Background
According to the latest globally statistics released in 2018, colorectal cancer (CRC) is estimated to be the third most commonly diagnosed malignant tumor (10.2% of the total cases) and the second leading cause of cancer-related deaths (9.2% of the total cancer-related deaths) [
1]. In China, the age-standardized incidence of CRC increased from 12.8 (2003) to 16.8 (2011) per 100,000 individuals [
2]. Although curative surgery is still the main treatment for CRC patients, most cases are diagnosed at advanced stage, or, worse, with metastasis. The prognosis is poor, even though many novel treatment strategies, such as targeted therapy and immunotherapy have been used. Therefore, development of novel treatment targets is important to help design novel therapies [
3]. In addition, there is also a great need to elucidate the molecular mechanisms of different subgroups of CRC, which can provide critical clues for the personalized treatment.
N-myc downstream regulated gene-1 (NDRG1), a member of the NDRG gene family, is pervasively expressed in epithelial tissues and is a cytoplasmic and nuclear protein [
4‐
6]. NDRG1 has been proved to play a key role in tumor proliferation, metastasis, differentiation, cell adhesion, cell-cycle modulation, and autophagy [
7‐
12]. Evidences have demonstrated that NDRG1 expression was significantly decreased in several malignant diseases, including gastric, colorectal, prostate, and breast cancer [
13‐
16]. Therefore, NDRG1 is recognized as “tumor suppressor”, which can induce tumor differentiation, inhibit invasion, metastasis, and cell proliferation [
13]. Besides, NDRG1 expression is closely associated with a positive survival of colon [
17], prostate [
18], breast [
15] and pancreatic cancer patients [
19]. It has been reported that locally advanced rectal cancer patients with NDRG1-positive expression could benefit from oxaliplatin-dominated chemotherapy [
20]. In the previous studies, our team has elaborately revealed part of the functions and underlying mechanisms of NDRG1 during CRC progression and metastasis [
10,
17,
21‐
24], however, there are limited studies regarding the mechanism of NDRG1 in proliferation in human solid tumors. Recent studies have shown NDRG1 could induce cancer cell G0/G1 arrest, inhibiting tumor proliferation [
13,
25]. Yet, its underlying mechanism is still obscure.
Dysregulated proliferation is a notable feature of tumorigenesis. Unlike the normal cells, whose proliferation is well balanced by growth and antigrowth signals, the tumor cells have their own growth signals, and their proliferation is precisely regulated by cell cycle regulators [
26]. p21, the inhibitor of the cyclin-dependent kinase and effector of the p53 tumor suppressor, is indispensable for cell-cycle progression, which can arrest most G1-phase in response to various stimuli [
27]. Various genes could promote the tumor proliferation and progression by inhibiting p21 [
28,
29]. In addition, there are studies suggesting that loss expression and/or function of p21 may contribute to tumorigenesis and metastasis [
27,
30].
Therefore, based on the functions of NDRG1 and p21 in diseases, there may be interactions between NDRG1 and p21. In this study, we demonstrated that NDRG1 could inhibit tumor proliferation through increasing p21 protein expression in vivo and in vitro.
Methods
Tissue specimens and immunochemistry (IHC)
A total of 89 CRC cases were recruited from Ruijin Hospital (Shanghai, China) in accordance with the guidelines set by the Ethical Committee of Ruijin Hospital. All cases got the written, informed consent before the study. Staging of CRC was performed according to the UICC guideline (8th Edition). The cohort of 89 tumor tissues and paired normal colonic tissues were fixed with formaldehyde and embedded with paraffin. Tissue array was constructed for further immunohistological assay. The staining score of each tissue was calculated based on the widely used German semi-quantitative scoring system by three independent pathologists as described previously [
22,
31,
32], score > 3 was considered positive expression.
Cell culture and transfection
The p53-wt HCT116, the p53-mutant SW1116 colon cancer cell lines and HEK 293 T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The NDRG1 overexpression and knockdown clones were established as described previously [
23]. All cells were cultured under a humidified atmosphere containing 5% CO
2 at 37 °C. The plasmid vector containing NEDD4, Flag-p21 and HA-Ubiquitin were constructed by Genechem Inc. (Shanghai, China). Lentivirus vectors containing Flag-p21 were constructed by Genechem Inc. (Shanghai, China). For a transient transfection, cells were seeded in a 6-well culture plate 24 h before transfection, then cells were transfected with corresponding si-RNA or vector, using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Sequences of siRNA used were as follows: p21: sense-1 5′-GAUGGAACUUCGACUUUGUTT-3′, antisense-1 5′-ACAAAGUCGAAGUUCCAUCTT-3′; sense-2 5′-CCUCUGGCAUUAGAAUUAUTT-3′, antisense-2 5′-AUAAUUCUAAUGCCAGAGGTT-3′; sense-3 5′-CAGGCGGUUAUGAAAUUCATT-3′, antisense-3 5′-UGAAUUUCAUAACCGCCUGTT-3′. NEDD4: sense-1 5′-GGAUGUUCCAACUCAUCUUTT-3′, antisense-1 5′-AAGAUGAGUUGGAACAUCCTT-3′; sense-2 5′-CCAAGAAGUCACAAAUCAATT-3′, antisense-2 5′-UUGAUUUGUGACUUCUUGGTT-3′; sense-3 5′-GCACAUCUCGGGUGCCUAUTT-3′, antisense-3 5′-AUAGGCACCCGAGAUGUGCTT-3′.
For colony formation, after dissociated into single cell, 500 tumor cells (HCT116) or 1000 tumor cells (SW1116) were seeded in six-well plate, which were cultured in an incubator until visible cloning appeared. Then these six-wells plates were washed, fixed with 4% formaldehyde, stained with crystal violet dye, calculated and analyzed.
For CCK-8 assay, six repeats of 1500 cells were seeded in 96-well plate and cultured in each group. The growth rates of the cells were determined at 24, 48, 72, 96 and 120 h using Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, Japan). The absorbance was measured at 450 nm using a model 3550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Western blot analysis
Cells were washed with PBS and then lysed with RIPA buffer. Insoluble materials were removed by centrifugation at 12,000 rpm for 15 min at 4 °C. Equal amounts of protein were separated using 10% SDS-PAGE, transferred to PVDF, and probed with the appropriate antibodies as indicated. Immunoreactive bands were visualized using an ECL kit (Amersham Biosciences, Piscataway, New Jersey, USA). The extraction of proteins from nuclear and cytoplasmic fractions were performed using Subcellular Protein Fractionation Kit from Thermo Scientific (78840; Waltham, MA, USA). Primary antibodies including: NDRG1 (Rabbit, catalog number ab124689) from Abcam; p21 (Rabbit, catalog number 2947) from Cell Signaling Technology; p21 (Mouse, catalog number ab80633) from Abcam; NEDD4 (Rabbit, catalog number 2740) from Cell Signaling Technology; NEDD4 (Rabbit, catalog number 21698-1-AP) from Proteintech; Flag (Rabbit, catalog number 14793) and Histone H3 (Rabbit, catalog number 4499) from Cell Signaling Technology. The secondary antibodies implemented include horseradish peroxidase-conjugated anti-goat (catalog number A5420), anti-rabbit (catalog number A6154), and anti-mouse (catalog number A4416) antibodies from Sigma-Aldrich.
RNA extraction and qPCR for mRNA expression analysis
RNA extraction and qPCR analysis were performed as described previously [
22,
33]. The primer sequences used for analysis are listed as follows: NDRG1 (5′-CTGCACCTGTTCATCAATGC-3′ and 5′-AGAGAAGTGACGCTGGAACC-3′); p21 (5′-AGGTGGACCTGGAGACTC-3′ and 5′-CGGCGTTTGGAGTGGTAG-3′); GAPDH (5′-TTCAACAGCAACTCCCACTCTT-3′ and 5′-TGGTCCAGGGTTTCTTACTCC-3′).
Immunoprecipitation and ubiquitylation assay
Cells were washed twice with ice-cold PBS and lysed by RIPA buffer containing protease inhibitors (Roche Diagnostics, Basel, Switzerland). Protein (300 mg) was incubated with specific antibody overnight at 4 °C. This mixture was added to 30 ul of beads (Protein A/G PLUS-Agarose, sc-2003) from Santa Cruz Biotechnology (Santa Cruz, CA) and incubated for 4 h at 4 ̊C. Then washed by ice-cold PBS, resuspended in loading buffer, and incubated over 90 °C for 10 min. The supernatant was separated on a 10% Bis-Tris gel. Then the mixture was detected by western blots.
UbiBrowser Database was used to screen possible E3 ligases related to NDRG1 or p21 (
http://ubibrowser.ncpsb.org/). The co-expression analysis of NDRG1 and p21 in GSE33114 and GSE37892 profiles was performed via the R2 Genomics Analysis and Visualization Platform (
http://r2.amc.nl). The co-expression analysis of NDRG1 and p21 in the cancer genomic atlas (TCGA) was performed in The Gene Expression Profiling Interactive Analysis (GEPIA,
http://gepia.cancer-pku.cn/index.html).
Ubiquitylation assay was performed as described previously. The cells were treated with Mg132 and then lysed by SDS-free RIPA buffer and immunoprecipitated with primary antibody followed by protein A/G plus agarose. Then, the supernatant was detected by immunoblotting using ubiquitin antibody (FK2, Enzo Life Sciences, New York, NY, USA).
Immunofluorescence
As described previously [
23], cells were incubated with the relative primary antibody overnight at 4 °C, followed by incubation with fluorescent secondary antibody for 2 h at room temperature. After final washes with phosphate-buffered saline (PBS), the coverslips were mounted using an antifade mounting solution containing 4,6-diamidino-2-phenylindole (DAPI; P36935, Invitrogen). Images were acquired on the confocal microscope (Carl Zeiss). Primary antibody:anti-rabbit p21 (catalog number 2947) from Cell Signaling Technology; anti-mouse p21 (catalog number ab80633) from Abcam for Immunofluorescence double-staining analysis; anti-rabbit NEDD4 (catalog number 2740) from Cell Signaling Technology.
Xenograft model
Mice were cultivated under standard conditions following institutional guidelines. A total of 5 × 106 HCT116 cells (NDRG1 overexpression, knockdown and corresponding controls) or SW1116 cells (sh-Control, SH-NDRG1 and SH-NDRG1/p21) were injected subcutaneously into nude mice (male BALB/c nu/nu nude mice, 4-week-old). Tumor size was measured every 7 days. Tumor volume (V) was determined by measuring the length and width of the tumor and using the formula V = (width*width*length) / 2. Twenty-eight days after injection, all the mice were sacrificed and then tumor grafts were excised, fixed with formalin and embedded with paraffin for further immunohistochemistry research.
Statistical analysis
IBM SPSS Statistical software (version 19.0) was utilized for statistical analysis. Differences were compared using a two-tailed Student t test. Differences with a P value < 0.05 were considered as statistically significant.
Discussion
In this study, we demonstrated that NDRG1 could inhibit tumor growth through increasing p21 protein expression. Our results indicated that NDRG1 overexpression stabilized p21 by decreasing its ubiquitylation, whereas NDRG1 silencing inhibited p21 expression by increasing ubiquitylation. NEDD4 was identified as a potential E3 ligase, which could target p21 for degradation. NDRG1 could emulatively antagonize NEDD4-mediated ubiquitylation of p21.
NDRG1, also known as Cap43, RTP, RIT42, and Drg-1 [
25], normally is present in cells originating from human epithelial tissues and plays an important role in keratinocyte differentiation, attenuating hypoxic injury, myelin sheath maintenance, and cell cycle regulation [
36‐
39]. Since the first time NDRG1 was recognized as metastasis suppressor in colon cancer [
40], accumulating evidences have demonstrated that NDRG1 was down-regulated in breast, prostate, pancreatic, and colorectal cancers [
14‐
16,
18], showing anti-tumor and anti-metastatic functions of NDRG1. However, its function in tumor development and progression still remains controversial, even in the same cancerous disease, such as hepatocellular carcinoma (HCC). Some studies reported that NDRG1 could promote HCC tumor growth, metastasis, and malignancy [
41‐
43], while others showed that NDRG1 expression was lower in HCC tumor tissues than in normal tissues and inhibited tumor growth [
25,
44]. In our previous studies, we have elaborately revealed the possible mechanisms of and related pathways of NDRG1 in inhibition of tumor metastasis [
10,
17,
21‐
23]. Recent studies also showed that NDRG1 could inhibit proliferation of gastric cancer, oral squamous cell carcinoma (OSCC) and hepatic tumors and it was associated with good patient survival [
13,
25,
45].
In agreement with previous findings, herein, we proved that NDRG1 could suppress the proliferation of CRC cell lines in vivo and in vitro. By analyzing the NDRG1 expression of 89 paired CRC samples using IHC, we showed that NDRG1 expression decreased in tumor tissues. There was a higher proportion of negative NDRG1 expression when tumor diameter > 3 cm. Tumor volume is correlated with proliferative abilities of tumor, suggesting that NDRG1 may be capable of modulation of the proliferative abilities of tumor. Moreover, xenograft models proved that NDRG1 overexpression significantly suppressed tumor growth, while NDRG1 knockdown increased growth in vivo. Ki-67 is commonly used as a proliferation marker, which is absent in G0 phase and is expressed throughout in G1, S, and G2/M phase of cell cycle [
25]. The IHC analysis of xenografts showed that positive Ki-67 was more present in the NDRG1 knockdown group, suggesting that more tumor cells tended to be in the G0 (or G0/G1) phase, while Ki-67 expression was much lower in the NDRG1 overexpression group, indicating that NDRG1 was closely related to tumor proliferation.
p21 is an universal cell-cycle inhibitor, controlled by p53 and/or p53-independent pathways, closely related to tumor proliferation by inducing G0/G1 arrest [
46]. In this study, NDRG1 promoted the expression of p21 protein in both HCT116 (p53-wt) and SW1116 (p53-mutant) cell lines, suggesting NDRG1 might regulated p21 in a p53-independent manner. Besides, xenograft model analysis also confirmed their positive relationship in vivo. In cell cycle analysis, consistent with findings of previous studies, NDRG1 overexpression induced G0/G1 arrest. p21 is the key regulator of G0/G1 arrest. This phenomenon was then restored through inhibiting p21 expression by si-RNA, and a similar “rescue” phenomenon was also observed in other proliferation-related assays such as CCK-8 and colony formation assays. Hence, NDRG1 may play a key role in inhibiting proliferation through up-regulating p21 expression.
There have been handful studies reported that NDRG1 can increase p21 protein expression in other diseases [
25,
47]. However, the underlying mechanisms are still obscure. Given that p21 is not stable in vivo, posttranslational modification (PTM) is a key regulatory mechanism for maintaining its steady-state level [
48]. Ubiquitylation is a critical PTM, which plays a significant role in all aspects of cell physiology and pathology, and the function of p21 is closely regulated by ubiquitylation [
49]. Ubiquitylation by several E3 ligases is the major regulatory mechanism of p21 protein [
50]. There are mainly three E3 ubiquitin ligase complexes responsible for ubiquitin-mediated degradation of p21: CRL4
CDT2, SCF
SKP2, and APC/C
CDC20. CRL4
CDT2 [
51] specifically induces p21 ubiquitylation and degradation at the S phase of cell cycle, while SCF
SKP2 [
48] promotes p21 degradation in both G1/S transition and S phase. APC/C
CDC20 [
52] is reported to drive p21 degradation during mitosis. In addition, other E3 ligases such as FBXO22 [
53], CHIP [
54], SPSB1 [
55], and RNF126 [
56] can target p21 for ubiquitin-mediated degradation. This study showed that NDRG1 overexpression could suppress p21 ubiquitylation, while NDRG1 knockdown increased ubiquitylation of p21. After screening in UbiBrowser Databas, we matched 20 overlapped E3 ligases that may simultaneously interact with both NDRG1 and p21, among which NEDD4 was selected. NEDD4, also known as neural precursor cell expressed developmentally downregulated protein 4, has been reported as E3 ligase for many substrates, including EGFR, PTEN, and ERBB4, and it was involved in tumor and other diseases [
57‐
59]. Not surprisingly, co-IP results showed that p21 could interact with NEDD4. p21 expression was up-regulated after si-NEDD4, and NEDD4 overexpression could increase the ubiquitylation of p21, indicating that p21 might be the substrate of NEDD4. Verma noted that NDRG1 could interfere with the combination between NEDD4 and its substrate in breast cancer [
35]. Further experiments showed that NDRG1 overexpression could inhibit combination between NEDD4 and p21, therefore, suppressing p21 ubiquitylation and increasing its protein expression. Consistent with another study [
47], our results indicated that the nuclear expression of p21 was significantly increased after NDRG1 overexpression, while NDRG1 knockdown decreased its nuclear expression, i.e., p21 translocation, by which NEDD4 was likely to interact with more cytoplasmic p21 and then induced p21 degradation.
As ubiquitylation is a complicated cascade, it’s obvious that NDRG1/NEDD4 cannot be the only factor to regulate p21 stability and ubiquitylation. In addition to E3 ligases we mentioned above, deubiquitylation by deubiquitylases is also indispensable for dynamic balance of p21 between stabilization and degradation. The study about deubiquitylation has been an emerging research field recently. Deng [
50] reported that USP11 could directly remove p21 polyubiquitylation and protect p21 from ubiquitin-mediated degradation. Cables1 could stabilize p21 by inhibiting PSMA3-mediated proteasomal degradation [
60]. Further studies are needed to clarify whether NDRG1 can regulate the functions of other E3 ligases and deubiquitylases. In this study, we showed that p21 could be the substrate of NEDD4 in CRC cell lines, added that NDRG1 was involved in the complex network regulating the p21 expression.
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