PROX1 and β-catenin are prognostic markers in pancreatic ductal adenocarcinoma

The Wnt/β-catenin pathway has a key role in regulating cellular processes and its aberrant signaling can lead to cancer development. The role of β-catenin expression in pancreatic ductal adenocarcinoma is somewhat controversial. Transcription factor PROX1 is a target of Wnt/β-catenin signaling and it is involved in carcinogenesis through alterations in its expression. The actions can be either oncogenic or tumor suppressive depending on the tissue. The aim of this study was to investigate PROX1 and β-catenin expression in pancreatic ductal adenocarcinoma (PDAC). Expression of PROX1 and β-catenin were evaluated in 156 patients by immunohistochemistry of tissue microarrays. Associations between tumor marker expression and clinicopathological parameters were assessed by the Fischer’s exact-test or the linear-by-linear association test. The Kaplan-Meier method and log-rank test were used for survival analysis. Uni- and multivariate survival analyses were carried out by the Cox regression proportional hazard model. High PROX1 expression was seen in 74 (48 %) tumors, and high β-catenin expression in 100 (65 %). High β-catenin expression was associated with lower tumor grade (p = 0.025). High PROX1 and β-catenin expression associated significantly with lower risk of death from PDAC in multivariate analysis (HR = 0.63; 95 % CI 0.42–0.95, p = 0.026; and HR = 0.54; 95 % CI 0.35–0.82, p = 0.004; respectively). The combined high expression of PROX1 and β-catenin also predicted lower risk of death from PDAC (HR = 0.46; 95 % CI 0.28–0.76, p = 0.002). In conclusion, high PROX1 and β-catenin expression were independent factors for better prognosis in pancreatic ductal adenocarcinoma.


Background
The Wnt/β-catenin signaling pathway has a role in regulating cellular processes including organ development and differentiation, and tissue homeostasis in adults [1]. It is widely established that its aberrant signaling can lead to cancer development [2]. β-catenin is a key molecule in this pathway. It is an intracellular protein that is localized in cell membrane, cytoplasm and nucleus. The binding of Wnt ligand to its receptors inhibits β-catenin phosphorylation, which allows β-catenin to escape from degradation. It accumulates in the cytoplasm, and translocates to the nucleus. After localizing to the nucleus, β-catenin activates a target gene expression through interacting mainly with members of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors (as reviewed in [3,4]). In colorectal cancer (CRC), most tumors have a mutation in a key regulatory factor of the Wnt/β-catenin pathway. Often the mutation is in adenomatous polyposis coli (APC) or protein β-catenin encoding gene (CTNNB1), which results in activation of the pathway [3].
In pancreatic ductal adenocarcinoma (PDAC), the role of the Wnt/β-catenin signaling pathway is controversial because of the variable and sometimes paradoxical effects in the pancreas. PDAC is a genetically heterogenous cancer with several key mutated genes including KRAS2, CDKN2A/p16, SMAD4/DPC4, and TP53 [5]. Although genetic alterations of the Wnt signaling pathway are involved in PDAC tumors [6], mutations of APC or CTNNB1 are less common [7]. Heiser et al. showed in mice that by introducing a β-catenin stabilizing mutation in CTNNB1 leads to pancreatic hypoplasia at an early phase of the developing pancreas. If this mutation is introduced in later phase in the developing pancreas, it results in enlargement of the exocrine pancreas without tumor formation [8].
An immunohistochemically positive expression of βcatenin has been reported earlier, but the results have remained somewhat controversial. Lowy et al. noted reduced membranous expression of β-catenin in PDAC correlating with loss of tumor differentiation [9]. However, there is evidence that the Wnt/β-catenin signaling pathway is upregulated in PDAC both by immunohistochemistry and polymerase chain reaction [7,10]. So far, the prognostic significance of β-catenin expression in PDAC has been investigated in a few studies with rather short follow-up times [11][12][13][14].
The transcription factor PROX1 has been shown to be a downstream target of the Wnt/β-catenin/TCF pathway in colorectal tumor neoplastic transformation and progression [15]. PROX1 is a transcriptional regulator and a part of the homeobox transcription factor family [16]. It has a key role in the development of the central nervous system [17], lens [18], liver [19], pancreas [19], lymphatic system [20], and heart [21]. But in addition, it is involved in oncogenesis through alterations in its expression. Depending on the tissue it can act either as a tumor suppressor or as an oncogene [22].
Recently, Wiener et al. constituted that PROX1 functions as a stem cell regulator in intestinal adenomas and in CRC, but not in the normal intestine [23]. In highgrade gliomas, and in colorectal cancer, high PROX1 tissue expression is associated with poor patient survival [24]. In esophageal squamous cell carcinoma PROX1 mediates the anti-proliferative effect by IFN-γ [25]. In hematological malignancies and in breast cancer PROX1 expression has been shown to be decreased [26,27]. In hepatocellular carcinoma, depletion of PROX1 causes a significant increase in cell proliferation, and patients with high PROX1 expression have better prognosis compared to patients with low expression [28]. Schneider et al. showed that PROX1 is less expressed in pancreatic cancer cells than in the normal exocrine pancreas [29]. They also noticed that the gene expression level of PROX1 was lower in patients who survived less than 6 months than in patients with longer survival [29]. However, to our knowledge, immunohistochemical prognostic studies of PROX1 tissue expression are lacking in PDAC.
The aim of this study was to examine tumor expression and prognostic value of PROX1 and β-catenin in PDAC.

Patients
This study is based on a series of 189 consecutive PDAC patients surgically treated in 2000-2011 at the Department of Surgery, Helsinki University Hospital. Only patients with verified PDAC were included in this study. Median age at operation was 64 (range 39-84) years. Twenty-one patients, who received neoadjuvant chemotherapy, were excluded from the study. Eight patients were eventually diagnosed with stage IV disease with distant metastases according to the American Joint Committee on Cancer Pancreatic Cancer Staging System [30], and four patients lacked data on stage. They were excluded from the study. Altogether, 156 patients were included in the study. Patients' records, the Finnish Population Registry and Statistics Finland were used to obtain survival data and cause of death of the patients. A description of the study cohort is in Table 5.

Preparation of tumor tissue microarrays and immunohistochemistry
Formalin-fixed and paraffin-embedded surgical tissue samples were collected from the archives of the Department of Pathology, Helsinki University Hospital. Experienced pathologists (J.H. and S.N.) re-evaluated all samples for confirmation of the histopathological diagnosis of PDAC. Representative regions of tumor specimens were defined and tumor areas were marked on hematoxylinand eosin-stained tumor slides for preparation of tissue microarray blocks (TMA). Two 1.0-mm cores were taken from each tumor block with a semiautomatic tissue microarrayer (Tissue Arrayer 1, Beecher Instruments Inc., Silver Spring, MD, USA). In order to evaluate TMA representativeness compared to whole tissue blocks, we examined altogether six spots per patient taken from different areas/parts of the tumor.
TMA blocks were freshly cut into 4-μm sections. After deparaffinization in xylene and rehydration through a gradually decreasing concentration of ethanol to distilled water, slides were treated in a PreTreatment module (Lab Vision Corp., Fremont, CA, USA) in Tris-HCl (pH 8.5) and Tris-EDTA (pH 9) buffer for 20 min at 98°C for antigen retrieval. Staining of sections was performed in an Autostainer 480 (Lab Vision Corp., Fremont, CA, USA) by the Dako REAL EnVision Detection system, Peroxidase/DAB +, Rabbit/Mouse (Dako, Glostrup, Denmark) for β-catenin, and by ImmPRESS HRP Polymer Detection Kit, Peroxidase, Anti-Goat IgG (Vector Laboratories, Burlingame, CA, USA) for PROX1. Tissues were incubated with beta-Catenin Antibody (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA; diluted to 1:500 = 5 μg/ml) for one hour at room temperature, and with Anti-human Prox1 Antibody (R&D Systems, Inc., Minneapolis, MN, USA; diluted to 1:1500 = 15 μg/ml) for overnight at room temperature. Samples of colon tissue and normal lymph node served as positive controls in each staining series (see Additional files 1 and 2). We also chose 13 whole tumor tissue blocks and corresponding lymph node metastases from the patient cohort to compare PROX1 expression in the tumor and its lymph node metastases.

Evaluation of stainings
Cytoplasmic stainings of PROX1 and β-catenin were scored as negative (0), weakly positive (1), moderately positive (2), or strongly positive (3) according to staining intensity. Also, β-catenin membranous staining was evaluated. In the samples, where no membranous staining was seen, there was no cytoplasmic staining either. The highest score of each sample was considered representative for analysis. Scoring was performed by two independent investigators (K.S. and J.H.) without knowledge of clinical data and outcome. In case of differing scores, consensus score was discussed and determined.
Associations between tumor marker expression and clinicopathological parameters were assessed by the Fischer's exact-test or the linear-by-linear association test. The Kaplan-Meier method and log-rank test were used for survival analysis. The Bonferroni correction was used for multiple comparisons by dividing the probability level by the number of comparisons. The Spearman correlation coefficient with bootstrapped (1000 resamples, bias corrected) confidence intervals was calculated to find out correlations between PROX1 and β-catenin expression. Uni-and multivariate survival analyses were carried out by the Cox regression proportional hazard model adjusted for age, gender, stage, metastasized lymph node ratio (LNR) ≥/<20 % (cut-off ≥/<20 %), perivascular invasion, and postoperative adjuvant therapy. Since stage and LNR are internally correlated to each other, a combination variable was formed for multivariate analyses (see Table 6). Interaction terms were considered. The Cox model assumption of constant hazard ratios over time was tested. For each testable variable at a time, a time-dependent covariate was included separately. All variables fulfilled the assumption. Stage and lymph node ratio were combined into a single variable to simplify the model. A p-value <0.05 was considered significant and all tests were two-sided.

Results
Immunohistochemical staining PROX1 expression was cytoplasmic and evenly distributed with no distinctive membranous staining. Cytoplasmic staining was scored as described above. In normal pancreatic tissue apparent nuclear staining is present although all the nuclei are not stained. In two cancer tissue samples we saw staining of the nuclei, and the cytoplasmic staining scores in these samples were 1, and 3. In all the other cancer specimens nuclei were negative. In the whole tumor specimens, there was no nuclear staining in the metastases; only negative or weak cytoplasmic staining was present ( Fig. 1).
β-catenin expression was distributed in the cell membrane and within the cytoplasm. Only in a few exceptions the staining was not uniform throughout the cell. With more intense membranous staining, also cytoplasmic staining was stronger. The cytoplasmic expression pattern showed two different types of staining; homogenous and granular. There was no distinct nuclear staining. Only three samples lacked membranous staining (Fig. 2). The membranous and cytoplasmic staining were very difficult to score separately. Because of this, cytoplasmic expression was used in statistical analyses.

Association between PROX1 and β-catenin expression and clinicopathological variables
There was a statistically significant association between PROX1 expression and age; patients in the low PROX1 expression group were younger than in the high PROX1 expression group (p = 0.038). PROX1 expression did not correlate with gender, stage, LNR, histological grade, perineural, or perivascular invasion (Table 1).
Patients with low β-catenin expression showed a significant association with higher tumor histological grade compared to patients with high expression (p = 0.025). No significant association was found between β-catenin and age, gender, stage, LNR, perineural, or perivascular invasion (Table 2).
Combined expression of PROX1 and β-catenin showed significantly poorer CSS for PDAC patients with low compared to high expression (p = 0.013). Between patients with moderate and low expression (p = 0.092), or with moderate and high expression (p = 0.435) no significant difference in CSS was seen (Fig. 7). Five-year CSS for patients with low combined expression was 10.  univariate analyses were stage, lymph node positivity, perivascular invasion, and postoperative adjuvant therapy (Table 5).
In multivariate analyses adjusted for age, gender, stage, LNR, perivascular invasion, and adjuvant therapy high β-catenin expression remained statistically significant for Fischer exact-test was used for 2×2 tables and linear-by-linear association test for tables with more than two rows. Missing data excluded from the analyses  (Table 6).

Discussion
We here show that high tissue expression of PROX1 and β-catenin independently predict better prognosis in PDAC. PROX1 expression is vital for pancreatic development. Loss of PROX1 in the pancreas leads to remarkable size reduction [31], and premature acinar cell differentiation and increased ductal cell proliferation [32]. Schneider et al. reported in 2006 that pancreatic cancer cells express less PROX1 mRNA than normal exocrine pancreatic cells [29]. They noticed that PROX1 gene expression levels were lower in patients with survival less than 6 months. Our study shows a similar tendency by immunohistochemistry although the difference in survival was not significant. To our knowledge, no prognostic studies on PROX1 protein expression in PDAC have been reported so far.
Increased PROX1 expression has been discovered to be associated with poor prognosis in CRC although it was not an independent prognostic factor in multivariate analysis [33]. These results are opposite to our results in PDAC. In CRC, high PROX1 expression was associated with high tumor grade. This finding was not confirmed in our study. PROX1 is required for the formation of lymphatic vasculature [20], and overexpression of PROX1 in blood endothelial cells induces lymphatic endothelial cell gene expression [34]. However, Schneider et al. suggested that active lymphangiogenesis is not needed for lymphovascular spread in pancreatic cancer [30]. Recent data shows that positive PROX1 expression correlates with positive lymph node metastases in CRC and gastric cancer [35,36]. It remains unclear whether the downregulation of PROX1 expression enhances the lymphatic metastatic spread of pancreatic ductal adenocarcinoma.
We evaluated the staining of PROX1 in the cytoplasm, whereas in the previous studies of CRC, hepatocellular carcinoma (HCC), and gliomas, only the staining in tumor cell nuclei were evaluated [24,28,33]. In a recent study of gastric cancer, also cytoplasmic PROX1 expression by IHC was evaluated and it correlated with mRNA amplification [37]. We noted nuclear staining only in two tumor specimens. However, nuclear staining is present in the normal pancreas. At some point, the nuclear expression decreases, and in cancerous tissue, only cytoplasmic expression is left. These findings suggest that PROX1 may not function as an active transcription factor in PDAC. The role of cytoplasmic PROX1 expression has been studied in papillary thyroid cancer (PTC) by Choi et al. [38]. They discovered that PROX1 becomes inactivated through mRNA downregulation by aberrant NOTCH signaling, and cytoplasmic mislocalization of PROX1 increases protein stability in PTC cells. In addition, Linear-by-linear association test was used here restoration of PROX1 impaired tumor formation and diminished invasiveness of PTC cells. Whether the downexpression in the nuclei results from the evolved pancreatic cancer, or results in pancreatic cancer formation, remains unknown. Because of the limitations of IHC, we can only speculate, whether cytoplasmic PROX1 in pancreatic tumor tissue is in active or inactive form. The main remaining question is what the role of cytoplasmic PROX1 expression is and what the signal is that leads to relocation to the cytoplasm [38]. This question needs further studies to clarify the role of cytoplasmic PROX1 expression in PDAC. Our results are in line with the findings of PROX1 expression earlier discovered in PDAC, but also show the different nature of CRC and PDAC.
In the normal pancreas β-catenin expression is predominantly localized in the membrane of ductal cells. In pancreatic cancer, down-regulation of membrane expression and increased cytoplasmic expression are seen [11][12][13][14]. In our series, mainly uniform, granular cytoplasmic and membranous staining were seen, but there were only three specimens lacking membranous staining.
A few studies have reported that reduced or abnormal membranous β-catenin expression predicts poor prognosis of PDAC patients [11,13]. Wang et al. found no prognostic impact of β-catenin cytoplasmic expression in PDAC [14]. Qiao et al. showed that reduced membranous and positive cytoplasmic expression of β-catenin associated with poorer survival in PDAC during one-year follow-up [12]. These results differ from ours but the follow-up times in previous studies are only one or two years, and the patient cohorts have been small (n = 43-48). In none of those studies nuclear positivity was reported. In a few studies reduced β-catenin expression correlated with tumor dedifferentiation, but the prognostic significance was not investigated [9,39]. However, the controversy remains as in gene array analysis it has been demonstrated, that inhibition of Wnt/β-catenin signaling pathway blocks proliferation and induces apoptosis of cultured PDAC cells [10]. Also, increased β-catenin expression and protein levels have been reported in pancreatic tumors [7]. There is a need for further studies to validate the role   [40]. Our study shows by IHC that β-catenin expression in PDAC is both membranous and cytoplasmic with no distinct nuclear staining, and high β-catenin expression predicts better prognosis. The combination of PROX1 and β-catenin expression was created, because they have been linked to the same signaling pathway and their activation/expression is increased in CRC [15,23,33]. Furthermore, Yu et al. showed recently in CRC that β-catenin-PROX1 signaling axis is regulated by a transcriptional coactivator deleted in breast cancer (DBC1) [41]. They concluded that DBC1 acts as a positive regulator and as a key factor of β-catenin-PROX1 signaling axis in CRC progression. We demonstrate by Fig. 6 Low β-catenin expression is a marker of poor prognosis in pancreatic ductal adenocarcinoma. Cancer-specific survival analysis according to the Kaplan-Meier method for β-catenin expression in pancreatic ductal adenocarcinoma Fig. 7 Concomitant positivity by β-catenin and PROX1. Cancer-specific survival analysis according to the Kaplan-Meier method for combined expression of β-catenin and PROX1. A categorization of three classes was created to study the two markers together: low (β-catenin, and PROX1, low), moderate (either β-catenin, or PROX1 high), and high (β-catenin, and PROX1 high) IHC that both PROX1 and β-catenin expression are decreased in PDAC patients, and their expression are correlated significantly. These results were opposite from those in CRC. Whether β-catenin and PROX1 function in the same, yet opposite, way, remains unclear. However, we did not find any significant prognostic effect with combined PROX1 and β-catenin expression compared to β-catenin expression alone. Further analyses are required to examine the activity of Wnt/β-catenin/ PROX1 signaling pathway in PDAC. In order to thoroughly analyze the effect of PROX1 and β-catenin IHC expression on prognosis, a multi-center study with larger patient cohorts would be needed.
The TMA technique allows analysis of large patient cohorts. On the other hand, smaller areas of the tumors are evaluated compared to whole tissue sections. By taking cores from different parts of the tumor, possible sampling error can be diminished. Only less than 2 % of the specimens were lost in this patient cohort because of technical reasons. The strength of this study is a quite large patient cohort with long follow-up time. Unfortunately, due to the long period of data collecting, some of the crucial clinicopathological parameters were not available. Also, one of the weaknesses of the study is the lack of knowledge of the reliable resection margin status (R0/R1), which is known to be an important prognostic factor [42]. This results from the fact that our study is retrospective, and only in the last few years clinicians and pathologists have drawn enough attention to this important matter. All histological specimens were reevaluated and only ductal adenocarcinomas were included in the study.   Abbreviations CI Confidence interval, HR Hazard ratio. Multivariate analysis included adjustment for age, gender, stage (IA-IIA, IIB and III) and lymph node ratio (≥/< 20 %), postoperative adjuvant therapy, and perivascular invasion