Med Oncol (2012) 29:3306–3313 DOI 10.1007/s12032-012-0265-1
ORIGINAL PAPER
Suppression of MMP-9 activity by NDRG2 expression inhibits clear cell renal cell carcinoma invasion Jian-Jun Ma • Ling-Min Kong • Cheng-Gong Liao Xue Jiang • Yong Wang • Ting-Yi Bao
•
Received: 23 April 2012 / Accepted: 23 May 2012 / Published online: 13 June 2012 Ó Springer Science+Business Media, LLC 2012
Abstract Members of the NDRG (N-Myc downstreamregulated) gene family have been shown to play a variety of roles in human malignancies. Recently, it was shown decreased expression in clear cell renal cell carcinoma (CCRCC) and inhibited cell proliferation, but the role of the NDRG2 in CCRCC invasion has not been described. We examined the expression of NDRG2 protein in CCRCC samples and the association between NDRG2 expression and CCRCC patients survival. Real-time RT-PCR and immunohistochemical analysis were used to measure NDRG2 expression in 60 paired CCRCC and adjacent normal tissues. Changes in cell invasion were detected by Jian-Jun Ma, Ling-Min Kong, and Cheng-Gong Liao have contributed equally to the work. J.-J. Ma (&) Y. Wang T.-Y. Bao (&) Department of Urology Tangdu Hospital, The Fourth Military Medical University, 1 Xinsi Road, Xi’an 710038, People’s Republic of China e-mail:
[email protected] T.-Y. Bao e-mail:
[email protected] L.-M. Kong State Key Laboratory of Cancer Biology, Cell Engineering Research Centre and Department of Cell Biology, Fourth Military Medical University, Xi’an 710032, People’s Republic of China C.-G. Liao Department of Oncology, Urumqi General Hospital of Lanzhou Military Command of PLA, Urumqi 830000, People’s Republic of China X. Jiang Department of Operation Room, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710032, People’s Republic of China
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up- or down-regulating NDRG2 by adenovirus or siRNA. We found that NDRG2 expression is significantly downregulated in CCRCC at mRNA and protein levels in a manner negatively associated with aggressive tumor behaviors, such as TNM stage (P = 0.003), Fuhrman’s grade (P = 0.024), tumor invasion (P = 0.001) and tumor recurrence (P = 0.004), as well as shorter patient survival rates (P = 0.0041). Furthermore, NDRG2 could suppress CCRCC cell invasion through regulating MMP-9 expression and activity. So, these results suggest that NDRG2 can inhibit extracellular matrix-based tumor cell invasion and thereby play important roles in suppressing tumor metastasis in CCRCC. NDRG2 expression may also be a significant prognostic indicator for CCRCC. Keywords NDRG2 CCRCC Invasion MMP-9 Survival Prognostic indicator
Introduction Clear cell renal cell carcinoma (CCRCC) is the most common malignancy of the kidney [1]. About a quarter of the CCRCC patients have metastatic disease at the time of diagnosis and eventually one-third of the patients presented with localized tumors at diagnosis relapse. Despite recent advances using multikinase inhibitors, disseminated CCRCC remains inherently treatment resistant [2]. Consequently, studies leading to a better understanding of the factors that determines the metastatic phenotype of CCRCC are warranted [3, 4]. N-myc downstream-regulated gene 2 (NDRG2) belongs to the NDRG family, a new family of differentiation-related genes composed of four members named NDRG1, NDRG2, NDRG3, and NDRG4. Members of this gene family are
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involved in cell growth, differentiation, stress, and hormonal responses [5]. Recently, it has been proposed that NDRG2 is a candidate tumor suppressor gene since it induces apoptosis in certain cancer cells and mRNA was down-regulated or absent in several human cancers and cancer cell lines [6–8]. In clinical specimens, CCRCC has low or undetectable levels of NDRG2 compared with normal adjacent tissue [9]. Low expression of NDRG2 is a positive indicator of clinical parameters relevant to metastasis. It has been demonstrated that NDRG2 inhibits the proliferation of the CCRCC cells and induces arrest at G1 phase [10]. However, the role of NDRG2 in regulating metastasis remains unclear. Tumor metastasis, one of the fundamental properties of malignant cancer cells and a major cause of cancer mortality, is a multistep process including adhesion to the extracellular matrix (ECM), proteolytic digestion of the ECMs, invasion of lymph and blood vessels and migration [11]. Matrix metalloproteinases (MMPs) are generally present in greater amounts and activated more often in malignant cancers. Thus, a positive correlation has been found between the expression of MMPs and poor prognosis in various types of cancer [12]. Among several MMPs, MMP-2 and MMP-9 are the critical enzymes known to degrade surrounding extracellular matrix components during cancer invasion and metastasis [13]. In this study, we first measured NDRG2 expression levels in different TNM Stage CCRCC tumor tissues compared with normal renal tissues. Then, we identified the role of NDRG2 in CCRCC cell invasion. Furthermore, we also investigated the downstream tumor invasive factors regulated by NDRG2. Finally, we analyzed the clinical values of NDRG2 expression level in predicting the prognosis of CCRCC patients. This is the first study to examine the role of NDRG2 in CCRCC invasion and prognosis analysis.
Materials and methods Tissues collection and immunohistochemical analysis Sixty paired tissue specimens of CCRCC tissues (T) and adjacent non-tumor (ANT) tissues were obtained from Department of Urology, Tangdu Hospital affiliated to Fourth Military Medical University (Xi’an, China). Five fresh normal renal tissues (NR) were also collected as normal control. Informed consent was obtained from each patient. All of the tissues were obtained at the time of surgery and immediately stored in liquid nitrogen until use. Immunohistochemistry was performed using the Histostain-SP kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. Mouse anti-NDRG2 Mab was purchased from Santa Cruz Biotechnology (Santa Cruz,
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CA, USA). Immunopositivity was independently evaluated by two pathologists. Expression of NDRG2 was evaluated according to the ratio of positive cells per specimen and staining intensity as described previously [9]. The ratio of positive cells per specimen was evaluated quantitatively and scored as 0 for staining of B 1 %, 1 for staining of 2–25 %, 2 for staining of 26–50 %, 3 for staining of 51–75 %, and 4 for staining [ 75 % of the cells examined. Intensity was graded as follows: 0, no signal; 1, weak; and 2, strong staining. A total score of 0–8 was calculated and graded as negative (-; score: 0–1), positive: weak (?; 2–4), and strong (??; 5–8). Cell lines and culture conditions The following cell lines were used in this study: human normal kidney tubule epithelial cell HK-2; human embryonic kidney cell HEK-293; human renal cancer cell lines: 786-O, A-498, Caki-1, RCC-9863, RCC-949. All cell lines were purchased from Shanghai Institute for Biological Sciences (Shanghai, China). All cell lines were routinely cultured in RPMI-1640 medium (Hyclone Laboratories, Logan, UT, USA) supplemented with 10 % fetal calf serum (Gibco BRL, Rockville, MD, USA) at 37 °C in a humidified atmosphere of 5 % CO2. Real-time quantitative RT-PCR Total RNA was isolated from tissues and cell lines by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed using the Prime-Script RT reagent kit (TaKaRa, Otsu, Japan). Real-time PCR was performed using SYBR Premix Ex TaqTM II kit (TaKaRa) according to the manufacturer’s protocol on an MX3005P QPCR system (Stratagene, La Jolla, CA, USA). The primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and NDRG2 were used as described previously [10]. All of the reactions were run in triplicate. The delta-Ct method [14] for relative quantification of gene expression was used to determine mRNA expression levels. NDRG2 infection and knockdown Cultured cells were infected with adenovirus expressing NDRG2 (pAd-GFP-NDRG2) or the negative control gene LacZ (pAd-GFP) [10] according to the manufacturer’s protocol to up-regulate the NDRG2 expression. Cells were transfected with NDRG2 siRNA or negative control siRNA using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol to down-regulate the NDRG2 expression. NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.
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Fig. 1 NDRG2 is over-expressed in CCRCC tissues. a Analysis of the expression pattern of NDRG2 in nontumor (ANT) and tumor (T) tissues of CCRCC using real-time RT-PCR. The NDRG2 expression value reflects the log 2 value for the ratio of NDRG2 mRNA level in ANT and T tissues relative to that in fresh normal
renal tissues (NR), which was used as the common control. b and c The expression of NDRG2 in metastasis tumors and advanced histologic grades. The relative expression value (T/NT) was calculated after normalizing the mRNA level of NDRG2 against that of b-actin in each sample. Grades I–IV, Fuhrman’s grades
In vitro invasion assay
(1:5000). The primary antibody against NDRG2, MMP-2, MMP-9, and tubulin was purchased from Santa Cruz Corporation (Santa Cruz, CA, USA). Finally, the blots were washed and the signals were visualized using the ECL plus Kit (Amersham, Buckinghamshire, UK).
MilliCell (12 mm diameter with 8 lm pores) chambers (Millipore, Bedford, MA, USA) were pre-coated with Matrigel (BD, Bedford, MA, USA) on the upper side. A total of 1 9 105 serum-starved CCRCC cells were added to the upper compartment in medium supplemented with 0.1 % serum, and the chambers were placed into 24-well plates with medium containing 10 % serum. After 24 h at 37 °C, invaded cells on the lower membrane surface were fixed and stained with 0.1 % crystal violet. Invasive activity was quantified by counting nine high-power fields (HPFs, 400 9) per chamber. Mean values were obtained from at least three individual chambers for each experimental point per assay. Gelatin zymography Gelatin zymography was performed using 10 % SDS– PAGE containing 1 mg/mL gelatin. Transfected CCRCC cells (1.0 9 105 per well) were cultured in 96-well plates. After attachment, the cells were washed and incubated in serum-free medium for 12 h. Then, the supernatants were collected and prepared in non-reducing loading buffer. After electrophoresis, SDS was removed using 2.5 % Triton X-100 to renature gelatinases. Gels were then incubated at 37 °C for 16 h in developing buffer (50 mM Tris–HCl (pH 7.8), 200 mM NaCl, 5 mM CaCl2, and 0.02 % Brij35) and then stained with Coomassie Blue R-250. MMPs activity was visualized as clear bands against the bluestained gelatin background.
Statistical analysis Each experiment was performed independently at least twice with similar results; one representative experiment was presented. All statistical analyses were performed using the SPSS 16.0 statistical software package (SPSS, Chicago, IL, USA). The significance of the data was determined using Student’s t-test. Pearson chi-square (v2) test was used to compare the difference in the distribution of demographic and clinical variables in NDRG2 negative/ weak and positive CCRCC patients. Follow-up time was limited to 5 years. Survival rates were compared by Kaplan–Meier test and log-rank test. All the statistical tests were two-sided, and a P value \ 0.05 was considered significant.
Western blot Western blots were performed according to standard protocols using Immobilon-P PVDF membranes (Millipore). For immunoblotting, membranes were incubated with the primary antibody (0.5 lg/mL) for 2 h, followed by a 1-h incubation with HRP-conjugated secondary antibody
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Fig. 2 The relative levels of NDRG2 in the five CCRCC and two normal renal cell lines. Student’s t-test was used to analyze the significant differences
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Fig. 3 Immunohistochemical analysis of NDRG2 protein expression in renal tissues and survival analysis of CCRCC patients in relation to NDRG2 expression pattern. a Immunohistochemical analysis. Upper left, normal renal tissue showing strong positive staining in cytoplasm and membranes of renal cell. Upper right, image of NDRG2positive CCRCC tissue. Lower, CCRCC tissues with weak and negative staining. b Correlation of the overall survival rate of CCRCC patients with NDRG2 expression pattern. Curves were estimated using the Kaplan– Meier method (P = 0.0041). Continuous line NDRG2 negative group; dotted line NDRG2 positive group
Results NDRG2 expression is significantly reduced in CCRCC tissues and cell lines The expression of NDRG2 was assessed by real-time RT-PCR in 60 CCRCC samples with their adjacent noncancerous tissues, among which 30 were low-grade (grades I and II) and 30 were high-grade (grade III and IV). Our results showed that NDRG2 was significantly reduced in CCRCC than that in adjacent tissues (Fig. 1a, P = 0.0001). We also confirmed that NDRG2 expression was significantly reduced in metastasis tumors (P = 0.0045) and advanced histologic grades (P = 0.0232, Fig. 1b, c). Therefore, we conclude that NDRG2 expression is significantly down-regulated in CCRCC at mRNA levels in a manner negatively associated with aggressive tumor behaviors.
We also detected the NDRG2 expression in CCRCC and normal renal cell lines. We performed real-time RT-PCR on a panel of five CCRCC and two normal renal cell lines. As shown in Fig. 2, NDRG2 levels of all cancer cell lines were lower than that of normal renal cell lines. NDRG2 expression in 786-O, Caki-1 and RCC-9863 cells was relatively high. In contrast, expression levels of NDRG2 in A-498 and RCC-949 cells were relatively low. Evaluation of the clinical significance of NDRG2 by immunohistochemical analysis of renal cancer tissues The expression of NDRG2 was assessed by immunohistochemistry in 60 CCRCC samples with their adjacent noncancerous tissues. Our results showed that NDRG2 was predominantly located in the cytoplasm and membrane of
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Table 1 Clinicopathologic features of CCRCC in relation to the NDRG2 expression Variable
NDRG2 expression
P value
Negative
Positive
\ 50
20
8
C 50 Sex
23
9
Female
29
12
Male
14
5
\5
17
10
C5
26
7
I–II
16
14
III–IV
27
3
1–2
16
12
3–4
27
5
Age, years 1.000
1.000
Tumor size, cm 0.251
TNM stage 0.003
Fuhrman’s grade 0.024
Tumor invasion Yes No Tumor recurrence
34
5
9
12
Yes
26
3
No
17
14
0.001
0.004
renal clear cell carcinoma cells (Fig. 3a). NDRG2 positive expression found in CCRCC specimens was 28.33 % (17/60), which was significantly lower than the 88.33 % (53/60) in their adjacent tissues (P \ 0.01). Associations between NDRG2 expression level and clinicopathological variables are summarized in Table 1. Of the 60 CCRCC examined, 17 exhibited a positive NDRG2 expression in tumor cells, whereas in 43 CCRCC, the NDRG2 staining was not detectable at all or at a much lower level. For statistical analysis, the CCRCC samples were categorized into two groups (negative or positive: weak and strong) according to the NDRG2 expression level. We found a significant negative relationship between the NDRG2 level and TNM stage (P = 0.003) or Fuhrman’s grade (P = 0.024; Table 1). Down-regulation of NDRG2 expression was also significantly associated with invasive tumor features such as tumor invasion (P = 0.001) and tumor recurrence (P = 0.004). However, NDRG2 expression was not significantly associated with age, sex, or tumor size. We next examined whether NDRG2 down-regulation is correlated with CCRCC patient survival. Kaplan–Meier analysis showed that higher expression of NDRG2 was related to better overall survival (Fig. 3b). The survival rate of NDRG2-negative patients was lower than that of
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Fig. 4 The expression of NDRG2 was detected by real-time RT-PCR after the siRNA-NDRG2 transfected in 786-O (a) and in pAd-GFPNDRG2 infected A-498 cells (b)
positive patients, as determined using the log-rank test (P = 0.0041). These results confirm that NDRG2 downregulation is associated with advanced and aggressive tumor behaviors that are relevant to tumor metastasis and survival in CCRCC. NDRG2 inhibits CCRCC cell invasion To investigate whether NDRG2 regulates CCRCC cell migration and invasion, we selected 786-O cells, which show relatively high NDRG2 expression, and A-498 cells, which show relatively weak NDRG2 expression, for further study. The behavior of pAd-GFP-NDRG2-infected A-498 cells and siRNA-NDRG2-transfected 786-O cells were assessed. First, we analyzed NDRG2 levels after infection or transfection by real-time RT-PCR and found that NDRG2 levels were significantly reduced by the NDRG2 siRNA (Fig. 4a) and elevated by the NDRG2 adenovirus expression vector (Fig. 4b). To evaluate the effect of NDRG2 on metastatic activity, we performed in vitro migration and invasion assays. siRNA-treated 786-O cells showed increased invasion compared with control cells (Fig. 5a, b). By contrast, restoration of NDRG2 expression significantly inhibited cell invasion (Fig. 5c, d). These observations indicated a negative role for NDRG2 in migration and invasion of human CCRCC cell lines.
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Fig. 5 NDRG2 inhibits CCRCC cell invasion. a Down-regulation of NDRG2 increases the invasion of 786-O cells. NDRG2-high HCC 786-O cells were transfected with NDRG2 siRNA or the negative control siRNA (SNC) using Lipofectamine 2000. The cells were subjected to invasion assays. c Over-expression of NDRG2 suppresses
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the invasion of A-498 cells. NDRG2-low CCRCC A-498 cells were infected with adenovirus expressing NDRG2 (pAd-GFP-NDRG2) or the negative control gene Lac Z (pAd-GFP). In (b) and (d), the histograms represent the quantification of cells having invaded through Matrigel. Data represent three independent experiments
Fig. 6 NDRG2 suppresses the MMP-9 expression and gelatinase activity. a and c mRNA levels of MMPs in NDRG2 over- or downexpression cells were examined by real-time RT-PCR. b and d Conditioned media (CM) were collected after incubation of cells in serum-free media for 48 h and then subjected to gelatin zymography for the detection of MMP-2 and MMP9 activities
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NDRG2 suppresses the MMP-9 expression and gelatinase activity To further investigate the signaling pathway, we measured whether the MMP-2 and MMP-9 expression levels were affected by NDRG2. Our results showed that NDRG2 could regulate the expression of MMP-9, but not the MMP-2 (Fig. 6a, c). Next, we examined the ability of secreted MMP to degrade ECM components using serum-free conditioned media (CM). Gelatin zymographic analyses revealed that A-498 wild-type and mock cells constitutively secreted gelatinolytic MMP-9, whereas, in A-498 NDRG2 cells, secretion of gelatinolytic MMP-9 was remarkably decreased (Fig. 6b, d). These results suggest that NDRG2 can inhibit tumor cell invasion and migration by inhibiting MMP-9 activity and thereby play important roles in suppressing tumor metastasis in CCRCC.
Discussion In our study, we demonstrated that NDRG2 expression is decreased in human CCRCC tissues and cell lines compared with matching adjacent nontumoral tissue and normal renal cell lines. The down-expression of NDRG2 correlates with CCRCC metastatic ability. NDRG2 suppress CCRCC cell migration and invasion through regulating MMP-9 activity. It is also shown that NDRG2 down-regulation is associated with advanced and aggressive tumor behaviors that are relevant to tumor metastasis and survival in CCRCC. The identification of NDRG2 as an important regulator of tumor cell migration and invasion in vitro emphasizes an essential role of this NDRG2 in mediating renal oncogenesis and tumor behavior. The treatment of renal cancer is challenging due to its strong resistance to conventional cancer therapy [15]. The development and progression of RCC is thought to mainly arise from changes in some key genes that are related to cell proliferation, apoptosis, and genomic stability [16]. Therefore, it is important to identify more genes specifically related to renal cell carcinoma, which may expand our understanding of this disease and assist in the development of new targets for the therapy and diagnostic indicators. Members of the human NDRG family, comprising NDRG1, NDRG2, NDRG3, and NDRG4, share 57–65 % amino acid identity. Although we do not yet fully understand the roles NDRG family members play, it is possible that these proteins have a crucial function in tumor differentiation and progression. Previous reports have established a complex role for NDRG2 in various cancers [17–20]. However, to date there has been no research on the prognostic role of NDRG2 in clear cell renal cell carcinoma. New molecular prognostic factors for CCRCC
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may contribute to a better assessment of survival probability and, consequently, the tailoring of treatments for individual patients. Our previous studies have demonstrated that NDRG2 down-expressed in CCRCC tissues and can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. NDRG2 may function as a tumor suppressor in CCRCC. In present studies of CCRCC patients, our results showed that NDRG2 expression was involved in tumor progression and the overall survival of the patients. Furthermore, loss of NDRG2 expression in CCRCC is significantly correlated with aggressive clinicopathologic features of CCRCC patients, and NDRG2 expression contributes to the suppression of renal cancer cell metastasis. Metastasis is a characteristic of highly malignant cancers with poor clinical outcome, and excess degradation of surrounding ECM is one of the hallmarks of tumor invasion and metastasis [21]. Among ECM-degrading enzymes, MMPs are the most important metastasis-promoting genes because MMPs affect basic, necessary steps in the metastatic cascade, including angiogenesis, proliferation, and apoptosis [22, 23]. As shown in our current study, up-regulation of NDRG2 could significantly inhibit MMP-9 expression. Decreased levels of MMP-9 may attenuate metastatic progression, including anchorage-independent growth, migration, invasion, and angiogenesis. So NDRG2 may affect as an invasion suppress factor in CCRCC. In conclusion, this study shows for the first time that NDRG2 is involved in CCRCC metastasis through regulation of MMP-9 expression. Our work provides convincing evidence that NDRG2 expression correlates closely with WHO grade and overall survival of patients with CCRCC, and that NDRG2 might be a novel prognostic marker that could be used as an adjunct to WHO grade to improve prognostication for an individual patient. This observation broadens our understanding of the molecular mechanisms of CCRCC metastasis and may lead to the development of new therapeutic approaches. Acknowledgments This study was supported by National Natural Science Foundation of China (81071764, 81101568 and 81101614) and Key Personnel Assistance Funds of Tangdu Hospital, Fourth Military Medical University. Conflict of interest The authors have no conflict of interest to disclose.
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