High Expression of HIF-1 α , BNIP 3 and PI 3 KC 3 : Hypoxia-Induced Autophagy Predicts Cholangiocarcinoma Survival and Metastasis

Cholangiocarcinoma (CCA), a bile duct cancer, arises from biliary epithelium within either the intrahepatic or the extrahepatic biliary tract (Patel, 2006). Recent epidemiological studies show that the incidence and mortality rates of CCA are increasing worldwide (Patel, 2006). Surgical intervention is the only promising curative treatment in the early stage of disease; unfortunately, the prognosis of advanced CCA is extremely poor and chemotherapy is the only feasible treatment for this stage of disease (Thongprasert et al., 2005). Therefore, the hallmark of molecular pathways regulating CCA progression need to be further addressed in order to provide new prognostic markers and possible therapeutic targets to improve clinical outcomes. Hypoxia is a common feature of solid tumors that alters imperative pathways resulting in an aggressive behavior in an extensive variety of tumors. Recent studies suggest that tumor hypoxia induced by oxygen depletion contributes to chemotherapeutic resistance of CCA cell lines (Seubwai


Introduction
Cholangiocarcinoma (CCA), a bile duct cancer, arises from biliary epithelium within either the intrahepatic or the extrahepatic biliary tract (Patel, 2006).Recent epidemiological studies show that the incidence and mortality rates of CCA are increasing worldwide (Patel, 2006).Surgical intervention is the only promising curative treatment in the early stage of disease; unfortunately, the prognosis of advanced CCA is extremely poor and chemotherapy is the only feasible treatment for this stage of disease (Thongprasert et al., 2005).Therefore, the hallmark of molecular pathways regulating CCA progression need to be further addressed in order to provide new prognostic markers and possible therapeutic targets to improve clinical outcomes.
Hypoxia is a common feature of solid tumors that alters imperative pathways resulting in an aggressive behavior in an extensive variety of tumors.Recent studies suggest that tumor hypoxia induced by oxygen depletion contributes to chemotherapeutic resistance of CCA cell lines (Seubwai
Autophagy is a homeostatically controlled pathway by which autophagosomes fuse with lysosomes for subsequent degradation and recycling of macronutrients and impaired organelles by lysosomal enzymes (Klionsky and Emr, 2000).Autophagy is up-regulated in response to stress conditions such as nutrient deprivation, growth factor depletion, and hypoxia (Levine and Kroemer, 2008).Autophagy requires a regulator that contains lipid kinase activity named class III phosphoinositide 3-kinase (PI3KC3), for its activation under metabolic stress (Byfield et al., 2005).It has been reported that autophagy was activated in CCA tissues of patients demonstrating by increasing the expression level of Beclin-1, a part of PI3KC3 complex (Hou et al., 2011).Interestingly, elevation of autophagy extends the survival in pancreatic cancer patients (Fujii et al., 2008) and inhibition of autophagy leads to apoptotic cell death in CCA cells (Hou et al., 2011).
In the present study, we aimed to investigate the role of hypoxia-induced autophagy on CCA survival and metastasis.Expressions of HIF-1α, BNIP3 and PI3KC3 were determined in human CCA tissues and the correlation between those proteins and clinicopathological data of CCA patients was analyzed.In addition, we demonstrated that the hypoxic-mimicking condition activated the autophagy leading to the control over tumor cell migration.

Patients and samples
CCA tissue microarrays (CCA-TMAs) were constructed from archival paraffin embedded tissue of intrahepatic CCA specimens originated from primary tumors of patients.All patients underwent liver resection at Srinagarind Hospital, Khon Kaen University, Thailand during 1999-2010.Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki and its later revision.The Human Research Ethics Committee, Khon Kaen University (#HE43201 and #HE471214), approved the research protocol.

Cell lines, chemicals, and reagents
Two human CCA cell lines, M139 and M214, were cultured in Ham's F-12 medium supplemented with 44 mM NaHCO 3 , penicillin (100 units/ml), streptomycin (100 mg/ml) and 10 % fetal bovine serum in a humidified atmosphere containing 5 % CO 2 .CoCl 2 was purchased from Sigma, St. Louis, MO, United States.All other chemicals used were of analytical grade.

Immunohistochemical analysis
The CCA-TMAs of 183 cases were deparaffinized with xylene, dehydrated before exposure to 3% hydrogen peroxide for inactivating endogenous peroxidase activity and blocked with 10% skim milk for 1 h.Sections were incubated overnight with the primary antibodies against HIF-1α, BNIP3 and PI3KC3 (1:25, 1:50 and 1:100, respectively; Abcam; MA, USA) at 4 o C, followed by peroxidase-conjugated Envision secondary antibodies (DAKO; Glostrup, Denmark) for an additional 1 h at room temperature.The presence of brown color corresponding to the peroxidase activity was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (Sigma-Aldrich; MO, USA), counterstained with Mayer's hematoxylin, dehydrated and mounted.The TMA sections were observed under a light microscope (Carl Zeiss, Germany) by using the high magnification power (X200); the 5 fields of each representative tumor section were evaluated.

Wound-induced migration assay
CCA cell lines were cultured in medium containing 10%FBS at 37 o C with 5% CO 2 in a 24-well plate until cells were confluent or nearly (>90%) confluent.Cell monolayers were scratched, then rinsed several times with 1x PBS to remove cell debris.Cells were incubated in medium containing CoCl 2 for 0, 8, 16, and 36 h.Cell migration in the wound area was monitored and visualized by microscopy and digitally photographed.The distance of the wound area was measured on the images and the migration area was calculated by using the formula: Migration area=(Area of original wound -Area of wound during healing)/ Area of original wound.

Statistical analysis
Statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL).The correlation of HIF-1α, BNIP3 and PI3KC3 with clinicopathological parameters of CCA patients was analyzed by Fisher's exact test.The association among those proteins was performed by Pearson's correlation analysis.Patients' survival was calculated from the time of surgical resection to death and the survival curves were constructed according to Kaplan-Meier, with a Log-Rank test.A multivariate analysis was performed by the Cox proportional hazard regression model.The significance of different data was determined by the Student's t-test.A P value of less than 0.05 was considered statistically significant.
Cumulative survivals of CCA patients with positive expression of HIF-1α (p=0.047) had significantly lower survival rates than those with negative expression (Figure 2A).The 5-year survival rates of patients who were positive with co-expressed HIF-1α and BNIP3 (p=0.032) or co-expressed HIF-1α and PI3KC3 (p=0.043) were significantly greater than that of the negative groups (Figure 2B and Figure 2C respectively).Results in Table 3 showed that the expression of HIF-1α was positively associated with BNIP3 (r= 0.3, p<0.01) and PI3KC3 (r= 0.2, p<0.01).Multivariate analysis was performed using the Cox proportional hazard model to investigate the independent value of each factor to predict overall survival.The results showed that HIF-1α expression (HR=1.4,p=0.048), lymph nodes metastasis (HR=1.7,p=0.002) and non-papillary histological type (HR=1.4,p=0.038) were independent prognostic risk factors for overall survival (Table 4).

CoCl 2 stabilized HIF-1α with induction of autophagicresponsive proteins in CCA cell lines
The CoCl 2 was added into cell culture with various concentrations and incubation times in order to determine the protein levels of HIF-1α, BNIP3, PI3KC3 and LC3 (an autophagosomal marker) using western blotting.As shown    CoCl 2 induced migration ability of CCA cells M139 and M214 cell lines were treated with 100 μM CoCl 2 and the migration ability were assessed.As shown in Figure 4A and 4B, cells treated with 100 μM CoCl 2 significantly migrated faster (1.7 times in M139 cells and 1.2 times in M214 cells) than untreated cells in which their wounds had closed at 36 h for M139 and 16 h for M214 cells.A metastasis marker, pFAK (phosphorylated focal adhesion kinase (Jiang et al., 2010)) and LC3-II were determined in CCA cells treated with CoCl 2 using western blotting.As depicted in Figure 4C and 4D, the 100 μM CoCl 2 treated cells increased the levels of LC3-II and pFAK when compared with untreated cells.

Discussion
Previous studies have suggested that hypoxia activates autophagy may promote tumor progression in many cancers (Bellot et al., 2009;Mazure and Pouysségur, 2010).We demonstrated that a hypoxia-inducible factor HIF-1α was positively expressed in CCA tissues in 66% of cases and was strongly correlated with shorter patients' survival.Our finding is consistent with previous studies revealing that HIF-1α correlated with shorter survival of breast cancer (Deb et al., 2014), lung cancer (Li et al., 2012), pancreatic cancer (Hoffmann et al., 2008), colorectal cancer (Baba et al., 2010) and Japanese type of intrahepatic CCA (Morine et al., 2010).In addition, it has     doi.org/10.7314/APJCP.2014.15.14.5873 Hypoxia-Induced Autophagy Predicts Cholangiocarcinoma Survival and Metastasis been revealed that HIF-1α was presented in intrahepatic CCA tissues of Thai patients (Pinlaor et al., 2005), and in this study, we found that HIF-1α had a high expression in CCA patients with a shorter survival.We then revealed that HIF-dependent gene product BNIP3 was positively expressed in CCA tissues with 44% of cases and the autophagy regulator PI3KC3 was positive in 46% of CCA cases.Patients who positively expressed two markers, HIF-1α plus BNIP3 or HIF-1α plus PI3KC3, showed the significant correlation with the shorter survivals.In addition, HIF-1α, BNIP3 or PI3KC3 were positively associated with lymph node metastasis.Furthermore, we also elucidated that HIF-1α was positively correlated with BNIP3 and PI3KC3 by Pearson's correlation analysis.This implies that HIF-1α might trigger autophagy in CCA tissues via its target gene BNIP3 cross-linking to autophagy via activation of PI3KC3 complex (Bellot et al., 2009), providing the role in tumor progression.In addition to HIF-1α alone, combination between HIF-1α and BNIP3 or HIF-1α and PI3KC3 could predict a survival and metastasis of CCA.

Table 3. Correlation Coefficients between Immunohistochemistry Scores of Hypoxic-(HIF-1α) and Autophagic-Responsive Proteins in CCA Tissues
We established the hypoxia-mimicking condition using CoCl 2 to mimic an oxygen-depleted atmosphere in two CCA cell lines to explore the cellular functions in CCA.Our results revealed that CoCl 2 generated a hypoxic condition and induced autophagy by increasing levels of HIF-1α, BNIP3, PI3KC3 and autophagosomal marker LC3-II in CCA cell lines.A chemical mimetic of hypoxia CoCl 2 increased autophagy has been demonstrated in several models such as in the H9c2 rat cardiomyoblast cell culture (Gallo et al., 2014) and human periodontal ligament cells (Song et al., 2012) resulting in an increasing LC3-II level that is similar to O 2 depletion induced hypoxia (Hu et al., 2012).The hypoxia induced LC3-II/LC3-I ratio has been shown that it activates autophagosome maturation (Gallo et al., 2014).We demonstrated that treatment with 100 µM CoCl 2 accelerated CCA cell migration by increasing the level of pFAK, a metastasis marker.Our results imply that a synchronized function of HIF-1α, BNIP3, PI3KC3 and LC3-II promotes CCA cell migration.Likewise, hypoxiamimetic agent CoCl 2 has previously been demonstrated that it has potential in modulating the invasive ability of primary and metastatic breast cancer cells (Fu et al., 2009).Knockdown HIF-1α by siRNA inhibiting cell migration and invasion under hypoxic environment was determined in malignant gliomas (Mendez et al., 2010).BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton (Agostinis et al., 2013).Furthermore, autophagy itself can promote tumor metastasis (Kenific et al., 2010).
In conclusion, our results with CCA tissues in patients and cell lines support that several molecular markers closely correlated with tumor hypoxic conditionassociated autophagy activated in CCA reflect severity of the disease, contribute to their survival and metastasis and provides as prognostic markers.a meta-analysis.Asian Pac J Cancer Prev, 14, 3607-12. Mazure NM, Pouysségur J (2010).Hypoxia-induced autophagy: cell death or cell survival?Curr Opin Cell Biol, 22, 177-80. Mendez O, Zavadil J, Esencay M, et al (2010).Knock down of HIF-1alpha in glioma cells reduces migration in vitro and invasion in vivo and impairs their ability to form tumor spheres.Mol Cancer, 9, 133.Morine Y, Shimada M, Utsunomiya T, et al (2010).

Figure 4 .Figure 3 .
Figure 4. CoCl 2 Induced Migration Ability in CCA Cells.CoCl 2 (100 μM) were added in A) M139 and B) M214 cell lines and incubated for 36 h or 16 h, respectively, and the migration of the cells towards the wound was visualized.Images were taken at various time points.Data were presented as mean±SEM, *p<0.05.Each culture was done in three independent experiments.Western blotting of LC3, pFAK and total FAK in C) M139 and D) M214 cell lines treated with 100 μM CoCl 2 .β-actin was used as an internal control.Each culture was done in three independent experiments

Table 2 . Correlation between Expression of Hypoxic-/ Autophagic-Responsive Proteins in Tumor Tissues and Clinicopathology of CCA Patients Demonstrated by Immunohistochemical Staining
*Fisher's exact probability was used for the comparison of variables that had two categories; **P value less than 0.05 was considered statistically significant