Cellular Physiology and Biochemistry

Download Fulltext PDF

Original Paper

Open Access Gateway

ZFX Promotes Proliferation and Metastasis of Pancreatic Cancer Cells via the MAPK Pathway

Song X.a,b,c,d · Zhu M.e · Zhang F.f · Zhang F.a,b,c,d · Zhang Y.a,b,c,d · Hu Y.a,b,c,d · Jiang L.a,b,c,d · Hao Y.a,b,c,d · Chen S.a,b,c,d · Zhu Q.a,b,c,d · Huang W.a,b,c,d · Lu J.a · Gu J.a · Gong W.a,b,c,d · Li M.a,b,c,d · Liu Y.a,b,c,d

Author affiliations

aDepartment of General Surgery and Laboratory of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
bShanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, China
cShanghai Research Center of Biliary Tract Disease, Shanghai, China
dInstitute of Biliary Tract Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, China
eDepartment of Hepatobiliary Surgery, The Third Affiliated Hospital to Wenzhou Medical University, Zhejiang, China
fDepartment of Gastroenterology, The First People’s Hospital of Xiaoshan, Zhejiang, China

Corresponding Author

Yingbin Liu and Maolan Li

Department of General Surgery and Laboratory of General Surgery,

Xinhua Hospital affiliated to Shanghai Jiao Tong Univ. School of Medicine, Shanghai, (PR China)

E-Mail liuyingbin@xinhuamed.com.cn; limaolan@xinhuamed.com.cn

Key Words: Zinc finger X-chromosomal proteinCell proliferationPancreatic cancerRNA interference

Related Articles for ""
Cell Physiol Biochem 2018;48:274–284
https://doi.org/10.1159/000491727

Abstract

Background/Aims: The role of ZFX in tumourigenesis is unclear. We aimed to study ZFX expression, regulation, and function and the clinical implications of this protein in human pancreatic cancer (PCa). Methods: One hundred and twenty patients with histologically confirmed PCa who underwent surgery were recruited for this study. Tumour samples and PCa cell lines were used to examine ZFX. Various cell functions related to tumourigenesis were assessed. In vivo mouse tumour xenografts were used to confirm the in vitro results. Results: Patients with ZFX-positive tumours had worse overall survival than patients with ZFX-negative tumours. The depletion of ZFX using lentiviral shRNAs significantly inhibited cell proliferation by inducing cell cycle arrest in G0/G1 phase and resulted in increased cell apoptosis and invasive repression. In vivo studies confirmed that ZFX promoted tumour growth. Mechanistically, MAPK pathway activation was involved in the oncogenic functions of ZFX. Conclusions: ZFX acts as a putative oncogene in PCa and could be a novel therapeutic target for this disease.

© 2018 The Author(s). Published by S. Karger AG, Basel

Introduction

Pancreatic cancer (PCa) is the most lethal solid tumour and is the fourth leading cause of cancer-related death, with an overall 5-year survival rate of < 5%[1, 2]. Due to local invasion, early metastasis, and resistance to standard chemotherapy, the median survival time of PCa patients is only 5-8 months despite recent advances in surgical techniques and medical management [3, 4]. In addition, although surgical resections are commonly performed, the long-term survival of PCa has not improved in two decades [5, 6]. Therefore, identification of effective early markers for the diagnosis and prognosis of the disease as well as novel therapeutic targets will be clinically valuable.

Zinc finger protein X-linked (ZFX), which is encoded on the X chromosome, contains an acidic transcriptional activation domain, a nuclear localization sequence, and a DNA binding domain consisting of 13 C2H2-type zinc fingers [7]. ZFX is highly conserved in vertebrates. Studies have demonstrated that ZFX plays a key role in controlling the self-renewal of embryonic and haematopoietic stem cells [8]. Recently, ZFX was shown to be involved various physiological functions, including proliferation, differentiation, cell cycle and cell death [9, 10]. Consistent with these critical roles, ZFX has been characterized as an oncogene in laryngeal squamous cell carcinoma, hepatocellular carcinoma, gastric carcinoma and colorectal cancer [11-14]. Clinicopathological analysis showed that high ZFX expression was correlated with poor prognosis in a variety of solid cancers, and increased ZFX expression was identified as an independent prognostic biomarker [11, 14, 15]. However, the biological function and molecular mechanisms of ZFX in PCa are unclear.

The present study aimed to elucidate the role of ZFX in PCa. To this end, we examined ZFX protein expression and localization in PCa tissues and investigated the phenotypic effects and mechanisms of ZFX in PCa.

Materials and Methods

Patients and clinicopathological data

The study was approved by the ethics committee of Xinhua Hospital, and all patients provided informed consent. Cancer tissue specimens were obtained from 120 PCa patients who underwent radical pancreaticoduodenectomy (without prior radiotherapy or chemotherapy) between 2014 and 2016 at the Department of General Surgery, Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, China. All diagnoses of PCa and lymph node metastasis were confirmed by histopathological examination, and adjacent control samples were confirmed to be free of tumour cells. All tissue samples were fixed in 4% formalin immediately after removal and embedded in paraffin for immunohistochemical staining.

Immunohistochemical analysis and evaluation of ZFX expression

Immunohistochemical staining was performed using a standard immunoperoxidase staining procedure, and ZFX expression in the benign and malignant specimens was evaluated as described by Li et al [16]. The sections were semi-quantitatively scored according to the extent of immunoreactivity as follows: 0, 0% immunoreactive cells; 1, < 5% immunoreactive cells; 2, 5–50% immunoreactive cells; and 3, > 50% immunoreactive cells. Additionally, the staining intensity was scored semi-quantitatively as follows: 0, negative; 1, weak; 2, intermediate; and 3, strong. The final immunoreaction score was defined as the sum of both parameters (extension and intensity), and the samples were classified as negative (0), weakly stained (1–2), moderately stained (3), and strongly stained (4–6). For statistical purposes, only moderate and strong final immunoreaction scores were considered positive; the other final scores were considered negative.

Cell culture

The PANC-1 and MIAPaca-2 were purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China). All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) foetal bovine serum (FBS) and routinely cultivated in a humidified incubator at 37°C with 5% CO2.

Lentivirus-mediated RNA interference

The guiding strand sequence of human small interfering RNA (siRNA) is 5’-GTCGGAAATTGATCCTTGTAA-3’. The negative control siRNA sequence is 5’-TTCTCCGAACGTGTCACGT-3’, with no significant sequence similarity to human gene sequences. Short hairpin RNAs (shRNAs) were synthesized and inserted into the pFH1UGW lentivirus vector containing a CMV-driven EGFP reporter gene and an H1 promoter upstream of restriction sites (NheI/PacI). Recombinant lentiviruses expressing ZFX siRNA or control siRNA (Lv-shZFX or Lv-shCon) were produced by co-transfecting HEK293 cells with pVSVG and pCMV⊿R 8.92 using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. The virus was harvested from the culture medium 2 days after transfection, concentrated by centrifugation, and used for subsequent studies.

Quantitative real-time PCR (qPCR)

Total RNA was extracted from cultured cells with TRIzol reagent (Invitrogen). The following primers were used to detect the expression of ZFX and β-actin (internal control):

ZFX (sense): 5’-GGCAGTCCACAGCAAGAAC-3’;

ZFX (antisense): 5’- TTGGTATCCGAGAAAGTCAGAAG-3’;

β-actin (sense): 5’- GTGGACATCCGCAAAGAC-3’;

β-actin (antisense): 5’- AAAGGGTGTAACGCAACTA-3’.

The protocol was performed according to the manufacturer’s instructions. The results were normalised to the expression of β-actin.

Western blot analysis

Total cell lysates were separated on a 10% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked and then probed with primary antibody against ZFX (1: 200 dilution, Abcam, Cambridge, UK) and GAPDH (1: 5000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the membranes were washed, they were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz, CA, USA) and visualised using the enhanced chemiluminescent detection reagent.

Cell proliferation assay

Cell proliferation was assessed with the CCK8 assay following the manufacturer’s instructions. In addition, the absorbance at 450 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA). A growth curve was prepared according to the absorbance values at 450 nm. The results reflect the average of three replicates under the same conditions.

Colony formation assay

Both non-transfected and transfected PANC-1 and MIAPaca-2 cells (400 cells/well) were seeded in 6-well plates. The culture medium was changed at regular time intervals. After 14 days of culture, adherent cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Colonies were stained with Giemsa solution for 15 min and then washed with water and air-dried. Cell colonies were counted by light microscopy. Experiments were performed in triplicate.

Cell cycle and apoptosis analyses

Forty-eight hours after lv-shRNA transfection, the cells were collected and stained using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Cell cycle analysis was performed after staining with propidium iodide. Both apoptosis and cell cycle distribution were quantified using a flow cytometer. The data presented are representative of at least three different experiments.

In vitro cell migration assay

Cell migration was assessed using 6.5-mm transwell chambers with a pore size of 8 µm (Corning). Transwell upper chambers were pre-coated with 50 µg/chamber of solubilized basement membrane. DMEM/10% FBS was added to the bottom chamber. PCa cells (2.5 ×104 per chamber) were seeded in serum-containing media in the upper well of the transwell chambers and incubated for about 20 h at 37°C and 5% CO2. The cells in the lower chamber (below the filter surface) were fixed in 70% ethanol, stained with 0.1 mg/ml crystal violet solution, and counted under a microscope (20× magnification). Five random visual fields were counted for each well, and the average was determined.

Wound healing assay

Cells were cultured for 72 h until confluency after the designated treatments in 6-well plates. Cells were then treated with mitomycin C (10 μg/ml) at 37°C in a 5% (v/v) CO2 incubator for 1 h to inhibit cell division. Three separate wounds through the cells were made by moving a sterile 200 μl pipet tip perpendicular to the demarcation line. Cells were gently rinsed with PBS and incubated with 2 ml serum-free media. Cells were photographed under a phase contrast microscope at 0 h (control) and 48 h. Each sample was assayed in triplicate.

Tumour xenografts in nude mice

Four- to 6-week-old nu/nu nude mice were purchased from the Shanghai Laboratory Animal Centre of the Chinese Academy of Sciences (Shanghai, China). All mice were housed under specific pathogen-free conditions following the guidelines of the Ethics Committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. For induction of subcutaneous PCa tumours, two groups (Lv-shCon group, and Lv-shZFX group) of PCa cells (1×106) in 0.1 mL serum-free DMEM were implanted subcutaneously into the left flank of the mice (5 mice/group). Tumour growth was monitored every 3 days and measured in 2 dimensions. Tumour volume was calculated using the formula width2 (mm2)×length (mm)/2, where width and length were the shortest and longest diameters, respectively. After approximately 4 weeks, mice were sacrificed, and primary tumours were collected for further immunohistochemical staining and western blot analysis.

Statistical analysis

Statistical analysis was conducted with SPSS software, version 22.0 (SPSS Inc., Chicago, IL, USA). The data are expressed as the mean±S.D. Independent Student’s t-tests and χ2 tests were used to assess the effects of ZFX in the cell lines. The Kaplan-Meier test was used for univariate survival analysis. The Cox proportional hazard model was used for multivariate analysis and for determining the 95% confidence interval. The results were considered to be significant at P< 0.05.

Results

ZFX is highly expressed in patients with PCa and correlated with poor prognosis

To determine the potential role of ZFX in PCa progression, we evaluated ZFX expression in PCa and adjacent control tissues by immunohistochemistry (Fig. 1A). ZFX was primarily located in the nucleus. Approximately 61.7% (74/120) of the PCa cases had positive ZFX staining in the tumour cells. In contrast, only 38.3% (46/120) of the cases had positive staining in the adjacent control tissues (Fig. 1A-1B, P< 0.001).

Fig. 1.

The enhanced expression of ZFX in PCa and the negative association with prognosis. Immunohistochemistry of ZFX expression (scale bar, 50 µm). The positive staining was predominantly localized to the cell cytoplasm. (A) Immunohistochemistry of ZFX in normal tissues and tumour tissues from PCa patients. (B) Average staining scores for ZFX in PCa tumours and in adjacent tissues (P< 0.001). (C) Kaplan–Meier plots of the overall survival of PCa patients based on ZFX expression (P< 0.001).

/WebMaterial/ShowPic/983856

Next, we assessed the correlation between ZFX expression levels and histopathological parameters in patients with PCa. As shown in Table 1, ZFX overexpression was significantly correlated with TNM stage (P< 0.001) and lymph node metastasis (P< 0.001) but not with sex, age, histopathological subtypes, tumour size, or tumour location, indicating a potential role of ZFX in promoting aggressive phenotypes in PCa.

Table 1.

Association between ZFX expression with the clinicopathological parameters of PCa. Notes: *, Statistically significant

/WebMaterial/ShowPic/983859

The survival information of the 120 PCa patients was obtained through phone calls. Kaplan–Meier survival analysis revealed that lymph node metastasis (P< 0.001) and TNM stage (P< 0.001) were significantly associated with the average survival time. The average survival time for ZFX-negative patients was significantly higher than that of patients with positive ZFX expression (P< 0.001) (Table 2, Fig. 1C). To obtain a more precise estimate of the prognosis, we used the Cox proportional hazards regression model. The results confirmed that ZFX expression, as well as the presence of lymph node metastasis and TNM stage, was negatively correlated with post-operative survival, suggesting that high ZFX expression is a risk factor (Table 3).

Table 2.

Univariate log-rank analysis of overall survival (OS). Notes: *, Statistically significant

/WebMaterial/ShowPic/983858
Table 3.

Multivariate analysis of overall survival (OS). Notes: *, Statistically significant. 1 HR, hazard ratio

/WebMaterial/ShowPic/983857

Ectopic expression of ZFX promotes tumourigenic properties of PCa cells

As unlimited growth, invasiveness, and metastasis are hallmarks of malignancy, we explored the role of ZFX in PCa progression. We silenced ZFX expression in PANC-1 and MIAPaca-2 cells, and the knockdown efficiency is shown in Fig. 2A. ZFX knockdown suppressed the growth of PANC-1 and MIAPaca-2 cells as shown by CCK8 assays (Fig. 2B). Furthermore, colony formation assays demonstrated that ZFX silencing significantly reduced the number of colonies (Fig. 2C). These results indicated that ZFX promotes cell growth in vitro. Next, to investigate whether ZFX affects the proliferation of PCa cells in vivo, we established xenograft mouse models. As shown in Fig. 2D, the tumour volume and weight of ZFX-depleted xenografts were significantly inhibited compared with the negative control group. Immunohistochemical analysis indicated that ZFX and Ki67 levels were substantially decreased in ZFX-depleted implanted tumour tissues (Fig. 2E).

Fig. 2.

ZFX exhibiting oncogenic properties in PCa cell lines. (A) The transfection efficiency was determined 2 days after incubation with lentivirus at an MOI of 40. Total protein and RNA were extracted 2 days after infection, and relative ZFX expression was determined using western blot analysis and RT-PCR. Data represent the mean±S.D. of three independent experiments. ***P< 0.001, compared with Lv-shCon. (B) Cellular proliferation of untransfected or transfected PANC-1 and MIAPaca-2 cells was measured using a CCK8 assay daily for 5 days. (C) PANC-1 cells and MIAPaca-2 cells were seeded at 500 cells/well, and the cells were allowed to form colonies. Colony numbers were counted and recorded. (D) Mice were treated with Lv-shCon and Lv-shZFX PCa cells for 4 weeks. Tumour volumes and weights were measured. (E) Immunohistochemical analysis shows a decrease in Ki67 and ZFX expression in Lv-shZFX cells compared with Lv-shCon cells. (F) Migration in PCa cell lines measured by transwell assays decreased after transfection. The number of migrating cells was calculated and is depicted in the bar chart (P< 0.01). (G) Wound closure was delayed in Lv-shZFX cells compared to Lv-shCon cells after 48 h (P< 0.01).

/WebMaterial/ShowPic/983855

Metastasis is frequently observed in patients with PCa [4], and therefore, we investigated whether ZFX might be involved in the migration of PANC-1 and MIAPaca-2 cells. In transwell migration assays, ZFX knockdown significantly impaired the migration of PANC-1 and MIAPaca-2 cells (Fig. 2F). Moreover, migration was also determined by wound healing assays. Knockdown of ZFX in PCa cells moderately decreased the migration rate (Fig. 2G). These results suggested that ZFX regulated the proliferation, survival and migration of PCa cells in vitro and further explained the aforementioned correlation of ZFX with poor prognosis of PCa patients.

Effects of ZFX silencing on cell cycle progression and apoptosis

To determine the potential mechanism underlying the role of ZFX in PCa cell growth, we assessed the cell cycle profile of the Lv-shCon and Lv-shZFX groups by FACS analysis 48 h after infection. As shown in Fig. 3A, the cell cycle analysis revealed G0/G1 phase arrest in PCa cells (Fig. 3A) after depletion of ZFX, induced cell cycle arrest in the G0/G1 phase. Furthermore, the effect of ZFX knocgkdown on the expression of cell cycle regulators was examined by real-time PCR and western blot analyses. As shown in Fig. 3B-3C, the protein and mRNA levels of CDK2, CDC25A, and cyclin D1 in the Lv-shZFX group were lower than those in the Lv-shCon group. These results indicated that ZFX knockdown induced cell cycle arrest in the G0/G1 phase, possibly by affecting the expression of cell cycle regulators.

Fig. 3.

Effects of ZFX silencing on cell cycle progression and apoptosis. (A) The cell cycle phases of treated cells were evaluated by flow cytometry after transfection for 48 h. The x-axis represents the periodic distribution, and the y- axis represents the number of cells. (B) The protein levels of cell cycle regulators, such as CDK2, CDC25A and cyclinD1, were examined by western blotting analysis. (C) The mRNA levels of cell cycle regulators, such as CDK2, CDC25A and cyclinD1, were examined by RT-PCR. (D) Untransfected and transfected PANC-1 and MIAPaca-2 cells were analysed by flow cytometry with annexin V–FITC/propidium iodide (PI) staining. The x- axis represents annexinV-FITC, and the y- axis represents propidium iodide (PI) staining. Annexin V versus PI plots from the gated cells show the populations corresponding to viable (annexin V/PI), necrotic (annexin V/PI+), early apoptotic (annexin V+/PI), and late apoptotic (annexin V+/PI+) cells. (E) The protein levels of bad, bax and bcl-2 were examined by western blotting analysis.

/WebMaterial/ShowPic/983854

To further investigate whether expression of ZFX affects apoptosis in PCa cells, we performed an apoptosis assay by FACS analysis. Transfection of ZFX shRNA resulted in a significant increase in the percentage of apoptotic cells compared with that of the Lv-shCon in both PCa cell lines (Fig. 3D). Moreover, knockdown of ZFX dramatically increased the expression of Bax and Bad and decreased the expression of Bcl-2 (Fig. 3E); these factors play important roles in apoptosis. The above data indicated that ZFX was involved in the regulation of PCa cell apoptosis and cell cycle progression.

ZFX regulates MAPK/ERK signalling in PCa

To elucidate the molecular mechanisms by which ZFX modulates the tumourigenic properties of PCa cells, we investigated the effects of ZFX on cell survival and cell cycle-related signalling molecules in PCa cells after Lv-shZFX infection. Western blot analysis showed that transfection with Lv-shZFX significantly decreased p-MEK and p-ERK in both PANC-1 and MIAPaca-2 cells but had no effect on p-STAT3 and p-JNK (Fig. 4A). Encouraged by the above results, we investigated whether targeting MAPK/ERK could attenuate the oncogenic function of ZFX. We treated PCa cells with the ERK inhibitor SCH772984 and found that this compound inhibited ZFX overexpression-induced cell proliferation and invasion (Fig. 4B-4C). As expected, SCH772984 also promoted the ZFX overexpression-inhibited apoptosis and G0/G1 phase cell accumulation (Fig. 4D-4E). Moreover, the downstream genes of the MAPK signalling pathway, including Foxo3 and PPARγ, were upregulated, which also supports activation of the MAPK pathway in PCa cells (Fig. 4A). Thus, the data indicated that the MAPK/ERK pathway might participate in the oncogenic function of ZFX.

Fig. 4.

ZFX regulates MAPK/ERK signalling in PCa. (A) Western blotting analysis of MAPK/ERK signalling-related proteins in both cell lines. GAPDH was used as a loading control. (B) Cellular proliferation of PANC-1 and MIAPaca-2 cells treated with or without the ERK inhibitor SCH772984 were measured using a CCK8 assay daily for 5 days. (C) Migration in PCa cell lines PANC-1 and MIAPaca-2 cells treated with or without the ERK inhibitor SCH772984 were measured by transwell migration assays. The number of migrating cells was calculated and is depicted in the bar chart. (D) PANC-1 and MIAPaca-2 cells treated with or without the ERK inhibitor SCH772984 were analysed by flow cytometry with annexin V-FITC/propidium iodide (PI) staining. The x- axis represents annexinV-FITC, and the y- axis represents propidium iodide (PI) staining. Annexin V versus PI plots from the gated cells show the populations corresponding to viable (annexin V/PI), necrotic (annexin V/PI+), early apoptotic (annexin V+/PI), and late apoptotic (annexin V+/PI+) cells. (E) The cell cycle phases of treated cells were evaluated by flow cytometry. The x- axis represents the periodic distribution, and the y- axis represents the number of cells.

/WebMaterial/ShowPic/983853

Discussion

PCa remains a lethal malignancy despite the progress made in elucidating its molecular mechanisms [17, 18]. Therefore, identification of novel methods that can effectively inhibit PCa cell growth and metastasis is urgently needed. Targeted therapies have shown promise in achieving these goals [19-21]. ZFX, also known as ZNF926, is a sex chromosome gene [21]. This protein is a novel member of the Kruppel C2H2-type ZNF protein family and encodes a zinc finger transcription factor that contains 13 C2H2 type ZNF motifs at the carboxyl terminus [22]. ZFX was originally identified as a factor that determines whether an embryo develops as a male or female. Further studies confirmed that ZFX was required for the self-renewal of haematopoietic and embryonic stem cells and could regulate cell survival [15, 22]. Furthermore, ZFX forms a unique transcription network with Cnot3, Trim28 and c-Myc that is required for self-renewal in embryonic stem cells, suggesting a potential role of ZFX in tumourigenesis [10, 11]. Recent studies have also linked ZFX upregulation to cancer development and progression in various types of cancer. ZFX facilitates the development of experimental basal cell carcinoma and medulloblastoma in mice initiated by deletion of the Hedgehog (Hh) inhibitory receptor Ptch1. These results indicate that ZFX is a common cell-intrinsic regulator of diverse Hh-induced tumours and also suggest new therapeutic targets for these malignancies [23]. Furthermore, knocking down ZFX dramatically inhibited the proliferation and metastasis of glioma cells. Based on these findings, ZFX plays an important role in carcinogenesis. However, to date, ZFX has never been linked to PCa.

In the present study, we found for the first time that significantly increased ZFX expression was detected in the vast majority of PCa tissues compared with adjacent control samples. Moreover, increased ZFX protein expression correlated not only with advanced tumour status but also with poor patient survival, suggesting the significance of ZFX in PCa progression. Notably, Cox multivariate analysis indicated that a high nuclear ZFX level was an independent factor for poor prognosis in PCa patients. Thus, ZFX could be a potential therapeutic target in PCa.

Additionally, we found that ZFX may play an important role in PCa cell apoptosis, cell cycle, metastasis and proliferation in vitro and in vivo. Knockdown of ZFX significantly impaired PCa cell growth in vitro and in vivo and increased the number of apoptotic PANC-1 and MIAPaca-2 cells; these results were further confirmed by enhanced expression of Bad and Bax and reduced expression of Bcl-2, key apoptosis-related factors. Notably, progression through the cell cycle depends on the activation of cyclin-dependent kinases (CDKs) and their regulatory subunits, the cyclins. In this study, we observed that ZFX knockdown inhibited PCa cell proliferation by inducing cell cycle arrest in G0/G1 phase while down-regulating CDK2, CDC25A, and Cyclin D1. Thus, ZFX may regulate the expression of cell cycle regulators. Additionally, consistent with the results of a previous study [24, 25], knockdown of ZFX substantially inhibited cell migration and invasion in PCa cells. These properties might contribute to the ZFX-associated aggressive biological behaviours of PCa.

We further elucidated the downstream signalling pathway responsible for ZFX oncogenic function in PCa and found that the ZFX-mediated tumour growth and metastasis could be, at least partly, attributed to the activation of MAPK/ERK signalling, which is critical for the initiation and progression of PCa. The ZFX-induced MAPK/ERK signalling in PANC-1 and MIAPaca-2 cells was confirmed because targeting MAPK/ERK could attenuate the oncogenic function of ZFX and the upregulation of MAPK/ERK target gene expression. However, the direct link between ZFX and the MAPK/ERK pathway should be further investigated in future studies.

In summary, the present study showed that ZFX might be involved in PCa progression and aggressiveness. The classification of patients according to ZFX expression levels provides a valuable tool with which to identify PCa patients with poor prognoses. Furthermore, we demonstrated that knockdown of ZFX inhibited PCa cell growth via the MAPK/ERK pathway. These findings will help elucidate the biological progression of PCa and could provide a new therapeutic target for PCa.

Acknowledgements

This study was supported by Shanghai Key Laboratory of Biliary Tract Disease Research Foundation(17DZ2260200), the National Natural Science Foundation of China (No. 81773043, 81172026, 81272402, 81301816, 81172029, 91440203, 81402403, 81502433 and 31501127) , the development fund for Shanghai talents(No.201608), the Shanghai Science and Technology Commission Key Basic Research Program (No. 16JC1400200), the Key Program of Shanghai Science and Technology Commission (No. 16411952501), the Program for Changjiang Scholars, the Multiple Central Clinical Research Program of Shanghai Jiao Tong University School of Medicine (No. DLY201507), the Precision Medicine Research Program of Shanghai Jiao Tong University School of Medicine (No. 15ZH4003) and the Shanghai Rising-Star Programme (No. 15QA1403100).

Disclosure Statement

No conflict of interests to disclose.


Related Articles:

References

  1. Delpero JR, Jeune F, Bachellier P, Regenet N, Le Treut YP, Paye F, Carrere N, Sauvanet A, Adham M, Autret A, Poizat F, Turrini O, Boher JM: Prognostic Value of Resection Margin Involvement After Pancreaticoduodenectomy for Ductal Adenocarcinoma: Updates From a French Prospective Multicenter Study. Ann Surg 2017; 266: 787-796.
  2. Chen Q, Yang L, Xiao Y, Zhu J, Li Z: Circulating microRNA-182 in plasma and its potential diagnostic and prognostic value for pancreatic cancer. Medical oncology 2014; 31: 225.
  3. Neoptolemos JP, Kleeff J, Michl P, Costello E, Greenhalf W, Palmer DH: Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature 2014; 514: 628-632.
  4. Chiaravalli M, Reni M, O’Reilly EM: Pancreatic ductal adenocarcinoma: State-of-the-art 2017 and new therapeutic strategies. Cancer Treat Rev 2017; 60: 32-43.
  5. Reiter JG,Iacobuzio DC: Pancreatic cancer: Pancreatic carcinogenesis - several small steps or one giant leap? Nat Rev Gastroenterol Hepatol 2016; 14: 7-8.
  6. Vakoc CR, Tuveson DA: Soils and Seeds That Initiate Pancreatic Cancer Metastasis. Cancer Discov 2017; 7: 1067-1068.
  7. Iovanna J, Dusetti N: Speeding towards individualized treatment for pancreatic cancer by taking an alternative road. Cancer Lett 2017; 410: 63-67.
  8. Weng H, Wang X, Li M, Wu X, Wang Z, Wu W, Zhang Z, Zhang Y, Zhao S, Liu S, Mu J, Cao Y, Shu Y, Bao R, Zhou J, Lu J, Dong P, Gu J, Liu Y: Zinc finger X-chromosomal protein (ZFX) is a significant prognostic indicator and promotes cellular malignant potential in gallbladder cancer. Cancer Biol Ther 2015; 16: 1462-1470.
  9. Yang H, Lu Y, Zheng Y, Yu X, Xia X, He X, Feng W, Xing L, Ling Z: shRNA-mediated silencing of ZFX attenuated the proliferation of breast cancer cells. Cancer Chemother Pharmacol 2014; 73: 569-576.
  10. Li K, Zhu ZC, Liu YJ, Liu JW, Wang HT, Xiong ZQ, Shen X, Hu ZL, Zheng J: ZFX knockdown inhibits growth and migration of non-small cell lung carcinoma cell line H1299. Int J Clin Exp Pathol 2013; 6: 2460-2467.
  11. Liu TY, Gong W, Tan ZJ, Lu W, Wu XS, Weng H, Ding Q, Shu YJ, Bao RF, Cao Y, Wang XA, Zhang F, Li HF, Xiang SS, Jiang L, Hu YP, Mu JS, Li ML, Wu WG, Shen BY, Jiang LX, Liu YB: Baicalein inhibits progression of gallbladder cancer cells by downregulating ZFX. PLoS One 2015; 10:e0114851.
  12. Lai KP, Chen J, He M, Ching AK, Lau C, Lai PB, To KF, Wong N: Overexpression of ZFX confers self-renewal and chemoresistance properties in hepatocellular carcinoma. Int J Cancer 2014; 135: 1790-1799.
  13. Fang J, Yu Z, Lian M, Ma H, Tai J, Zhang L, Han D: Knockdown of zinc finger protein, X-linked (ZFX) inhibits cell proliferation and induces apoptosis in human laryngeal squamous cell carcinoma. Mol Cell Biochem 2012; 360: 301-307.
  14. Wu S, Lao XY, Sun TT, Ren LL, Kong X, Wang JL, Wang YC, Du W, Yu YN, Weng YR ,Hong J, Fang JY: Knockdown of ZFX inhibits gastric cancer cell growth in vitro and in vivo via downregulating the ERK-MAPK pathway. Cancer Lett 2013; 337: 293-300.
  15. Xu S, Duan P, Li J, Senkowski T, Guo F, Chen H, Romero A, Cui Y, Liu J, Jiang SW: Zinc Finger and X-Linked Factor (ZFX) Binds to Human SET Transcript 2 Promoter and Transactivates SET Expression. Int J Mol Sci 2016; 17.
  16. Li M, Zhang S, Wang Z, Zhang B, Wu X, Weng H, Ding Q, Tan Z, Zhang N, Mu J, Yang J, Shu Y, Bao R, Ding Q, Wu W, Cao Y, Liu Y: Prognostic significance of nemo-like kinase (NLK) expression in patients with gallbladder cancer. Tumour Biol 2013; 34: 3995-4000.
  17. Palanivelu C, Senthilnathan P, Sabnis SC, Babu NS, Srivatsan GS, Anand VN, Nalankilli VP, Praveen RP, Parthasarathy R, Rajapndian S: Randomized clinical trial of laparoscopic versus open pancreatoduodenectomy for periampullary tumours. Br J Surg 2017; 104: 1443-1450.
  18. Chan E, Arlinghaus LR, Cardin DB, Goff L, Berlin JD, Parikh A, Abramson RG, Yankeelov TE, Hiebert S, Merchant N, Bhaskara S, Chakravarthy AB: Phase I trial of vorinostat added to chemoradiation with capecitabine in pancreatic cancer. Radiother Oncol 2016; 119: 312-318.
  19. Delpero JR, Jeune F, Bachellier P, Regenet N, Le Treut YP, Paye F, Carrere N, Sauvanet A, Adham M, Autret A, Poizat F, Turrini O, Boher JM: Prognostic Value of Resection Margin Involvement After Pancreaticoduodenectomy for Ductal Adenocarcinoma: Updates From a French Prospective Multicenter Study. Ann Surg 2017; 266: 787-796.
  20. Liu H, Sun B, Wang S, Liu C, Lu Y, Li D, Liu X: Circulating Tumor Cells as a Biomarker in Pancreatic Ductal Adenocarcinoma. Cell Physiol Biochem 2017; 42: 373-382.
  21. Zhou B, Sun C, Hu X, Zhan H, Zou H, Feng Y, Qiu F, Zhang S, Wu L, Zhang B: MicroRNA-195 Suppresses the Progression of Pancreatic Cancer by Targeting DCLK1. Cell Physiol Biochem 2017; 44: 1867-1881.
  22. Sanpaolo E, Miroballo M, Corbetta S, Verdelli C, Baorda F, Balsamo T, Graziano P, Fabrizio FP, Cinque L, Scillitani A, Muscarella LA, Guarnieri V: EZH2 and ZFX oncogenes in malignant behaviour of parathyroid neoplasms. Endocrine 2016; 54: 55-59.
  23. Zhu Z, Li K, Xu D, Liu Y, Tang H, Xie Q, Xie L, Liu J, Wang H, Gong Y, Hu Z, Zheng J: ZFX regulates glioma cell proliferation and survival in vitro and in vivo. J Neurooncol 2013; 112: 17-25.
  24. Palmer CJ, Galan JM, Weisberg SP, Lei L, Esquilin JM, Croft GF, Wainwright B, Canoll P, Owens DM, Reizis B: Zfx facilitates tumourigenesis caused by activation of the Hedgehog pathway. Cancer Res 2014; 74: 5914-5924.
  25. Yin J, Jiang Y, Wu H, Wang J, Zhang S, Liu H: Overexpression of ZFX and its involvement in squamous cell carcinoma of the tongue. Oncol Rep 2015; 33: 141-8.
  26. Tan Z, Zhang S, Li M, Wu X, Weng H, Ding Q, Cao Y, Bao R, Shu Y, Mu J, Ding Q, Wu W, Yang J, Zhang L, Liu Y: Regulation of cell proliferation and migration in gallbladder cancer by zinc finger X-chromosomal protein. Gene 2013; 528: 261-266.

Author Contacts

Yingbin Liu and Maolan Li

Department of General Surgery and Laboratory of General Surgery,

Xinhua Hospital affiliated to Shanghai Jiao Tong Univ. School of Medicine, Shanghai, (PR China)

E-Mail liuyingbin@xinhuamed.com.cn; limaolan@xinhuamed.com.cn

Article / Publication Details

First-Page Preview
Abstract of Original Paper

Received: February 05, 2018
Accepted: May 20, 2018
Published online: July 13, 2018
Issue release date: August 2018

Number of Print Pages: 11
Number of Figures: 4
Number of Tables: 3

ISSN: 1015-8987 (Print)
eISSN: 1421-9778 (Online)

For additional information: https://www.karger.com/CPB

Open Access License / Drug Dosage / Disclaimer

This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Delpero JR, Jeune F, Bachellier P, Regenet N, Le Treut YP, Paye F, Carrere N, Sauvanet A, Adham M, Autret A, Poizat F, Turrini O, Boher JM: Prognostic Value of Resection Margin Involvement After Pancreaticoduodenectomy for Ductal Adenocarcinoma: Updates From a French Prospective Multicenter Study. Ann Surg 2017; 266: 787-796.
  2. Chen Q, Yang L, Xiao Y, Zhu J, Li Z: Circulating microRNA-182 in plasma and its potential diagnostic and prognostic value for pancreatic cancer. Medical oncology 2014; 31: 225.
  3. Neoptolemos JP, Kleeff J, Michl P, Costello E, Greenhalf W, Palmer DH: Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature 2014; 514: 628-632.
  4. Chiaravalli M, Reni M, O’Reilly EM: Pancreatic ductal adenocarcinoma: State-of-the-art 2017 and new therapeutic strategies. Cancer Treat Rev 2017; 60: 32-43.
  5. Reiter JG,Iacobuzio DC: Pancreatic cancer: Pancreatic carcinogenesis - several small steps or one giant leap? Nat Rev Gastroenterol Hepatol 2016; 14: 7-8.
  6. Vakoc CR, Tuveson DA: Soils and Seeds That Initiate Pancreatic Cancer Metastasis. Cancer Discov 2017; 7: 1067-1068.
  7. Iovanna J, Dusetti N: Speeding towards individualized treatment for pancreatic cancer by taking an alternative road. Cancer Lett 2017; 410: 63-67.
  8. Weng H, Wang X, Li M, Wu X, Wang Z, Wu W, Zhang Z, Zhang Y, Zhao S, Liu S, Mu J, Cao Y, Shu Y, Bao R, Zhou J, Lu J, Dong P, Gu J, Liu Y: Zinc finger X-chromosomal protein (ZFX) is a significant prognostic indicator and promotes cellular malignant potential in gallbladder cancer. Cancer Biol Ther 2015; 16: 1462-1470.
  9. Yang H, Lu Y, Zheng Y, Yu X, Xia X, He X, Feng W, Xing L, Ling Z: shRNA-mediated silencing of ZFX attenuated the proliferation of breast cancer cells. Cancer Chemother Pharmacol 2014; 73: 569-576.
  10. Li K, Zhu ZC, Liu YJ, Liu JW, Wang HT, Xiong ZQ, Shen X, Hu ZL, Zheng J: ZFX knockdown inhibits growth and migration of non-small cell lung carcinoma cell line H1299. Int J Clin Exp Pathol 2013; 6: 2460-2467.
  11. Liu TY, Gong W, Tan ZJ, Lu W, Wu XS, Weng H, Ding Q, Shu YJ, Bao RF, Cao Y, Wang XA, Zhang F, Li HF, Xiang SS, Jiang L, Hu YP, Mu JS, Li ML, Wu WG, Shen BY, Jiang LX, Liu YB: Baicalein inhibits progression of gallbladder cancer cells by downregulating ZFX. PLoS One 2015; 10:e0114851.
  12. Lai KP, Chen J, He M, Ching AK, Lau C, Lai PB, To KF, Wong N: Overexpression of ZFX confers self-renewal and chemoresistance properties in hepatocellular carcinoma. Int J Cancer 2014; 135: 1790-1799.
  13. Fang J, Yu Z, Lian M, Ma H, Tai J, Zhang L, Han D: Knockdown of zinc finger protein, X-linked (ZFX) inhibits cell proliferation and induces apoptosis in human laryngeal squamous cell carcinoma. Mol Cell Biochem 2012; 360: 301-307.
  14. Wu S, Lao XY, Sun TT, Ren LL, Kong X, Wang JL, Wang YC, Du W, Yu YN, Weng YR ,Hong J, Fang JY: Knockdown of ZFX inhibits gastric cancer cell growth in vitro and in vivo via downregulating the ERK-MAPK pathway. Cancer Lett 2013; 337: 293-300.
  15. Xu S, Duan P, Li J, Senkowski T, Guo F, Chen H, Romero A, Cui Y, Liu J, Jiang SW: Zinc Finger and X-Linked Factor (ZFX) Binds to Human SET Transcript 2 Promoter and Transactivates SET Expression. Int J Mol Sci 2016; 17.
  16. Li M, Zhang S, Wang Z, Zhang B, Wu X, Weng H, Ding Q, Tan Z, Zhang N, Mu J, Yang J, Shu Y, Bao R, Ding Q, Wu W, Cao Y, Liu Y: Prognostic significance of nemo-like kinase (NLK) expression in patients with gallbladder cancer. Tumour Biol 2013; 34: 3995-4000.
  17. Palanivelu C, Senthilnathan P, Sabnis SC, Babu NS, Srivatsan GS, Anand VN, Nalankilli VP, Praveen RP, Parthasarathy R, Rajapndian S: Randomized clinical trial of laparoscopic versus open pancreatoduodenectomy for periampullary tumours. Br J Surg 2017; 104: 1443-1450.
  18. Chan E, Arlinghaus LR, Cardin DB, Goff L, Berlin JD, Parikh A, Abramson RG, Yankeelov TE, Hiebert S, Merchant N, Bhaskara S, Chakravarthy AB: Phase I trial of vorinostat added to chemoradiation with capecitabine in pancreatic cancer. Radiother Oncol 2016; 119: 312-318.
  19. Delpero JR, Jeune F, Bachellier P, Regenet N, Le Treut YP, Paye F, Carrere N, Sauvanet A, Adham M, Autret A, Poizat F, Turrini O, Boher JM: Prognostic Value of Resection Margin Involvement After Pancreaticoduodenectomy for Ductal Adenocarcinoma: Updates From a French Prospective Multicenter Study. Ann Surg 2017; 266: 787-796.
  20. Liu H, Sun B, Wang S, Liu C, Lu Y, Li D, Liu X: Circulating Tumor Cells as a Biomarker in Pancreatic Ductal Adenocarcinoma. Cell Physiol Biochem 2017; 42: 373-382.
  21. Zhou B, Sun C, Hu X, Zhan H, Zou H, Feng Y, Qiu F, Zhang S, Wu L, Zhang B: MicroRNA-195 Suppresses the Progression of Pancreatic Cancer by Targeting DCLK1. Cell Physiol Biochem 2017; 44: 1867-1881.
  22. Sanpaolo E, Miroballo M, Corbetta S, Verdelli C, Baorda F, Balsamo T, Graziano P, Fabrizio FP, Cinque L, Scillitani A, Muscarella LA, Guarnieri V: EZH2 and ZFX oncogenes in malignant behaviour of parathyroid neoplasms. Endocrine 2016; 54: 55-59.
  23. Zhu Z, Li K, Xu D, Liu Y, Tang H, Xie Q, Xie L, Liu J, Wang H, Gong Y, Hu Z, Zheng J: ZFX regulates glioma cell proliferation and survival in vitro and in vivo. J Neurooncol 2013; 112: 17-25.
  24. Palmer CJ, Galan JM, Weisberg SP, Lei L, Esquilin JM, Croft GF, Wainwright B, Canoll P, Owens DM, Reizis B: Zfx facilitates tumourigenesis caused by activation of the Hedgehog pathway. Cancer Res 2014; 74: 5914-5924.
  25. Yin J, Jiang Y, Wu H, Wang J, Zhang S, Liu H: Overexpression of ZFX and its involvement in squamous cell carcinoma of the tongue. Oncol Rep 2015; 33: 141-8.
  26. Tan Z, Zhang S, Li M, Wu X, Weng H, Ding Q, Cao Y, Bao R, Shu Y, Mu J, Ding Q, Wu W, Yang J, Zhang L, Liu Y: Regulation of cell proliferation and migration in gallbladder cancer by zinc finger X-chromosomal protein. Gene 2013; 528: 261-266.
ppt logo Download Figures (.pptx)

Figures
Thumbnail
Thumbnail
Thumbnail
Thumbnail
Tables
Thumbnail
Thumbnail
Thumbnail

Article Tools
Article Details

2018, Vol.48, No. 1

August 2018

Open Access Gateway

Article

Recommend This
Open Access Gateway
Additional Information

Xiaoling Song, Minghui Zhu and Fahong Zhang contributed equally to this work.

TOP