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Overexpression of DIXDC1 correlates with enhanced cell growth and poor prognosis in human pancreatic ductal adenocarcinoma
Overexpression of DIXDC1 correlates with enhanced cell growth and poor prognosis in human pancreatic ductal adenocarcinoma Xiaohong Li MD, Ying Xiao MD, Shaoqing Fan MD, Mingbing Xiao MD, Xiaotong Wang MD, Xiaolin Zhu MD, Xudong Chen PhD, Chunsun Li MD, Guijuan Zong MD, Guoxiong Zhou PhD, Chunhua Wan PhD PII: DOI: Reference:
S0046-8177(16)30167-8 doi: 10.1016/j.humpath.2016.07.015 YHUPA 3963
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
31 March 2016 18 July 2016 20 July 2016
Please cite this article as: Li Xiaohong, Xiao Ying, Fan Shaoqing, Xiao Mingbing, Wang Xiaotong, Zhu Xiaolin, Chen Xudong, Li Chunsun, Zong Guijuan, Zhou Guoxiong, Wan Chunhua, Overexpression of DIXDC1 correlates with enhanced cell growth and poor prognosis in human pancreatic ductal adenocarcinoma, Human Pathology (2016), doi: 10.1016/j.humpath.2016.07.015
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ACCEPTED MANUSCRIPT Overexpression of DIXDC1 correlates with enhanced cell growth and poor prognosis in human pancreatic ductal adenocarcinoma Xiaohong Li MD1 ·Ying Xiao MD2,3 · Shaoqing Fan MD2 · Mingbing Xiao MD2· Xiaotong Wang MD2· Xiaolin PhD3,5
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Xiaohong Li and Ying Xiao are contributed equally to this work.
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Zhu MD2,3 · Xudong Chen PhD4 · Chunsun Li MD4· Guijuan Zong MD4 · Guoxiong Zhou PhD2 · Chunhua Wan
1. Department of General Surgey, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
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2. Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China 3 Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, Jiangsu, China
4. Department of Pathology, Affiliated Cancer Hospital of Nantong University, Nantong 226001, Jiangsu, China 5. Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226001,
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Jiangsu, China
Guoxiong. Zhou ()
Chunhua Wan ()
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e-mail: [email protected]
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e-mail: [email protected]
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The authors declare no conflicts of interest.
Running head: DIXDC1 correlates with poor prognosis of PDAC
ACCEPTED MANUSCRIPT Abstract
DIX domain containing 1 (DIXDC1), a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain, is involved in the progression of multiple cancers. However, the role of DIXDC1 in human pancreatic
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ductal adenocarcinoma (PDAC) remains unclear. In this study, we investigated the role and prognostic value of
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DIXDC1 in the development of human PDAC. Western blot analysis revealed that DIXDC1 was highly expressed in PDAC tissues and cell lines. Immunohistochemistry on 165 paraffin-embedded sections showed that high expression of DIXDC1 was significantly correlated with tumor size (p = 0.002), histological
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differentiation (p = 0.001), tumor node metastasis (TNM) stage (p = 0.001), and the proliferation marker Ki-67 (p = 0.000). Importantly, Kaplan-Meier analysis revealed that high expression of DIXDC1 was obviously
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correlated with worsened overall survival (p<0.001). In vitro, using serum starvation–refeeding experiment, our results suggested that DIXDC1 was upregulated in proliferating PDAC cells, together with the percentage of cells at the S phase, and was correlated with the expression of Cyclin D1. In addition, depletion of DIXDC1 decreased PCNA and Cyclin D1 levels. Accordingly, CCK-8, colony formation, and flowcytometry analyses revealed that knocking down DIXDC1 induced growth impairment and G1/S cell cycle arrest in PDAC cells, while overexpression of DIXDC1 led to accelerated cell proliferation and cell cycle progression. On the basis of
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these results, we proposed that DIXDC1 could play an important role in the tumorigenesis of PDAC and serve as a potential therapeutical target to prevent PDAC progression.
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Keywords
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DIXDC1 · Pancreatic ductal adenocarcinoma (PDAC) · Prognosis· Cell proliferation
Introduction
Pancreatic cancer is the fourth leading cause of cancer-related death in developed countries with a dismal overall 5-year survival rate of 5 % [1, 2]. Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancer and has a dismal prognosis [3, 4]. The poor prognosis is mainly attributed to late diagnosis and resistance to chemotherapy [5]. Although intensive investigations have been done to develop advanced diagnostics and new treatment of PDAC, many aspects of the exact molecular mechanisms underlying PDAC remain to be clarified [6]. Therefore, there is an urgent need to find novel valuable prognostic biomarkers and therapeutic targets for early diagnosis and effective treatment of PDAC. DIX domain containing 1 (DIXDC1) is a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain. DIXDC1 has been identified as a positive regulator in Wnt signaling pathway [7]. It has been demonstrated that the DIX domain is important in connecting the Wnt-signaling factors Axin, Dishevelled and β-catenin [8-10]. In the Wnt signaling pathway, the DIX domain is involved in the initiation of both homomeric and heteromeric complexes between Axin and Dishevelled (Dvl) that eventually form the multiprotein
ACCEPTED MANUSCRIPT complexes of adenomatous polyposis coli (APC), glycogen synthase kinase 3 beta (GSK-3β), and β-catenin, which regulate T-cell-factor (TCF) signaling [9, 11]. Despite the fact that the importance of DIX domain in the transduction of Wnt signaling has been well-confined, the role of DIXDC1 in human cancer development remains poorly understood. In colon cancer, up-regulation of DIXDC1 might target p21 and Cyclin D1 to
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promote cell proliferation and tumorigenesis through activating the PI3K/Akt pathway [11]. In non-small cell
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lung cancer (NSCLC), overexpression of DIXDC1 promoted tumor invasion and migration through PI3K-Akt/AP-1-dependent activation of metalloproteinases [12]. In gastric cancer, upregulated expression of DIXDC1 promoted cell invasion and metastasis by activating the Wnt Signaling pathway [13]. These studies
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imply that DIXDC1 may be involved in cell proliferation and invasion during the development of multiple tumors. However, the expression and pathological significance of DIXDC1 in pancreatic cancer remain obscure.
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In this study, we for the first time demonstrated that DIXDC1 was significantly overexpressed in human PDAC specimens and cell lines. Meanwhile, we explored the correlation of DIXDC1 with various clinical and pathological parameters as well as the prognosis in patients with PDAC using immunohistochemistry and western blot analyses. Moreover, we employed small interfering RNA (siRNA) oligos and flag-DIXDC1to further explore the role of DIXDC1 in regulating cell cycle progression and cell proliferation in PDAC. These results showed that DIXDC1 might be a novel prognostic marker and play a potential role in anti-proliferative
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therapy of PDAC.
Patients and tissue samples
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Methods
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The paraffin-embedded pathologic specimens from 165 patients with PDAC were obtained from the Surgery Department, at the Affiliated Hospital of Nantong University. All patients underwent surgery without
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preoperative systemic chemotherapy between 2006 and 2010. Eighty-six patients were men while seventy-nine were women, and their average age was 57 year (range, 38-84). Clinicopathologic characteristics of these patients include Gender, Age, Location, Tumor size, Histological differentiation, Lymph node metastasis, Nerve invasion, TNM stage and Ki-67 expression. Eight pairs of fresh PDAC and adjacent normal samples were frozen in the liquid nitrogen and maintained at−80 °C until used for Western blot analysis. Approval for this study was obtained from patients’ consents and the Ethics Committee of Affiliated Hospital of Nantong University.
Western Blot Tissues and harvested cells were immediately homogenized in a homogenization buffer (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 % TritonX-100, 1 % NP-40) supplemented with complete protease and phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany) and then centrifuged at 13,000 g for 30 min to collect the supernatant. Total protein concentrations were detected by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Before gel electrophoresis, the supernatant was diluted with 2×SDS loading buffer and boiled for 15 min. An equivalent amount of protein from each sample was separated by 10% sodium dodecyl
ACCEPTED MANUSCRIPT sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with 5% non-fat milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20), the membranes were incubated overnight at room temperature with the following primary antibodies: DIXDC1 (1:500, Santa Cruz, USA), proliferating cell nuclear antigen (PCNA) (1:1000, Santa Cruz, USA), Cyclin D1
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(1:500, Santa Cruz, USA), and β-actin (1:500, Santa Cruz, USA) and glyceraldehyde-3-phosphate
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dehydrogenase (GAPDH) (1:1000, Santa Cruz, USA). After three times of washes, the membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (1:5,000; Pierce). The membranes were visualized using an enhanced chemiluminescent (ECL) detection reaction (NEN Life Science Products,
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Boston, MA, USA).Western blot analyses were used to examine the levels of protein. All target proteins were
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normalized to β-actin or GAPDH to determine the expression differences.
Immunohistochemistry
Immunohistochemistry was used to assess the clinical significance of DIXDC1 in PDAC progression. In brief, the PDAC tissue microarray were deparaffinized using a graded ethanol series, and were processed in 10 mM citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 3 min to retrieve the antigen. Endogenous peroxidase activity was blocked by immersion in 3 % hydrogen peroxide for 20 min at room temperature. Then,
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the sections were incubated with anti-DIXDC1 antibody (Sigma-Aldrich, MO, USA, diluted 1:100) overnight at 4 °C and anti-Ki-67 antibody (Millipore, Bedford, MA, USA diluted 1:500) for 2 h at room temperature were incubated in the sections after rinsing in PBS (pH 7.2). Negative control slides were incubated in parallel using a
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nonspecific immunoglobulin IgG (Sigma-Aldrich, MO, USA) at the same concentration as the primary antibody. All slides were processed using the peroxidase-anti-peroxidase complex method (Dako, Hamburg, Germany).
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After rinsing in PBS, the sections were counterstained with hematoxylin, dehydrated, and cover-slipped. Finally, the sections were examined with a Leica CTR5000 microscope (Leica Microsystems, Wetzlar, Germany). All of
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the immunostained sections were independently evaluated by three pathologists (two well-trained pathologists and a senior pathologist) in a blinded manner with no knowledge of the clinical and pathological information of the patients. The interobserver variation was evaluated using Bland-Altman plots and the scores were identified to be statistically equal among the pathologists. The mean scores of the three pathologists were used as the overall score in every patient. For the assessment of DIXDC1 and Ki-67 expression, five high-power fields were randomly chosen, and more than 500 cells were counted in each section. For determining the expression of DIXDC1, the intensity was assessed as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). Scores representing the percentage of tumor cells stained positive were as follows: 0, <1 %; 1, 1-25 %; 2, 26-50 %; 3, 51-75 %; and 4, >75 %. Then, we multiplied the two scores and divided them into two groups: high expressers (>4) and low expressers (0 to 4). When evaluating the Ki-67 protein immunoreaction, staining was scored in a semi-quantitative fashion. A cut-off value of 50% positively stained nuclei in 5 high-power fields was used to identify Ki67 staining: high-expression group (≥50%) and low-expression group (<50%).
Cell cultures and transient transfection The human pancreatic cancer cell lines Panc-1, BxPC-3, and CFPAC-1 and human normal pancreatic ductal
ACCEPTED MANUSCRIPT epithelial cell line HPDE6-C7 were purchased from the Cell Bank of Type Culture Collection Committee, China Academy of Sciences (Shanghai, China). Panc-1, CFPAC-1, and HPDE6-C7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM; GIBCO-BRL, Grand Island, NY, USA). BxPC-3 cells were maintained in RPMI-1640 medium (GIBCOBRL, Grand Island, NY, USA). All cells were supplemented with
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10 % fetal bovine serum (FBS; Gibco) in a humidified atmosphere of 5 % CO2 at 37 °C. Transient transfection
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was used to decrease or increase the levels of DIXDC1. Control-siRNA, DIXDC1-siRNA#1, #2 oligos and pcDNA3-flag-DIXDC1 were purchased from Genechem (Shanghai). Cell transfection was performed using
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Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Flow cytometric analysis
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Flow cytometric analysis was used to explore whether DIXDC1 was involved in cell cycle control of PDAC cells. After harvest, cells were fixed in 70% ethanol at -20 °C overnight and then incubated with 1 mg/ml RNaseA in Phosphate Buffered Saline (PBS) for 30 min at 37 °C. Then, the cells were stained with 0.5 mg/ml propidium iodide (PI; Sigma, St Louis, MO) in PBS-Triton in dark for an additional 20 min at 4 °C. At last, the cells were analyzed by using a flow cytometry analyzer (BD Biosciences, San Jose, CA, USA) and Cell Quest
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programs (Becton-Dickinson, USA).
Cell proliferation assay
Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s
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instructions. In brief, Panc-1 cells were plated at a density of 2×104 cells/well in a 96-well plate in a volume of 100 μl DMEM complete medium overnight. Later, 10 μl of CCK-8 (Dojindo, Kumamoto, Japan) reagent was
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added to each well and the cells were incubated for 2 h at 37 °C. Immediately following the incubation, we quantified the absorbance at a test wavelength of 450 nm and a reference wavelength of 630 nm on an
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automated plate reader (BioTek Instruments, Winooski, VT).
Plate colony formation assay Plate colony formation assay was used to measure cell proliferation. Panc-1 cells were seeded at 200 cells/well in 6-well plates after transfecting DIXDC1 siRNA#2 or Flag-DIXDC1. After 10 days of culture, the cell colonies (≥50 cells/colony)) were counted by staining with 0.5% crystal violet.
Statistical analysis Statistical analysis was performed using the SPSS 17.0 software package. The association between DIXDC1 and Ki-67 expression, clinicopathological features were computed using the χ2 test. Survival date was analyzed by using Kaplan-Meier survival curves and log-rank test. Multivariate analysis was performed using Cox’s proportional hazards model, and the risk ratio and its 95% confidence interval were recorded for every marker. All experiments were repeated at least three times. The values were expressed as mean ± SD, and P < 0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT Results DIXDC1 was overexpressed in PDAC tissues and cell lines To explore the potential role of DIXDC1 in PDAC progression, western blot analysis was employed to examine the expression profile of DIXDC1 in eight pairs of matched PDAC and adjacent normal tissues. The result
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showed that the expression of DIXDC1 was upregulated in PDAC tissues, compared with adjacent normal
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tissues (Fig. 1a). We next examined the expression of DIXDC1 in four human pancreatic ductal epithelial cell lines. We found that the expression of DIXDC1 was significantly higher in three PDAC cell lines Panc-1, BxPC-3, and CFPAC-1, compared with normal human pancreatic ductal epithelial cell line, HPDE6-C7 (Fig.
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1b). Furthermore, to confirm the clinical significance of DIXDC1 in PDAC progression, immunohistochemical analysis of DIXDC1 expression was carried out on tissue microarray that contained 165 samples from PDAC
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patients. As expected, the expression of DIXDC1 and Ki-67 were frequently elevated in tumorous tissues compared with the paired normal ones，especially in the ductal epithelial cells (Fig. 2a-d). 58.8% of PDAC samples exhibited high DIXDC1 expression. However, only 37.5% of adjacent pancreatic tissues showed clear evidence of DIXDC1 expression. Moreover, 63.0% of PDAC samples exhibited high Ki-67 expression. In contrast, only 36.3% of the adjacent pancreatic tissues showed clear evidence of Ki-67 expression (Table 1). Furthermore, we used Spearman’s correlation coefficient to investigate the correlation between DIXDC1 and
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Ki-67 expression. We found there was a positive correlation between DIXDC1 and Ki-67 expression, with a correlation coefficient of 0.469 (P<0.001, Fig. 3a). Collectively, these findings implicated that DIXDC1 may be
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involved in proliferation of PDAC
Expression of DIXDC1 correlates with clinicopathological factors of PDAC
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Next, we evaluated the association between DIXDC1 expression and the clinicopathological features of 165 PDAC patients. The patients’ clinicopathological data were summarized in Table 2. We found high expression of
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DIXDC1 was significantly correlated with tumor size (P= 0.002), histological differentiation (P= 0.001), TNM stage (P= 0.001), and Ki-67 (P= 0.000). However, there was no statistical correlation between DIXDC1 expression and other prognostic factors such as age and the location of tumors. These findings implicated that DIXDC1 expression might contribute to malignant proliferation of PDAC, highlighting the involvement of DIXDC1 in the regulation of PDAC progression.
Prognostic significance of DIXDC1 expression in PDAC patients In addition, Kaplan-Meier analysis and log-rank test were performed to analyze the association between DIXDC1 expression and 165 patients’ survival. As shown in Fig. 3b, high expression of DIXDC1 was obviously correlated with worsened overall survival (p<0.001). Moreover, univariate analysis showed that tumor size (p= 0.008), histological differentiation (p= 0.004), TNM stage (P= 0.000), DIXDC1 expression (P= 0.000), and Ki-67 expression (P= 0.000) were independent prognostic factors of overall survival in PDAC patients (Table 3). Furthermore, multivariate analysis using the Cox proportional hazards model showed that DIXDC1 (P= 0.003) was an independent prognostic indicator of overall survival (Table 3). Thus, DIXDC1 could be a valuable prognostic indicator for PDAC patients’ survival.
ACCEPTED MANUSCRIPT Expression of DIXDC1 was upregulated in proliferating PDAC cells Because the upregulation of DIXDC1 was associated with tumor size, histological differentiation, TNM stage, and Ki-67 expression in PDAC specimens, we speculated that DIXDC1 might play a regulatory role in the proliferation of PDAC cells. Thus, we analyzed the expression profile of DIXDC1 in PDAC cells under
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different proliferating statuses. Flow cytometry analysis showed that Panc-1 and BxPC-3 cells were arrested in
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the G0/G1 phase following serum deprivation for 72 h. After serum-refeeding, the cells were released from G1 phase and entered into S phase (Fig. 4a, b). Western blot analysis showed that the expression of DIXDC1 was increased gradually after serum stimulation in the both cell lines. Meanwhile, the expression of cell cycle
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protein Cyclin D1 was analyzed (Fig. 4c, d), which showed that DIXDC1 expression showed a similar tendency with that of Cyclin D1 following serum refeeding. These results suggested that DIXDC1 might function as a
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positive regulator of cell cycle progression in PDAC cells.
Depletion of DIXDC1 retarded cell cycle progression and inhibited PDAC cell proliferation To further elucidate the regulatory role of DIXDC1 in PDAC cell proliferation, we used two different siRNA oligos to knock down endogenous DIXDC1 in Panc-1 and BxPC-3 cells. As predicted, both of the two DIXDC1 siRNA oligos, especially siRNA#2, markedly decreased the cellular level of DIXDC1 in PDAC cells, compared
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with cells transfected with control siRNA (Fig. 5a). Meanwhile, western blot analysis showed that depletion of DIXDC1 caused the downregulation of Cyclin D1 and PCNA in the two cell lines (Fig. 5a). Moreover, CCK-8 and colony formation assays both showed that DIXDC1-depleted PDAC cells exhibited significantly decreased
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proliferation, compared with negative control siRNA-transfected cells (Fig. 5b, c). To explore whether DIXDC1 was involved in cell cycle control of PDAC cells, we next assessed the effect of DIXDC1 interference on cell
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cycle distribution using flow cytometric analysis. Panc-1 and BxPC-3 cells were transfected with DIXDC1 siRNA#2 or control siRNA. 48 h after transfection, the cells were harvested and subjected to flow cytometric
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analysis. As shown in Fig. 5d, the proportion of cells in the G1 phase were significantly increased in PDAC cells transfected with DIXDC1 siRNA#2 and a corresponding reduction in the percentage of cells in the S phase was observed, suggesting that depletion of DIXDC1 led to G1-S arrest and cell growth impairment. Collectively, these results suggested that interference of DIXDC1 retarded cell cycle progression, leading to impaired proliferation of PDAC cells.
Overexpression of DIXDC1 resulted in cell proliferation and accelerated cell cycle progression in PDAC cells To verify the notion that DIXDC1 was involved in the regulation of PDAC proliferation, a normal pancreatic ductal cell line (HPDE6-C7) that expresses relatively low level of DIXDC1 was employed to transfect with empty vector or Flag-DIXDC1 plasmid. As shown in Fig. 6a, western blot analysis demonstrated that overexpression of DIXDC1 caused the upregulation of Cyclin D1 and PCNA in PDAC cells (Fig. 6a). CCK-8 assay showed that ectopic DIXDC1 expression significantly enhanced the growth of PDAC cells, compared with control cells (Fig. 6b). Similar results were obtained using colony formation assay (Fig. 6c). Meanwhile, flow cytometry analysis demonstrated that the proportion of PDAC cells in the S phase was elevated in the
ACCEPTED MANUSCRIPT presence of ectopic DIXDC1 and a corresponding reduction in cells at the G1 phase was observed accordingly (Fig. 6d). Taken these results together, we concluded that DIXDC1 expression accelerated cell cycle progression,
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leading to the proliferation of PDAC cells.
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Discussion
Pancreatic cancer is one of the most aggressive human malignancies and the 4th leading cause of cancer related death, with a 5-year survival rate of approximate 5 % [14]. Despite the fact that great efforts have been made in
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the understanding of the etiology, pathogenesis, and epidemiology of PDAC, many aspects of the molecular mechanisms underlying PDAC development remain obscure [15]. Thus, the identification of novel molecular
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markers that can be used as therapeutic targets of PDAC is a primary task in the field of PDAC research. DIXDC1 is a DIX (Dishevelled-Axin) domain containing protein and a positive regulator of the Wnt pathway in zebrafish neural patterning [16]. Meanwhile, the activation of the canonical Wnt pathway stabilizes DIXDC1 through inhibiting ubiquitin-dependent degradation of DIXDC1[17]. In addition to a positive role in the transduction of the canonical Wnt pathway, recent researches showed that DIXDC1 play diverse roles in assorted biological processes, including axis formation and neural patterning during embryonic development,
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somatic cell growth and differentiation[16]. Moreover, the importance of DIXDC1 in carcinogenesis has attracted the attention of many researchers in recent years. Recently, it was shown in colon carcinoma that upregulated DIXDC1 might target p21 and Cyclin D1 to promote cell proliferation and tumorigenesis through
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activating the PI3K/Akt pathway [11]. A recent study found that overexpression of DIXDC1 promoted the invasion and migration of NSCLC through PI3K-Akt/AP-1-dependent activation of metalloproteinases [12]. In
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gastric cancer, upregulated expression of DIXDC1 promoted cell invasion and metastasis by activating the Wnt Signaling pathway [13]. However, the expression and significance of DIXDC1 in human pancreatic cancer
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development remain virtually unknown. In the present study, we first found that DIXDC1 was upregulated in PDAC cell lines and tissues, compared with normal pancreatic tissues and pancreatic ductal epithelial cells. Immunohistochemistry analysis revealed that DIXDC1 expression was correlated with tumor size, histological differentiation, TNM stage, and Ki-67 of PDAC. Univariate and multivariate analyses demonstrated that DIXDC1 expression could be an independent prognostic factor to predict the survival of PDAC patients. In addition, we demonstrated that DIXDC1 depletion could result in cell cycle arrest and impaired proliferation of PDAC cells. Taken together, these results revealed that DIXDC1 might be a novel prognostic marker and therapeutic target of PDAC. It has been well documented that aberrant activation of the Wnt signaling pathway is associated with the development of a variety of human cancers, including pancreatic cancer [18-20]. Moreover, studies found that DIXDC1 affected the progression of human cancers through the regulation of PI3K/Akt pathway and Wnt/β-catenin pathway [17]. PI3K/Akt pathway has been implicated in the regulation of the proliferation of cancer cells via modulating the expression of cell cycle regulators, such as Cyclin D1 and p21 [21]. Meanwhile, DIXDC1 could inhibit the phosphorylation of β-catenin and promote the translocation of β-catenin to the nucleus through stabilizing β-catenin [7]. The accumulation of β-catenin in the nucleus activates various Wnt
ACCEPTED MANUSCRIPT target genes, such as Cyclin D1, which are involved in cell proliferation [22-24]. We found that PDAC cell lines transfected with DIXDC1 siRNA showed an apparently reduced population of cells in the S phase, whereas cells in the G1 phase was markedly elevated. Coincidently, depletion of DIXDC1 significantly downregulated the expression of Cyclin D1, suggesting that Cyclin D1 might be a downstream effecter of DIXDC1 in the
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regulation of PDAC cell proliferation. These date suggested that overexpression of DIXDC1 might play an
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important role in the cell cycle progression of PDAC cells through activating PI3K/Akt signaling pathway and Wnt/β-catenin pathway.
In summary, we for the first time showed that the expression of DIXDC1 was significantly increased in the
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human PDAC. Upregulated expression of DIXDC1 predicted dismal prognosis in PDAC patients. Moreover, depletion of DIXDC1 obviously inhibited the proliferation, cell cycle progression of PDAC cells. Therefore,
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DIXDC1 might serve as a novel molecular target for the detection and treatment of PDAC, pending further analyses in larger studies and functional in vivo experiments.
Acknowledgments This work was supported by the National Natural Scientific Foundation of China (No.81572397, 81200918) and Jiangsu Province’s Outstanding Medical Academic Leader program (No.
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LJ101135).
Conflict of interest We declare that we have no conflicts of interest.
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Figure legends
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[24] Tetsu O, McCormick F. Beta-catenin regulates expression of cyclin d1 in colon carcinoma cells. Nature
Fig.1 DIXDC1 was upregulated in human PDAC tissues and cell lines. a Eight representative paired samples of PDAC tissue (T) and adjacent normal tissues (N) were used for western blot analysis. DIXDC1 expression levels were higher in eight representative paired samples of PDAC tissues (T) compared with paired adjacent normal tissues (N). GAPDH was included as a loading control. The bar chart showed the ratio of DIXDC1 protein to GADPH. Mean±SD of three independent experiments (*P<0.05 compared with control nontumorous adjacent tissues). b The DIXDC1 expression is increased in different PDAC cell lines compared with normal pancreatic ductal cell (HPDE6-C7). The bar chart showed the ratio of DIXDC1 protein to GAPDH. Mean±SD of three independent experiments (*P<0.05).
Fig.2 Immunohistochemical evaluate of DIXDC1 and Ki-67 expression in paraffin-embedded PDAC (T) tissues and adjacent non-cancerous (N) tissues. High expression of DIXDC1 (a) and Ki-67 (b) was detected in PDAC specimen (SP×100, ×400). However, low levels of DIXDC1 expression (c) and Ki-67 (d) were seen in the paired normal tissues (SP×100, ×400).
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Fig.3 The relation between DIXDC1 and clinical characteristics. a Linear regression analysis of the expression of DIXDC1 and the proliferation index Ki-67 in 165 PDAC specimens. Scatterplot of Ki-67 versus DIXDC1 with regression line showed a correlation between them using the Spearman’s correlation coefficient. b
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Kaplan–Meier survival analysis of 165 pancreatic cancer patients based on DIXDC1 expression level.
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According to the DIXDC1 percentages, patients were divided into high DIXDC1 expressers (score >4) and low DIXDC1 expressers (score ≤4). Patients in the high-expression DIXDC1 group had a significantly shorter
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overall survival (P< 0.001).
Fig.4 DIXDC1 was upregulated in proliferating PDAC cells. a, b Flow cytometry analysis of cell cycle
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distribution in Panc-1(a) and BxPC-3 (b) cells. Cells were synchronized at the G1 phase after serum starvation (S) for 72 h, then progressed into cell cycle by adding medium containing 10 % FBS for the indicated times (R4 h, R8 h, R12 h, R24 h). c, d Western blot analysis of the expression of DIXDC1, Cyclin D1 in PDAC cells that serum starvation for 72 h and serum re-addition in indicated times (R4 h, R8 h, R12 h, R24 h). GAPDH was included as a loading control. The bar chart below demonstrated the ratio of DIXDC1 and Cyclin D1 protein to
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GAPDH by densitometry in Panc-1 and BxPC-3 cells. Mean±SD of three independent experiments(*, # p<0.05).
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Fig.5 DIXDC1 knockdown suppressed PDAC cell proliferation and retarded cell cycle progression. a Western blot analysis of the expression of DIXDC1, Cyclin D1, PCNA in PDAC cells transfected with control-siRNA
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and different DIXDC1-targeting siRNA. GAPDH was included as a loading control. b Cell proliferation was measured using CCK-8 assay. Panc-1 and BxPC-3 cells that have been transfected with RACK1-siRNA#2 exhibited inhibited cell proliferation. c Silencing endogenous DIXDC1 suppressed cell growth of Panc-1 and
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BxPC-3 determined by colony formation assays. d DIXDC1 depletion resulted in a decrease of S transition of Panc-1 and BxPC-3 cells, whereas an increased population in the G1 phase. Mean±SD of three independent experiments (*, p<0.05).
Fig.6 Overexpression of DIXDC1 promoted PDAC cell proliferation and cell cycle progression. a HPDE6-C7 cells were transiently transfected with Flag-DIXDC1 plasmid as described above for 48 h. Western blot analysis of ectopic expression of DIXDC1. b Cell proliferation was measured using CCK-8 assay. HPDE6-C7 cells transfected with Flag- DIXDC1 exhibited enhanced cell proliferation. c Colony formation assays showed that increased endogenous DIXDC1 enhanced cell growth. d DNA content between Flag- DIXDC1 and empty vector treated cells was compared by flow cytometry. Mean±SD of three independent experiments (*, p <0.05).
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Table 1 Up-regulation of DIXDC1 in PDAC tissues compared to adjacent normal tissue DIXDC1 Tumor Adjacent normal P value expression (n=165) tissue(n=165) Low 68 103 0.000* High 97 62 Statistical analyses were carried out using Pearson χ2 test *P<0.05 was considered significant
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Table 2 Correlation between DIXDC1 expression and clinicopathologic speciments Characteristic Total RACK1(n%) χ2 value Low High n=68 n=97 Gender 0.021 Male 103 42 61 Female 62 26 36 Age(years) 2.206 ＜60 98 45 53 ≥60 67 23 44 Location 1.147 Head-neck 81 30 51 Body-tail 75 34 41 Total 9 4 5 Size(cm) 12.569 ＜2 30 21 9 2-4 96 33 63 ＞4 39 14 25 Histological 15.003 differentiation Well 29 19 10 Moderate 83 37 46 Poor 53 12 41 Lymph node 0.119 metastasis Yes 73 29 44 No 92 39 53 Nerve invasion 2.017 Yes 64 22 42 No 101 46 55 TNM stage 11.343 Ⅰ-Ⅱ 101 52 49 Ⅲ-Ⅳ 64 16 48 Ki67 expression 20.624 High 104 29 75 Low 61 39 22 Statistical analyses were carried out using Pearson χ2 test *P<0.05 was considered significant
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Table 3 Univariate analysis and multivariate analyses of the contribution of various potential prognostic factors to survival in patients with PDAC Characteristic Univariate Cox regression Multivariate Cox regression P P HR 95%CI HR 95%CI value value Gender 0.946 0.690-1.296 0.729 Age 1.031 0.736-1.443 0.861 Location 1.086 0.779-1.515 0.627 Size 1.366 1.086-1.718 0.008* 1.074 0.830-1.391 0.588 Histological 1.435 1.124-1.830 0.004* 1.003 0.765-1.316 0.982 differentiation Lymph node metastasis 1.044 0.737-1.480 0.809 Nerve invasion 1.131 0.808-1.583 0.474 TNM stage 2.333 1.658-3.281 0.000* 1.507 1.008-2.254 0.046* Ki67 2.770 1.907-4.022 0.000* 1.817 1.139-2.898 0.012* DIXDC1 2.346 1.630-3.376 0.000* 1.802 1.216-2.699 0.003* Statistical analyses were performed by Cox proportional hazards regression *P<0.05 was considered significant
S0046-8177(16)30167-8 doi: 10.1016/j.humpath.2016.07.015 YHUPA 3963
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
31 March 2016 18 July 2016 20 July 2016
Please cite this article as: Li Xiaohong, Xiao Ying, Fan Shaoqing, Xiao Mingbing, Wang Xiaotong, Zhu Xiaolin, Chen Xudong, Li Chunsun, Zong Guijuan, Zhou Guoxiong, Wan Chunhua, Overexpression of DIXDC1 correlates with enhanced cell growth and poor prognosis in human pancreatic ductal adenocarcinoma, Human Pathology (2016), doi: 10.1016/j.humpath.2016.07.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Overexpression of DIXDC1 correlates with enhanced cell growth and poor prognosis in human pancreatic ductal adenocarcinoma Xiaohong Li MD1 ·Ying Xiao MD2,3 · Shaoqing Fan MD2 · Mingbing Xiao MD2· Xiaotong Wang MD2· Xiaolin PhD3,5
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Xiaohong Li and Ying Xiao are contributed equally to this work.
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Zhu MD2,3 · Xudong Chen PhD4 · Chunsun Li MD4· Guijuan Zong MD4 · Guoxiong Zhou PhD2 · Chunhua Wan
1. Department of General Surgey, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
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2. Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China 3 Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, Jiangsu, China
4. Department of Pathology, Affiliated Cancer Hospital of Nantong University, Nantong 226001, Jiangsu, China 5. Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226001,
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Jiangsu, China
Guoxiong. Zhou ()
Chunhua Wan ()
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e-mail: [email protected]
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e-mail: [email protected]
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The authors declare no conflicts of interest.
Running head: DIXDC1 correlates with poor prognosis of PDAC
ACCEPTED MANUSCRIPT Abstract
DIX domain containing 1 (DIXDC1), a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain, is involved in the progression of multiple cancers. However, the role of DIXDC1 in human pancreatic
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ductal adenocarcinoma (PDAC) remains unclear. In this study, we investigated the role and prognostic value of
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DIXDC1 in the development of human PDAC. Western blot analysis revealed that DIXDC1 was highly expressed in PDAC tissues and cell lines. Immunohistochemistry on 165 paraffin-embedded sections showed that high expression of DIXDC1 was significantly correlated with tumor size (p = 0.002), histological
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differentiation (p = 0.001), tumor node metastasis (TNM) stage (p = 0.001), and the proliferation marker Ki-67 (p = 0.000). Importantly, Kaplan-Meier analysis revealed that high expression of DIXDC1 was obviously
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correlated with worsened overall survival (p<0.001). In vitro, using serum starvation–refeeding experiment, our results suggested that DIXDC1 was upregulated in proliferating PDAC cells, together with the percentage of cells at the S phase, and was correlated with the expression of Cyclin D1. In addition, depletion of DIXDC1 decreased PCNA and Cyclin D1 levels. Accordingly, CCK-8, colony formation, and flowcytometry analyses revealed that knocking down DIXDC1 induced growth impairment and G1/S cell cycle arrest in PDAC cells, while overexpression of DIXDC1 led to accelerated cell proliferation and cell cycle progression. On the basis of
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these results, we proposed that DIXDC1 could play an important role in the tumorigenesis of PDAC and serve as a potential therapeutical target to prevent PDAC progression.
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Keywords
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DIXDC1 · Pancreatic ductal adenocarcinoma (PDAC) · Prognosis· Cell proliferation
Introduction
Pancreatic cancer is the fourth leading cause of cancer-related death in developed countries with a dismal overall 5-year survival rate of 5 % [1, 2]. Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancer and has a dismal prognosis [3, 4]. The poor prognosis is mainly attributed to late diagnosis and resistance to chemotherapy [5]. Although intensive investigations have been done to develop advanced diagnostics and new treatment of PDAC, many aspects of the exact molecular mechanisms underlying PDAC remain to be clarified [6]. Therefore, there is an urgent need to find novel valuable prognostic biomarkers and therapeutic targets for early diagnosis and effective treatment of PDAC. DIX domain containing 1 (DIXDC1) is a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain. DIXDC1 has been identified as a positive regulator in Wnt signaling pathway [7]. It has been demonstrated that the DIX domain is important in connecting the Wnt-signaling factors Axin, Dishevelled and β-catenin [8-10]. In the Wnt signaling pathway, the DIX domain is involved in the initiation of both homomeric and heteromeric complexes between Axin and Dishevelled (Dvl) that eventually form the multiprotein
ACCEPTED MANUSCRIPT complexes of adenomatous polyposis coli (APC), glycogen synthase kinase 3 beta (GSK-3β), and β-catenin, which regulate T-cell-factor (TCF) signaling [9, 11]. Despite the fact that the importance of DIX domain in the transduction of Wnt signaling has been well-confined, the role of DIXDC1 in human cancer development remains poorly understood. In colon cancer, up-regulation of DIXDC1 might target p21 and Cyclin D1 to
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promote cell proliferation and tumorigenesis through activating the PI3K/Akt pathway [11]. In non-small cell
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lung cancer (NSCLC), overexpression of DIXDC1 promoted tumor invasion and migration through PI3K-Akt/AP-1-dependent activation of metalloproteinases [12]. In gastric cancer, upregulated expression of DIXDC1 promoted cell invasion and metastasis by activating the Wnt Signaling pathway [13]. These studies
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imply that DIXDC1 may be involved in cell proliferation and invasion during the development of multiple tumors. However, the expression and pathological significance of DIXDC1 in pancreatic cancer remain obscure.
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In this study, we for the first time demonstrated that DIXDC1 was significantly overexpressed in human PDAC specimens and cell lines. Meanwhile, we explored the correlation of DIXDC1 with various clinical and pathological parameters as well as the prognosis in patients with PDAC using immunohistochemistry and western blot analyses. Moreover, we employed small interfering RNA (siRNA) oligos and flag-DIXDC1to further explore the role of DIXDC1 in regulating cell cycle progression and cell proliferation in PDAC. These results showed that DIXDC1 might be a novel prognostic marker and play a potential role in anti-proliferative
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therapy of PDAC.
Patients and tissue samples
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Methods
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The paraffin-embedded pathologic specimens from 165 patients with PDAC were obtained from the Surgery Department, at the Affiliated Hospital of Nantong University. All patients underwent surgery without
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preoperative systemic chemotherapy between 2006 and 2010. Eighty-six patients were men while seventy-nine were women, and their average age was 57 year (range, 38-84). Clinicopathologic characteristics of these patients include Gender, Age, Location, Tumor size, Histological differentiation, Lymph node metastasis, Nerve invasion, TNM stage and Ki-67 expression. Eight pairs of fresh PDAC and adjacent normal samples were frozen in the liquid nitrogen and maintained at−80 °C until used for Western blot analysis. Approval for this study was obtained from patients’ consents and the Ethics Committee of Affiliated Hospital of Nantong University.
Western Blot Tissues and harvested cells were immediately homogenized in a homogenization buffer (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 % TritonX-100, 1 % NP-40) supplemented with complete protease and phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany) and then centrifuged at 13,000 g for 30 min to collect the supernatant. Total protein concentrations were detected by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Before gel electrophoresis, the supernatant was diluted with 2×SDS loading buffer and boiled for 15 min. An equivalent amount of protein from each sample was separated by 10% sodium dodecyl
ACCEPTED MANUSCRIPT sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with 5% non-fat milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20), the membranes were incubated overnight at room temperature with the following primary antibodies: DIXDC1 (1:500, Santa Cruz, USA), proliferating cell nuclear antigen (PCNA) (1:1000, Santa Cruz, USA), Cyclin D1
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(1:500, Santa Cruz, USA), and β-actin (1:500, Santa Cruz, USA) and glyceraldehyde-3-phosphate
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dehydrogenase (GAPDH) (1:1000, Santa Cruz, USA). After three times of washes, the membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (1:5,000; Pierce). The membranes were visualized using an enhanced chemiluminescent (ECL) detection reaction (NEN Life Science Products,
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Boston, MA, USA).Western blot analyses were used to examine the levels of protein. All target proteins were
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normalized to β-actin or GAPDH to determine the expression differences.
Immunohistochemistry
Immunohistochemistry was used to assess the clinical significance of DIXDC1 in PDAC progression. In brief, the PDAC tissue microarray were deparaffinized using a graded ethanol series, and were processed in 10 mM citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 3 min to retrieve the antigen. Endogenous peroxidase activity was blocked by immersion in 3 % hydrogen peroxide for 20 min at room temperature. Then,
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the sections were incubated with anti-DIXDC1 antibody (Sigma-Aldrich, MO, USA, diluted 1:100) overnight at 4 °C and anti-Ki-67 antibody (Millipore, Bedford, MA, USA diluted 1:500) for 2 h at room temperature were incubated in the sections after rinsing in PBS (pH 7.2). Negative control slides were incubated in parallel using a
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nonspecific immunoglobulin IgG (Sigma-Aldrich, MO, USA) at the same concentration as the primary antibody. All slides were processed using the peroxidase-anti-peroxidase complex method (Dako, Hamburg, Germany).
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After rinsing in PBS, the sections were counterstained with hematoxylin, dehydrated, and cover-slipped. Finally, the sections were examined with a Leica CTR5000 microscope (Leica Microsystems, Wetzlar, Germany). All of
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the immunostained sections were independently evaluated by three pathologists (two well-trained pathologists and a senior pathologist) in a blinded manner with no knowledge of the clinical and pathological information of the patients. The interobserver variation was evaluated using Bland-Altman plots and the scores were identified to be statistically equal among the pathologists. The mean scores of the three pathologists were used as the overall score in every patient. For the assessment of DIXDC1 and Ki-67 expression, five high-power fields were randomly chosen, and more than 500 cells were counted in each section. For determining the expression of DIXDC1, the intensity was assessed as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). Scores representing the percentage of tumor cells stained positive were as follows: 0, <1 %; 1, 1-25 %; 2, 26-50 %; 3, 51-75 %; and 4, >75 %. Then, we multiplied the two scores and divided them into two groups: high expressers (>4) and low expressers (0 to 4). When evaluating the Ki-67 protein immunoreaction, staining was scored in a semi-quantitative fashion. A cut-off value of 50% positively stained nuclei in 5 high-power fields was used to identify Ki67 staining: high-expression group (≥50%) and low-expression group (<50%).
Cell cultures and transient transfection The human pancreatic cancer cell lines Panc-1, BxPC-3, and CFPAC-1 and human normal pancreatic ductal
ACCEPTED MANUSCRIPT epithelial cell line HPDE6-C7 were purchased from the Cell Bank of Type Culture Collection Committee, China Academy of Sciences (Shanghai, China). Panc-1, CFPAC-1, and HPDE6-C7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM; GIBCO-BRL, Grand Island, NY, USA). BxPC-3 cells were maintained in RPMI-1640 medium (GIBCOBRL, Grand Island, NY, USA). All cells were supplemented with
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10 % fetal bovine serum (FBS; Gibco) in a humidified atmosphere of 5 % CO2 at 37 °C. Transient transfection
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was used to decrease or increase the levels of DIXDC1. Control-siRNA, DIXDC1-siRNA#1, #2 oligos and pcDNA3-flag-DIXDC1 were purchased from Genechem (Shanghai). Cell transfection was performed using
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Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Flow cytometric analysis
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Flow cytometric analysis was used to explore whether DIXDC1 was involved in cell cycle control of PDAC cells. After harvest, cells were fixed in 70% ethanol at -20 °C overnight and then incubated with 1 mg/ml RNaseA in Phosphate Buffered Saline (PBS) for 30 min at 37 °C. Then, the cells were stained with 0.5 mg/ml propidium iodide (PI; Sigma, St Louis, MO) in PBS-Triton in dark for an additional 20 min at 4 °C. At last, the cells were analyzed by using a flow cytometry analyzer (BD Biosciences, San Jose, CA, USA) and Cell Quest
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programs (Becton-Dickinson, USA).
Cell proliferation assay
Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s
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instructions. In brief, Panc-1 cells were plated at a density of 2×104 cells/well in a 96-well plate in a volume of 100 μl DMEM complete medium overnight. Later, 10 μl of CCK-8 (Dojindo, Kumamoto, Japan) reagent was
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added to each well and the cells were incubated for 2 h at 37 °C. Immediately following the incubation, we quantified the absorbance at a test wavelength of 450 nm and a reference wavelength of 630 nm on an
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automated plate reader (BioTek Instruments, Winooski, VT).
Plate colony formation assay Plate colony formation assay was used to measure cell proliferation. Panc-1 cells were seeded at 200 cells/well in 6-well plates after transfecting DIXDC1 siRNA#2 or Flag-DIXDC1. After 10 days of culture, the cell colonies (≥50 cells/colony)) were counted by staining with 0.5% crystal violet.
Statistical analysis Statistical analysis was performed using the SPSS 17.0 software package. The association between DIXDC1 and Ki-67 expression, clinicopathological features were computed using the χ2 test. Survival date was analyzed by using Kaplan-Meier survival curves and log-rank test. Multivariate analysis was performed using Cox’s proportional hazards model, and the risk ratio and its 95% confidence interval were recorded for every marker. All experiments were repeated at least three times. The values were expressed as mean ± SD, and P < 0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT Results DIXDC1 was overexpressed in PDAC tissues and cell lines To explore the potential role of DIXDC1 in PDAC progression, western blot analysis was employed to examine the expression profile of DIXDC1 in eight pairs of matched PDAC and adjacent normal tissues. The result
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showed that the expression of DIXDC1 was upregulated in PDAC tissues, compared with adjacent normal
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tissues (Fig. 1a). We next examined the expression of DIXDC1 in four human pancreatic ductal epithelial cell lines. We found that the expression of DIXDC1 was significantly higher in three PDAC cell lines Panc-1, BxPC-3, and CFPAC-1, compared with normal human pancreatic ductal epithelial cell line, HPDE6-C7 (Fig.
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1b). Furthermore, to confirm the clinical significance of DIXDC1 in PDAC progression, immunohistochemical analysis of DIXDC1 expression was carried out on tissue microarray that contained 165 samples from PDAC
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patients. As expected, the expression of DIXDC1 and Ki-67 were frequently elevated in tumorous tissues compared with the paired normal ones，especially in the ductal epithelial cells (Fig. 2a-d). 58.8% of PDAC samples exhibited high DIXDC1 expression. However, only 37.5% of adjacent pancreatic tissues showed clear evidence of DIXDC1 expression. Moreover, 63.0% of PDAC samples exhibited high Ki-67 expression. In contrast, only 36.3% of the adjacent pancreatic tissues showed clear evidence of Ki-67 expression (Table 1). Furthermore, we used Spearman’s correlation coefficient to investigate the correlation between DIXDC1 and
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Ki-67 expression. We found there was a positive correlation between DIXDC1 and Ki-67 expression, with a correlation coefficient of 0.469 (P<0.001, Fig. 3a). Collectively, these findings implicated that DIXDC1 may be
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involved in proliferation of PDAC
Expression of DIXDC1 correlates with clinicopathological factors of PDAC
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Next, we evaluated the association between DIXDC1 expression and the clinicopathological features of 165 PDAC patients. The patients’ clinicopathological data were summarized in Table 2. We found high expression of
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DIXDC1 was significantly correlated with tumor size (P= 0.002), histological differentiation (P= 0.001), TNM stage (P= 0.001), and Ki-67 (P= 0.000). However, there was no statistical correlation between DIXDC1 expression and other prognostic factors such as age and the location of tumors. These findings implicated that DIXDC1 expression might contribute to malignant proliferation of PDAC, highlighting the involvement of DIXDC1 in the regulation of PDAC progression.
Prognostic significance of DIXDC1 expression in PDAC patients In addition, Kaplan-Meier analysis and log-rank test were performed to analyze the association between DIXDC1 expression and 165 patients’ survival. As shown in Fig. 3b, high expression of DIXDC1 was obviously correlated with worsened overall survival (p<0.001). Moreover, univariate analysis showed that tumor size (p= 0.008), histological differentiation (p= 0.004), TNM stage (P= 0.000), DIXDC1 expression (P= 0.000), and Ki-67 expression (P= 0.000) were independent prognostic factors of overall survival in PDAC patients (Table 3). Furthermore, multivariate analysis using the Cox proportional hazards model showed that DIXDC1 (P= 0.003) was an independent prognostic indicator of overall survival (Table 3). Thus, DIXDC1 could be a valuable prognostic indicator for PDAC patients’ survival.
ACCEPTED MANUSCRIPT Expression of DIXDC1 was upregulated in proliferating PDAC cells Because the upregulation of DIXDC1 was associated with tumor size, histological differentiation, TNM stage, and Ki-67 expression in PDAC specimens, we speculated that DIXDC1 might play a regulatory role in the proliferation of PDAC cells. Thus, we analyzed the expression profile of DIXDC1 in PDAC cells under
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different proliferating statuses. Flow cytometry analysis showed that Panc-1 and BxPC-3 cells were arrested in
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the G0/G1 phase following serum deprivation for 72 h. After serum-refeeding, the cells were released from G1 phase and entered into S phase (Fig. 4a, b). Western blot analysis showed that the expression of DIXDC1 was increased gradually after serum stimulation in the both cell lines. Meanwhile, the expression of cell cycle
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protein Cyclin D1 was analyzed (Fig. 4c, d), which showed that DIXDC1 expression showed a similar tendency with that of Cyclin D1 following serum refeeding. These results suggested that DIXDC1 might function as a
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positive regulator of cell cycle progression in PDAC cells.
Depletion of DIXDC1 retarded cell cycle progression and inhibited PDAC cell proliferation To further elucidate the regulatory role of DIXDC1 in PDAC cell proliferation, we used two different siRNA oligos to knock down endogenous DIXDC1 in Panc-1 and BxPC-3 cells. As predicted, both of the two DIXDC1 siRNA oligos, especially siRNA#2, markedly decreased the cellular level of DIXDC1 in PDAC cells, compared
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with cells transfected with control siRNA (Fig. 5a). Meanwhile, western blot analysis showed that depletion of DIXDC1 caused the downregulation of Cyclin D1 and PCNA in the two cell lines (Fig. 5a). Moreover, CCK-8 and colony formation assays both showed that DIXDC1-depleted PDAC cells exhibited significantly decreased
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proliferation, compared with negative control siRNA-transfected cells (Fig. 5b, c). To explore whether DIXDC1 was involved in cell cycle control of PDAC cells, we next assessed the effect of DIXDC1 interference on cell
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cycle distribution using flow cytometric analysis. Panc-1 and BxPC-3 cells were transfected with DIXDC1 siRNA#2 or control siRNA. 48 h after transfection, the cells were harvested and subjected to flow cytometric
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analysis. As shown in Fig. 5d, the proportion of cells in the G1 phase were significantly increased in PDAC cells transfected with DIXDC1 siRNA#2 and a corresponding reduction in the percentage of cells in the S phase was observed, suggesting that depletion of DIXDC1 led to G1-S arrest and cell growth impairment. Collectively, these results suggested that interference of DIXDC1 retarded cell cycle progression, leading to impaired proliferation of PDAC cells.
Overexpression of DIXDC1 resulted in cell proliferation and accelerated cell cycle progression in PDAC cells To verify the notion that DIXDC1 was involved in the regulation of PDAC proliferation, a normal pancreatic ductal cell line (HPDE6-C7) that expresses relatively low level of DIXDC1 was employed to transfect with empty vector or Flag-DIXDC1 plasmid. As shown in Fig. 6a, western blot analysis demonstrated that overexpression of DIXDC1 caused the upregulation of Cyclin D1 and PCNA in PDAC cells (Fig. 6a). CCK-8 assay showed that ectopic DIXDC1 expression significantly enhanced the growth of PDAC cells, compared with control cells (Fig. 6b). Similar results were obtained using colony formation assay (Fig. 6c). Meanwhile, flow cytometry analysis demonstrated that the proportion of PDAC cells in the S phase was elevated in the
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Discussion
Pancreatic cancer is one of the most aggressive human malignancies and the 4th leading cause of cancer related death, with a 5-year survival rate of approximate 5 % [14]. Despite the fact that great efforts have been made in
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the understanding of the etiology, pathogenesis, and epidemiology of PDAC, many aspects of the molecular mechanisms underlying PDAC development remain obscure [15]. Thus, the identification of novel molecular
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markers that can be used as therapeutic targets of PDAC is a primary task in the field of PDAC research. DIXDC1 is a DIX (Dishevelled-Axin) domain containing protein and a positive regulator of the Wnt pathway in zebrafish neural patterning [16]. Meanwhile, the activation of the canonical Wnt pathway stabilizes DIXDC1 through inhibiting ubiquitin-dependent degradation of DIXDC1[17]. In addition to a positive role in the transduction of the canonical Wnt pathway, recent researches showed that DIXDC1 play diverse roles in assorted biological processes, including axis formation and neural patterning during embryonic development,
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somatic cell growth and differentiation[16]. Moreover, the importance of DIXDC1 in carcinogenesis has attracted the attention of many researchers in recent years. Recently, it was shown in colon carcinoma that upregulated DIXDC1 might target p21 and Cyclin D1 to promote cell proliferation and tumorigenesis through
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activating the PI3K/Akt pathway [11]. A recent study found that overexpression of DIXDC1 promoted the invasion and migration of NSCLC through PI3K-Akt/AP-1-dependent activation of metalloproteinases [12]. In
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gastric cancer, upregulated expression of DIXDC1 promoted cell invasion and metastasis by activating the Wnt Signaling pathway [13]. However, the expression and significance of DIXDC1 in human pancreatic cancer
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development remain virtually unknown. In the present study, we first found that DIXDC1 was upregulated in PDAC cell lines and tissues, compared with normal pancreatic tissues and pancreatic ductal epithelial cells. Immunohistochemistry analysis revealed that DIXDC1 expression was correlated with tumor size, histological differentiation, TNM stage, and Ki-67 of PDAC. Univariate and multivariate analyses demonstrated that DIXDC1 expression could be an independent prognostic factor to predict the survival of PDAC patients. In addition, we demonstrated that DIXDC1 depletion could result in cell cycle arrest and impaired proliferation of PDAC cells. Taken together, these results revealed that DIXDC1 might be a novel prognostic marker and therapeutic target of PDAC. It has been well documented that aberrant activation of the Wnt signaling pathway is associated with the development of a variety of human cancers, including pancreatic cancer [18-20]. Moreover, studies found that DIXDC1 affected the progression of human cancers through the regulation of PI3K/Akt pathway and Wnt/β-catenin pathway [17]. PI3K/Akt pathway has been implicated in the regulation of the proliferation of cancer cells via modulating the expression of cell cycle regulators, such as Cyclin D1 and p21 [21]. Meanwhile, DIXDC1 could inhibit the phosphorylation of β-catenin and promote the translocation of β-catenin to the nucleus through stabilizing β-catenin [7]. The accumulation of β-catenin in the nucleus activates various Wnt
ACCEPTED MANUSCRIPT target genes, such as Cyclin D1, which are involved in cell proliferation [22-24]. We found that PDAC cell lines transfected with DIXDC1 siRNA showed an apparently reduced population of cells in the S phase, whereas cells in the G1 phase was markedly elevated. Coincidently, depletion of DIXDC1 significantly downregulated the expression of Cyclin D1, suggesting that Cyclin D1 might be a downstream effecter of DIXDC1 in the
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regulation of PDAC cell proliferation. These date suggested that overexpression of DIXDC1 might play an
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important role in the cell cycle progression of PDAC cells through activating PI3K/Akt signaling pathway and Wnt/β-catenin pathway.
In summary, we for the first time showed that the expression of DIXDC1 was significantly increased in the
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human PDAC. Upregulated expression of DIXDC1 predicted dismal prognosis in PDAC patients. Moreover, depletion of DIXDC1 obviously inhibited the proliferation, cell cycle progression of PDAC cells. Therefore,
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DIXDC1 might serve as a novel molecular target for the detection and treatment of PDAC, pending further analyses in larger studies and functional in vivo experiments.
Acknowledgments This work was supported by the National Natural Scientific Foundation of China (No.81572397, 81200918) and Jiangsu Province’s Outstanding Medical Academic Leader program (No.
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LJ101135).
Conflict of interest We declare that we have no conflicts of interest.
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Figure legends
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[24] Tetsu O, McCormick F. Beta-catenin regulates expression of cyclin d1 in colon carcinoma cells. Nature
Fig.1 DIXDC1 was upregulated in human PDAC tissues and cell lines. a Eight representative paired samples of PDAC tissue (T) and adjacent normal tissues (N) were used for western blot analysis. DIXDC1 expression levels were higher in eight representative paired samples of PDAC tissues (T) compared with paired adjacent normal tissues (N). GAPDH was included as a loading control. The bar chart showed the ratio of DIXDC1 protein to GADPH. Mean±SD of three independent experiments (*P<0.05 compared with control nontumorous adjacent tissues). b The DIXDC1 expression is increased in different PDAC cell lines compared with normal pancreatic ductal cell (HPDE6-C7). The bar chart showed the ratio of DIXDC1 protein to GAPDH. Mean±SD of three independent experiments (*P<0.05).
Fig.2 Immunohistochemical evaluate of DIXDC1 and Ki-67 expression in paraffin-embedded PDAC (T) tissues and adjacent non-cancerous (N) tissues. High expression of DIXDC1 (a) and Ki-67 (b) was detected in PDAC specimen (SP×100, ×400). However, low levels of DIXDC1 expression (c) and Ki-67 (d) were seen in the paired normal tissues (SP×100, ×400).
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Fig.3 The relation between DIXDC1 and clinical characteristics. a Linear regression analysis of the expression of DIXDC1 and the proliferation index Ki-67 in 165 PDAC specimens. Scatterplot of Ki-67 versus DIXDC1 with regression line showed a correlation between them using the Spearman’s correlation coefficient. b
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Kaplan–Meier survival analysis of 165 pancreatic cancer patients based on DIXDC1 expression level.
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According to the DIXDC1 percentages, patients were divided into high DIXDC1 expressers (score >4) and low DIXDC1 expressers (score ≤4). Patients in the high-expression DIXDC1 group had a significantly shorter
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Fig.4 DIXDC1 was upregulated in proliferating PDAC cells. a, b Flow cytometry analysis of cell cycle
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distribution in Panc-1(a) and BxPC-3 (b) cells. Cells were synchronized at the G1 phase after serum starvation (S) for 72 h, then progressed into cell cycle by adding medium containing 10 % FBS for the indicated times (R4 h, R8 h, R12 h, R24 h). c, d Western blot analysis of the expression of DIXDC1, Cyclin D1 in PDAC cells that serum starvation for 72 h and serum re-addition in indicated times (R4 h, R8 h, R12 h, R24 h). GAPDH was included as a loading control. The bar chart below demonstrated the ratio of DIXDC1 and Cyclin D1 protein to
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GAPDH by densitometry in Panc-1 and BxPC-3 cells. Mean±SD of three independent experiments(*, # p<0.05).
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Fig.5 DIXDC1 knockdown suppressed PDAC cell proliferation and retarded cell cycle progression. a Western blot analysis of the expression of DIXDC1, Cyclin D1, PCNA in PDAC cells transfected with control-siRNA
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and different DIXDC1-targeting siRNA. GAPDH was included as a loading control. b Cell proliferation was measured using CCK-8 assay. Panc-1 and BxPC-3 cells that have been transfected with RACK1-siRNA#2 exhibited inhibited cell proliferation. c Silencing endogenous DIXDC1 suppressed cell growth of Panc-1 and
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BxPC-3 determined by colony formation assays. d DIXDC1 depletion resulted in a decrease of S transition of Panc-1 and BxPC-3 cells, whereas an increased population in the G1 phase. Mean±SD of three independent experiments (*, p<0.05).
Fig.6 Overexpression of DIXDC1 promoted PDAC cell proliferation and cell cycle progression. a HPDE6-C7 cells were transiently transfected with Flag-DIXDC1 plasmid as described above for 48 h. Western blot analysis of ectopic expression of DIXDC1. b Cell proliferation was measured using CCK-8 assay. HPDE6-C7 cells transfected with Flag- DIXDC1 exhibited enhanced cell proliferation. c Colony formation assays showed that increased endogenous DIXDC1 enhanced cell growth. d DNA content between Flag- DIXDC1 and empty vector treated cells was compared by flow cytometry. Mean±SD of three independent experiments (*, p <0.05).
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Table 1 Up-regulation of DIXDC1 in PDAC tissues compared to adjacent normal tissue DIXDC1 Tumor Adjacent normal P value expression (n=165) tissue(n=165) Low 68 103 0.000* High 97 62 Statistical analyses were carried out using Pearson χ2 test *P<0.05 was considered significant
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Table 2 Correlation between DIXDC1 expression and clinicopathologic speciments Characteristic Total RACK1(n%) χ2 value Low High n=68 n=97 Gender 0.021 Male 103 42 61 Female 62 26 36 Age(years) 2.206 ＜60 98 45 53 ≥60 67 23 44 Location 1.147 Head-neck 81 30 51 Body-tail 75 34 41 Total 9 4 5 Size(cm) 12.569 ＜2 30 21 9 2-4 96 33 63 ＞4 39 14 25 Histological 15.003 differentiation Well 29 19 10 Moderate 83 37 46 Poor 53 12 41 Lymph node 0.119 metastasis Yes 73 29 44 No 92 39 53 Nerve invasion 2.017 Yes 64 22 42 No 101 46 55 TNM stage 11.343 Ⅰ-Ⅱ 101 52 49 Ⅲ-Ⅳ 64 16 48 Ki67 expression 20.624 High 104 29 75 Low 61 39 22 Statistical analyses were carried out using Pearson χ2 test *P<0.05 was considered significant
p value
0.884
0.137
0.563
0.002*
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0.730
0.156
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Table 3 Univariate analysis and multivariate analyses of the contribution of various potential prognostic factors to survival in patients with PDAC Characteristic Univariate Cox regression Multivariate Cox regression P P HR 95%CI HR 95%CI value value Gender 0.946 0.690-1.296 0.729 Age 1.031 0.736-1.443 0.861 Location 1.086 0.779-1.515 0.627 Size 1.366 1.086-1.718 0.008* 1.074 0.830-1.391 0.588 Histological 1.435 1.124-1.830 0.004* 1.003 0.765-1.316 0.982 differentiation Lymph node metastasis 1.044 0.737-1.480 0.809 Nerve invasion 1.131 0.808-1.583 0.474 TNM stage 2.333 1.658-3.281 0.000* 1.507 1.008-2.254 0.046* Ki67 2.770 1.907-4.022 0.000* 1.817 1.139-2.898 0.012* DIXDC1 2.346 1.630-3.376 0.000* 1.802 1.216-2.699 0.003* Statistical analyses were performed by Cox proportional hazards regression *P<0.05 was considered significant