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Epithelial–Mesenchymal Transition Induced by SMAD4 Activation in Invasive Growth Hormone-Secreting Adenomas

SMAD4 and Epithelial–Mesenchymal Transition
  • Xiaosong Shan , Qian Liu , Zhenye Li , Chuzhong Li , Hua Gao and Yazhuo Zhang EMAIL logo
Published/Copyright: June 14, 2018
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Open Chemistry
From the journal Open Chemistry Volume 16 Issue 1

Abstract

Background

The detection and treatment of invasive growth hormone-secreting pituitary adenoma (GHPA) remains challenging. Several transcription factors promoting the epithelial–mesenchymal transition (EMT) can act as cofactors for the transforming growth factor-beta (TGF-ß)/SMAD4. The goal of this study was to investigate the association of SMAD4 expression and clinicopathologic features using a tissue microarray analysis (TMA). The levels of SMAD4 and the related genes of EMT in GHPAs were analyzed by q-PCR and western blot. SMAD4 was strongly expressed in 15/19 cases (78.9%) of invasive GHPA and 10/42 cases (23.8%) of noninvasive GHPA (χ2=10.887, p=0.000). In the high SMAD4 group, a headache was reported in 16/25 cases (64%) compared with 13/36 cases (36.1%) in the low SMAD4 group (χ2=4.565, p=0.032). The progression-free survival (PFS) in the high group was lower than that in the low group (p=0.026). qRT-PCR and western blot analysis further revealed a significant downregulation of E-cadherin and upregulation of N-cadherin and vimentin in the invasive GHPA group. SMAD4 was associated with increased levels of invasion of GH3 cells, as determined by a transwell test. SMAD4 downregulated E-cadherin levels and increased the levels of N-cadherin and vimentin. Our data provide evidence that SMAD4 is a potential prognosis biomarker and a therapeutic target for patients with invasive GHPA.

Keywords: SMAD4; Growth Hormone-secreting Pituitary Adenoma; Invasive; Epithelial–Mesenchymal Transition; GH3 cells

1 Introduction

Pituitary adenomas are relatively benign tumors that account for 10–15% of intracranial tumors [1, 2]. Growth hormone-secreting adenomas (GHPAs) are the second most common hormone-secreting pituitary adenomas and represent 10–15% of pituitary adenomas [3]. GHPAs are characterized by an excessive production of growth hormone and the consequent increase in insulin-like growth factor 1 (IGF-1). This condition typically results in acromegaly, a disorder characterized by enlargement of the hands, feet and facial lines [4]. Although GHPAs are usually regarded as benign lesions, many can invade the sphenoid, cavernous sinus, or dura mater becoming malignant. The goal of GHPA treatment is to normalize the growth hormone and IGF-1 levels in order to prevent tumor growth [5]. Surgery provides 60–80% biochemical remission in microadenomas or noninvasive macroadenomas [6,7,8]. However, cure rate in invasive GHPAs is low due to the failure of complete resection [9, 10]. The remission rate in invasive GHPAs remains unsatisfactory even when surgical intervention is followed by radiological or pharmacological therapy.

Over the past 20 years, major advances have been made in cancer research, especially in the understanding of the molecular mechanisms of tumor development and the role of IGF-1. The mechanisms mediating invasive behavior in pituitary adenomas are complex [11, 12]. Epithelial–mesenchymal transition (EMT), a process involved in tumor invasion, occurs when an epithelial cell differentiates into a more motile mesenchymal cell. EMT markers such as N-cadherin, vimentin, and Snail are significant indicators of the appearance of cystic lesions, tumor progression, bone destruction, and endocrine functions. N-cadherin is a well-known EMT biomarker for invasive tumors, including growth hormone adenomas. Healthy cells express the E-cadherin-catenin complex on their plasma membrane; this complex plays a key role in cellular adhesion by maintaining tissue structure. When E-cadherin switches to N-cadherin this function is lost, resulting in cell invasion and dissemination of carcinoma cells [13, 14].

The growth factor-β (TGF-β) performs a dual role in carcinogenesis, being a tumor suppressor in the early stages and a tumor promoter in the later stages of carcinogenesis [15, 16]. TGF-β/SMAD signaling pathway is believed to play a major role in EMT. In this pathway, the activated complex SMAD2/SMAD3 binds the mediator SMAD4 and is translocated to the nucleus, where it further binds regulatory elements and induce the transcription of key genes associated with EMT, such as Snail1, ZEB1/2, and Twist. Despite the importance of TGF-β/SMAD pathway in EMT, the roles of TGF-β and SMAD4 in tumor progression remain controversial. Consistent with the role of TGF-β as a tumor suppressor is the observation that mutation of SMAD4 [17, 18] stimulates thyroid carcinogenesis and progression. Loss or weak expression of SMAD4 strongly correlates with invasion, metastasis, high histopathological grade, and poor prognosis of patients with diverse cancers as well [19,20,21,22,23]. Conversely, other studies suggest a tumor-promoting activity of TGF-β [24]. Likewise, the precise mechanism underlying the role of SMAD4 in the oncogenic response remains unclear. SMAD4 has been shown to act as an oncogene in tumorigenesis in previous studies [25,26,27]. Disrupting the association between SMAD4 and SMAD receptors also leads to the inhibition of EMT in human mammary epithelial cells [28]. Furthermore, knockdown of SMAD4 through RNA interference results in the inhibition of EMT [29,30,31]. In our previous work, we have suggested that TGF-β/SMAD signaling may be associated with the development and invasion of nonfunctioning pituitary adenomas [32].

Elucidating the potential role of SMAD4 in GHPAs may contribute to the improved diagnosis and treatment of invasive GHPA patients. In the present study, we first assessed SMAD4 expression in a cohort of GHPA patients and investigated the correlation between the expression of SMAD4 and the clinicopathological features of GHPA. Then, the markers of EMT were analyzed to determine the underlying mechanism of SMAD4 overexpression in GH3 cells.

2 Materials and methods

2.1 Patients and sample collection

The study was carried out on 61 patients with GHPA admitted at the Department of Neurosurgery in Beijing Tiantan Hospital (Capital Medical University) from April 2009 to November 2014. GHPAs were classified histologically according to the 2007 World Health Organization histologic classification of pituitary adenomas [33]. The cases that we selected met the following strict criteria: i) none of the patients had undergone somatostatin-analog therapy or radiotherapy during the preoperative period; ii) patients with recurrence were excluded; iii) adequate anamnestic and clinical data were available; and iv) the follow-up duration was a minimum of 2 years. The present study did not include a normal pituitary control group because of insufficient cases. Specimens were classified into two groups: non-invasive and invasive. The biological behavior of tumors was assessed based on preoperative magnetic resonance imaging (MRI) and computed tomography. GHPAs that were classified as Knosp classification grade III-IV and Hardy classification grade III-IV were defined as invasive.

GHPAs were resected by trans-sphenoidal surgery. Part of each specimen was formalin-fixed and paraffin-embedded to allow for analysis by tissue microarray (TMA), and the remainder was immediately snap-frozen in liquid nitrogen prior to further study. Postoperative sella MRI scans were performed within 72 h of surgery to evaluate the residual mass. Serial sella MRI scans were performed at 6-month intervals in the first 2 postoperative years and at 12-month intervals thereafter. MRI scans were performed immediately whenever the clinical symptoms occurred or reoccurred. Recurrence was identified based on one or more of the following parameters: i) for patients with total resection: the presence of a new tumor in the first postoperative sella MRI; ii) for patients with subtotal resection: the emergence of a new growth area in the first postoperative sella MRI compared with the sella MRI scan; and iii) recurrence or aggravation of postoperative clinical symptoms accompanied by rising levels of growth hormone.

The present study was performed in accordance with the ethical standards of the Ethics Committee of Beijing Tiantan Hospital (Beijing, China). Written informed consent was obtained from all patients for surgery and for participation in the study.

2.2 TMA construction

Formalin-fixed paraffin-embedded tissue blocks were sliced and stained with hematoxylin and eosin (H&E). Three 2.0-mm diameter core biopsies were selected from the paraffin-embedded tissue blocks. The cores were transferred to TMAs and randomly ordered using the Leica Bond-III fully automated arrayer from Leica Biosystems (Aperio, CA, USA). The pathologists were blinded to the identity of the TMA slides. The TMAs were cut into 4-μm sections using a serial microtome and placed in a water bath at 50°C. They were then applied onto positively-charged glass slides. The slides were dewaxed and then rehydrated through graded alcohols into water. After mounting, the slides were dried at room temperature for 24–48 h and stored in a freezer at –80°C until further testing. To minimize the loss of antigenicity, the microarray slides were processed within 1 week of cutting.

2.3 Immunohistochemistry (IHC)

Prior to IHC, all TMA slides were H&E stained to evaluate the tumor content and quality. IHC tests with rabbit monoclonal anti-SMAD4 antibody (1:100, Abcam) were performed on the sections from all TMAs using the Leica Bond-III (Aperio, CA, USA) automated, random, and continuous access slide-staining system. IHC protocol F was selected in the machine, and 3 min ER1 (epitope retrieval) was set as the HIER (heat-induced epitope retrieval) parameter. The primary antibodies were detected by the BondTM polymer refine detection system (Leica Biosystems, DS9800). The expression of slides was examined by an Aperio AT2 digital scanner (Leica Biosystems). The optimal titer of the primary antibodies was determined based on the pre-experimental results.

2.4 Evaluation of immunohistochemical staining

The sections were examined and scored by two pathologists who were unaware of the clinical records of the patients. Staining for SMAD4 was present in the cytoplasm and nucleus, and staining in either location was considered to indicate positive expression. Five fields were randomly selected at a ×200 magnification. The results were calculated using the Aperio AT2 system (Leica Biosystems) with digital slide viewing software. The staining intensity of SMAD4 was scored as follows: 0, no; 1, weak; 2, moderate; and 3, high intensity. The proportion of positively stained cells was scored as follows: 0 (<5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The total score was calculated by multiplying the positive proportion score by the intensity score.

2.5 Cell culture and plasmid transfection

Rat GHPA cells (GH3) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum in a humidified incubator at 37°C in 5% CO2. The culture medium was replaced every other day. The experimental GH3 cells were divided into three groups based on transfection: (1) the SMAD4 transfection groups, in which GH3 cells were transfected with p-CMV6-AC-GFP-SMAD4 (1μg plasmid in 1*106 GH3 cells); (2) the negative control transfection group, in which GH3 cells were transfected with p-CMV6-AC-GFP; and (3) the untransfected group. The transfection was established using LipofectamineTM 3000 according to the manufacturer’s protocol (Invitrogen, CA, USA). The SMAD4 transfection group of cells was harvested 24, 48, 72, and 96 h after the transfection for further analysis. The untransfected group and the negative control transfection group of cells were harvested 72 h after transfection. Plasmids p-CMV6-AC-GFP-SMAD4 (GFP-tagged) that expresses full-length human SMAD4 and p-CMV6-AC-GFP were purchased from OriGene Technologies (Beijing, China).

2.6 RNA extraction and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays

Total RNA was extracted from the frozen GHPAs specimens (40–60 mg) and the experimental GH3 cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The quality of total RNA was examined by UV spectrophotometry. The cDNA was synthesized using the Superscript First-Strand Synthesis system with Superscript II reverse transcriptase following the manufacturer’s instructions (Invitrogen Life Technologies). RT-PCR was performed using an Applied Biosystems 7500 Fast system and Platinum SYBR-Green/ROX qPCR supermix-UDG (Invitrogen Life Technologies). The qRT-PCR reactions were performed using a 20 μL mix comprising 2×master mix (10 μL), forward primers (1 μl, 10 μmol/L), reverse primers (1 μL, 10 μmol/L), sample cDNA (1 μL), and double distilled water (ddH2O; 7 μL). The amplification conditions were 50°C for 120 sec and 95°C for 120 sec, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The fluorescence intensity values of PCR products were read after completion of the extension step. The expression of mRNA was determined from the threshold cycle (TC). The relative expression levels of the tested genes were normalized relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and calculated from the TC value using the 2-ΔΔTC method for quantification [34].

2.7 Protein preparation and western blot analysis

GHPA specimens or the experimental GH3 cells were homogenized in ice-cold radio immunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). The homogenate was centrifuged at 12,000 g and 4°C for 30 min, then protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). After denaturing at 95°C for 5 min in loading buffer, 40 μg of the protein samples per lane were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel, then transferred to nitrocellulose membranes. The membranes were incubated with blocking buffer made of 5% nonfat milk in Tris-buffered saline Tween-20 (TBST) at room temperature for 1 h. Different blots were incubated with the corresponding primary antibodies against SAMD4 (1:2000, CST, USA), E-cadherin (1:2000, CST), N-cadherin (1:2000, CST), Vimentin (1:2000, CST) and GAPDH (1:8000, Sigma) at 4°C overnight, followed by three 10 min washes with TBST. Subsequently, membranes were incubated with secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA) at room temperature for 1 h. Blots were visualized by enhanced chemiluminescence, and densitometry was performed with an Amersham Imager 6000 (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The final data were subjected to grayscale scanning and semi-quantitative analysis using Quantity One software (Bio-Rad, Hercules, CA, USA), and GAPDH levels were assessed as a loading control.

2.8 Cell migration and invasion assays

The migration and invasion of GH3 cells were measured using modified Transwell chambers (Corning, USA) coated with fibronectin and Matrigel in 24-well culture plates (BD Biosciences). GH3 cells (2×105 cells) of the SMAD4 transfection group, the negative control transfection group, and the untransfected control group after transient transfection for 72 h in serum-free DMEM, were respectively added onto membranes coated with fibronectin and Matrigel. After 24 h, migratory cells adhering to the lower membrane were fixed in 4% paraformaldehyde and stained using Harris stain. The average number of migratory cells was quantified by counting five random high-powered fields (100×) under a fluorescent microscope (ZEISS, Germany). All experiments were performed in triplicate.

2.9 Statistical analysis

χ2 and Fisher’s tests were used to analyze the significance of categorical variables. Differences between subgroups were analyzed using the Student’s t-test for normally distributed continuous values. Variables that showed a relationship between SMAD4 expression levels and clinicopathologic characteristics were subjected to univariate analyses. All P-values were two-sided and P-values <0.05 were considered statistically significant. SPSS software version 17.0 was used for statistical analyses.

Results

3.1 Sample characteristics of the cohort

In this study, we retrospectively analyzed the clinicopathological features of 61 GHPA patients by standard H&E staining and IHC using an anti-SMAD4 antibody, as shown in Table 1. The patients’ ages ranged from 16 to 75 years (median age: 39.5 years) including 32 males and 29 females. The largest tumor diameter ranged from 1.1 to 7.1 cm (median volume: 4.2 cm). All specimens were classified into either the invasive GHPA group (19 cases) or the non-invasive GHPA group (42 cases) according to the Knosp and Hardy classification systems. Of the tumors, 40 were gross total resections and 21 were subtotal resections. The serum GH levels of the preoperative patients ranged from 0.13 to 40 ng/mL (22.9±13.22 ng/mL) and from 0.27 to 22.4 ng/mL (9.41±7.05 ng/mL) after surgery.

Table 1

Clinicopathological data in 61 GHPAs cases.

VariableInvasiveNon-Invasive
Case1942
Age(years)Range19-5916-75
Mean±SD38.94±8.8543.9±3.34
GenderMale1220
Female722
HGH (ng/mL)23.32±14.322.72±12.9
Tumor sizecm333.6±23.23.88±1.01
Headache1415
Recurrence79
SMAD4H-Scores≥61510
H-Score6<432

3.2 Tissue microarray analysis

SMAD4 protein expression was investigated in the 61 GHPA specimens by TMA, as shown in Figure 1. The results were categorized as either high level (H-Score ≥ 6) or low level (H-Score < 6) expression. Using univariate analysis, SMAD4 expression showed no association with age, gender, tumor size, or preoperative serum GH levels. High SMAD4 expression was detected in 15/19 (78.9%) invasive GHPA cases and 10/42 (23.8%) noninvasive GHPA cases according to the H-Score analysis (χ2=16.44, p=0.000) (Table 2). In the high SMAD4 expression group, a headache occurred in 16/25 cases (64%), compared with only 13/36 cases (36.1%) in the low SMAD4 expression group (χ2=4.565, p=0.033). The follow-up interval for the 61 cases ranged from 2 to 7 years (mean = 3.6 years). In 16 cases (26.3%), tumor recurrence was detected during the follow-up period.

Table 2

Association of SMAD4 levels and clinicopathological data in 61 GHPAs patients.

VariableSMAD4P-Value
H-Score≥6H-Score<6
Age≥451517p=0.326
<451019
GenderMale1220p=0.561
Female1316
HGH(ng/mL)23.03±13.7522.82±14.04p>0.05
Tumor size15.89±23.8711.08±26.15p>0.05
HeadacheYes1613p=0.032
No923
InvasionYes154p=0.000
No1032
RecurrenceYes97p=0.148
No1629

Figure 1 IHC analysis of SMAD4 expression in the GHPAs. A-C: 50×; D-F: 400× magnification. bar=60μm.
Figure 1

IHC analysis of SMAD4 expression in the GHPAs. A-C: 50×; D-F: 400× magnification. bar=60μm.

3.3 qRT-PCR and western blot analysis of the expression of SMAD4 and the genes related to EMT in GHPA specimens

The mRNA levels of SMAD4 in the 19 invasive GHPA specimens were nearly three-fold higher than those of the 42 non-invasive specimens (p<0.001). To investigate the expression of EMT-related genes in the GHPAs, we analyzed the epithelial marker E-cadherin as well as the mesenchymal markers N-cadherin and vimentin. Our results showed a significant downregulation of E-cadherin mRNA (0.141-fold, p<0.001) and upregulation of both the N-cadherin (8.87-fold, p<0.001) and Vimentin (5.24-fold, p<0.001) mRNA levels in the invasive GHPAs compared with the non-invasive group, as shown in Table 3 and Figure 2A.

Table 3

The mRNA levels of SMAD4, E-CAD, N-CAD and vimentin in 61 GHPAs patients.

NumberCategorySMAD4 (ΔCT)E-CAD (ΔCT)N-CAD (ΔCT)Vimentin (ΔCT)
1Invasive-1.293-0.61079-1.81642-1.56792
2Invasive-1.92385-0.42722-2.192780.372415
3Invasive-1.587341.188364-1.80819-0.14489
4Invasive1.7199580.5714213.37209-0.88194
5Invasive-2.122633.668694-1.367511.388776
6Invasive1.883064.3905322.9432250.968368
7Invasive1.7302744.2341722.472812-0.17352
8Invasive-3.23187-0.238261-2.4367190.287138
9Invasive2.068822.6834761.2871822.381739
10Invasive-2.11923.6283470.2871921.562841
11Invasive1.668232.36882-2.382191-0.382126
12Invasive1.493821.283021-1.648736-1.283276
13Invasive-2.8922454.281293-0.281362-2.038263
14Invasive-0.2872251.937266-2.182351-0.273164
15Invasive-3.82382-1.223184-2.628711-0.382134
16Invasive-2.3326882.3266844.286371-1.382142
17Invasive-1.8821323.281163-0.2736183.281724
18Invasive2.0184553.8267832.832813-2.028172
19Invasive1.6632984.2871221.271386-1.76831
20Non-Invasive1.774347-1.610182.6178813.229507
21Non-Invasive2.015728-0.415213.7989733.372608
22Non-Invasive2.089746-1.630876.4941722.04606
23Non-Invasive3.134202-1.110136.0530793.166369
24Non-Invasive2.548817-1.022824.6607182.442292
25Non-Invasive2.700965-0.547883.5699561.762701
26Non-Invasive-1.2298322.1182935.428319-0.128321
27Non-Invasive3.0287013.6293644.8129383.381529
28Non-Invasive1.832922-2.128236-1.2812762.896134
29Non-Invasive2.028813-1.1128375.2873913.729716
30Non-Invasive-2.228612-2.1822634.289162-1.273821
31Non-Invasive4.132422-3.623864-1.7210824.012371
32Non-Invasive2.632133-0.2822174.398264-0.274812
33Non-Invasive2.8826123.6278222.3821963.278516
34Non-Invasive3.118227-0.1822934.1873923.193725
35Non-Invasive4.22821-1.283376-2.0182743.726351
36Non-Invasive2.1822310.2673811.8297224.283714
37Non-Invasive3.0163220.2871343.8267160.372871
38Non-Invasive-1.022386-0.2783914.7628943.298417
39Non-Invasive-1.339821-1.2827333.8273912.384612
40Non-Invasive3.208835-2.3782163.6286113.748219
41Non-Invasive-2.7762314.282671-1.6289420.382182
42Non-Invasive4.129833-1.483255-0.281126-1.328376
43Non-Invasive3.102132-3.2751285.7298123.642813
44Non-Invasive1.2283240.2387192.61273562.0274325
45Non-Invasive3.2876342.2173926.3287123.172831
46Non-Invasive-0.238712-2.1172694.2873264.482136
47Non-Invasive3.228766-1.2818276.12367162.09152
48Non-Invasive4.286624-0.2811644.5287192.347619
49Non-Invasive0.441029-0.821284-0.17028123.291723
50Non-Invasive0.310719-3.182923-1.283298-0.128362
51Non-Invasive1.458597-2.1829363.738721-1.392134
52Non-Invasive0.361096-1.2987314.6276843.681472
53Non-Invasive0.1356150.2811295.2017244.0126329
54Non-Invasive-0.5524-0.212933.7287213.729172
55Non-Invasive0.855096-0.8219274.0382172.738124
56Non-Invasive0.654499-0.2846212.483901-0.281462
57Non-Invasive0.2080532.3712543.782143-1.283581
58Non-Invasive1.294783-3.820187-0.238173.672193
59Non-Invasive-0.52388-2.3218211.2834714.012834
60Non-Invasive-0.21548-0.2734910.2638711.293744
61Non-Invasive-0.97771-1.2839276.0162393.569214

Figure 2 PCR and western blot analysis of SMAD4 and the genes related to EMT in the GHPA specimens. A: The mRNA levels of SMAD4, E-cadherin (E-CAD), N-cadherin (N-CAD), and Vimentin in the 61 GHPA specimens. B: The protein levels of SMAD4, E-CAD, N-CAD, and Vimentin in the 61 GHPA specimens. C: Statistical analysis of the western blot data.
Figure 2

PCR and western blot analysis of SMAD4 and the genes related to EMT in the GHPA specimens. A: The mRNA levels of SMAD4, E-cadherin (E-CAD), N-cadherin (N-CAD), and Vimentin in the 61 GHPA specimens. B: The protein levels of SMAD4, E-CAD, N-CAD, and Vimentin in the 61 GHPA specimens. C: Statistical analysis of the western blot data.

Western blot analysis was used to detect SMAD4 expression in the 19 invasive specimens and 42 noninvasive specimens. The protein levels of SMAD4, E-cadherin, N-cadherin, and vimentin in the invasive group were 3.83-fold, 0.31-fold, 9.2-fold, and 3.8-fold those of the non-invasive group, respectively, as shown in Figure 2B,C. According to the median mRNA levels, the PFS in the high SMAD4 group was lower than that in the low SMAD4 group (p=0.026), as shown in Figure 3.

Figure 3 PSF of 61 GHPAs according to the SMAD4 levels.
Figure 3

PSF of 61 GHPAs according to the SMAD4 levels.

3.4 Overexpression of SMAD4 regulated the genes related to EMT in GH3 cells

The efficiency of overexpression of SMAD4 was first measured in GH3 cells. The mRNA levels of SMAD4 were 2.77, 5.69, 60.55, and 90.61-fold that of the control group at 24, 48, 72, and 96 h after transfection, respectively (Figure 4A). Western blot analysis showed that the SMAD4 protein levels in GH3 cells were 1.1, 1.5, 3.8, and 4.1-fold that of the control group at 24, 48, 72, and 96 h after transfection (Figures 4B and 4C). EMT in tumor cells was characterized by decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers N-cadherin and vimentin. Our findings showed significant downregulation of E-cadherin mRNA levels and upregulation of N-cadherin and vimentin mRNA levels after overexpression of SMAD4 compared with the control group (p<0.001). Furthermore, the data showed significant downregulation of E-cadherin and upregulation of N-cadherin and vimentin compared with the negative control transfection group according to western blot analysis, as shown in Figure 4 (p<0.001). The following experiments were performed 72 h after transfection in GH3 cells.

Figure 4 PCR and western blot analysis of the expression of SMAD4 and genes related to EMT in GH3 cells. A: The mRNA levels of SMAD4 and genes related to EMT closely correlated with transfection time. B: The protein levels of SMAD4, E-CAD, N-CAD, and Vimentin in GH3 cells after overexpression of SMAD4. The levels of SMAD4 and Vimentin peaked after 72 h of transfection. C: Statistical analysis of western blot data.
Figure 4

PCR and western blot analysis of the expression of SMAD4 and genes related to EMT in GH3 cells. A: The mRNA levels of SMAD4 and genes related to EMT closely correlated with transfection time. B: The protein levels of SMAD4, E-CAD, N-CAD, and Vimentin in GH3 cells after overexpression of SMAD4. The levels of SMAD4 and Vimentin peaked after 72 h of transfection. C: Statistical analysis of western blot data.

3.5 Overexpression of SMAD4 promoted the invasion of GH3 cells

Consistent with our observations in the GHPA specimen assays, we measured cell invasion and migration by performing Transwell assays in vitro and found that overexpression of SMAD4 expression greatly increased the migratory and invasive behaviors of GH3 cells, along with elevated N-cadherin and vimentin levels. The number of positive cells was 761±159 after 72 h transfection in GH3 cells, and 184±57 in the control group (Figure 5). The transmembrane invasion assay revealed that overexpression of SMAD4 significantly increased the invasion of GH3 cells, which positively correlated with SMAD4 levels.

Figure 5 Overexpression of SMAD4 promoted the invasion of GH3 cells. A: Positive cells in the Transwell test. bar=60μm B: The statistical results of the Transwell test. The number of positive cells overexpressing SMAD4 was nearly 4-fold that of the control group.
Figure 5

Overexpression of SMAD4 promoted the invasion of GH3 cells. A: Positive cells in the Transwell test. bar=60μm B: The statistical results of the Transwell test. The number of positive cells overexpressing SMAD4 was nearly 4-fold that of the control group.

4 Discussion

The aim of this study was to determine whether there is a relationship between the expression of SMAD4 and clinicopathological features in patients with GHPAs. In tumor specimens, we found a significant upregulation of SMAD4 levels in invasive GHPAs compared with noninvasive GHPAs by TMAs. This finding was confirmed by RT-PCR and western blot analysis. To the best of our knowledge, this is the first study to demonstrate a correlation between SMAD4 expression and GHPAs.

Our results indicate that upregulation of SMAD4 may promote the malignant potential of GHPAs. This discovery was unexpected in light of the dogma that SMAD4 is a potent tumor suppressor. The SMAD4 gene (also known as DPC4), which was initially identified as a candidate tumor suppressor in various human tumors and is deleted in pancreatic carcinoma, is located at locus 4 on chromosome 18q21.1 [35]. Mutations resulting in loss of SMAD4 expression or weak SMAD4 expression strongly are correlated with poor prognosis [36,37,38]. However, SMAD4 exerts a tumor-promoting role in hepatocellular carcinoma and breast cancer [39, 40]. Therefore, the role of SMAD4 is likely more complex than simply a tumor suppressor. SMAD4 may have various functions in different tumor types and at different stages of tumor development, which is consistent with the dichotomous function of TGF-β signaling.

Various steps are involved in the progression of tumor cells, including invasion, which requires EMT. E-cadherin is typically expressed in epithelial cells and provides a physical link between adjacent cells and intracellular cytoskeleton [14]. Downregulated expression of E-cadherin destroys the intracellular junction and thus epithelial cells acquire the ability to migrate, which facilitates tumor invasion and metastasis [41]. Tumor cells that express mesenchymal markers such as N-cadherin and vimentin have a greater tendency to be invasive and metastasize than tumor cells only expressing epithelial markers [42, 43]. In this study, we found that mRNA and protein expression levels of E-cadherin were significantly reduced in invasive GHPAs compared with non-invasive GHPAs. This finding is in agreement with previous studies [44, 45]. We also found that the mRNA and protein expression levels of N-cadherin and vimentin are significantly upregulated in invasive GHPAs compared with non-invasive GHPAs. Therefore, our study confirmed previous suggestions that EMT plays an important role in pituitary adenoma progression [46, 47]. Our analysis of the changes in EMT biomarkers in invasive GHPAs strengthens this conclusion.

TGF-β/SMAD signaling has been identified to play a significant role during the activation of EMT in various cancers [48]. SMAD4 is indispensable for TGF-β-induced EMT [49, 50]. It has previously been shown that ectopic SMAD4 expression levels could affect the activation of EMT. Yang and colleagues reported that SMAD4 mRNA levels positively correlate with N-cadherin mRNA levels in human NSCLC tissue [51]. In vitro studies showed that overexpression of SMAD4 enhance TGF-β-induced EMT in NMuMG cells [52] and PC3 cells [31]. To further investigate the mechanism, we analyzed the invasion of GH3 cells in an in vitro model by altering the levels of SMAD4 using transfection technology, and assessed whether EMT was induced using EMT biomarkers. Firstly, western blot analysis revealed a low level of SMAD4 expression in wildtype GH3 cells. Following transfection of the SMAD4 gene into GH3 cells, a Transwell assay revealed that upregulation of SMAD4 significantly promoted the invasion of GH3 cells, indicating that invasion was positively correlated with SMAD4 expression. Further studies showed that the overexpression of SMAD4 downregulated the level of E-cadherin, and upregulated the levels of N-cadherin and vimentin. These results suggested that the overexpression of SMAD4 promoted the invasion of GH3 cells by inducing EMT. An increasing number of studies have shown that tumor cell invasion is suppressed when EMT is inhibited by targeting SAMD4 [31, 53,54,55]. Our study demonstrated the relationship between the expression of SMAD4 and the induction of EMT in tumor progression, revealing the mechanism underlying the role of SMAD4 in the oncogenic response. Taken together, our findings provide new insight into the potential role of SMAD4 in promoting the invasion of GHPAs and suggest that SMAD4 is a potential molecular target for developing new therapeutic strategies to combat invasive GHPAs via the suppression of EMT.

A previous study showed that SMAD4 overexpression in hepatocellular carcinoma was strongly associated with overexpression of TGF-β receptors [56], indicating activation or dysregulation of the TGF-β signaling pathway during tumorigenesis. Inducing EMT in tumors may contribute to the switch in TGF-β signaling from a tumor-suppressive to a tumor-promoting pathway. The expression of other upstream markers of the TGF-β pathway or downstream markers regulated by SMAD4 that affect EMT in GHPAs should be investigated in future studies.

In summary, our study demonstrated that the overexpression of SMAD4 is positively linked to the invasion and recurrence of GHPAs. Furthermore, the mechanism of action of SMAD4 may be associated with induction of EMT. Our in vitro studies indicated that upregulation of SMAD4 promote invasion of GH3 cells by inducing EMT. Therefore, SMAD4 may be an effective biomarker for predicting the invasion and recurrence of GHPAs, and a potential therapeutic target for patients with invasive GHPAs.

Acknowledgements

This study was supported by the National High-tech Research and Development Program of China (863 Program, 2015AA020504), National Natural Science Foundation of China (81601205), Beijing Natural Science Foundation of China (7162035), and the Beijing High-level Talent Plan (2015-3-040).

  1. Conflict of interest: Authors declare no conflict of interest.

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Received: 2018-02-07
Accepted: 2018-03-29
Published Online: 2018-06-14

© 2018 Xiaosong Shan et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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  77. Topological study of the para-line graphs of certain pentacene via topological indices
  78. A brief insight into the prediction of water vapor transmissibility in highly impermeable hybrid nanocomposites based on bromobutyl/epichlorohydrin rubber blends
  79. Comparative sulfite assay by voltammetry using Pt electrodes, photometry and titrimetry: Application to cider, vinegar and sugar analysis
  80. MicroRNA delivery mediated by PEGylated polyethylenimine for prostate cancer therapy
  81. Reversible Fluorescent Turn-on Sensors for Fe3+ based on a Receptor Composed of Tri-oxygen Atoms of Amide Groups in Water
  82. Sonocatalytic degradation of methyl orange in aqueous solution using Fe-doped TiO2 nanoparticles under mechanical agitation
  83. Hydrotalcite Anchored Ruthenium Catalyst for CO2 Hydrogenation Reaction
  84. Production and Analysis of Recycled Ammonium Perrhenate from CMSX-4 superalloys
  85. Topical Issue on Agriculture
  86. New phosphorus biofertilizers from renewable raw materials in the aspect of cadmium and lead contents in soil and plants
  87. Survey of content of cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, mercury, sodium and zinc in chamomile and green tea leaves by electrothermal or flame atomizer atomic absorption spectrometry
  88. Biogas digestate – benefits and risks for soil fertility and crop quality – an evaluation of grain maize response
  89. A numerical analysis of heat transfer in a cross-current heat exchanger with controlled and newly designed air flows
  90. Freshwater green macroalgae as a biosorbent of Cr(III) ions
  91. The main influencing factors of soil mechanical characteristics of the gravity erosion environment in the dry-hot valley of Jinsha river
  92. Free amino acids in Viola tricolor in relation to different habitat conditions
  93. The influence of filler amount on selected properties of new experimental resin dental composite
  94. Effect of poultry wastewater irrigation on nitrogen, phosphorus and carbon contents in farmland soil
  95. Response of spring wheat to NPK and S fertilization. The content and uptake of macronutrients and the value of ionic ratios
  96. The Effect of Macroalgal Extracts and Near Infrared Radiation on Germination of Soybean Seedlings: Preliminary Research Results
  97. Content of Zn, Cd and Pb in purple moor-grass in soils heavily contaminated with heavy metals around a zinc and lead ore tailing landfill
  98. Topical Issue on Research for Natural Bioactive Products
  99. Synthesis of (±)-3,4-dimethoxybenzyl-4-methyloctanoate as a novel internal standard for capsinoid determination by HPLC-ESI-MS/MS(QTOF)
  100. Repellent activity of monoterpenoid esters with neurotransmitter amino acids against yellow fever mosquito, Aedes aegypti
  101. Effect of Flammulina velutipes (golden needle mushroom, eno-kitake) polysaccharides on constipation
  102. Bioassay-directed fractionation of a blood coagulation factor Xa inhibitor, betulinic acid from Lycopus lucidus
  103. Antifungal and repellent activities of the essential oils from three aromatic herbs from western Himalaya
  104. Chemical composition and microbiological evaluation of essential oil from Hyssopus officinalis L. with white and pink flowers
  105. Bioassay-guided isolation and identification of Aedes aegypti larvicidal and biting deterrent compounds from Veratrum lobelianum
  106. α-Terpineol, a natural monoterpene: A review of its biological properties
  107. Utility of essential oils for development of host-based lures for Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae), vector of laurel wilt
  108. Phenolic composition and antioxidant potential of different organs of Kazakh Crataegus almaatensis Pojark: A comparison with the European Crataegus oxyacantha L. flowers
  109. Isolation of eudesmane type sesquiterpene ketone from Prangos heyniae H.Duman & M.F.Watson essential oil and mosquitocidal activity of the essential oils
  110. Comparative analysis of the polyphenols profiles and the antioxidant and cytotoxicity properties of various blue honeysuckle varieties
  111. Special Issue on ICCESEN 2017
  112. Modelling world energy security data from multinomial distribution by generalized linear model under different cumulative link functions
  113. Pine Cone and Boron Compounds Effect as Reinforcement on Mechanical and Flammability Properties of Polyester Composites
  114. Artificial Neural Network Modelling for Prediction of SNR Effected by Probe Properties on Ultrasonic Inspection of Austenitic Stainless Steel Weldments
  115. Calculation and 3D analyses of ERR in the band crack front contained in a rectangular plate made of multilayered material
  116. Improvement of fuel properties of biodiesel with bioadditive ethyl levulinate
  117. Properties of AlSi9Cu3 metal matrix micro and nano composites produced via stir casting
  118. Investigation of Antibacterial Properties of Ag Doped TiO2 Nanofibers Prepared by Electrospinning Process
  119. Modeling of Total Phenolic contents in Various Tea samples by Experimental Design Methods
  120. Nickel doping effect on the structural and optical properties of indium sulfide thin films by SILAR
  121. The effect mechanism of Ginnalin A as a homeopathic agent on various cancer cell lines
  122. Excitation functions of proton induced reactions of some radioisotopes used in medicine
  123. Oxide ionic conductivity and microstructures of Pr and Sm co-doped CeO2-based systems
  124. Rapid Synthesis of Metallic Reinforced in Situ Intermetallic Composites in Ti-Al-Nb System via Resistive Sintering
  125. Oxidation Behavior of NiCr/YSZ Thermal Barrier Coatings (TBCs)
  126. Clustering Analysis of Normal Strength Concretes Produced with Different Aggregate Types
  127. Magnetic Nano-Sized Solid Acid Catalyst Bearing Sulfonic Acid Groups for Biodiesel Synthesis
  128. The biological activities of Arabis alpina L. subsp. brevifolia (DC.) Cullen against food pathogens
  129. Humidity properties of Schiff base polymers
  130. Free Vibration Analysis of Fiber Metal Laminated Straight Beam
  131. Comparative study of in vitro antioxidant, acetylcholinesterase and butyrylcholinesterase activity of alfalfa (Medicago sativa L.) collected during different growth stages
  132. Isothermal Oxidation Behavior of Gadolinium Zirconate (Gd2Zr2O7) Thermal Barrier Coatings (TBCs) produced by Electron Beam Physical Vapor Deposition (EB-PVD) technique
  133. Optimization of Adsorption Parameters for Ultra-Fine Calcite Using a Box-Behnken Experimental Design
  134. The Microstructural Investigation of Vermiculite-Infiltrated Electron Beam Physical Vapor Deposition Thermal Barrier Coatings
  135. Modelling Porosity Permeability of Ceramic Tiles using Fuzzy Taguchi Method
  136. Experimental and theoretical study of a novel naphthoquinone Schiff base
  137. Physicochemical properties of heat treated sille stone for ceramic industry
  138. Sand Dune Characterization for Preparing Metallurgical Grade Silicon
  139. Catalytic Applications of Large Pore Sulfonic Acid-Functionalized SBA-15 Mesoporous Silica for Esterification
  140. One-photon Absorption Characterizations, Dipole Polarizabilities and Second Hyperpolarizabilities of Chlorophyll a and Crocin
  141. The Optical and Crystallite Characterization of Bilayer TiO2 Films Coated on Different ITO layers
  142. Topical Issue on Bond Activation
  143. Metal-mediated reactions towards the synthesis of a novel deaminolysed bisurea, dicarbamolyamine
  144. The structure of ortho-(trifluoromethyl)phenol in comparison to its homologues – A combined experimental and theoretical study
  145. Heterogeneous catalysis with encapsulated haem and other synthetic porphyrins: Harnessing the power of porphyrins for oxidation reactions
  146. Recent Advances on Mechanistic Studies on C–H Activation Catalyzed by Base Metals
  147. Reactions of the organoplatinum complex [Pt(cod) (neoSi)Cl] (neoSi = trimethylsilylmethyl) with the non-coordinating anions SbF6– and BPh4–
  148. Erratum
  149. Investigation on Two Compounds of O, O’-dithiophosphate Derivatives as Corrosion Inhibitors for Q235 Steel in Hydrochloric Acid Solution
https://doi.org/10.1515/chem-2018-0061
Keywords for this article
SMAD4; Growth Hormone-secreting Pituitary Adenoma; Invasive; Epithelial–Mesenchymal Transition; GH3 cells
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  117. Properties of AlSi9Cu3 metal matrix micro and nano composites produced via stir casting
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  119. Modeling of Total Phenolic contents in Various Tea samples by Experimental Design Methods
  120. Nickel doping effect on the structural and optical properties of indium sulfide thin films by SILAR
  121. The effect mechanism of Ginnalin A as a homeopathic agent on various cancer cell lines
  122. Excitation functions of proton induced reactions of some radioisotopes used in medicine
  123. Oxide ionic conductivity and microstructures of Pr and Sm co-doped CeO2-based systems
  124. Rapid Synthesis of Metallic Reinforced in Situ Intermetallic Composites in Ti-Al-Nb System via Resistive Sintering
  125. Oxidation Behavior of NiCr/YSZ Thermal Barrier Coatings (TBCs)
  126. Clustering Analysis of Normal Strength Concretes Produced with Different Aggregate Types
  127. Magnetic Nano-Sized Solid Acid Catalyst Bearing Sulfonic Acid Groups for Biodiesel Synthesis
  128. The biological activities of Arabis alpina L. subsp. brevifolia (DC.) Cullen against food pathogens
  129. Humidity properties of Schiff base polymers
  130. Free Vibration Analysis of Fiber Metal Laminated Straight Beam
  131. Comparative study of in vitro antioxidant, acetylcholinesterase and butyrylcholinesterase activity of alfalfa (Medicago sativa L.) collected during different growth stages
  132. Isothermal Oxidation Behavior of Gadolinium Zirconate (Gd2Zr2O7) Thermal Barrier Coatings (TBCs) produced by Electron Beam Physical Vapor Deposition (EB-PVD) technique
  133. Optimization of Adsorption Parameters for Ultra-Fine Calcite Using a Box-Behnken Experimental Design
  134. The Microstructural Investigation of Vermiculite-Infiltrated Electron Beam Physical Vapor Deposition Thermal Barrier Coatings
  135. Modelling Porosity Permeability of Ceramic Tiles using Fuzzy Taguchi Method
  136. Experimental and theoretical study of a novel naphthoquinone Schiff base
  137. Physicochemical properties of heat treated sille stone for ceramic industry
  138. Sand Dune Characterization for Preparing Metallurgical Grade Silicon
  139. Catalytic Applications of Large Pore Sulfonic Acid-Functionalized SBA-15 Mesoporous Silica for Esterification
  140. One-photon Absorption Characterizations, Dipole Polarizabilities and Second Hyperpolarizabilities of Chlorophyll a and Crocin
  141. The Optical and Crystallite Characterization of Bilayer TiO2 Films Coated on Different ITO layers
  142. Topical Issue on Bond Activation
  143. Metal-mediated reactions towards the synthesis of a novel deaminolysed bisurea, dicarbamolyamine
  144. The structure of ortho-(trifluoromethyl)phenol in comparison to its homologues – A combined experimental and theoretical study
  145. Heterogeneous catalysis with encapsulated haem and other synthetic porphyrins: Harnessing the power of porphyrins for oxidation reactions
  146. Recent Advances on Mechanistic Studies on C–H Activation Catalyzed by Base Metals
  147. Reactions of the organoplatinum complex [Pt(cod) (neoSi)Cl] (neoSi = trimethylsilylmethyl) with the non-coordinating anions SbF6– and BPh4–
  148. Erratum
  149. Investigation on Two Compounds of O, O’-dithiophosphate Derivatives as Corrosion Inhibitors for Q235 Steel in Hydrochloric Acid Solution
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