Ann Surg Oncol (2012) 19:2763–2772 DOI 10.1245/s10434-011-2204-3

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

NDRG2 Is Involved in the Oncogenic Properties of Renal Cell Carcinoma and Its Loss Is a Novel Independent Poor Prognostic Factor After Nephrectomy Zhe Long Liang, MD1, Kyeongah Kang, PhD2, Sukjoon Yoon, PhD2, Song Mei Huang, MD1, Jae Sung Lim, MD3, Jin Man Kim, MD1, Jong-Seok Lim, PhD2, and Hyo Jin Lee, MD4 1

Department of Pathology, Cancer Research Institute, and Infection Signaling Network Research Center, Chungnam National University School of Medicine, Daejeon, Republic of Korea; 2Department of Biological Science and the Research Center for Women’s Disease, Sookmyung Women’s University, Seoul, Republic of Korea; 3Department of Urology and Cancer Research Institute, Chungnam National University School of Medicine, Daejeon, Republic of Korea; 4Department of Internal Medicine and Cancer Research Institute, Chungnam National University School of Medicine, Daejeon, Republic of Korea

ABSTRACT Background. Although NDRG2 is a candidate tumor suppressor, its exact role in renal cell carcinoma (RCC) is not fully understood. We investigated the functional role of NDRG2 and its clinical relevance in RCC tumorigenesis. Methods. NDRG2 expression and its clinical implications in clear cell RCC were evaluated. Biological function was assessed by a proliferation assay, anchorage-independent growth assay, and wound healing and transwell migration assays in RCC cell lines overexpressing NDRG2 coupled with an investigation of the effects of NDRG2 expression on the epithelial–mesenchymal transition (EMT). Results. NDRG2 was differentially expressed in patients with RCC. A loss of NDRG2 was significantly associated with a higher proportion of tumors [10 cm and a high

Zhe Long Liang and Kyeongah Kang contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1245/s10434-011-2204-3) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2012 First Received: 29 August 2011; Published Online: 14 January 2012 J.-S. Lim, PhD e-mail: [email protected] H. J. Lee, MD e-mail: [email protected]

nuclear grade. Furthermore, multivariate analyses indicated that a loss of NDRG2 was an independent poor prognostic factor for patient survival (recurrence-free survival, hazard ratio 7.901; disease-specific survival, hazard ratio 15.395; overall survival, hazard ratio 11.339; P \ 0.001 for all parameters). NDRG2 expression inhibited the anchorageindependent growth and migration of RCC cells. NDRG2 expression also modulated the expression of EMT-related genes such as Snail, Slug, and SIP1, and it decreased EMT signaling in RCC cells. Finally, NDRG2 recovered E-cadherin expression in E-cadherin-negative RCC cells. Conclusions. These results indicate that a lack of NDRG2 is associated with oncogenic properties through the loss of its role as a tumor suppressor, and that NDRG2 is an independent poor prognostic factor predicting survival in clear cell RCC, suggesting that it can serve as a novel prognostic biomarker.

Renal cell carcinoma (RCC) is one of the most lethal genitourinary malignancies.1 Among RCC patients, 20–30% experiences a local or distant recurrence within 5 years after an initial curative nephrectomy. RCC has a dismal prognosis after metastasis has occurred.2,3 Therefore, predicting which patients will develop disease recurrence after surgery for localized RCC is important. Several prognostic models, based mainly on clinicopathologic parameters, exist for nonmetastatic RCC.4–6 Although risk grouping using these features is possible, marked heterogeneity exists among patients with similar prognoses. In addition to clinicopathologic prognostic

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features, molecular markers may play a crucial role in prognosis.7,8 N-myc downstream-regulated gene 2 (NDRG2) belongs to the NDRG family, which has 4 identified members (NDRG1–4) that are expressed in brain, heart, skeletal muscle, and kidneys.9,10 NDRG2 has been implicated in cell growth, differentiation, and neurodegeneration. NDRG2 is also a candidate tumor suppressor gene because it induces apoptosis in several cancer cells, and its transcription is downregulated or absent in cancer cell lines and some human cancers.11,12 Additionally, the protein expression of NDRG2 in gliomas and meningiomas is greatly decreased in high-grade compared to low-grade tumors.13 Furthermore, NDRG2 expression is positively correlated with survival in patients with high-grade gliomas.14 NDRG2 inhibits the growth of clear cell RCC (CCRCC) cell lines and induces a cell cycle arrest at G1 in vitro.15 Moreover, NDRG2 is downregulated in CCRCC tissues compared to adjacent nonneoplastic tissues, suggesting that it plays an important role in carcinogenesis of CCRCC.12 However, the exact role of NDRG2 in CCRCC is not fully understood, and the differential expression and clinical relevance of NDRG2 have not been investigated. We investigated NDRG2 expression as a prognostic marker in tumor specimens from patients with CCRCC and studied the relationship between NDRG2 expression and clinicopathologic features and patient survival. We also investigated the role of NDRG2 expression in CCRCC cell lines in vitro.

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from recipient blocks and placed on 3-amino-propyltriethoxysilane-coated slides that were dried for 2 h before staining at 57°C. All procedures were performed at room temperature, as described previously.16 Prepared polyclonal mouse anti-NDRG2 antibodies (raised against human NDRG2) were diluted 1:3200 with backgroundreducing diluents (Dako). The slides were incubated overnight at 4°C in a humid chamber then washed with TBS-T (Tris-buffered saline and 0.05% Tween-20). The slides were then incubated for 30 min with an EnVision anti-mouse (Dako) polymer. The reaction products were visualized after 5 min in diaminobenzidine plus a substrate-chromogen solution. The slides were counterstained with Meyer’s hematoxylin and mounted. Negative controls were prepared by excluding the primary antibody or by using preimmune IgG1 to evaluate nonspecific staining. Immunostaining was evaluated by 2 independent pathologists (JMK, ZLL) who were blinded to the patients’ clinicopathologic details. The tumors were classified as follows on the basis of the staining intensity: 0, no staining; 1, weak; 2, intermediate; and 3, strong. In cases of heterogeneous staining within samples, the higher score was chosen if [50% of the cells showed a higher staining intensity. Scores from 2 tumor cores in the same patient were averaged to obtain a mean score for all patients. Cases with no staining were assigned to the NDRG2-negative group, whereas those with a score of 1 and 2 were assigned to the NDRG2-intermediate group and those with a score of 3 to the NDRG2-high group. Cell Lines and NDRG2 Overexpression

MATERIALS AND METHODS Patients and Tumor Samples We investigated 112 consecutive patients with CCRCC who underwent nephrectomy for sporadic, localized RCC (N0M0) at Chungnam National University Hospital between 1999 and 2006 (Supplementary Table S1). Clinicopathologic data were obtained through medical record reviews. T classification was defined according to 2002 American Joint Committee on Cancer criteria; the nuclear grade was defined according to Fuhrman’s grading system. Tumor samples were collected from tissue blocks used for routine pathologic examination. This study was approved by the local ethics committee.

The SN12C, 786-O, and ACHN human RCC cell lines were provided by Dr. Sukjoon Yoon of Sookmyung Women’s University. The cells were cultured in RPMI 1640 (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) at 37°C in a humidified 5% CO2 incubator. RCC cell lines were transfected with 10 lg of pCMV/ Taq2B or pCMV/Taq2B-NDRG2 with SuperFect Transfection Reagent (Qiagen, Hilden, Germany). After 2 days, the cells were selected with complete medium conditioning with 1 mg/ml G418 (Duchefa, Haarlem, The Netherlands). Mixed populations of the transfected cells were analyzed. NDRG2 expression was confirmed by reverse transcription– polymerase chain reaction (RT-PCR) and Western blot test. Anchorage-independent Growth Assay

Immunohistochemical Staining of NDRG2 Tissue microarrays were constructed from archival, original formalin-fixed, paraffin-embedded tissue blocks. Sections (3 lm thick) were cut for immunohistochemistry

Cells (5 9 103) were uniformly suspended in 2 ml of medium containing 0.3% agar and 10% fetal bovine serum and applied to presolidified 1% agar (1.5 ml) in a 6-well plate. After 14 days, all colonies were inspected by phase-contrast

NDRG2 in Renal Cell Carcinoma

microscopy and photographed. Colony size was calculated according to the formula V = p/6 9 D1 9 D2, where D1 is the larger diameter and D2 is the smaller diameter.

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methylene blue. Those cells that migrated to the lower side of the filter were identified with 10% acetic acid. Absorbance was measured with a microplate reader (PerkinElmer Victor, Fremont, CA).

RT-PCR Statistical Analysis RNA was isolated with TRI Reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized from 5 lg of total RNA with M-MLV Reverse Transcriptase (Promega, Madison, WI). The cDNAs were amplified over 32–37 cycles of PCR. b-actin (loading control) was subjected to 21 cycles of PCR. The primer sequences are given in Supplementary Table S2. All products were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining. Western Blot Analysis Cells were lysed in protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) and their proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to PVDF membranes. The membranes were incubated with specific antibodies and washed with TBS-T. The antibodies used were specific for epithelial cadherin (E-cadherin), Snail, phosphatase and tensin homolog (PTEN), Akt, phospho-Akt (S473), IjBa, b-catenin, NDRG2, and actinin. The antibodies for PTEN, phosphoAkt (S473), and IjBa were purchased from Cell Signaling Technology (Beverly, MA), while the antibodies for E-cadherin, Snail, Akt, NDRG2, and actinin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The b-catenin antibody was purchased from BD Biosciences (San Jose, CA). After incubation with the diluted secondary antibody coupled to horseradish peroxidase, the antigen-antibody complexes were detected by enhanced chemiluminescence and exposed with the LAS 3000 imaging system (Fujifilm Corp., Tokyo, Japan). Wound Healing and Transwell Migration Assay Cells were grown to confluence in 6-well culture plates. A wound was created with a sterile pipette tip at an angle of approximately 30°. After washing, the culture medium was changed. Cell migration was monitored with a microscope for 15–45 h depending on the growth rate of each cell line. The in vitro migration assay was performed with 24-well transwell units and 8.0-lm pore-size polycarbonate filters (SPL Lifescience, Pocheon, Korea). Cells (3 9 105) were added to the upper part of the transwell and incubated for 48 h at 37°C. Cells attached to the upper surface of the filter were completely removed by wiping with a cotton swab, and the filters were stained with 0.5%

Paired Student t-tests were used to compare differences in biological behavior between the mock and NDRG2expressing cells. Linear by linear association was used to assess the correlation between NDRG2 expression and clinicopathologic features. Recurrence-free survival (RFS), disease-specific survival (DSS), and overall survival (OS) were estimated by the Kaplan-Meier method and the log rank test. RFS was measured from the date of nephrectomy to the date of recurrence or death from RCC. DSS was measured from the date of nephrectomy to the date of death from RCC only. OS was measured from the date of surgery to death from all causes. A Cox’s proportional hazards model analysis was conducted to analyze the effect of NDRG2 expression on survival. P values of \0.05 were considered statistically significant. All analyses were conducted by SPSS 13.0 software (SPSS, Chicago, IL). RESULTS Correlation Between NDRG2 Expression and Clinicopathologic Features NDRG2 was positively stained in the cytoplasm and membranes of the nonneoplastic renal tubular epithelium, whereas CCRCC cells showed diverse cytoplasmic and membrane staining patterns (Fig. 1a–d). Seven cases (6.3%) showed no staining, whereas 115 cases (93.8%) showed staining (1, 35 cases; 2, 33 cases; and 3, 37 cases). Our results indicate that NDRG2 is expressed differentially in CCRCCs and adjacent noncancerous normal tissues, and that NDRG2 is not expressed in some cell populations in CCRCC tumors. We next investigated the correlation between NDRG2 expression and various clinicopathologic parameters (Table 1). NDRG2-negative tumors tended to be of a higher T stage compared to those in the NDRG2intermediate and -high groups (85.7% vs. 55.2%). Furthermore, the NDRG2-negative group was significantly associated with a higher proportion of tumors [10 cm (P = 0.041) and a higher nuclear grade (P = 0.044) and tumor recurrence (P = 0.001). Correlation Between NDRG2 Expression and Survival The 5-year RFS, DSS, and OS rates were 83.0, 88.4, and 81.3%, respectively, for the entire study population. The

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FIG. 1 Representative photomicrographs of immunohistochemical staining for NDRG2 in normal (a) and clear RCCs (b–d), and correlation between NDRG2 expression and survival rates in RCC patients (e–g). a NDRG2 expression in normal kidney tubules

(9200). b Strong staining intensity (9400). c Intermediate staining intensity (9400). d No staining intensity (9400). e Recurrence-free survival (P \ 0.001). f Disease-specific survival (P \ 0.001). g Overall survival (P \ 0.001)

survival curves based on NDRG2 expression are depicted in Fig. 1e–g. The 5-year RFS rates in the NDRG2-high, -intermediate, and -negative groups were 89.2, 85.3, and 28.6%, respectively (P \ 0.001), while the rates of 5-year DSS for the NDRG2-high, -intermediate, and -negative groups were 94.6, 91.2, and 28.6%, respectively (P \ 0.001). The rates of 5-year OS were 89.2, 83.8, and 14.3%, respectively (P \ 0.001). On univariate analyses, Fuhrman nuclear grade and the absence of NDRG2 expression were significant risk factors affecting RFS, DSS, and OS (Table 2). Furthermore, multivariate analyses indicate that Fuhrman nuclear grade (hazard ratio 2.990; 95% confidence interval 1.145–7.806; P = 0.025) and the absence of NDRG2 expression (hazard ratio 7.901; 95% confidence interval 2.713–23.008; P \ 0.001) were independent risk factors predicting RFS in RCC patients. The absence of NDRG2 expression remained an independent prognostic factor for DSS and OS.

negative RCC cell lines SN12C, 786-O, and ACHN. The NDRG2-transfected cells showed exclusive, strong mRNA and protein expression of NDRG2 (Fig. 2a). The NDRG2-positive cells exhibited no significant difference in morphology (data not shown) or proliferation (Fig. 2b). We investigated anchorage-independent growth in the 3 cell lines because it is a distinct feature of transformed cells. As shown in Fig. 2c, parental and mock cells of SN12C, 786-O, and ACHN proliferated quickly and formed sizable colonies in soft agar. However, the NDRG2-positive cells produced fewer and smaller colonies. The inhibition of anchorage-independent growth by NDRG2 expression was not due to differences in cell growth rates (Fig. 2b). Therefore, these data suggest that NDRG2 expression inhibits the anchorage-independent growth of RCC cells.

Inhibition of Anchorage-independent RCC Cell Growth by NDRG2 Expression To explore the effect of NDRG2 overexpression in RCC cells, NDRG2 was transfected into the NDRG2-

NDRG2 Expression Modulates Epithelial– Mesenchymal Transition (EMT)-Related Gene Expression and Suppresses EMT Signaling As shown in Fig. 3a, wild-type RCC cells significantly expressed EMT-regulating genes, including Snail, Slug, and SIP1. In contrast, NDRG2-positive cells usually

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TABLE 1 Associations of the expression of NDRG2 with clinicopathologic parameters Feature

NDRG2 expression Negative (n = 7) n (%)

P

Intermediate (n = 68) n (%)

High (n = 37) n (%)

B70 year 4 (57.1)

55 (80.9)

34 (91.9)

[70 year 3 (42.9)

13 (19.1)

3 (8.1)

Age 0.060

Sex Male

5 (71.4)

49 (72.1)

25 (67.6)

Female

2 (28.6)

19 (27.9)

12 (32.4)

0.731

ECOG PS 0

3 (42.9)

32 (47.1)

14 (37.8)

C1

4 (57.1)

36 (52.9)

23 (62.2)

Tumor size B10 cm 5 (71.4)

65 (95.6)

35 (94.6)

2 (28.6)

3 (4.4)

2 (5.4)

T1

1 (14.3)

28 (41.2)

19 (51.4)

T2/3

6 (85.7)

40 (58.8)

18 (48.6)

[10 cm

0.594

0.041

T stage 0.064

Fuhrman grade G1/2

3 (42.9)

59 (86.8)

31 (83.8)

G3/4

4 (57.1)

9 (13.2)

6 (16.2)

No

2 (28.6)

58 (85.3)

33 (89.2)

Yes

5 (71.4)

10 (14.7)

4 (10.8)

0.044

NDRG2 Expression Suppresses RCC Cell Migration

Recurrence 0.001

ECOG Eastern Cooperative Oncology Group, PS performance status TABLE 2 Univariate and multivariate analyses of the association of prognosis with clinicopathologic parameters and NDRG2 expression in patients with RCC

showed reduced Snail, Slug, and SIP1 mRNA expression. Moreover, NDRG2 expression induced an increase of E-cadherin expression in E-cadherin-negative cell lines (Fig. 3b). NDRG2 also downregulated Snail protein expression in the 3 cell lines (Fig. 3b). These data indicate NDRG2 expression modulates the expression of EMTrelated genes and recovers E-cadherin expression in E-cadherin-negative RCC cells. We next evaluated whether NDRG2 expression regulates EMT signaling. As shown in Fig. 3c, the SN12C and ACHN cells were PTEN positive, but the 786-O cells, which had a PTEN mutation, showed reduced PTEN expression. Regardless of the PTEN mutation status, NDRG2 overexpression increased PTEN expression in all 3 RCC cell lines. Akt activation was noted in all 3 cell lines and was not influenced by NDRG2 expression. Furthermore, NDRG2 induced a decrease in b-catenin and inhibited NF-jB activation by stabilizing IjB (Fig. 3c). The decrease in b-catenin by NDRG2 in SN12C cells was mainly due to the disappearance of nuclear b-catenin; its cytoplasmic expression was maintained at a high level (data not shown). These results indicate NDRG2 expression suppresses signaling pathways involved in the EMT in RCC cells.

Variable

To evaluate the effect of NDRG2 expression on cellular function, we performed wound healing and in vitro transwell migration assays. When the ability to heal wounds was Univariate P Hazard ratio (95% confidence interval)

Multivariate P Hazard ratio (95% confidence interval)

Recurrence-free survival ECOG PS (C1)

0.739 (0.349–2.111)

0.858

Tumor size ([10 cm)

3.440 (1.000–11.836)

0.050

Pathologic T stage (T2, T3)

2.386 (0.859–6.627)

0.095

Fuhrman nuclear grade (G3, G4)

3.472 (1.366–8.827)

NDRG2 expression (negative)

8.954 (3.189–25.141)

0.009 \0.001

2.990 (1.145–7.806) 7.901 (2.713–23.008)

0.025 \0.001

Disease-specific survival ECOG PS (C1)

0.895 (0.301–2.664)

0.842

Tumor size ([10 cm)

3.026 (0.670–13.661)

0.150

Pathologic T stage (T2, T3)

2.803 (0.771–10.189)

0.117

Fuhrman nuclear grade (G3, G4)

4.737 (1.590–14.117)

0.005

NDRG2 expression (negative)

20.272 (6.419–64.016)

3.070 (0.949–9.932)

\0.001 15.395 (4.479–52.916)

0.061 \0.001

Overall survival

ECOG Eastern Cooperative Oncology Group, PS performance status

ECOG PS (C1)

0.840 (0.357–1.977)

0.689

Tumor size ([10 cm) Pathologic T stage (T2, T3)

1.773 (0.413–7.614) 1.688 (0.681–4.185)

0.441 0.258

Fuhrman nuclear grade (G3, G4)

2.799 (1.128–6.943)

0.026

NDRG2 expression (negative)

13.235 (4.981–35.168)

1.998 (0.764–5.230)

\0.001 11.339 (4.067–31.614)

0.158 \0.001

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FIG. 2 NDRG2 expression inhibits anchorage-independent growth of 3 RCC cell lines but not their growth rate. a NDRG2 expression was examined by RT-PCR (top 2 lanes) and Western blot (bottom 2 lanes) analyses. b Cells were seeded in a plate and were collected daily for 4 days. Cell numbers were determined with the trypan blue exclusion assay. c Cells (5 9 103) were uniformly suspended in 2 ml of the medium containing 0.3% agar and 10% fetal bovine serum,

then applied onto the presolidified 1% agar (1.5 ml) in a 6-well plate. After incubation for 14 days, colonies on soft agar were observed under a phase-contrast microscope, and the diameter of representative 5 colonies was measured. The results represent the means ± SD of [3 samples. **P \ 0.01, ***P \ 0.001 (values are compared with mock cells)

compared among parental, mock, and NDRG2-expressing cells, NDRG2 expression inhibited wound healing in all 3 RCC cell lines (Fig. 4a, c, e). Migration was also decreased significantly with NDRG2 overexpression compared to parental and mock cells in all cell lines (Fig. 4b, d, f). Thus, NDRG2 expression may reduce the migratory ability of RCC cells.

DISCUSSION We investigated and clarified the clinical implications of NDRG2 expression in RCC. Recent data have showed that NDRG2 expression was downregulated or lost in several malignant tumors compared to normal or benign tissues,

NDRG2 in Renal Cell Carcinoma

a

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FIG. 3 NDRG2 expression modulates expression of EMT-related genes in RCC cells and activation of signaling pathways promoting EMT is attenuated by NDRG2 expression. a Parental, mock, and NDRG2-expressing cells of 3 RCC cells were collected for RNA

preparation, respectively. mRNA levels were measured by RT-PCR. b, c Equal amounts of whole lysates were subjected to electrophoresis on SDS-PAGE gels, and Western blot analysis was performed. Data are representative of the repeated results from [3 experiments

suggesting that NDRG2 plays a crucial role in carcinogenesis and tumor progression.17 Among 7 gastric cancer and 2 noncancer cell lines, only 2 gastric cancer cell lines expressed NDRG2, whereas NDRG2 was highly expressed in normal gastric tissues. Moreover, a loss of NDRG2 expression is an independent poor prognostic factor in patients with gastric carcinoma with shorter survival.18 A similar pattern has been reported for colon, breast, thyroid, and esophageal cancer, as well as oral squamous cell carcinoma.17,19–22 NDRG2 expression is decreased or absent in CCRCC cell lines and tissues.12,15 However, the correlation between NDRG2 expression, clinicopathologic features, and patient survival has not been investigated. In this study, we found that NDRG2 was differentially expressed in CCRCC and that NDRG2 expression was lost in some cell populations. Interestingly, we found that a loss of NDRG2 tended to result in a higher T stage and was

associated with a higher proportion of tumors [10 cm, a higher nuclear grade, and disease recurrence in patients with RCC. Furthermore, multivariate analyses clearly indicated that a loss of NDRG2 expression was an independent poor prognostic factor for RFS, DSS, and OS, suggesting that NDRG2 can be used as a marker to predict tumor recurrence and survival in patients with CCRCC. Transformed cells have the ability to grow unattached to a semisolid medium, a feature called anchorage-independent growth. Because anchorage-independent growth provokes various cellular changes, including a loss of contact inhibition, motility exchange, and adhesiveness, the ability to grow independently from anchorage is a marker of tumorigenesis and metastatic potential.23,24 Interestingly, in this study, NDRG2 expression in 3 RCC cell lines induced not only a reduction in colony number but also a reduction in colony size in a soft agar assay, indicating a

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FIG. 4 Suppression of renal cell migration by NDRG2 expression. a, c, e Confluent monolayers of parental, mock, and NDRG2expression cells were wounded and incubated up to 45 h. Images were photographed when the wounds were made (top lane) and after incubation for 15 to 45 h (bottom lane). b, d, f A fixed number of cells (3 9 105 cells) was plated onto the upper part of the transwell chamber. After incubation for 48 h, cells migrating to the lower

surface of the membrane were stained with methylene blue solution and were observed with phase-contrast microscopy. Migrating cells to the lower side of filter were also quantified by staining with methylene blue and dissolving with 10% acetic acid. Absorbance was measured with a microplate reader set (PerkinElmer Victor, Fremont, CA). The results represent the means ± SD of duplicates. *P \ 0.05 (values are compared with mock cells)

suppressive effect of NDRG2 expression on anchorageindependent cell growth. Tumor progression and metastasis are a multi-step process that includes cell adhesion to the extracellular matrix, proteolytic digestion of the extracellular matrix, invasion into the lymph and blood vessels, and migration. In particular, the EMT facilitates tumor cell invasion, spread, and metastasis.25 Thus, the EMT is a critical mechanism of tumor progression, and suppressing the EMT is important for preventing tumor metastasis. In the present study, we examined the inhibitory effect of NDRG2 expression on EMT-promoting transcription factors. A remarkable reduction in Snail, Slug, and SIP1 mRNA expression was seen in NDRG2-positive RCC cells, whereas ZEB1, Twist, and vimentin expression did not

change markedly (data not shown). Thus, NDRG2 expression modulates specific transcription factors involved in the EMT in RCC tumor cells. Additionally, the inhibition of Snail, Slug, and SIP1 resulted in a recovery of E-cadherin expression in E-cadherin-negative SN12C and 786-O cells. E-cadherin is a dominant mediator of cell interactions, and low levels of E-cadherin expression are associated with a poor prognosis during carcinogenesis.26 Thus, NDRG2 may play an essential role in inhibiting RCC progression into a malignant form. Oncogenic transformation is usually associated with various signaling pathways regulating the EMT. Abnormal PI3K/Akt activation is often present in diverse tumors and induces modifications in cell morphology, tumorigenicity,

NDRG2 in Renal Cell Carcinoma

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and invasiveness.27–29 In contrast, PTEN, an important tumor suppressor, is an upstream negative regulator of PI3K/Akt leading to glycogen synthase kinase (GSK)-3b phosphorylation.30,31 The inhibition of GSK-3b leads to the accumulation of unphosphorylated b-catenin in the cytoplasm, which avoids degradation and translocates to the nucleus.32 In this study, NDRG2 expression resulted in decreased b-catenin, mainly as a result of the disappearance of nuclear b-catenin. This observation is comparable with previous results showing that NDRG2 overexpression in mouse melanoma cells inhibited TCF/b-catenin signaling induced by a GSK-3b inhibitor.33 Furthermore, the inactivation of NF-jB, represented by an increased level of IjBa, was observed in all 3 NDRG2-expressing RCC cell lines. NF-jB is activated independently and/or dependently of GSK-3b during the EMT, and constitutive NF-jB expression has been shown in cancer cell lines and tissue samples from patients with cancer.34 Similarly, we recently showed that NDRG2 affects malignant mouse melanoma cell invasion by suppressing NF-jB activity.35 Additionally, NDRG2 expression did not affect Akt activation in the 3 RCC cell lines, but it induced PTEN upregulation, indicating an inhibitory effect on malignant cell growth. PTEN expression was clearly enhanced by NDRG2 expression, even in 786-O cells, which do not express PTEN because of a mutation. Collectively, our results suggest that NDRG2 expression attenuates the expression of transcription factors associated with the EMT and interferes with Wnt/b-catenin signaling and NF-jB activation in RCC cell lines. In conclusion, our results indicate that NDRG2 was lost in some patients with CCRCC, and that its loss was significantly associated with oncogenic properties and tumor progression. In vitro experiments clearly demonstrated that NDRG2 expression suppressed the malignant transformation by inhibiting the EMT and cellular signaling linked to tumor progression. Therefore, a loss of NDRG2 is an independent poor prognostic factor for survival in patients with RCC, suggesting that it can serve as a novel molecular marker. ACKNOWLEDGMENT This work was supported by a grant of the Research Center for Women’s Diseases through the National Research Foundation of Korea (NRF) (R11-2011-0001386), and the NRF grants funded by the Ministry of Education, Science and Technology of Korea (MEST) (No. 2011-0006229). CONFLICT OF INTEREST were disclosed.

No potential conflicts of interest

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