The Clinicopathological and Prognostic Impact of 14-3-3 Protein Isoforms Expression in Human Cholangiocarcinoma by Immunohistochemistry

Cholangiocarcinoma (CCA) is an adenocarcinoma arising from the epithelial tissue of the intra-hepatic (10%), hepatic hilar (25%) or extrahepatic (65%) bile ducts (Lim, 2003). It is accounting for approximately 3% of all gastrointestinal malignancies (Wijaya and Abdullah, 2011). Recent data show that the incidence of cholangiocarcinoma have been increasing in several areas worldwide (Shu et al., 2011), especially the incidence of intrahepatic CCA (Patel, 2001). Though several risk factors for developing cholangiocarcinoma have been identified, the pathogenesis of the disease remains poorly understood. Among gastrointestinal (GI) cancers, CCA is the most difficult to detect and diagnose (Mosconi et al., 2009), patients often have advanced stage disease at the time of diagnosis (Fava, 2010), leading to a poor clinical outcome (Tompkins et al., 1981; Tio et al., 1991; Miyakawa et al., 2009), the patient survival is usually measured in months, therefore it is of great importance to identify novel candidate markers and potential early indicators of this disease as well as molecules that may


Introduction
Cholangiocarcinoma (CCA) is an adenocarcinoma arising from the epithelial tissue of the intra-hepatic (10%), hepatic hilar (25%) or extrahepatic (65%) bile ducts (Lim, 2003).It is accounting for approximately 3% of all gastrointestinal malignancies (Wijaya and Abdullah, 2011).Recent data show that the incidence of cholangiocarcinoma have been increasing in several areas worldwide (Shu et al., 2011), especially the incidence of intrahepatic CCA (Patel, 2001).Though several risk factors for developing cholangiocarcinoma have been identified, the pathogenesis of the disease remains poorly understood.Among gastrointestinal (GI) cancers, CCA is the most difficult to detect and diagnose (Mosconi et al., 2009), patients often have advanced stage disease at the time of diagnosis (Fava, 2010), leading to a poor clinical outcome (Tompkins et al., 1981;Tio et al., 1991;Miyakawa et al., 2009), the patient survival is usually measured in months, therefore it is of great importance to identify novel candidate markers and potential early indicators of this disease as well as molecules that may
The 14-3-3 proteins are highly conserved eukaryotic proteins which are primarily found in high levels in neurones but which have also been shown to be expressed in a wide range of other cells and tissues.These proteins comprise seven distinct isoforms (β, σ, γ, θ, δ, ε, η), named from their reversephase high performance liquid chromatography elution profile.
14-3-3 proteins are involved in most of the cellular processes (Shankardas et al., 2008), including regulation of several metabolic pathways, redox regulation, transcription RNA processing, protein synthesis, folding and degradation, cell cycle, apoptosis, cytoskeletal organization and cellular trafficking by binding to phosphorylated sites in diverse target proteins (over 300 phosphoproteins identified).Considering the number of binding partners, it is not surprising that 14-3-3 proteins play crucial roles in regulating multiple cellular processes.In addition to their important roles in many normal physiology processes, 14-3-3 proteins have attracted much recent interest in the etiopathogenesis of human cancers owing to their involvement in the prevention of apoptosis.
It has been reported that 14-3-3 proteins are overexpressed in some human diseases and have relations with them, particularly in cancers, including stomach cancers, breast cancer, and oral squamous cell carcinomas.However, its protein expression and clinicopathologic correlation in cholangiocarcinoma have not been reported.
To study the possible role of 14-3-3 proteins in cholangiocarcinoma, we were interested to examine the expression of these proteins on a total of 86 cases of surgically resected cholangiocarcinoma by immunohistochemistry with a specific anti-14-3-3 antibody.In addition, we investigate the correlation between 14-3-3 proteins expression and the patients' clinical outcome.

Cases and tissues
Eighty-six surgically resected samples were selected from patients pathologically diagnosed with cholangiocarcinoma with no preceding therapy at the Department of General Surgery, Peking Union Medical College Hospital (PUMCH), Beijing, China, from January 2000 to September 2010.This project was approved by the ethical committees of the hospital and informed consent was obtained from the participating patients.Cholangiocarcinoma was classified as intrahepatic or extrahepatic carcinoma depending on radiologic findings by computed tomography, magnetic resonance imaging, endoscopic retrograde cholangiopancreatography, or magnetic resonance cholangiopancreatography.Tumors arising in the intrahepatic bile ducts were classified as intrahepatic carcinoma (n=8) and in the extrahepatic bile ducts were classified as extrahepatic carcinoma (n=76), including Klatskin's tumors that occur at the bifurcation of the left and right hepatic ducts.Tumors were histologically divided into 3 groups (28 well differentiated, 44 moderately differentiated, and 14 poorly differentiated), according to their degree of papillary or tubular formation.If more than 1 type was found, the predominant type was recorded.The following histologic features were also examined: tumor location, tumor size, pathologic differentiation, lymphatic permeation, lymph node metastasis, and tumor stage.The tumor histologic stage was defined as stage I (n = 4), stage II (n = 28), stage III (n = 27), stage IVA (n=24) or stage IVB (n = 3) by histologic examination based on the pTNM classification proposed by the International Union Against Cancer.
Tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin for histologic examination.A paraffin block used for routine pathologic diagnosis and containing representative tumor histology was selected in each case, and 3 µmthick paraffin sections were prepared from this block for immunohistochemical analysis.Frozen tissues were obtained immediately after surgical resection and stored frozen at -80 ℃.

Tissue preparation
The paraffin-embedded tissue sections of cholangiocarcinoma were dewaxed and rehydrated.
After incubation with 3% hydrogen peroxide in absolute methanol to block endogenous peroxidase activity for 10 minutes, antigen retrieval was carried out according to the method shown in Table 1.The tissue sections were incubated at 37 °C for one hour in a wet container with the specific antibodies against seven 14-3-3 isoforms (Santa Cruz, CA), diluted in primary antibody diluting buffer (Dako).Then, the slides were incubated with a secondary antibody (Dako) for 50 minutes at room temperature.The tissue sections were visualized with 3, 3-diaminobenzidine (DAB) and counterstained with Mayer's hematoxylin.Finally, the slides were viewed with an Axiophot microscope (CarlZeiss, Germany) coupled with a ProgRes charge-coupled device (CCD) camera (Jenoptik, Germany).

Statistical analysis
All statistical analyses were done using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL).To trace correlations between 14-3-3 proteins expression and several clinicopathologic parameters, data were crosstabulated and Fisher exact test was done.The correlation of 14-3-3 proteins staining with patient survival was evaluated using life tables constructed from survival data with Kaplan-Meier plots.Comparisons of the different groups were done with the log-rank test.The end point in the present study was overall survival ranging from date of surgery until date of death or, if no information was documented, until date of last follow up information (=censored).Results were considered significant when P < 0.05.

Immunohistochemical staining for 14-3-3 proteins in cholangiocarcinoma
We first did seven 14-3-3 isoforms immunohistochemistry staining on cancer tissues obtained from a cohort of 86 cholangiocarcinoma patients with different clinical stages and histologic types and grades.Immunohistochemical results are summarized in Figure 1.

Correlation between 14-3-3 proteins expression and clinicopathologic parameters
The correlative analysis between 14-3-3 proteins expression and the clinicopathologic characteristics of patients with cholangiocarcinoma is summarized in Table 2.
High expression of 14-3-3 β and σ were significantly correlated with lymph node metastasis and tumor stage of those with cholangiocarcinoma.In 44 cases of cholangiocarcinoma with positive lymph node metastasis, the total positive staining rates of 14-3-3 β and σ were 100% and 81.8%.In 42 cases without lymph node metastasis, the total positive staining rates of these two isoforms were 88.1% and 35.7%.These results indicated that the patients with positive immunohistochemistry staining of 14-3-3 β and σ were presented a higher frequency of lymph node and distant metastasis (P = 0.024, 0.000, Figure 2), and a more advanced clinical stage (P = 0.017, 0.000).

Discussion
The 14-3-3 proteins have emerged as critical regulators of cellular responses in eukaryotic organisms (Zang et al., 2010), they can form homodimers or heterodimers that allow them to function as an adapter, linker, scaffold or coordinator in assembling signaling complexes (Wilker et al., 2004).Because of its importance in the regulation of key signal transduction pathways, deregulation of 14-3-3 has been associated with pathological consequences (Porter et al., 2006;Kilani et al., 2007).Recent studies have suggested that 14-3-3 proteins are potential oncogenes (Tzivion et al., 2006).To our knowledge, it is the first time to evaluate the expression of seven different 14-3-3 isoforms using immunohistochemistry method and their possible roles in conjunction with clinical outcome of the patients in cholangiocarcinoma, it is important to clarify the relationship between 14-3-3 family proteins with the carcinogenesis of cholangiocarcinoma.In tumoral tissue, six isoforms, 14-3-3 β, σ, γ, θ, δ and η, were present in abundance.These data indicate that these isoforms may be involved in bile duct tumorigenesis.Because 14-3-3 proteins participate in important cellular events including cell cycle checkpoint, signal transduction, apoptosis and cell division, it may be that these processes are dysregulated in these tumors.It has been found recently that human cruciform binding protein belongs to theβ, ε,σ,δandγisoforms of the 14-3-3 family (Todd et al., 1998).These five 14-3-3 proteins form a complex that binds to cruciform DNA to stimulate DNA replication.Given the transformed cells need more DNA replication to support the cells proliferation, the elevated number and quantity of isoforms that we found in cholangiocarcinoma may reflect the cancer cell growth requirements.
14-3-3 σ was identified as an epithelial-specific marker (Guweidhi et al., 2004), functionally, it is associated with control of the G2/M checkpoint in the cell cycle.The expression of 14-3-3 σ is induced downstream of the activation of p53 and results in a G2/M arrest.Loss of 14-3-3 σ expression has been reported in primary gastric (Suzuki et al., 2000), hepatocellular (Iwata et al., 2000), and breast adenocarcinoma (Jeanteur, 2000).In contrast, 14-3-3 σ expression is up-regulated in head and neck squamous cell carcinoma and in chemoresistant pancreatic adenocarcinoma cells (Sinha et al., 1999).In our study, 14-3-3 σ was overexpressed in cholangiocarcinoma.The biological significance of 14-3-3 σ expression in cholangiocarcinoma may be its anti-apoptotic effect like in pancreatic carcinogenesis, achieved by inhibiting the proapoptotic proteins BAD and BAX (Zha et al., 1996;Samuel et al., 2001;Nomura et al., 2003).Therefore, due to diversity of 14-3-3 σ functions, its role in cancerogenesis might be restricted to the particular type of tumor.
Furthermore, we found that 14-3-3 β and σoverexpression were mainly found in more advanced tumor stages and predicts poor overall survival in this patient group indicating that 14-3-3 β and σ may be associated with an aggressive biological characteristics in cholangiocarcinoma progression, which play an important role in prognosis and/or recurrence, although it could be a relatively early event during their carcinogenesis.
14-3-3 δ is one of the major transforming growth factor-b-induced proteins, which can promote epithelialmesenchymal transition of epithelial cells in cancer cell transformation (Keshamouni et al., 2006).In the present study, the upregulation of 14-3-3 δ was observed in cholangiocarcinoma.This result suggests that the δ isoform of 14-3-3 is dysregulated in tumors.14-3-3 δ was proposed to be involved in apoptosis through multiple interactions with proteins of the core mitochondrial machinery, proapoptotic transcription factors, and their upstream signaling pathways.Our study showed that 14-3-3 δ expression is positively correlated with pathologic differentiation, and tumor stage.Thus, increased expression of 14-3-3 δ contributes to cholangiocarcinoma development and progression, and the detection of the 14-3-3 δ aberrations might be a useful biomarker to identify poor prognoses in patients with cholangiocarcinoma.More importantly, our data first indicate that 14-3-3 δ expression is positively correlated with tumor location, there were more 14-3-3 δ expression in intrahepatic carcinoma than extrahepatic carcinoma.
In our study, we also found the overexpression of 14-3-3 θ, γ, η in cholangiocarcinoma compared with adjacent normal bile ducts.These data indicate that the increased expression of the three 14-3-3 isoforms may contribute to bile duct tumorigenesis.Previous study has proved that a region from α-helix 7 to the C terminus of 14-3-3 θ was crucial for the interaction with Bax, the caspasedependent dissociation of Bax from 14-3-3 θ acts as an initial trigger for apoptotic mitochondrial changes.The overexpression of 14-3-3 θ can lead to cell adhesion to tenascin-C and an enhanced growth rate of tumor cells (Martin et al., 2003), it was overexpressed in human astrocytomas and lung adenocarcinoma and squamous cell carcinoma.Furthermore, we also found the expression of 14-3-3 θ is improved with the increasing of pathological grades of cholangiocarcinoma, like 14-3-3 δ.One reasonable explanation for this phenomenon may be that comparing to the lower-grade cholangiocarcinoma, the higher-grade cholangiocarcinoma have relatively insufficient blood supply and are more susceptive to apoptosis-inducing signals theoretically, in order to be resistant to these proapoptotic signals and to gain survival advantages.One possible option for high grades astrocytomas cells is to produce more 14-3-3 θ and δ, and which in turn exert their anti-apoptotic functions.
The overexpression of 14-3-3 ε contributes to remodeling of extracellular matrix in skin through increasing MMP-2 expression via p38 MAPK signaling (Lee et al., 2009).However, we noticed that 14-3-3 ε protein had similar expression levels between cholangiocarcinoma tissue and adjacent normal bile ducts in our studies, which was different from its higher expression in renal carcinoma (Liang et al., 2009).This difference was mainly due to the following reasons.The tissue-specific interactions with 14-3-3 ε are different, in combination with the potential heterodimerization, which were mainly responsible for its differed expression level in specific tumors.In addition, the methodology to compare 14-3-3 ε expression was different in other the group's report and our studies.
In summary, this study documents the first analysis of 14-3-3 proteins expression in cholangiocarcinoma.The results show that 14-3-3 is expressed in majority of cases, although with different intensity and distribution.The results also suggest that up-regulated expression of 14-3-3 β, σ, γ, θ, δ, η isoforms may be related to occurrence or progression of cholangiocarcinoma, and target 14-3-3 may be a novel promising strategy for the treatment of cholangiocarcinoma and targets for therapy.