family history of HCC, and short OS [ 16]. In the present study, we analyzed the PPM1D protein and confirmed the positive correlation between PPM1D and HCC-specific OS. However, we did not find any significant associations between PPM1D and the following features, except for serum AFP level: age, gender, tumor size, hepatitis B infection, tumor nodule number, tumor capsule, vascular invasion, tumor stage, and tumor differentiation. These results must be validated in further studies with large sample sizes.

Although the clinical significance of PPM1D was previously examined, the tumorigenic potentials of PPM1D in HCC have not been investigated. In this study, we used gain-of-function and loss-of-function experiments to explore the effects of PPM1D manipulation on HCC phenotypes. We provided solid evidence showing that overexpressing PPM1D can promote proliferation and invasion of HCC cells. Using two siRNAs to specifically silence PPM1D expression in Hep3B and MHCC97L cells, we demonstrated that the downregulation of PPM1D expression can inhibit the growth, migration, and invasion capabilities of HCC cells. These results were further confirmed in vivo, that is, the sizes of tumor xenografts derived from siPPM1D-pretreated MHCC97L cells markedly decreased.

P53 gene is a tumor suppressor that acts as a cellular gatekeeper for growth and division [ 28]. PPM1D amplification or overexpression can promote tumor development by abrogating the tumor-suppressive activity of p53 [ 6]. More than 50% of human cancers contain P53 mutations [ 28], but breast cancer with PPM1D amplification rarely harbors P53 mutations [ 29]. In the present study, the P53 status of the studied cohort remains unknown, which is one of the limitations of our research. The association between PPM1D and P53 in HCC tumorigenesis must be investigated in further studies.

In conclusion, the present study demonstrated that overexpression of PPM1D is associated with poor prognosis of patients with HCC. Results from in vitro and in vivo experiments indicated that PPM1D exerts tumorigenic functions during hepatocarcinogenesis. However, the underlying mechanisms of PPM1D in HCC remain unclear. Further studies on the molecular mechanisms and dysfunctional signaling pathways related to PPM1D are urgently needed to elucidate the role of PPM1D in HCC.

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