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Keywords:

  • cholangiocarcinoma;
  • protein kinase A regulatory subunit 1 alpha;
  • cAMP-dependent protein kinase

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real-time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/β-catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1A's potential as a drug target for CCA therapy.

Cholangiocarcinoma (CCA) is a malignant transformation of cholangiocytes, the epithelial cells lining the bile duct. The incidence of CCA is relatively rare in the western countries, but it is found frequently in Southeast Asia, especially Northeastern Thailand where the highest incidence of CCA has been reported.1 Presently, curative surgical resection, radiotherapy and chemotherapy are the treatment options for CCA patients.2 However, the majority of patients undergoing curative resection still develop recurrent disease at the surgical site and overall survival is poor.3 Moreover, radiation therapy or current chemotherapy does not appreciably improve long-term survival rates.4 Therefore, CCA treatment remains challenging. The details of its molecular carcinogenesis have been elusive making it difficult to identify possible therapeutic targets.

cAMP-dependent protein kinase (PKA) is a well-known member of the serine-threonine protein kinase superfamily, which is activated by the second messenger cAMP and is involved in controlling a variety of cellular processes.5 PKA isozyme switching as reflected by the proportion of two PKA regulatory subunits, protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) and protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII), has been implicated in the initiation and progression of many tumors. Based on its role in cancer, PKA has been suggested as a possible new molecular target for therapy.6–10

We reported the overexpression of PRKAR1A in the liver fluke (Opirthorchis viverrini) and N-nitrosodimethylamine (NDMA)-induced hamster CCA tumors.11 We also observed PKA isozyme switching, which indicated that the PRKAR1A/PKAI pathway might have contributed to the induction of biliary cell transformation and proliferation in O. viverrini and NDMA-induced progressive cholangiocarcinogenesis. However, the mechanism by which PRKAR1A/PKAI contributes in CCA development is not yet clear. Therefore, our study was aimed at investigating possible molecular mechanisms by which PRKAR1A/PKAI promotes human CCA development. This information could help determine if PRKAR1A/PKAI is a suitable target for inhibiting CCA cell growth.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients and tumor collection

Between January 2003 and December 2004, 39 surgically resected specimens of CCA were selected from the tissue bank of the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Thailand. The informed consent was obtained from each subject before surgery, and Human Research Ethics Committee, Khon Kaen University approved the research protocol (#HE43201 and #HE471214).

Tumor tissues were obtained during surgery and immediately dissected by the pathologist; sections were fixed and paraffin embedded for diagnostic and immunohistochemical studies and others were frozen and stored in liquid nitrogen until further use for the molecular studies.

Cell lines and cell culture

Human CCA cell lines, M156, OCA17, KKU100 and M214, were cultured in HAM-F12 (Gibco/BRL, Grand Island, NY) supplemented with 10% inactivated fetal bovine serum, 2 mg/ml sodium bicarbonate and 1% antibiotic–antimycotic solution (Life Technologies, Gaithersburg, MD). All cultured cell lines were incubated at 37°C in a humidified incubator maintained with an atmosphere of 5% CO2.

Inhibitors and antibodies

The isoquinoline H89, which inhibits PKA, and the site-selective cAMP analogs, 8-Cl cAMP and 8-Br cAMP, were purchased from Calbiochem (La Jolla, CA). Inhibitors were dissolved in water at a stock concentration of 10 mM and stored at −20°C until used. Antibodies used for Western blotting were as follows: PRKAR1A (BD Transduction Laboratories™, San Jose, CA), PRKAR2B (BD Transduction Laboratories™), cyclin D1 (Cell Signaling Technology, Danvers, MA), cdk4 (Cell Signaling Technology), ApoptoPak™ Miniature Set (anti-Bcl-2, anti-Bak, anti-Bax; Millipore, Billerica, MA), Bcl-X (BD Transduction Laboratories™, San Jose, CA), Phospho-p44/42 mitogen-activated protein kinase (MAPK; Erk1/2; Thr202/Tyr204) (Cell Signaling Technology), p44/42 MAPK (Erk1/2) (Cell Signaling Technology), phosphor-AKT (Cell Signaling Technology), AKT (Cell Signaling Technology) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).

RNA extraction and cDNA synthesis

Total RNA was extracted by Trizol® reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Reverse transcription reaction consisted of 2.5 μg total RNA and random hexamers (2.5 μM) that were mixed together and then heated at 70°C for 10 min. Each reaction mixture contained the first strand cDNA synthesis buffer (1×; 75 mM KCl, 50 mM Tris-Cl pH 8.3, 3 mM MgCl2), 10 mM DTT, 0.5 mM each dNTPs and 200 units reverse transcriptase (Promega Corp., Madison, WI). Reverse transcription was carried using a DNA thermal cycler (GeneAmp PCR system 2400, Perkin-Elmer Applied Biosystems, Waltham, MA). The thermal conditions were 25°C for 10 min, 37°C for 1 hr and 95°C for 5 min.

Real-time PCR and relative quantification of PRKAR1A and PRKAR2B expression

Real-time PCR was performed using TaqMan® Gene Expression assay kit and the ABI 7500 real time PCR system (Applied Biosystems, Foster City, CA). PCR was performed in the relative quantification of PRKAR1A and PRKAR2B gene expression using the comparative cycle threshold (CT) method, and GAPDH expression was used as the endogenous control. Fold change of gene expression in tumor and adjacent nontumor tissues was calibrated with normal liver tissues.

Western blot analysis

Protein extract (50 μg) from the cell lysates was solubilized in SDS buffer and boiled for 5 min. Samples were separated on 12.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) at room temperature for 1 hr or 4°C overnight and incubated in primary antibody for 1 hr at room temperature. Primary antibody was used as polyclonal antibody to human PRKAR1A (1:1,000; Calbiochem, Gibbstown, NJ). After rinsing with TBS containing 0.1% polyoxyethylene sorbitan monolaurate (Tween-20 or TBS-T), membranes were incubated in horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biochemical, Santa Cruz, CA) at room temperature for 1 hr. After rinsing with TBS-T, membranes were exposed to the ECL Plus Western Blotting Detection System (GE Healthcare, UK) for 5 min. Human GAPDH was used as a loading control. The immunoblot and intensity were analyzed by ImageQuant™ analysis system (GE Healthcare). In addition, F9 (mouse embryonic carcinoma cell line) whole-cell lysate was used as a positive control for PRKAR2B antibody.

Immunohistochemistry

Immunohistochemistry (IHC) was performed for the PKA regulatory subunits (PRKAR1A and PRKAR2B) according to the standard method.12 Briefly, sections of liver tissues were deparaffinized in xylene for 5 min (three times), rehydrated in 100% alcohol for 5 min (three times) and then placed with 0.5% H2O2–methanol for 5 min with shaking to block endogenous peroxidase activity and this reaction was stopped in water for 2–3 min. Thereafter, the tissue sections were irritated in 0.1 M citrate buffer in a microwave oven for antigen retrieval. Nonspecific binding was blocked by normal horse serum in PBS for 20 min. Sections were incubated with the primary antibody, rabbit anti-PRKAR1A IgG; 1:1,000 (or rabbit anti-PRKAR2B; 1:1,000), then incubated in moisture chamber for 1 hr. After that, sections were incubated with peroxidase-conjugated Envision™ secondary antibody (DAKO, Denmark). After washing in working PBS for 5 min (three times), the color was developed with 0.1% diaminobenzidine tetrahydrochloride solution for 10 min and then counterstained with Mayer's hematoxylin. The sections were dehydrated with stepwise increasing concentrations of ethanol, cleared with xylene and mounted with permount. The stained sections were reviewed and digital photomicrographs were obtained with a Carl Zeiss Axio Scope.A1 microscopy, Stockholm Sverige, Sweden).

The intensity of PRKAR1A and PRKAR2B expression was semiquantitatively classified into four groups on the basis of the percentage of positive tumor cells as: 0, negative; +1, 1–25%; +2, 26–50%; +3, > 50%, respectively. The intensity of PRKAR1A staining was scored as low (0 and +1) and high (2+ and 3+) expression.

Stable PRKAR1A-knockdown CCA cell lines

A Mission™ TRC-Hs (Human) clone set of sequence-verified shRNA lentiviral plasmid vectors against PRKAR1A, TRCN0000039938-42, were obtained from the JHU High Throughput Biology. VSV-G pseudotyped virus was produced by cotransfecting 293T cells with a shRNA transducing vector and two packaging vectors: psPAX2 and PMD2.G using Lipofectamine2000 (Invitrogen). Infectious virus was harvested at 36 and 48 hr after transfection and filtered through a 0.22-μm pore size cellulose acetate filter.

M156 and OCA17 cells (1 × 105) were plated in six-well plates (BD Falcon™, San Jose, CA). After 24 hr, 100 μl of nonconcentrated virus was added in DMEM containing 8 μg/ml polybrene. Cells were incubated at 37°C and 5% CO2. Growth media was replaced 24 hr post-transduction. PRKAR1A-knockdown cells were selected with puromycin containing media. The efficiency of shRNA transduction was assessed by western blot as described above. The second transduction was performed in OCA17 to get PRKAR1A knockdown completely.

PKA activity assay

PKA activity was determined using a nonradioactive PKA activity assay kit (Promega) according to the manufacturer's recommendations. Protein extract (75 μg) from stable PRKAR1A-knockdown CCA cell lines (M156 and OCA17) and their empty viral transduction control were incubated with PKA reaction mixture, which contain PKA-specific peptide substrate (Kemptide, LRRASLG) at 30°C for 30 min. The reactions were terminated by placing the tube at 95°C for 30 min. The migration of phosphorylated peptide toward the cathode (+) was detected using 0.8% agarose gel at 100 V for 15 min. The gel image was analyzed and quantified using ImageQuant™ Imager (GE Healthcare).

Cell proliferation assay

Cell proliferation was determined by using alamarBlue® assay (Invitrogen). Briefly, cells (5 × 102) were seeded in black clear bottom 96-well plates (BD Falcon™) and incubated overnight. Then, 20 μl of 1× alamarBlue® was added and the volume in each well was made up to 200 μl with the growth medium. After 72 hr incubation, fluorescence was measured on a Perkin Elmer Wallac 1420 Multilable counter (Perkin Elmer, Turku, Finland) with a 540-nm excitation filter and a 590-nm emission filter. Experiment was done three times in sextuplicate. The effect of PKA inhibitor and cAMP analogs on CCA cell lines' proliferation was also determined as described above but the test substances were added at designed concentration.

Apoptosis assays

Apoptosis assays were performed using the caspase-Glo® 3/7 assay (Promega) according to manufacturer's instructions. Briefly, cells (5 × 103) were plated in 100 μl of media and incubated for 72 hr. Caspase-Glo® 3/7 reagent (50 μl) was added and mixed to each well, followed by a 1 hr incubation at room temperature. Luminescence was measured on a Victor3 multiwell plate reader (Perkin Elmer) as per the manufacturer's instructions. Experiments were performed twice with three replicates per experiment.

Flow cytometry

Cell cycle distribution was determined by staining DNA with propidium iodide (PI; Sigma, St. Louis, MO). Briefly, stable PRKAR1A-knockdown CCA cell lines (M156 and OCA17) and their empty viral transduction control were seeded in six-well plates at a cell density of 2 × 105 cells per well for 72 hr. Cells were washed in ice-cold PBS, then fixed in 70% ice ethanol overnight, after which they were suspended in PBS containing 40 μg/ml PI, 0.1 mg/ml RNase (Sigma) and 0.1% Triton-X-100 at 37°C for 30 min. Next, the cell cycle distribution was determined with flow cytometry (Beckman Coulter, Fullerton, CA) and analyzed by using CXP analysis software (Beckman Coulter).

Cell apoptosis was measured by using Annexin-V-FLUOS staining kit (Roche, Penzberg, Germany) containing Annexin-V-FLUOS and PI staining. Cells were plated as described above for 72 hr and then washed with ice-cold PBS. Cell pellets were then suspended in 100 μl of Annexin-V-FLUOS labeling solution and incubated at room temperature for 15 min. Cellular apoptosis was detected with flow cytometry (Beckman Coulter) and analyzed by using CXP analysis software (Beckman Coulter).

Phospho-kinase array

Profiling of kinases and phosphorylation of their protein substrates were analyzed by using Human Phospho-Kinase Array Kit (ARY-003, R&D systems, Minneapolis, MN) according to the manufacturer's instructions using protein extracts from stable PRKAR1A-knockdown CCA cell lines (M156 and OCA17) in comparison to their empty viral transduction control. Cell lysates (750 μg per membrane) were incubated overnight with nitrocellulose membrane containing 46 kinase phosphorylation sites and seven different controls printed in duplication. Then membranes were incubated first with detection antibody cocktail and later with streptavidin–horseradish peroxidase. The signals were detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). The array images were analyzed and quantified using ImageQuant™ Imager (GE Healthcare).

Statistical analysis

Statistical analyses were performed using SPSS software. Expression of PKA subunits was assessed for association with various clinical and pathological parameters using the χ2-test. Patient survival was calculated from the time of surgical resection to death, and survival curves were calculated according to Kaplan-Meier with a log-rank test. Results from cell proliferation and apoptosis experiments were represented as mean ± SD and the significance of differences was addressed by Student's t-test. A p value < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patient characteristics

Of the 39 intrahepatic CCA patients studied, 71.8% were male, resulting in a male to female ratio of 2.5:1. The mean age was 55.1 ± 8.9 (range, 37–74 years). Most of the patients were in an advanced stage, with 51% having metastasis. In this series, the histopathological type was classified as poorly differentiated- (33.3%), moderately differentiated- (7.7%), well differentiated- (33.3%) and papillary (25.7%)-type CCA.

Expression of PKA mRNA subunits in human CCA tissues

Quantifiable levels of PRKAR1A/PKAI and PRKAR2B/PKAII were detected in 39 human CCA as well as in adjacent nontumorous tissues of the same patient and normal liver of cadaveric donors by real-time PCR. The results showed a significantly higher level of PRKAR1A mRNA expression in tumor tissues compared to adjacent nontumorous tissues (Fig. 1a). Conversely, the expression of PRKAR2B mRNA is remarkably decreased (Fig. 1b). Moreover, the PRKAR1A/PRKAR2B ratio is also significantly higher in tumor than that of the nontumor tissues (Fig. 1c). The results indicated that mRNA expression of PRKAR1A/PKAI was increased, whereas PRKAR2B/PKAII was downregulated in human CCA tissues. However, there was no correlation between PRKAR1A, PRKAR2B and their ratio with clinicopathological as well as survival data (Table 1).

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Figure 1. Expression of PKA regulatory subunits mRNA in human CCA tissues. (a) PRKAR1A mRNA, (b) PRKAR2B mRNA and (c) PRKAR1A/PRKAR2B ratio in malignant and adjacent nontumorous tissues of CCA was determined by real-time PCR as described in Material and methods section. Fold change of gene expression in tumor and adjacent nontumor tissues was calibrated with normal liver tissues. *p < 0.05 compared to adjacent nontumor tissues.

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Table 1. Pathological features of CCA patients and PRKAR1A/PRKAR2B mRNA ratio in primary tumor tissues
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Expression of PKA protein subunits in human CCA tissues

Consecutive slides of five normal liver controls from cadaveric donors showed a strong immunopositivity for PRKAR2B subunit in normal bile duct epithelia as well as hepatocytes, whereas weak immunoreactivity was detected for PRKAR1A (Fig. 2a). Conversely, most of the CCA samples (89%) showed a strong immune reactivity for PRKAR1A subunit, whereas PRKAR2B was weakly detected (Fig. 2a). Moreover, there was an increased PRKAR1A expression in biliary cells at the transition zone where normal bile duct cells are adjacent to neoplastic cells (Fig. 2b). No correlation was observed in expression of regulatory subunits with clinicopathological or survival data (Table 2).

Table 2. Pathological features of CCA patients, and expression of PRKAR1A protein in primary tumor tissues
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Expression of PKA mRNA and protein subunits in CCA cell lines

For the CCA cell lines, M156, OCA17, KKU100 and M214, real-time PCR showed high PRKAR1A/PRKAR2B mRNA ratio (Fig. 3a). In accordance with mRNA level, Western blot analysis demonstrated the strong expression of PRKAR1A in all CCA cell lines studied, whereas no expression of PRKAR2B was detected (Fig. 3b). We selected for further investigations M156 and OCA17 as representative of nonpapillary- and papillary-type CCA, respectively. These tumor subtypes exhibit different prognosis and survival. 13

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Figure 2. Expression of PKA regulatory subunits protein in human CCA tissues. (a) Immunohistochemistry (IHC) staining for the PRKAR1A and PRKAR2B was performed in normal liver (a′ and b′) and human cholangiocarcinoma tissues, with representative examples of different histopathological subtypes as poorly differentiated- (c′ and d′), well differentiated- (e′ and f′) and papillary (g′ and h′)-type CCA, respectively. The black arrow indicated normal cholangiocyte. (b) IHC staining for PRKAR1A protein in normal biliary cell being transformed to neoplastic cell (transitional zone). Digital photomicrographs were obtained with Carl Zeiss Axio Scope.A1 microscopy. An original magnification is 40× for a′ to f′ whereas 10× for g′, h′ and b. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 3. Expression of PKA regulatory subunits mRNA and protein in CCA cell lines. (a) The relative PRKAR1A/PRKAR2B mRNA ratio and (b) protein level of PRKAR1A and PRKAR2B in human CCA cell lines, as determined by real-time PCR and Western blot analysis, respectively. F9 (mouse embryonic carcinoma cell line) whole cell lysate was used as a positive control for PRKAR2B antibody.

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PRKAR1A knockdown reduces growth of CCA cells

The contribution of PRKAR1A/PKA in CCA cell proliferation was further confirmed on both M156 and OCA17 cell lines. The near-total abrogation of PRKAR1A protein (Fig. 4a), which leads to a decrease of PKA activity (Figs. 4b and 4c), was associated with significant inhibition of proliferation in both cell lines (Fig. 4d). In addition, flow cytometry demonstrated the increase in accumulation of cells at G0/G1 phase on PRKAR1A silencing (Fig. 4e). Moreover, the downregulation of PRKAR1A expression appears to have resulted in decreased expression of cell cycle regulation proteins including cyclin D1 and CDK4 (Fig. 4f), which are known to play important roles in the G1/S transition.

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Figure 4. PRKAR1A knockdown reduces the growth of CCA cells. (a) Western blot analysis of M156 and OCA17 cell lines after transfection with a stable PRKAR1A knockdown using lentiviral transfection compared to their empty viral transfection control. (b) PKA activity in PRKAR1A silencing M156 and OCA17 determined by nonradioactive PKA activity assay and (c) the gel image was analyzed and quantified using ImageQuant™ Imager compared to their controls. Cell proliferation (d) and cell cycle distribution (e) were determined using alamarBlue® assay and flow cytometry, respectively. (f) Western blot analysis of cell cycle regulation proteins including cyclin D1 and cdk4, respectively. Data in (c), (d), and (e) are mean ± SD of three independent experiments. *p < 0.05 compared to control cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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PRKAR1A knockdown enhances apoptosis induction

To test if PRKAR1A knockdown inhibited cell growth at least in part by inducing apoptosis, we tested if mediators of apoptosis, caspase 3/7, were induced. As shown in Figure 5a, decreased PRKAR1A protein expression with slightly increased caspase 3/7 activity were observed in both cell lines. Moreover, flow cytometry using Annexin-V-FLUOS staining confirmed the apoptosis induction by PRKAR1A abrogation (Figs. 5b and 5c). We also examined the effect of PRKAR1A knockdown on the expression of antiapoptotic, Bcl-2 and Bcl-X proteins as well as proapoptotic proteins, Bak and Bax (Fig. 5d). Induction of Bcl-2 expression was seen in M156 cell line but not in OCA17 cell line, whereas no expression of Bcl-X was found in both lines (data not shown). Conversely, Bax protein was markedly upregulated in M156 cell line while increased Bak protein expression was observed in OCA17 cell line. The data confirmed the above finding that apoptosis induction by silencing PRKAR1A expression also contributes to CCA cell growth inhibition.

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Figure 5. Specific PRKAR1A knockdown enhances apoptosis induction of CCA cell lines. (a) Caspase 3/7 activity assay and (b) flow cytometry using Annexin-V-FLUOS staining were used to determined apoptosis induction in stable PRKAR1A knockdown CCA cell lines. (c) Fold change in apoptosis induction is compared to their empty viral transfection controls. (d) Western blot analysis of apoptotic protein expression in CCA cell lines. Data in (a) and (c) are mean ± SD of two independent experiments. *p < 0.05 compared to control cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Profiling of kinases and phosphorylation of their protein substrates in stable PRKAR1A silencing CCA cell lines

We hypothesized that PRKAR1A/PKAI contributes to the growth of CCA cells. To identify the cellular signaling network activated by PRKAR1A/PKAI, we investigated the profile of kinases and phosphorylation of their protein substrate in M156 and OCA17. We compared stable PRKAR1A silencing to an empty viral transduction control for each cell line. In M156 cell line (Figs. 6a and 6c), the results of a phospho-kinase array analysis showed a decrease in the MAPKs signaling cascade's phosphorylation, including MEK1/2, ERK1/2, p38α, MSK1/2, RSK1/2/3 and HSP27 as well as the reduction of Akt (S473) and β-catenin phosphorylation. Phosphorylated cAMP response element binding protein (CREB), which is responsible for modulating effects of protein kinase A, was not significantly reduced. Conversely, an induction of Akt (T308), Src, c-Jun, STAT4, Chk-2 and e-Nos phosphorylation was found. In OCA17 cell lines (Figs. 6b and 6c), a reduction of MAPKs (MEK1/2, ERK1/2, p38α, MSK1/2 and RSK1/2/3), CREB, Src, Akt (Akt, TOR and p70 S6 kinase), JAK-STAT (STAT2, STAT3 and STAT6) phosphorylation was observed. In addition, a decreased in 5′ adenosine monophosphate-activated protein kinase (AMPKα1 and AMPKα2), which plays a role in cellular energy homeostasis was seen. As similar as M156 cell line, there was an induction of c-Jun and e-Nos phophorylation in OCA17 cell line. The results indicated that PRKAR1A/PKA may control CCA cell growth mainly via MAPKs as well as in crosstalk with the PI3K/AKT, JAK/STAT and Wnt/β-catenin signaling pathways.

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Figure 6. PRKAR1A/PKAI contribution in CCA cell growth and survival is associated with multipathway signaling. The phospho-kinase array detects phosphorylated proteins in PRKAR1A silencing in (a) M156 and (b) OCA17 compared to empty viral transfection control of each cell line. The lower panels are the profiles created by quantifying the mean spot pixel densities. Array signal from scanned X-ray film images were analyzed using ImageQuant™ Imager. (c) Western blot analysis confirmed the decrease in phosphorylation status of Erk1/2 (T202/Y204) and Akt (S473), respectively.

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Small molecule inhibitors of PKA inhibited CCA cells growth

We examined if specific PKA inhibitors, H89 and cAMP analogs (8-Cl cAMP and 8-Br cAMP), would inhibit CCA cells' proliferation. Four CCA cell lines (M156, OCA17, KKU100 and M214) were treated with a range of concentration of the small molecules and assessed for proliferation. The results showed that PKA inhibitor, H-89 inhibited CCA cell lines' proliferation in a dose-dependent manner (Fig. 7a). However, the cAMP analogs, 8-Cl cAMP and 8-Br cAMP, slightly inhibited CCA cell growth when treated with 10 μM of drugs (Figs. 7b and 7c), while 100 μM concentration of both compounds exhibited 10–20% cell growth inhibition in all the four CCA cell lines (data not shown). This supports the siRNA experiments, which revealed near-total suppressed PRKAR1A expression also inhibited about 10–20% of CCA cell's proliferation (Fig. 4b).

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Figure 7. The PKA inhibitor and cAMP analogs reduces CCA cell's proliferation. Four CCA cell lines were treated with H89 (a), 8-Cl cAMP (b) and 8-Cl cAMP (c) with the indicated concentration, and cell proliferation was assessed using alamarBlue® assay. Data are represented as mean ± SD of three independent experiments.*p < 0.05 compared to untreated cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The involvement of PKA pathway in neoplastic transformation and tumor growth is implicated in many types of cancer. We have found the overexpression of PRKAR1A in the tumor tissues of O. viverrini and NDMA-induced hamster CCA 11 as well as PKA isozyme switching and PRKAR1A/PKAI pathway might contribute to the induction of biliary cells' transformation and proliferation in O. viverrini and NDMA-induced progressive cholangiocarcinogenesis. Others have reported that PKA isozyme switching, with dramatic changes in the proportion of the two PKA regulatory subunits PRKAR1A and PRKAR2B, has been associated with many tumor types, a poorer prognosis, and as a target for therapy. 6–10 A recent study indicated that high expression of PRKAR1A was associated with a diminished response to radiation therapy in prostate cancer, suggesting that such patients would benefit from therapy that reduces PRKAR1A levels. 14 This has led us to investigate the pattern of expression of the different subtypes of PKA subunits in human cholangiocarcinoma as well as to elucidate the intracellular signaling of PKA and identify their potential role in the control of CCA cell proliferation.

Our study finds that human CCA is characterized by the altered ratio of expression for the two PKA regulatory subunits, PRKAR1A and PRKAR2B. Real-time PCR showed a significantly higher level of PRKAR1A mRNA expression in tumor tissues than that in adjacent nonmalignant tissues and vice versa in case of PRKAR2B mRNA. Moreover, the PRKAR1A/PRKAR2B ratio is also significantly higher in tumor than that of nontumor tissues. The results indicated that the mRNA expression of PRKAR1A/PKAI was increased, whereas PRKAR2B/PKAII was downregulated in human CCA tissues. In addition, the increased PRKAR1A/PRKAR2B mRNA ratio was also found in all CCA cell lines studied.

IHC demonstrated a strong immunoreactivity for the PRKAR1A protein in all human CCA included in our study, as well as in the four human CCA cell lines investigated as shown by Western blot analysis. In contrast, normal biliary and hepatocyte weakly expressed PRKAR1A subunit, whereas stronger expression of PRKAR2B was observed. No PRKAR2B expression was detected in all CCA cell lines. Moreover, one particular slide showed the increased PRKAR1A expression in biliary cells at the transition zone where normal bile duct cells transitioned to neoplastic cells. These data indicated a high PRKAR1A/PRKAR2B ratio in CCA when compared to the normal counterpart, consistent with previous data from a variety of human cancer cell lines where transformation coincides with a sharp increase in PRKAR1A. 8, 15 However, no correlation was observed in regulatory subunits' expression neither at mRNA nor protein level with clinical, pathological or survival data.

To confirm the contribution of PRKAR1A/PKA in CCA cell growth, we have performed cell proliferation assay and found a significant growth reduction in both CCA cell lines transduced with siRNA directed against PRKAR1A as compared to those transduced with empty viral control. Moreover, this appears to lead to an increased number of cells accumulating in the G0/G1 phase, which is consistent with the decrease expression of G1–S transition regulatory proteins such as cyclin D1 and cdk4. Our results are consistent with the previous studies that suppressed PRKAR1A expression by antisense oligodeoxynucleotides and produced growth inhibition in human cancer cells both in vitro and in vivo. 15–20 We also found that PRKAR1A silencing slightly induced CCA cell apoptosis as indicated by phenotype, caspase 3/7 activity, annexin V positive staining as well as modulation of Bcl-2 family protein expression.

We further sought the interrelation and crosstalk between PRKAR1A/PKA and the other intracellular signaling pathways. We have demonstrated that PRKAR1A silence leads to decrease in PKA activity, with an associated decrease in MAPKs, PI3K/Akt, JAK/STAT and Wnt/β-catenin pathway signaling, which revealed the crosstalk between PKA and those particular involving pathways. Our kinase antibody array analysis indicated that all three major families of MAPKs and their downstream signaling molecules were reduced. Indeed, the control of cell proliferation by a signal transduction network between the PKA and MAPKs pathways in many cell types including normal biliary epithelium is largely known. 21 Our study demonstrated that cAMP stimulates the secretory and proliferative capacity of rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway. 21 Here we demonstrated that the interplay between PKA and MAPKs pathways might also contribute to CCA cell growth and survival. Moreover, CREB phosphorylation was reduced in the OCA17 cell line, whereas no change was observed in the M156 cell line. However, CREB can be activated by protein kinase C signaling pathway. 22

In addition, the decrease of the kinase and substrate proteins of the PI3K/AKT, JAK/STAT and Wnt/β-catenin pathways was found secondary to PRKAR1A silencing in CCA cell lines. This indicates for the first time the interaction between PKA and those signaling pathways in regulating CCA cells growth and survival. The serine/threonine kinase Akt (or protein kinase B) controls key cellular processes such as glucose metabolism, cell cycle progression, and apoptosis, 23 and activated-Akt can contribute to tumorigenesis in vivo in lymphoid, breast, ovarian, prostate, brain tissues and CCA. 24, 25 There was a reduction of Akt phosphorylation at both sites (S473 and T308) in OCA17 cell line, whereas decreased phosphorylation at S473 but increased at T308 in M156 cell line. Full activation of Akt requires phosphorylation at two sites. 26 Moreover, reduction of kinase phosphorylation in the Akt downstream targets, TOR and p70 S6, was seen in the OCA17 cell line. Our study demonstrated that disturbing PKA signaling pathway caused decrease of PI3K/AKT activation in CCA cell lines.

The JAK/STAT signaling participates in the regulation of cell proliferation, differentiation, survival, motility and apoptosis in different organs including liver. 27 During the last decades, the data on expression and functions of cytokine/growth factor receptors in different liver cell types (hepatocytes, cholangiocytes, Kupffer and stellate cells) have rapidly grown. It highlights the importance of JAK-STAT signaling in normal liver physiology and pathology. Moreover, deregulation of this pathway is closely associated with tumorigenesis. Although the role of the JAK-STAT cascade is poorly deciphered both in normal cholangiocytes and in CCA cells, a number of papers have been published the role of prolactin (Prl) and prolactin receptor (PrlR) in the regulation of cholangiocyte either in normal and obstructive cholestasis conditions which seems to be mediated by the activation of the JAK/STAT cascade. 28, 29 In addition, IL-6 signaling was aberrant in CCA cells with prolonged and sustained STAT-3 phosphorylation; a mechanism likely accounting for upregulation of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) expression which leads to cancer resistance via the tumor necrosis factor-related apoptosis-inducing ligand. 30, 31 In our study, there was decreased phosphorylation of STAT2, STAT3 and STAT6 in the OCA17 cell line but increased phosphorylation of STAT4 in the M156 cell line. As there are a lot of members belonging to the STAT protein family, the exact role of the JAK-STAT pathway should be further investigated in CCA.

Phosphorylation of β-catenin, the chief downstream effector of the canonical Wnt signaling pathway, was downregulated in M156, whereas no change was found in OCA17 cell line indicating activation of different signaling pathways in various subtypes of CCA, which exhibit different prognosis and survival. 13 Recent evidences also suggest the alteration of Wnt/β-catenin pathway is implicated in cholangiocarcinogenesis. 32–35

We also investigated if the small molecule inhibitors of PKA would inhibit CCA growth. Proliferation of CCA cell lines was significantly suppressed in the presence of the small molecule inhibitor of PKA, the isoquinoline H89 in a dose-dependent manner. However, others have demonstrated that H89 can inhibit other kinases and have a relatively large number of PKA-independent effects, 36, 37 therefore off-target effects of the H89 on the growth inhibition of CCA cells cannot be excluded. In addition, the site-selective cAMP analogs, 8-Cl cAMP and 8-Br cAMP, also showed a slight growth inhibitory effect; however, high concentrations of drugs were needed. 8-Cl cAMP has completed several phase I clinical studies and recently entered phase II as an anticancer agent. 10 The above studies together with our in vitro results indicate that PKA is a potential therapeutic target in CCA.

In conclusion, our data demonstrates that overexpression of PRKAR1A, with a consequential switch to a high PRKAR1A/PRKAR2B ratio, is a common occurrence in CCA and provides the signaling pathway by which PRKAR1A/PKA is involved in cholangiocarcinogeneis. This work also indicates that abrogation of PRKAR1A expression induce growth inhibition and apoptosis of CCA cells. Importantly, we showed that a PKA inhibitor as well as cAMP analogs potentiate the growth inhibitory effect of antineoplastic drugs and should be further investigated as a therapeutic strategy against CCA, either as single-drug or in combination with other antitumor drugs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by the Thailand Research Fund (Grant No. MRG4980174) to W.L., the grant from Liver Fluke and Cholangiocarcinoma Research Center to S.J. and P.Y., the grant of Faculty of Medicine (Grant No. i50121), the Virginia & D.K. Ludwig Fund for Cancer Research, Khon Kaen University (Grant No. 50-03-1-01-007 and 51-03-1-07-001) to N.N., and the Research Strengthening Grant from BIOTEC-NSTDA, Thailand.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References