NUP153 overexpression suppresses the proliferation of colorectal cancer by negatively regulating Wnt/ β -catenin signaling pathway and predicts good prognosis

Abstract

BACKGROUND

: Nucleoporin NUP153 (NUP153) is well known to be involved in the regulating of nuclear transport. Although NUP153 is associated with several cancers, its role in colorectal cancer (CRC) and the underlying mechanism are still unknown.

OBJECTIVE:

The aim of this study was to access the effect of NUP153 on the prognosis of patients with CRC, and cancer cell proliferation.

METHODS:

The expression levels of NUP153 in CRC tissues and matched normal colon tissues were examined by real-time quantitative PCR and immunohistochemistry. Then the association between NUP153 levels with clinical variables as well as survival time was investigated. Moreover, overexpression of NUP153 in HCT116 cells was established to study its influence on cell proliferation in vitro, and a xenograft model was performed to explore this effect in vivo.

RESULTS:

We found that NUP153 was highly expressed in adjacent normal tissues than in cancer tissues, and elevated NUP153 expression was negatively associated with pathological grade ( $P=$ 0.015), T stage ( $P=$ 0.048) and distant metastasis ( $P=$ 0.006). Kaplan-Meier analysis revealed that patients with higher NUP153 expression had a longer overall survival (OS) ( $P=$ 0.01) and recurrence free disease (RFS) ( $P=$ 0.001). Logistic regression analysis further identified NUP153 as an independent prognostic safe factor for OS and recurrence. Moreover, NUP153 overexpression suppressed CRC cells proliferation and inhibited tumor growth in a xenograft model. Its mechanistic investigations showed that NUP153 overexpression inhibited $\beta$ -catenin transcriptional activity and down-regulated the mRNA expression levels of Wnt downstream proteins-Axin2, cyclinD1, c-myc and lef-1.

CONCLUSIONS:

NUP153 might be a promising prognostic factor, a potential tumor suppressor and therapeutic target in human CRC through an interaction with the Wnt/ $\beta$ -catenin signaling pathway.

Keywords

NUP153 colorectal cancer suppressor gene prognosis proliferation Wnt/β-catenin pathway

1. Introduction

Colorectal cancer (CRC) is one of the most common gastrointestinal cancer worldwide, with 37,000 newly diagnosed cases and 19,000 estimated deaths in China in 2015 [1, 2]. Although new methods for CRC diagnosis and treatment have continuously emerged, the survival rate of patients in advanced CRC remains low [3, 4]. Many tumor related gene mutations are identified to associate with prognosis of patients with CRC. Thus, understanding the role of regulatory genes and its related pathway in CRC is essential to optimizing current therapeutic strategies.

Successive acquisition of genetic alterations in Wnt pathway are the driving force for several diseases, especially for CRC [5, 6]. Several key molecular gene mutations in this pathway causing CRC formation have been identified [7]. Namely that, adenomatous polyposis coli (APC) mutations occurs in 90% of spontaneous colorectal cancer [8]. Activating Axin2 mutations occurs in some cases of CRC patients. Therefore, Wnt/ $\beta$ -catenin signaling pathway is recognized to be the most related pathway to CRC development, and modification for mutation genes in this pathway is a research hot for CRC therapy strategy. Recently, our work found that silencing NUP153 could up-regulate the conduction of the canonical Wnt pathway in a RNA interference library [9]. Another two papers directly showed that NUP153 overexpression reduced the expression levels of the downstream proteins like Axin2, Lef1 in the Wnt pathway [10, 11]. Based on these findings, therefore, we speculated that NUP153 might be a regulatory gene of importance in the Wnt pathway influencing CRC progression.

NUP153 is an essential part of the structure of mammalian nuclear pore complexes (NPCs) and is a key part of NPCs anchoring on the surface of the nuclear membrane and plays an important role in nuclear transport [12, 13]. More studies about NUP153 are focusing on embryo development, HIV and chromosomal translocation [14, 15, 16]. For example, Toda et al. reported that NUP153 interacted with sox2 to enable bimodal gene regulation and maintenance of neural progenitor cells, which could control cell fate [17]. Nanni et al. indicated that NUP153 was an epigenetic regulator, associating with chromatin and regulating cardiac gene expression in dystrophic mdx hearts [18]. Furthermore, NUP153 is reported to be associated with several cancers. In human breast carcinoma, silencing NUP153 in MDA231 cell line significantly reduced the speed of directional cell migration [19]. Chromosomal rearrangements of NUP153 were related with its increased expression in urothelial and retinoblastoma cancer [20]. Shain et al. revealed a possible oncogenic functions of NUP153 by regulating TCF $\beta$ signaling in pancreatic cancer [21]. However, there is no information about the effects of NUP153 on CRC.

Here we explored the role of NUP153 in colorectal cancer. First we found that NUP153 highly expressed in adjacent normal tissues compared with the cancer tissues, and patients with higher NUP153 expression had a good prognosis. Also, our work uncovered that NUP153 suppressed the proliferation of CRC and inactivated the Wnt/ $\beta$ -catenin signaling pathway. This study demonstrated that NUP153 might be a favorable biomarker in CRC and inhibit CRC progression via inhibiting Wnt pathway.

2. Materials and methods

2.1 Human samples

With the approval of the Ethnic Committee of Fifth Hospital of Shanghai, Fudan University. A total of 18 primary CRC (I–II: 11, III–IV: 7; T1–2: 9, T3–4: 9; Tumor diameter: 5.00 $\pm$ 2.15 mm) and adjacent normal tissues were extracted their mRNA. The human colorectal cancer tissue microarrays (HColA180Su15) used in this study contain 101 CRC specimens and 79 corresponding non-cancerous tissues were purchased from Shanghai Outdo Biotech Co., LTD. (Shanghai, China).

2.2 Cell culture and materials

Plasmids of NUP153, Topflash vector, p-cDNA3.1, renilla luciferase pRL-TK reporter vector were got from East China Normal U niversity. Two hundred ninety three T and HCT116 cells were maintained in DMEM with 10% fetal bovine serum (Gibco) in 37 ${}^{\circ}$ C, 5% CO ${}_{2}$ incubator.

2.3 RNA extraction and qRT-PCR

Total cellular or tissue RNA was extracted from the different groups using trizol reagent (Invitrogen, MA, USA), the cDNAs were amplified by PCR using Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) in the Applied Bio-systems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The thermal cycler protocols included 3 min at 95 ${}^{\circ}$ C and 40 cycles of 5 s at 95 ${}^{\circ}$ C and 30 s at 60 ${}^{\circ}$ C. The primers sequences of those proteins (NUP153, Axin2, c-myc, cylinD1 and Lef-1) were listed in Table 1. GAPDH was used as an internal control.

Table 1
Human primers set for qRT-PCR analysis

Gene	Direction	Primer sequence
GAPDH	Forward	TCAAGAGGCGAACACACAAC
	Reverse	GGCCTTTTCATTGTTTTCAA
NUP153	Forward	GGCCTCAGCGGACATCCTC
	Reverse	CCTCGTTGGCCAAAGAAAAG
C-MYC	Forward	TCAAGAGGCGAACACACAAC
	Reverse	GGCCTTTTCATTGTTTTCAA
Axin2	Forward	TACACTCCTTATTGGGCGATCA
	Reverse	TTGGCTAATCGTAAAGTTTTGGT
Cycind1	Forward	GTGGCCTCTAAGATGAAGGAGA
	Reverse	GGAAGTGTTCAATGAAACGTGT
Lef-1	Forward	TGCCAAATATGAATAACGACCCA
	Reverse	GAGAAAAGTGCTCGTCACTGT

2.4 Immunohistochemistry

Immunohistochemistry for NUP153 expression in CRC and adjacent non-tumor samples was performed using standard methods. Colorectal cancer tissue microarray (TMA) was deparaffinized by xylene, followed by rehydration through a graded series of ethanol, and then incubated with blocking solution for 30 min at room temperature. Next, tissue sections were successively incubated with rabbit anti-NUP153 diluted 1:100 (ab96462; abcam; US) overnight at 4 ${}^{\circ}$ C and the secondary anti-rabbit IgG antibody (1:500; abcam; US) at room temperature for 1 h.

2.5 Western blots

Cell lysis buffer was used to extracted proteins. Subsequently, those proteins were boiled with SDS sample buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred to PVDF membranes (Millipore, USA). After transmembrane, membranes contained proteins were respectively incubated with anti-NUP153 antibody (1:500; ab96462; abcam; US) overnight at 4 ${}^{\circ}$ C and the second antibody (Cell Signaling Technology) at 25 ${}^{\circ}$ C for 2 h. Finally, the proteins was detected.

2.6 MTT assay

HCT116-NUP153 cells were seeded into 96-well plates at a density of 4,000 cells per well and cultured for 24 h at 37 ${}^{\circ}$ C in a CO ${}_{2}$ incubator. After 12 h, 24 h, 48 h and 72 h, 50 uL of 10 mg/mL 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were added to each well. Then the plates were incubated for another 2 h. The supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in 500 $\mu$ l of DMSO. The absorbance was measured by a microplate reader at a wavelength of 570 nm. The experiments were repeated independently for three times.

2.7 Clone formation assay

HCT116-NUP153 cells were seeded into 6-well plates at a density of 1,000 cells per well and cultured for 8 days at 37 ${}^{\circ}$ C. When the clones were formed, the culture was terminated and the cells were carefully washed with phosphate buffered saline twice and stained with 0.1% crystal violet solution for 30 min. The number of colonies were counted directly by a microscope or by the naked eye. Plate clone formation efficiency $=$ (number of colonies/number of cells inoculated) $\times$ 100%. The experiments were repeated independently for three times.

2.8 Xenograft experiment

Nude mice aged 5 weeks were purchased from the Experimental Animal Center of East China Normal University. To demonstrate the effect of NUP153 on inhibit tumor growth in vivo, HCT116-NUP153 cells were implanted into nude mice at 2 $\times$ 10 ${}^{6}$ cells in 100 $\mu$ l per spot. Six days after injection, the tumor volumes were continuously measured every 4 days until 30 days after injection by the formula: 0.5 $\times$ length $\times$ width ${}^{2}$ . All procedures involving animals in this study were approved by the East China Normal University Institutional Animal Care and Use Committee.

2.9 Luciferase reporter assay

HCT116-NUP153 cells were plated at a density of 4 $\times$ 10 ${}^{4}$ cells/well in 24-well plates, and transfected using lipofectamine 2000 (Invitrogen, Inc., MA, USA) according to the manufacturer’s protocol. For the measurement of TCF/ $\beta$ -catenin activity, topflash vector (375 ng per well) and renilla luciferase reporter vector (37.5 ng per well) were co-transfected into HCT116 cells. After 48 h, wnt3a-conditional media was used to activate the Wnt pathway lasting for 24 h. TCF/ $\beta$ -catenin activity was measured using the dual luciferase reporter system and normalized to renilla luciferase activity.

Figure 1.

The expression levels of NUP153 in colorectal cancer (CRC). (A) qRT-PCR analysis showed that the expression levels of NUP153 mRNA highly expressed in adjacent normal tissues compared with cancer tissues. (B) IHC analysis for the expression pattern of NUP153 protein in CRC. Strong expression in normal colorectal mucosa. The staining of NUP153 protein was mainly located in the membrane of cells. Scale bars $=$ 200 $\mu$ m. (C) The semi-quantitative analysis of NUP153 immunohisochemical staining in human normal and cancer tissues. Abbreviations: Cancer, colorectal cancer tissues; Normal, adjacent normal colon tissues; qRT-PCR, quantitative real-time polymerase chain reaction; IHC, immunohistochemistry.

2.10 Statistical analysis

All statistical analyses were performed using SPSS17.0 (Chicago, IL, USA). Graphpad Prism 5.0 (La Jolla, CA, USA) was used for graphs. Student t test was used to compare the expression levels of NUP153 in cancer tissues and matched normal non-cancer tissues. Chi-square test was used to analyze the correlation between NUP153 expression and clinicopatholigical features. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Logistic regression analysis was performed to assess the effects of NUP153 on patients’ OS and tumor recurrence.

3. Results

3.1 Expression of NUP153 in colorectal cancer samples

To explore the expression pattern of NUP153 in cancer tissues. We first detected the expression levels of NUP153 mRNA in 18 pairs of CRC tumor tissue with matched normal tissue. The result showed that NUP153 was highly expressed in normal tissues than cancer tissues (Fig. 1A). Furthermore, we purchased a colorectal cancer tissue microarray (TMA) with complete pathological information. Beside 101 cases of cancer tissues, 79 matched normal tissues spots were found on this TMA. As showed in colorectal adenocarcinoma cells, NUP153 was found to be stained mainly in the membrane of cells (Fig. 1B). Meanwhile, two pathologists with no knowledge of the clinical characteristics of the patients were employed to score staining intensity (0–4) and extent (0–100) of NUP153. As showed in Fig. 1C, NUP153 expression was markedly reduced in the cancer tissues compared with the normal tissues. 69.3% colorectal cancer tissues showed negative staining for NUP153, while 84.8% normal tissues showed obvious positive staining.

Table 2
Association between NUP153 and clinicopatholigical characteristics in TMA cohort

Expression	NUP153		$p$ -value
	High	Low
n (%)	31 (30.7%)	70 (69.3%)
Gender
Male	17 (54.8%)	33 (47.1%)	0.476
Female	14 (45.2%)	37 (52.9%)
Age (year)
$\leqslant$ 65	13 (41.9%)	31 (44.3%)	0.826
$>$ 65	18 (58.0%)	39 (55.7%)
Pathological grade
II	21 (67.7%)	29 (41.4%)	0.015*
III	10 (32.3%)	41 (58.6%)
Tumor size (cm)
$\leqslant$ 5	16 (51.6%)	39 (55.7%)	0.703
$>$ 5	15 (48.4%)	31 (44.3%)
T stage
T1–2	19 (61.3%)	28 (40.0%)	0.048*
T3–4	12 (38.7%)	42 (60.0%)
Tumor location
Right colon	7 (22.6%)	17 (24.3%)	0.981
Transverse colon	11 (35.5%)	28 (40.0%)
Left colon	13 (41.9%)	35 (50%)
Tumor classification
Mass type	11 (35.5%)	22 (31.4%)	0.159
Ulcerative type	15 (48.4%)	24 (34.3%)
Infiltration type	5 (16.1%)	24 (34.3%)
Lymph node status
Negative	12 (38.7%)	36 (51.4%)	0.238
Positive	19 (61.3%)	34 (48.6%)
Metastasis
Yes	22 (71.0%)	29 (41.4%)	0.006*
No	9 (29.0%)	41 (58.6%)

*P-values $<$ 0.05 were considered statistically significant. The Pearson chi-square test was used for between-group comparisons.

3.2 Correlation between NUP153 expression and clinicopatholigical features in TMA cohort

According to the information of IHC, we further investigated the association between NUP153 expression and clinicopatholigical features. As show in Table 2, higher NUP153 expression was positively associated with better tumor outcomes: pathological grade ( $P=$ 0.015), T stage ( $P=$ 0.048), distant metastasis ( $P=$ 0.006). However, no significant associations were found between NUP153 expression and age, gender, tumor size, tumor location, tumor classification, lymph node status ( $P>$ 0.05).

3.3 Correlation between NUP153 expression with OS and RFS

In order to explore whether patients with higher NUP153 expression could prolong their survival time, we then analyzed the association between their survival information and NUP153 expression. As showed in Fig. 2A, patients with higher NUP153 expression was found to have a favorable 9-years OS (log-rank $P<$ 0.05). A Cox proportional hazard model also revealed that a low expression of NUP153 was found to increase the hazard ratio (HR) for death in colorectal cancer patients (HR 1.517; 95% CI, 1.297–2.012, $P=$ 0.01). Meanwhile, patients with higher NUP153 expression also had a favorable RFS ( $P<$ 0.001) (Fig. 2B).

Figure 2.

Kaplan-Meier curves for OS rates between CRC patients with high and low NUP153 expression. (A and B) Survival analysis showed that patients with higher NUP153 expression was significantly had a longer OS and RFS ( $P=$ 0.01, $P=$ 0.001, respectively). Abbreviations: HR, hazard ratio; CI, confidence interval.

3.4 NUP153 functioned as an independent safe prognostic indicator

The influence of NUP153 expression on OS and recurrence were then analyzed based on TMA data. Univariate logistic regression analysis showed that higher NUP153 expression ( $P=$ 0.020), tumor clinical stage ( $P=$ 0.032), T stage ( $P=$ 0.023), histological differentiation ( $P=$ 0.009) were positively correlated with OS. While, vascular invasion ( $P=$ 0.042), distant metastasis ( $P=$ 0.017) were negatively correlated with OS. Multivariate logistic analysis further revealed that higher NUP153 expression ( $P=$ 0.041), early T stage ( $P=$ 0.036) and better histological differentiation ( $P=$ 0.013) of tumor had a favorable effects on OS (Table 3). With regard to recurrence, NUP153 expression was also uncovered to be an independent safe prognostic factor (Table 4). In summary, NUP153 overexpression could improve the prognosis in OS and recurrence.

3.5 Overexpression of NUP153 inhibited the proliferation of CRC

In order to determine the function of NUP153 in CRC cells, we overexpressed NUP153 in HCT116 human colon adenocarcinoma cell line. Protein levels were markedly increased after transfection of NUP153 plasmid (Fig. 3A). The MTT experiment showed that cell growth was markedly reduced in HCT116 cells in responsive to NUP153 overexpression (Fig. 3B). We further used soft agar colony formation assay to compare cell grow of HCT116 cells with or without NUP153 plasmid (Fig. 3C and D). Consistent with MTT results, overexpression of NUP153 significantly reduced cell proliferation compared with the control, indicating a tumor suppressor potential for NUP153. In xenograft experiment, compared with control group, the results showed that the tumor weights and volumes of node mice injected with HCT-NUP153 cells were significantly smaller (Fig. 3E–G).

Table 3
Univariate and multivariate analyses for over survival (OS) in TMA cohort

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Age
$\leqslant$ 65	1
$>$ 65	1.307	0.982–1.716	0.071
Gender
Male	1
Female	0.872	0.713–1.018	0.279
Clinical stage
1–2	1			1
3–4	1.735	1.302–2.324	0.032*	1.413	0.994–1.877	0.092
T stage
T1–2	1			1
T3–4	1.862	1.347–2.624	0.023*	1.674	1.143–2.325	0.036*
Histological differentiation
Well	1			1
Poor	2.141	1.537–2.861	0.009*	2.107	1.481–2.674	0.013*
Vascular invasion
Absent	1			1
Present	1.612	1.071–2.241	0.042*	1.018	0.942–1.224	0.134
Distant metastasis
Absent	1			1
Present	1.912	1.519–2.662	0.017*	1.571	1.079–2.247	0.045*
NUP153 expression
High	1			1
Low	1.883	1.452–2.731	0.020*	1.614	1.083–2.398	0.041*

Table 4

Univariate and multivariate analyses for recurrence in TMA cohort

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Clinical stage
1–2	1			1
3–4	1.893	1.452–2.478	0.027*	1.719	1.273–2.348	0.037*
T stage
T1–2	1			1
T3–4	1.013	0.878–1.761	0.119	1.127	0.924–1.679	0.194
Histological differentiation
Well	1			1
Poor	1.652	1.136–2.421	0.043*	1.134	0.972–1.651	0.101
NUP153 expression
High	1			1
Low	1.883	1.457–2.635	0.022*	1.712	1.293–2.442	0.038*

Figure 3.

NUP153 overexpression suppressed the proliferation of CRC. (A) Western blot blanket analysis of NUP153 protein blanket expression in HCT116 cells treated with or without NUP153 plasmid. (B) After 72 hours post transfection with or without NUP153, HCT116 cell viability was monitored by MTT assay at the indicated times. (C and D) Clone formation assay and statistical analysis for HCT116 cells transfected with or without NUP153, respectively. The experiments were repeated independently for three times. (E–G) Cells were subcutaneously injected into the flank of nude mice. Tumor volumes and weights were measured on the indicated days to evaluate the effects of NUP153 on tumor growth. Data points are showed as the mean $\pm$ SD tumor volume. ** $P<$ 0.01, *** $P<$ 0.001. Abbreviations: NUP153, group of HCT116-NUP153; NC, normal control; SD, standard deviation.

Figure 4.

NUP153 overexpression in HCT116 cells inhibited the Wnt/ $\beta$ -catenin signaling pathway in vitro. (A) Topflash activity showed that NUP153 overexpression inhibited the $\beta$ -catenin transcriptional activity in HCT116 cells. (B) Luciferase activity in fopflash remained unaffected, confirming a lack of nonspecific activation of the reporter system. (C) In immunofluorescence assay, the nucleus was stained blue, $\beta$ -catenin was stained red. The results showed that overexpression of NUP153 (Low) inhibited the amount of $\beta$ -catenin transported into the nucleus less than the control group (Upper). (D) qRT-PCR assay showed that the mRNA expression levels of Axin2, CyclinD1, C-MYC, Lef-1 were significantly down-regulated in responsive to NUP153 overexpression. Abbreviations: wnt3a, wnt3a condition medium; NUP153, group of HCT116-NUP153.

3.6 NUP153 overexpression inactivated the Wnt/

\beta

-catenin signaling pathway

According to the previous studies, we had found that NUP153 could inactivate the Wnt/ $\beta$ -catenin signaling pathway through large sample data screening. To confirm this effect of NUP153 on the Wnt pathway with experimental evidence, we first used topflash reporter assay to explore the influence of NUP153 on regulating the transcriptional activity of $\beta$ -catenin. The result showed that overexpression of NUP153 in HCT116 cells significantly reduced this activity (Fig. 4A). While Luciferase activity in fopflash remained unaffected, confirming a lack of nonspecific activation of the reporter system (Fig. 4B). And immunofluorescence assay also confirmed the above result (Fig. 4C). Meanwhile, to further investigate whether NUP153 overexpression could down-regulate the expression levels of the downstream proteins of the Wnt pathway. QRT-PCR analysis was used to test those proteins’ relative mRNA expression levels. Our data showed that the mRNA expression of Axin2, cyclinD1, c-myc, Lef-1 were markedly reduced in NUP153 overexpression group than the control group (Fig. 4D). Thus, NUP153 overexpression inhibited the classical Wnt/ $\beta$ -catenin signaling pathway in vitro.

4. Discussion

Colorectal cancer (CRC) develops through a series of genetic and epigenetic changes that cause the transformation of normal mucosa to a premalignant polyp, and finally to a cancer [22, 23, 24]. These regulatory genes are found to be closely associated with prognosis of patients, and also provide strong evidences for disease diagnosis and treatment [25, 26]. Some studies have reported that the role of the human NUP153 gene accounts for different type of cancers. However, the role for NUP153 in CRC tumorigenesis remains unknown.

In this study, our work first discovered that NUP153 was significantly down-regulated in colorectal cancer tissues, which suggested that NUP153 might be a potential suppressor gene in human CRC. Currently, cancer recurrence and metastasis remain to be the leading death cause for CRC patients. As reported, the 5-years survival time of patients with early tumor stage was up to 90%, while only 14% in those with distant metastasis [27]. According to our study, the results showed that higher NUP153 expression was positively related with some better tumor outcomes: pathological grade, T stage and distant metastasis. These findings supported that NUP153 might work as favorable biomarker in screening out those with risk factors. In order to demonstrate to effects of NUP153 on survival time, we followed up all of the patients for 9 years, and found out that patients with lower NUP153 expression had a risk in OS and RFS. Next, logistic regression analysis demonstrated that higher NUP153 expression increased the favorable prognosis of patients with CRC in OS and recurrence, which identified NUP153 as an independent safe prognostic indicator. As a result, we have revealed the clinical significance of NUP153 in CRC formation, which might function as a useful therapeutic target for CRC in the future. Just as KRAS mutation status, which is a strong predictive marker of resistance to EGFR-targeted therapy in patients with metastatic colorectal cancer [26, 28, 29].

NUP153 was reported to associate with cancer cells invasion, proliferation, and metastasis [30]. Both estrogens and NO cooperate and contribute to aggressiveness in prostate cancer through Nup153 upregulation [31]. Our work showed that NUP153 overexpression markedly suppressed colorectal cancer cell proliferation, which uncovered that NUP153 could inhibit the progression of CRC formation. Consistent with the result, our xenograft model experiment also confirmed that NUP153 could inhibit tumor growth in vivo. Thus, these findings support that NUP153 might be a potential biomarker and a therapeutic target of CRC through its interaction with various signaling pathway involved in this disease.

Wnt/ $\beta$ -catenin signaling pathway has been identified as the most relevant target-based approach for CRC treatment [32]. However, it is still unclear if this signaling pathway is related with NUP153 to inhibit colorectal cancer formation. Through RNAi library, three papers respectively mentioned that NUP153 overexpression down-regulated Wnt/ $\beta$ -catenin signaling pathway [9, 10, 11]. However, the conclusion was limited with further experimental evidences. As a result, we supposed that the Wnt pathway might be a one of important approaches for NUP153 to inhibit CRC formation. Our data showed that NUP153 markedly suppressed the transcriptional activity of $\beta$ -catenin in HCT116 cells. Moreover, qRT-PCR result further demonstrated that NU153 overexpressed in HCT116 cell could down-regulate the expression levels of Axin2, cyclinD1, c-myc and lef-1 in Wnt pathway. These findings indicate that Wnt/ $\beta$ -catenin signaling pathway may be one of the important effectors of NUP153 downstream.

NUP153 expression was generally down-regulated in CRC, and patients with higher NUP153 expression was correlated with a longer OS and RFS. Meanwhile, NUP153 was identified as an independent safe prognostic factor in OS and tumor recurrence. Furthermore, overexpression of NUP153 was found to inhibit the proliferation of CRC via inactivating Wnt/ $\beta$ -catenin signaling pathway. We believed that NUP153 is likely to be an essential factor in protecting against human CRC formation, whose effects are mediated by Wnt/ $\beta$ -catenin signaling pathway.

Footnotes

Acknowledgments

This study was funded by Science and Technology Commission of Shanghai Municipally (No. 17411967 600), Science and Technology Commission of Minhang Municipally (No. 2015MW07).

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NUP153 overexpression suppresses the proliferation of colorectal cancer by negatively regulating Wnt/ β -catenin signaling pathway and predicts good prognosis

Abstract

BACKGROUND

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Human samples

2.2 Cell culture and materials

2.3 RNA extraction and qRT-PCR

Table 1 Human primers set for qRT-PCR analysis

2.5 Western blots

2.6 MTT assay

2.7 Clone formation assay

2.8 Xenograft experiment

2.9 Luciferase reporter assay

3. Results

3.1 Expression of NUP153 in colorectal cancer samples

Table 2 Association between NUP153 and clinicopatholigical characteristics in TMA cohort

3.3 Correlation between NUP153 expression with OS and RFS

3.5 Overexpression of NUP153 inhibited the proliferation of CRC

Table 3 Univariate and multivariate analyses for over survival (OS) in TMA cohort

4. Discussion

Footnotes

Acknowledgments

References

Table 1
Human primers set for qRT-PCR analysis

Table 2
Association between NUP153 and clinicopatholigical characteristics in TMA cohort

Table 3
Univariate and multivariate analyses for over survival (OS) in TMA cohort