Overexpression of Protein Phosphatase 4 Correlates with Poor Prognosis in Patients with Stage II Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with less than 5% 5-year survival rate (1). Surgical resection remains the only hope for long time survival in patients with PDAC. However, at the time of diagnosis, only 15% to 20% of patients with PDAC are surgically resectable. Even for the patients who underwent pancreatectomy, the disease commonly recurs and the prognosis is poor with the long-term survival rate of 10% to 20% (2). Therefore, identifying new markers and therapeutic targets are urgently needed.

Protein phosphatases are divided into 3 major groups based on their substrate specificity: serine/threonine phosphatases, tyrosine phosphatases, and dual-specificity phosphatases (3). Protein serine/threonine phosphatases have been further classified into 5 major groups: protein phosphatase type 1, 2A, 3, 5, and 7, based on their biochemical characteristics, sensitivity to specific inhibitors, ability to dephosphorylate specific substrates, and the requirement for divalent cations (4). Protein phosphatase 4 (PP4, also known as PPX) is a PP2A-like phosphatase, and shares 65% amino acid sequence identity with PP2A (5). Like PP2A, PP4 is a holoenzyme composed of catalytic subunit (PP4C), structural and regulatory subunits PP4R1 and PP4R2. PP2A has been shown to regulate cellular processes as diverse as metabolism, signal transduction, DNA replication, transcription, translation, cell cycle, development, and transformation (6). However, many of these functional studies of PP2A have relied upon the use of the phosphatase inhibitor okadaic acid. PP4 contains a putative binding domain for okadaic acid and okadaic acid inhibits PP4 with a similar range of concentration (IC₅₀ = 0.1 nmol/L in vitro) to PP2A (7, 8). Therefore, some of the previously identified functions assigned to PP2A may in fact be the functions of PP4, rather than PP2A.

Recent study showed that PP4C is overexpressed in human breast and lung cancer (9). Inhibition of PP4C expression sensitized breast and lung cancer cells to cisplatin treatment (9), suggesting the functions of PP4 overexpression in cancer progression. However, the expression and the prognostic significance of PP4 expression in patients with PDAC have not been studied. In this study, we examined PP4C protein expression by immunohistochemistry in 133 patients with stage II PDAC and their matched benign pancreatic tissue samples (N = 113). In addition, we measured PP4 mRNA expression levels by real-time reverse transcriptase (RT)-PCR and PP4 protein expression by Western blotting. We correlated PP4C expression with survival and other clinicopathologic parameters in patients with stage II PDAC. Our data suggest that PP4 plays an important role in the progression of PDAC. Targeting PP4 signaling pathway may represent a new approach for the treatment of PDAC.

Our study population consisted of 133 patients with stage II PDAC who underwent pancreatectomy and did not receive any form of neoadjuvant chemoradiation therapy at our institution from 1990 to 2010. One patient each with stage IA, IB, or III disease and 2 patients with stage IV disease were excluded from this study because the case number was too small to be representative for these disease stages. One hundred and thirteen patients (85.0%) underwent pancreaticoduodenectomy, 18 patients (13.5%) underwent distal pancreatectomy, and 2 patients (1.5%) received total pancreatectomy. There were 78 male and 55 female patients with age ranging from 24.9 to 84.8 years (median age: 64.6 years). Postoperatively, 25 patients (18.8%) received adjuvant chemotherapy alone, 73 patients (54.9%) received combined chemoradiation therapy, and 35 patients (26.3%) did not receive adjuvant therapy. The study was approved by the Institutional Review Board of the University of Texas MD Anderson Cancer Center (Houston, TX).

The tissue microarrays used in this study were constructed using formalin-fixed, paraffin-embedded archival tissue blocks from the pancreatectomy specimens from 133 patients with stage II PDAC. The matched hematoxylin and eosin (H&E)-stained slides were reviewed to identify the representative areas for tumor and benign pancreas. For each patient, 2 cores of tumor and 1 core of paired benign pancreatic tissue were sampled from representative areas using a 1.0-mm punch. The tissue microarrays were constructed using a tissue microarrayer (Beecher Instruments) as described previously (10).

Immunohistochemical staining for PP4C was carried out on 5-μm unstained sections from the tissue microarray blocks. To retrieve the antigenicity, the tissue sections were treated at 100°C in a steamer containing 10 mmol citrate buffer (pH 6.0) for 35 minutes. The sections were then immersed in 3% hydrogen peroxidase for 20 minutes to block the endogenous peroxidase activity and were incubated in 2.5% blocking serum for 30 minutes to reduce nonspecific binding. The sections were then incubated with a rabbit polyclonal antibody against PP4C (Novus Biologicals) at a 1:300 dilution at 4°C overnight, washed, and then incubated with secondary antibody at room temperature for 60 minutes. Standard avidin-biotin immunohistochemical analysis of the sections was conducted according to the manufacturer's recommendations (Vector Laboratories). The immunohistochemically stained slides for PP4C were reviewed independently by 2 pathologists (D. Chatterjee and Huamin Wang), who graded PP4C expression in both tumor and their paired benign pancreatic samples. Because the immunohistochemical staining for PP4C showed diffuse staining in PDAC samples that were positive for PP4C, PP4C expression was scored on the basis of the staining intensity as negative, weak, moderate, and strong. All cases with a discrepancy in the grading of PP4C expression between the pathologists were rereviewed together and the consensus results for PP4C expression were used. For the statistical analysis, we categorized our cases into 2 groups based on the staining intensity for PP4C: PP4C-high (moderate or strong staining for PP4C in tumor cells) and PP4-low (negative or weak staining for PP4C in tumor cells).

All tissue samples used for Western blottings were selected by a pathologist from pancreatectomy specimens based on frozen sections prepared from frozen tissue blocks. Antibodies against PP4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology Inc. Protein expression was analyzed by 10% SDS-PAGE, which was electroblotted onto polyvinylidene difluoride membranes (Novex), blocked in 5% skim milk in ×1 TBS, and probed with the primary antibodies as indicated in the figures. Proteins were detected using an enhanced chemiluminescence (ECL) kit (Amersham-Pharmacia Biotech).

Frozen optimum cutting temperature (OCT)-embedded human samples of primary PDAC and benign pancreatic tissue were microdissected and total RNA isolation was conducted as described previously (11). One microgram of total RNA was reverse transcribed using the avian myeloblastosis virus reverse transcriptase kit (Promega) according to the manufacturer's protocol. Briefly, total RNA was denatured for 5 minutes at 70°C and cooled for 5 minutes on ice, reverse transcriptase was added to a total volume of 20 μL, and reverse transcription was conducted at 42°C for 60 minutes. Quantitative RT-PCR (qRT-PCR) was carried out using a set of primers specific for PP4C (QuantiTect Primer Assays, Qiagen, Inc.), according to the manufacturer's instructions. To correct for quantitative differences between samples and possible PCR artifacts, we used primers specific for ribosomal protein S6 (RPS6; forward, 5′-AAGGAGAGAAGGATATTCCTGGAC-3′; reverse, 5′-AGAGAGATTGAAAAGTTTGCGGAT-3′) as an internal control for each sample. Amplification was carried out using a thermal cycler (Bio-Rad Laboratories, Inc.) for 40 cycles consisting of 20 seconds at 95°C (denaturation), 1 minute at 60°C (annealing), and 1 minute at 60°C (extension). Each qRT-PCR was carried out in triplicate, and the mean value was used to calculate the ratio of PP4 to RPS6, with a value of 1 used as the control. All assays were repeated 3 times.

Patient follow-up information through June 2011 was extracted from the prospectively maintained institutional pancreatic cancer database in the Department of Surgical Oncology at our institution. The clinical and follow-up information were verified in all cases by reviewing the medical record and if necessary, by review of the U.S. Social Security Index. For the patients who were alive or died from other causes during follow-up, the follow-up time ranged from 7.1 to 213.8 months with a median follow-up of 38.2 months. For the patients who died of PDAC during follow-up, the follow-up time ranged from 4.2 to 82.9 months with a median follow-up of 18.1 months. Disease-free survival (DFS) was calculated as the time from the date of surgery to the date of first recurrence in patients with recurrence or to the date of last follow-up in patients without recurrence. The overall survival (OS) was calculated as the time from the date of diagnosis to the date of death or the date of last follow-up if death did not occur. The Fisher exact tests were used to compare categorical data. DFS and OS curves were constructed using the Kaplan–Meier method, and the log-rank test was used to evaluate the statistical significance of differences. Univariate Cox regression analysis was used to examine the prognostic significance of PP4C and clinicopathologic parameters. Cox proportional hazards models were fitted for multivariate analysis. After interactions between variables were examined, a backward stepwise procedure was used to derive the best-fitting model. Statistical analysis was conducted using Statistical Package for Social Sciences software (for Windows 12.0, SPSS Inc.). We used a 2-sided significance level of 0.05 for all statistical analyses.

For the PDAC samples that were positive for PP4C, diffuse cytoplasmic and focal nuclear staining for PP4C was observed in the tumor cells (Fig. 1A and B). No expression of PP4C was detected in tumor stromal cells (Fig. 1A–D). Among the 133 PDAC cases, negative, weak, moderate, and strong staining for PP4C was detected in 15 (11.3%), 43 (32.3%), 44 (33.1%), and 31 (23.3%) PDAC samples, respectively.

The paired benign pancreatic tissue was available in 113 cases. Fifteen percent (17 of 113) of the paired benign pancreatic tissue showed moderate to strong staining for PP4C. The frequency of moderate to strong staining for PP4C was significantly lower in benign pancreatic tissue than 56.4% (75 of 133) in PDAC samples (Fig. 2A, P < 0.001). When we compared the PP4C expression level in a PDAC sample to its paired benign pancreatic tissue from the same patient, we found that 73.5% (83 of 113) of the PDAC samples showed higher expression level of PP4C than their paired benign pancreatic tissue by immunohistochemistry. To further confirm our immunohistochemical staining results, we measured PP4C protein expression levels in 9 frozen PDAC samples and their paired benign pancreatic tissue samples by Western blotting. Compared with the paired benign pancreatic tissue samples, increased PP4C protein expression was detected in 9 of 9 (100%) PDAC samples (Fig. 2B). In addition, we also examined PP4C mRNA expression in 15 PDAC and 15 benign pancreatic tissue samples. We found that PP4C mRNA expression level was significantly higher in PDAC samples than benign pancreatic tissue samples by qRT-PCR (Fig. 2C). These data show that PP4C was overexpressed in PDAC and suggest that PP4C overexpression in PDAC is due to the upregulation of PP4C mRNA expression.

Clinicopathologic correlations of PP4C expression in patients with stage II PDAC are summarized in Table 1. In patients whose tumor was PP4C-high, 62.6% (47 of 75) developed distant metastasis during follow-up, which was significantly higher than those patients whose tumor was PP4C-low (41.4%, 24 of 58; P = 0.02). However, we did not observe any significant association between PP4C overexpression and lymph node metastasis, locoregional recurrence and other clinicopathologic features (Table 1). The patients whose tumors were PP4C-high had shorter DFS and OS than those whose tumors were PP4C-low. The median DFS was 10.9 months [95% confidence interval (CI), 8.7–13.2 months] for patients whose tumor were PP4C-high compared with 18.4 months (95% CI, 13.4–23.4 months) for patients whose tumors were PP4C-low (P = 0.001, log-rank method, Fig. 3A). The median OS was 20.9 months (95% CI, 14.9–26.9 months) for patients whose tumors were PP4C-high, which was significantly shorter than 36.2 months (95% CI, 21.2–51.2 months) in patients whose tumors were PP4C-low (P = 0.004, log-rank method, Fig. 3B). In univariate analysis, both DFS and OS correlated significantly with tumor size, lymph node metastasis (stage), and PP4C overexpression. In addition, OS also showed significant correlation with resection margin status (P = 0.04, Table 2). No significant correlations of either DFS or OS with the other clinicopathologic parameters were found (P > 0.05). In multivariate analysis, high level of PP4C expression (PP4C-high) was associated with shorter DFS (P = 0.006) and OS (P = 0.02) independent of tumor size, resection margin status, and lymph node metastasis (stage; Table 3).

In this study, we examined the expression of PP4C in 133 patients with stage II PDAC and their matched benign pancreatic tissue samples. We found that PP4C was overexpressed in PDAC samples compared with benign pancreatic tissue. PP4C overexpression was associated with shorter DFS and OS and was an independent prognostic factor for patients with stage II PDAC. Our data show that PP4C may be used as a prognostic factor in patients with stage II PDAC who underwent pancreatectomy.

It has been shown that PP4 is involved in the regulation of microtubule growth or organization at the centrosomes (12) and the centrosome maturation in mitosis and meiosis (13). Nakada and colleagues reported that depletion of PP4C results in a prolonged checkpoint arrest in human cells, suggesting that PP4c plays a critical role in dephosphorylating γ-H2AX after DNA damage (14). Lee and colleagues showed that PP4 dephosphorylates replication protein A subunit 2 (RPA2) and plays an essential role in DNA damage response and repair via homologous recombination (15). Previous studies have also shown that PP4 interacts with the members of the Rel/NF-κB family of transcription factors c-Rel, RelA, and p50 and activates NF-κB–mediated transcription (16). PP4 dephosphorylates Thr435 of RelA, which is required for NF-κB activation induced by cisplatin and MEK/ERK (17). In addition, PP4 has been found to be a positive regulator of the c-jun-NH₂-kinase (JNK) pathway in TNF-α signaling (18) and regulates hematopoietic progenitor kinase 1 (HPK1) activity in a T-cell receptor (TCR)-dependent manner (19). PP4 interacts with insulin receptor substrate 4 (IRS4) and HDAC3, downregulates IRS4, and inhibits HDAC3 activity (20, 21). Recently, PP4 has been shown to be overexpressed in human breast and lung cancer and regulates the sensitivity of breast and lung cancer cells to cisplatin treatment (9). PP4 activates JNK-1 in prostate carcinoma cell lines, PC-3 and LNCaP, and increases the activities of c-Jun/activator protein (AP-1) and EGR-1 (22). In addition, PP4 has also been shown to regulate the survival of both leukemic T cells and untransformed human peripheral blood T cells and has been shown to play an important role in the development and progression of leukemia (23). These data suggest that PP4 may play an important role in human malignancies. However, the expression and the function of PP4 in the tumorigenesis and progression of human cancers remain unclear. In this study, we showed that that PP4C expression levels were significantly higher in PDAC samples than their paired benign pancreatic tissue samples. Consistent with our immunohistochemical staining results, we showed that both PP4C protein and mRNA expression levels were significantly higher in PDAC samples than those in benign pancreatic tissue by either Western blotting analysis or real-time qRT-PCR. Therefore our data showed that PP4C was overexpressed in human PDAC samples. Increased PP4 mRNA expression in PDAC samples may suggest that PP4C overexpression in PDAC may be due to upregulation of PP4C gene transcription. Our findings were consistent with the previous report that PP4C is overexpressed in human breast and lung cancers compared with benign breast or lung tissue respectively. PP4C protein has been shown to be expressed in 44 of 46 (95.7%) breast cancer samples compared with 37.8% (14 of 37) in benign breast tissue (P < 0.01). In the same study, Wang and colleagues showed that PP4C protein is expressed in 52 of 57 (91.2%) lung cancer samples, which is significantly higher than 67.7% (21 of 31) in benign lung tissue (P < 0.01; ref. 9). Because the expression of PP4C is significantly higher in PDAC samples than the paired benign pancreas tissue in our study, immunohistochemical staining for PP4C may be used as a marker for the diagnosis of invasive PDAC.

We showed that overexpression of PP4C correlated with distant metastasis in patients with stage II PDAC. We also showed for the first time that overexpression of PP4C was associated with both shorter DFS and shorter OS and was a prognostic factor for both DFS and OS independent of tumor size, resection margin status, and lymph node status (stage) in patients with stage II PDAC. Consistent with our findings is the recent study, which showed that PP2A protein levels in the mononuclear cells at the time of the diagnosis of chronic myelogenous leukemia (CML) can consistently predict patients who will progress to blast crisis. Significantly higher levels of PP2A protein expression were detected in patients who will later progress to blast crisis than those patients who do not progress (P < 0.0001; ref. 24). PP2A promotes the growth and survival of pancreatic cancer cells. Li and colleagues showed that inhibition of PP2A induces growth inhibition in pancreatic cancer cells through the activation of JNK pathway (25, 26). Inhibition of PP2A also induces apoptosis in pancreatic cancer cells through the activation of NF-κB by persistent inhibitor of IκB kinase (IKK)α phosphorylation (27). In the light of these reports and the fact that PP4 is involved in the activation of both JNK and NF-κB pathways (16–18), overexpression of PP4C in PDAC may play an important role in the progression of PDAC and in predicting the prognosis in patients with stage II PDAC. Inhibition of PP4 either alone or in combination with other treatment modalities may be more effective for pancreatic cancer treatment.

One limitation of this study is that only patients with stage II PDAC were included in this study. This was mainly due to the number of patients of other disease stages being too small to be represented in our patient population. Only 1 patient each with stage IA, IB, or III PDAC and 2 patients with stage IV disease were identified in our patient population. However, our findings are important in that PP4C overexpression may be used as a prognostic marker for patients with stage II PDAC because vast majority of the patients with surgically resectable PDAC have stage II disease and little is known about the prognostic markers in this group of patients.

In conclusion, our study showed that PP4C is overexpressed in PDAC. High level of PP4C expression in PDAC samples is associated with poor prognosis and is an independent prognostic marker for patients with stage II PDAC. Therefore, targeting PP4C-signaling pathways may represent a novel target for the treatment of pancreatic cancer.

No potential conflicts of interest were disclosed.

Conception and design: S. Weng, Hua Wang, Huamin Wang

Development of methodology: S. Weng, Hua Wang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Weng, Hua Wang, W. Chen, M.H. Katz, H.F. Gomez, J.L. Abbruzzese, J.B. Fleming, Huamin Wang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Weng, Hua Wang, W. Chen, D. Chatterjee, Huamin Wang

Writing, review, and/or revision of the manuscript: S. Weng, Hua Wang, M.H. Katz, J.E. Lee, P.W. Pisters, Huamin Wang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Hua Wang, H.F. Gomez, J.B. Fleming

Study supervision: Hua Wang

The study was supported by the NIH grant (1R21CA149544-01A1) and G.S. Hogan Gastrointestinal Cancer Research Fund at The University of Texas MD Anderson Cancer Center.

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.