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Department of Digestive Medicine, Second Affiliated Hospital, Anhui Medical College, Anhui 230000, ChinaDepartment of Hepatobiliary Medicine, Shanghai Eastern Hepatobiliary Surgery Hospital, Naval Medical University, Shanghai 200438, China
Recently, some studies have suggested a link between AQP1 and cancer progression.
Aims
The aim of the present study was to investigate the influence of AQP1 on the clinicopathology and prognosis of intrahepatic cholangiocarcinoma (ICC) patients.
Methods
We retrospectively detected the expression of AQP1 protein in 307 patients with ICC who underwent partial hepatectomy. Western blot analysis was used to detect AQP1 protein levels in stable AQP1 overexpression and knockdown cell lines. The influence of AQP1 on the invasion and metastasis ability of ICC cells was assessed by wound-healing and Transwell assays in vitro as well as by a splenic liver metastasis model in vivo.
Results
Positive membranous AQP1 expression was identified in 34.2% (105/307) of the ICC specimens. Survival data revealed that positive AQP1 expression was significantly associated with favourable disease-free survival (DFS) and overall survival (OS) (p = 0.0290 and p = 0003, respectively). Moreover, high AQP1 expression inhibited the invasion and migration of ICC cells in vitro as well as inhibited liver metastasis in nude mice. Mechanistically, high AQP1 expression in ICC cells increased the levels of E-cadherin but decreased the levels of the Snail transcription factor.
Conclusions
AQP1 expression is associated with a favourable prognosis in ICC patients. AQP1 inhibits ICC cell invasion, metastasis, and epithelial-mesenchymal transition (EMT) through downregulation of Snail expression.
Although intrahepatic cholangiocarcinoma (ICC) occurs more rarely than hepatocellular carcinoma (HCC), it is the second most common primary hepatic malignancy after HCC [
. Currently, liver resection is considered to be the preferred treatment option for ICC, offering the possibility of disease cure. Unfortunately, compared to HCC, ICC is generally less resectable and curable because only a minority of patients are considered ideal candidates for curative resection.. The overall prognosis of ICC is generally poor because most ICC patients are typically diagnosed with advanced stages and miss the opportunity for surgery. Moreover, due to the highly aggressive biological behaviour of ICC, early recurrence and/or local or distant metastasis occurred in most of ICC patients after surgery. In addition, conventional chemotherapy with gemcitabine plus cisplatin is not effective in improving the long-term survival of ICC patients. Fibroblast growth factor receptor (FGFR) and isocitrate dehydrogenase (IDH) 1 inhibitors have recently been studied in ICC with encouraging results [
. Therefore, there is an ongoing demand for new biomarkers as possible sources of novel therapeutic targets for ICC.
Aquaporins (AQPs) are transmembrane proteins that weigh between 28 and 30 kDa that efficiently and selectively transport water and small solutes across cell membranes. AQPs play a vital role in fluid homoeostasis, bile formation and the maintenance of physiological functions of various organs. To date, 13 AQPs (AQP0–12) have been identified in mammals [
. Some recent studies have shown a close relationship between AQP1 and multiple human cancers, such as colon cancer, gastric cancer, lung cancer, prostate cancer, breast cancer, nasopharyngeal cancer, ovarian cancer and extrahepatic cholangiocarcinoma [
]. The expression of AQP1 is significantly increased in these cancer tissues compared to paracancerous or normal tissues. Moreover, the expression of AQP1 has been reported to correlate with poor prognosis and clinical characteristics, such as high histological grade, vascular invasion and lymph node metastasis [
In ICC, however, Aishima et al. found that large tumour sizes (>40 mm) and poorly differentiated tumours have a significantly higher proportion of AQP1-negative than AQP1-positive tumours. High AQP-1 expression is negatively associated with lymph node metastasis and is an independent prognostic factor according to multivariate survival analysis, and downregulation of AQP 1 is associated with aggressive progression of ICC [
]. The molecular mechanism underlying the biological function of AQP1 in ICC is still unclear. Therefore, the aim of the present study was to elucidate the effect of AQP1 on the clinicopathology and prognosis of ICC patients in a large cohort to elucidate the potential molecular mechanisms by which AQP1 regulates the biological behaviour of ICC cells, thereby providing potential novel therapeutic targets for ICC.
2. Materials and methods
2.1 Patients, samples and clinicopathological data
The present study included a retrospective cohort of 307 patients with ICC who primarily underwent their first surgical resection at Eastern Hepatobiliary Surgery Hospital (EHBH), Naval Medical University, Shanghai, China, between January 2008 and June 2010. All ICC patients had AQP1 protein expression data measured by immunohistochemistry. The demographics and clinicopathologic information were retrospectively obtained from the patients’ medical records. Informed consent was obtained from each patient before surgery for the use of their data for research, and the project was approved by the EHBH Ethical Committee, China. The data do not contain any information that could identify the patients.
2.2 Cell culture and transfection
CCLP1, TFK-1, HCCC9810, RBE, HUCCT1 and SK-CHK-1 cells (Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% antibiotic/antifungal solution (Sigma‒Aldrich, St. Louis, MO, USA). All cells were cultured in a 37 °C incubator containing humidified air with 5% CO2.
2.3 Lentivirus infection
Lentiviruses expressing AQP1 shRNA, AQP1 overexpression and negative control were constructed by Genechem Co., Ltd. (Shanghai, China). CCLP1 and HUCCT1 cells were infected with the lentiviruses at a multiplicity of infection (MOI) of 20 and with 6 µg/mL polybrene for 8 h.
2.4 RNA collection, cDNA synthesis, and real-time PCR analysis
Total RNA was extracted from cell lines using TRIzol (Invitrogen), and cDNA was synthesized using random hexamers (Roche) and SuperScript II Reverse Transcriptase (Invitrogen). PCR amplification was performed in a buffer containing 2 μl of cDNA, 1 × PCR buffer, 1.5 mmol/L MgCl2, 0.8 mmol/L deoxynucleotide triphosphatase, 0.2 μmol/L each primer and 1 unit of Taq DNA polymerase (Roche, Pleasanton, CA, USA). qRT-PCR was performed using the ABI 7900 Fast Real-Time PCR System (Applied Biosystems) and a SYBR Green PCR kit (Takara Bio Inc.). The primer sequences used for PCR were as follows: 18S, 5′-GCCCTACCCACAAAGCCT-3′ and 5′-GTGGCTTTCTGTTGCTGTTTCA-3′; FN1, 5′-GAGAATGGACCTGCAAGCCCA-3′ and 5′-AGTGCAAGTGAT-GCGTCCGC-3′; E-cadherin, 5′-TTGCACCGGTCGACAAAGGAC-3′ and 5′-TGGATTCCAGAAACGGAGGCC-3′; Snail, 5′-CTGGGTGCCC-TCAAGATGCA-3′ and 5′-CCGGACATGGCCTTGTAGCA-3′; and AQP1, 5′-CTGGGCATCGAGATCATCGG-3′ and 5′-ATCCCACAGCCAGT-GTAGTCA-3′.
2.5 Western blotting assay
Whole cell extracts were lysed with RIPA buffer (50 mmol/L Tris, pH 7.4; 150 mmol/L NaCl, 1% NP-40; 0.1% SDS; and 0.5% sodium deoxycholate) supplemented with a mixture of protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (Sigma‒Aldrich, St. Louis, MO, USA), and lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA) was used to measure protein concentrations. Equal amounts of protein were separated by SDS‒PAGE in TG buffer, transferred to nitrocellulose (NC) membranes (Millipore, Burlington, MA, USA), incubated with specific primary antibodies and incubated with fluorescein-coupled secondary antibodies (IRDye® 800CW Goat anti-Rabbit IgG, LI-COR, 925–32,211), which were then detected by the Odyssey CLx Imaging System (LI-COR). The anti-Snail antibody was purchased from Cell signaling Technology, and the anti-E-cadherin, anti-actin, anti-FN1, anti-vimentin and anti-AQP1 antibodies were purchased from Proteintech.
2.6 Immunohistochemistry
Tumour tissue specimens from mice and patients were fixed in formalin for 12 h, dehydrated, embedded in paraffin and cut into 4-µm sections. Tissue sections were dewaxed, rehydrated and immersed in methanol containing 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. The sections were then heated in a pressure cooker containing 10 mM ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) for 2 min. After cooling, the sections were incubated in 1% blocking serum for 30 min to reduce nonspecific binding. The sections were then incubated with the primary anti-AQP1 polyclonal antibody (1:250) overnight at 4 °C. The sections were then incubated with the biotinylated secondary antiserum followed by horseradish peroxidase-conjugated streptavidin-biotin complex. Finally, the sections were developed with diaminobenzidine (DAB) and restained with haematoxylin.
According to the German Immune Response Score, the immune response score (IRS) evaluates stained sections in an uninformed manner without prior clinical information. Briefly, the IRS assigns subscores to the immune response distribution (0–4) and intensity (0–3), which are then multiplied to produce an IRS score. The percentage of positive cells was scored as follows: 0, <5%); 1, 5–25%; 2, 25–50%; 3, 50–75%; and 4, > 75%. Staining intensity was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The final AQP1 expression score was calculated using the value of the percentage positivity score plus the staining intensity score, ranging from 0 to 12. We estimated the IRS by averaging the values in eight fields of view for each sample at 400x magnification. AQP1 expression was defined as follows: low expression (score 0–6/0–3) and high expression (score > 6/4–6). Immunohistochemical analysis and scoring were performed by two independent investigators.
2.7 Mice and in vivo study
All animal procedures were approved by the Institutional Animal Care and Use Committee of the Naval Medical University (Shanghai, China). Eight-week-old male nude mice were purchased from SLAC Ltd. (Shanghai, China) and were housed under standard conditions according to the requirements of the Naval Medical University Animal Care Facility and the NIH guidelines. Approximately 1 × 107 HUCCT1-NC, HUCCT1-ovAQP1 and HUCCT1-shAQP1 cells were suspended in 0.2 ml of PBS and injected into the lungs of mice via the tail vein. The mice were euthanized after 90 days, and the lungs were analysed by immunohistochemical staining.
2.8 Cell migration and assay
CCLP1 and HUCCT1 cells were seeded into 6-well plates and grown to confluence. Linear wounds were gently scored using the tip of a 200-µl sterile yellow pipette. After rinsing with PBS to remove debris, the cells were incubated with serum-free medium at 37 °C and 5% CO2 for 24 h. Images of the wound area were obtained using phase contrast microscopy, and the images were analysed to quantify migration. The wound closure for each sample was quantified as the area covered by cells over a 24-hour period.
Diluted basal matrix gel (BD Biosciences, Franklin Lakes, NJ, USA) was added to the inner chamber of the Transwell (Corning, NY, USA, 3421) and polymerized at 37 °C for 30 min. Cells (1 × 104) in 500 µL of serum-free DMEM were added to Transwell chambers, either pretreated with polymerized gel or left untreated, in a 24-well plate, and 700 µl of medium containing 10% foetal bovine serum was added to the lower chamber. After 24 h, the cells were removed from the upper surface of the membrane with a cotton swab, fixed with 10% formaldehyde for 10 min, stained with 1% crystalline violet for 30 min and observed by microscopy. Each experiment was repeated three times.
2.9 Statistical analysis
Disease-free survival (DFS) was measured from the date of surgery to the date of recurrence. Overall survival (OS) time was defined as the period from the date of hepatectomy to the date of death. Follow-up of patients was continued until death or June 1, 2022, whichever occurred first. Statistical analyses were performed using SPSS (version 26.0 for Windows; SPSS Inc., Chicago, IL, USA). Qualitative variables were analysed using the Pearson χ2 test or Fisher's exact test. OS and DFS were calculated using the Kaplan–Meier method and Life Table method. A comparison between groups was conducted using Student's t-test and the Mann‒Whitney test. A value of p<0.05 was considered statistically significant.
3. Results
3.1 Demographic characteristics and clinical outcomes
The demographic, biochemical and pathological data of 307 patients with ICC are shown in Table 1. The median age was 56.0 years old (range, 26–82 years old). There was a predominance of males with a male-to-female ratio of 2.1:1. Of the 307 patients, 143 patients (46.58%) were seropositive for HBsAg (hepatitis B surface of antigen), 98 patients (31.92%) had cirrhosis, 20 patients (6.51%) had hepatolithiasis and 19 patients (6.19%) had liver schistosomiasis. The tumour stage distribution according to the 8th edition of the AJCC/UICC staging system was as follows: stage I, 118 patients (38.43%); stage II, 92 patients (29.97%); stage III, 55 patients (17.92%); and stage IV, 42 patients (13.68%).
Table 1Clinicopathological features of ICC patients according to the expression of AQP1.
The median follow-up time was 12.6 months (range, 0.6 to 172.3 months) for all patients. Recurrence occurred in 190 patients (228 patients (74.3%) had available data about recurrence). The median DFS was 5.2 months (range, 0.7 to 69.8 months), and the postoperative 1-, 3- and 5-year DFS rates were 31.1%, 18.4% and 6.6%, respectively. The median survival time was 12.6 months (range, 0.6 to 172.3 months), and the cumulative 1-, 3- and 5-year survival rates were 51.8%, 27.0% and 16.9%, respectively (Fig. 1A).
Fig. 1AQP1 is differentially expressed in ICC samples and low AQP1 expression correlates with aggressive clinicopathological features.
A: Overall survival (OS) and disease-free survival (DFS) in 307 ICC patients from Eastern Hospital for Hepatobiliary Surgery (EHBH). B, C: A total of 307 cases of ICC tissue were obtained, and representative images of AQP1 staining in ICC immunohistochemistry are shown. C: Quantification map of AQP1 expression. D: OS according to AQP1 expression. E: DFS according to AQP1 expression.
3.2 Association of AQP1 protein expression with clinicopathological features and survival of ICC patients
Positive membranous AQP1 expression was identified in 34.2% (105/307) of the patients (Fig. 1B). Kaplan‒Meier analysis revealed that positive AQP1 expression was significantly associated with favourable DFS and OS (p = 0.0290 and p = 0.0003, respectively) (Fig. 1C, 1D). The association between AQP1 expression and the clinicopathological characteristics of ICC patients was analysed. Compared to patients in group I (AQP1-positive expression), patients in group II (AQP1-negative expression) had a lower proportion of ALT>41 U/L (p = 0.027), AST>37 U/L (p = 0.004), r-GT>61 U/L (p = 0.002), CEA>10 µg/L (p = 0.017), poor tumour differentiation (p = 0.003), microvascular invasion (p = 0.006), local extrahepatic metastasis (p = 0.027) and high level staging (p = 0.026) (Table 1). Survival analysis also demonstrated that AST>37 U/L, r-GT>61 U/L, CEA>10 µg/L, microvascular invasion, local extrahepatic metastasis and high-level TNM staging were significant prognostic factors for the poor survival rates of ICC patients (p<0.05) (Supplementary Figure 1).
3.3 AQP1 suppresses cell migration and invasion in vitro
To elucidate the role of AQP1 in ICC metastasis and invasion, we stably overexpressed or knocked down AQP1 in CCLP1 and HUCCT1 cells. The wound-healing assays showed that silencing AQP1 promoted cell migration ability but that overexpression of AQP1 inhibited tumour migration ability. Transwell assays using CCLP1 and HUCCT1 cells showed that knockdown of AQP1 increased the metastasis of ICC cells but that overexpression of AQP1 inhibited migratory capacity (Fig. 2A-E). These results suggested that AQP1 inhibits the migration and invasion of ICC cells in vitro.
Fig. 2AQP1 suppresses cell migration and invasion in vitro.
A-D: Wound-healing assays indicated that knockdown of AQP1 increased the migration ability of CCLP1 and HUCCT1 cells. The quantification of cell migration is presented in histograms B and D. E-G: Differences in the migration abilities of AQP1-deficient and -overexpressing CCLP1 or HUCCT1 cells as shown in representative images (E) and histogram quantification (F and G). H-J: Invasion assays indicated that knockdown of AQP1 increased CCLP1 and HUCCT1 cell invasion as shown in representative images (H) and histograms quantification (I and J). The quantification of cell invasion is presented in the column chart. K: Representative images and quantification of lung metastases derived from HUTTT1-shAQP1 and HUCCT1-NC cells by tail vein injection. Arrows indicate the tumours. Representative images of tumour pulmonary metastases with haematoxylin & eosin (H&E) staining. The results are representative of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001.
To confirm the role of AQP1 in cell metastasis and invasion in vivo, we established a model of lung metastasis by injecting tumour cells (HUTTT1-shAQP1 and HUCCT1-NC) into nude mice via the tail vein. The number of lung metastases was significantly increased in the HUCCT1-shAQP1 group compared to the HUTTT1-NC group 90 days after injection (Fig. 2F). These results indicated that AQP1 deficiency increases ICC metastasis.
3.4 AQP1 inhibits EMT through inhibition of Snail expression
According to the above results, we speculated that AQP1 may regulate tumour metastasis and invasion through EMT. To test this hypothesis, we investigated the expression level of AQP1 in ICC cell lines by western blot analysis, which demonstrated that AQP1 was highly expressed in most cholangiocarcinoma cell lines (Fig. 3A, B). Therefore, we selected CCLP1 and HUCCT1 cells to investigate the specific role of AQP1 in the EMT process. AQP1 silencing elevated the expression levels of two classical mesenchymal markers of EMT, namely, vimentin and fibronectin 1 (FN1), but suppressed the expression levels of the E-cadherin epithelial cell marker (Fig. 3C). Consistently, overexpression of AQP1 yielded the opposite experimental results, indicating that AQP1 inhibits EMT. Interestingly, AQP1 overexpression significantly decreased Snail protein levels. Subsequently, we examined the transcriptional regulation of FN1, E-cadherin, vimentin and Snail by AQP1. qRT‒PCR analysis demonstrated that knockdown of AQP1 increased FN1 and vimentin mRNA levels but downregulated E-cadherin mRNA levels. Correspondingly, overexpression of AQP1 significantly increased epithelial markers but decreased Snail mRNA levels (Fig. 3D, E). We next examined the expression levels of AQP1, E-cadherin, vimentin and Snail by immunohistochemistry in the tumour tissues of ICC patients. The expression levels of E-cadherin were higher in the AQP1-positive expression group than in the AQP1-negative expression group, and the expression levels of vimentin and snail were lower in the AQP1-positive expression group than in the AQP1-negative expression group (Fig. 3F). Thus, these results indicated that AQP1 inhibits EMT by suppressing the expression of the Snail transcription factor.
Fig. 3AQP1 inhibits EMT through inhibition of Snail expression.
A and B: Western blot analysis of the expression of AQP1 in CCLP1, TFK-1, HCCC9810, RBE, HUCCT1 and SK-CHK-1 ICC cell lines. Histogram quantification shown in B. C-E: CCLP1 or HUCCT1 cells were infected with lentiviruses expressing AQP1 shRNA, AQP1 overexpression or negative control, and the interference and overexpression efficiencies were determined by Western blot and qRT‒PCR analyses. Western blot and qRT‒PCR analyses were used to detect the expression of AQP1, fibronectin 1, vimentin, E-cadherin and Snail in AQP1-deficient and -overexpressing CCLP1 and HUCCT1 cells. F: The expression levels of AQP1, E-cadherin, vimentin and Snail in the tumour tissues of ICC patients were measured by immunohistochemistry. *P<0.05, **P<0.01 and ***P<0.001.
According to the relevant literature, AQPs are a family of small-molecule membrane proteins whose primary function is to facilitate the passive transport of water across the cytoplasmic membrane in response to osmotic gradients generated by active solute transport [
]. According to the relevant literature, AQPs are a family of small-molecule membrane proteins whose primary function is to facilitate the passive transport of water across the cytoplasmic membrane in response to osmotic gradients generated by active solute transport [
. However, the present study showed that patients with high AQP1 expression had significantly higher overall survival and disease-free survival than those with low AQP1 expression. Low AQP1 expression was associated with microvascular invasion, lymphatic metastasis, extrahepatic metastasis and TNM stage features. Silencing AQP1 promoted the metastasis and invasion of ICC cells, suggesting that AQP1 is involved in the malignant biological behaviour of ICC. These results revealed that AQP1 is involved in the determination of postoperative survival time in ICC patients, thereby indicating that AQP1 a molecular marker of prognosis in ICC patients.
According to the current literature, high expression of AQP1 plays a role in promoting tumour growth or metastasis in breast cancer, osteosarcoma, colorectal cancer and lung cancer [
Combined pharmacological administration of AQP1 ion channel blocker AqB011 and water channel blocker Bacopaside II amplifies inhibition of colon cancer cell migration.
, but high expression of AQP1 played an inhibitory role in the metastasis of ICC in the present study. Notably, there are only a few studies that have clearly demonstrated that high AQP1 expression plays a prognostic role in ICC patients [
]. Thus, further studies are needed to determine why AQP1 exhibits different biological behaviours in different tumour types.
EMT is a cellular phenotype transition that promotes embryonic development and tumour progression, and EMT is a process in which epithelial cells lose their connectivity and polarity [
Combined snail and E-cadherin predicts overall survival of cervical carcinoma patients: comparison among various epithelial-mesenchymal transition proteins.
]. Snail has been shown to be an important inducer of EMT, and Snail is an important factor in breast cancer lymph node metastasis. Thus, we propose that AQPQ inhibits EMT by mediating the expression of Snail, which affects ICC metastasis and invasion. Unfortunately, we were unable to elucidate the exact mechanism by which AQP1 regulates Snail, indicating that further studies are needed.
5. Conclusions
In conclusion, the present study demonstrated that AQP1 expression is associated with a favourable prognosis in ICC patients. AQP1 inhibits ICC cell invasion, metastasis and EMT through downregulation of Snail expression. These results revealed that AQP1 plays an important role in the progression of ICC, suggested that it may be a molecular marker for predicting patient prognosis and a potential target for the treatment of ICC.
Data Availability
The data and materials in this study are available from the corresponding author on request.
Conflict of interest
We confirm no potential conflict of interest or financial dependence regarding this study.
Acknowledgements
This project is supported by National key scientific instrument and equipment development grant 2012YQ220113 (H.H.) and Natural Science Foundation of China (NSFC) grants 81672371 (H.H.).
A statement of financial support
This project is supported by National key scientific instrument and equipment development grant 2012YQ220113 (H.H.), Mengchao Program of Eastern Hepatobiliary Surgery Hospital, and Natural Science Foundation of China (NSFC) grants 81672371 (H.H.).
Combined pharmacological administration of AQP1 ion channel blocker AqB011 and water channel blocker Bacopaside II amplifies inhibition of colon cancer cell migration.
Combined snail and E-cadherin predicts overall survival of cervical carcinoma patients: comparison among various epithelial-mesenchymal transition proteins.
number of available data; ICC: intrahepatic cholangiocarcinoma; AQP1: Aquaporin 1; HBsAg: hepatitis B surface antigen; HCV: hepatitis C virus; M: male; F: female; TBIL: total bilirubin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALB: serum albumin AFP: alpha-fetoprotein; ALP: alkaline phosphatase; r-GT: r-glutamyl transferase; CA19–9: carbohydrate antigen 19–9; CEA: carcinoembryonic antigen; CK: cytokeratin; IHC: immunohistochemistry. Bold type indicates statistical significance (p<0.05).
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