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Author Manuscript Published OnlineFirst on March 9, 2017; DOI: 10.1158/0008-5472.CAN-16-2339 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Title: Pancreatic cancer progression relies upon mutant p53-induced

oncogenic signaling mediated by NOP14

Yongxing Du1,§, Ziwen Liu1, §, Lei You1, Pengjiao Hou2, Xiaoxia Ren1, Tao Jiao2, Wenjing

Zhao1, Zongze Li1, Hong Shu1, Changzheng Liu2,*, Yupei Zhao1,*

1 Department of General Surgery, Peking Union Medical College Hospital,

Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing

100730, PR China

2 Department of Biochemistry and Molecular Biology, State Key Laboratory of

Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese

Academy of Medical Sciences，School of Basic Medicine, Peking Union

Medical College, Beijing 100005, PR China

§ Authors share co-first authorship.

* To whom correspondence should be addressed: Yupei Zhao, E-mail:

[email protected], Fax: 86-10-65124875; Changzheng Liu, E-mail:

[email protected], Fax: 86-10-65253005.

Running Title: NOP14 primes mutp53-driven cancer progression

Key words: Pancreatic ductal adenocarcinoma, Mutant p53, NOP14, Cancer

metastasis

Abbreviations:

Mutp53, Mutant p53; PDAC, Pancreatic ductal adenocarcinoma; NOP14,

NOP14 nucleolar protein; rRNA, ribosomal RNA; PDGFRb, Platelet-derived

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 9, 2017; DOI: 10.1158/0008-5472.CAN-16-2339 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

growth factor receptor b; MAPK3, Mitogen-activated protein kinase 3; CDK2,

Cyclin dependent kinase 2; MMP9, Matrix metallopeptidase 9; RhoA, Ras

homolog family member A; p53, Tumor protein p53; P21, Cyclin dependent

kinase inhibitor 1A; H&E, hematoxylin-eosin; ATCC, the American Type

Culture Collection; DMEM, Dulbecco’s modified Eagle medium; cDNA,

complementary DNA; messenger RNA; siRNA, small interfering RNA; UTR,

untranslated region; qRT-PCR, quantitative reverse-transcription polymerase

chain reaction; Scr, scramble; miRNA, microRNA; MYO10, myosin X; SLUG,

Snail family transcriptional repressor 2; TGFB1, Transforming growth factor

beta 1; ZEB1, zinc finger E-box binding homeobox 1; EGFR, Epidermal growth

factor receptor; ITGB1, Integrin subunit beta 1; SMAD2, SMAD family member

2; TWIST1, Twist family bHLH transcription factor 1; ZNF652, Zinc finger

protein 652; CCNG2, Cyclin G2; SHARP1, Basic helix-loop-helix family

member e41; TP63, Tumor protein p63; PERP, PERP TP53 apoptosis effector ;

DAB2IP, DAB2 interacting protein; S100A4, S100 calcium binding protein A4;

PIM1, Pim-1 proto-oncogene, serine/threonine kinase; E2F1, E2F transcription

factor 1; TP53I3, Tumor protein p53 inducible protein 3; c-Myc, v-myc avian

myelocytomatosis viral oncogene homolog; BCL2, BCL2 apoptosis regulator;

NIH, National Institutes of Health; PBS, Phosphate Buffered Saline; SCID mice,

Severe combined immunodeficient mice; HRP, Horseradish peroxidase; ActD,

Actinomycin D; si-Con, si-control; RNA-seq, whole transcriptome sequencing;

miRNA-seq, microRNA sequencing; pri-miRNA, primary microRNA;

pre-miRNA, microRNA precursor; IHC, Immunohistochemistry; OS, overall

survival.

Financial Support: This research was supported by the National Nature

Science Foundation of China (2013, 81272767, to Z. W. Liu; 2015, 81572459,

to Z. W. Liu; 2015, 81570780, to C. Z. Liu; and 2016, 81672443 to Y. P. Zhao).

Corresponding authors: Yupei Zhao, Department of General Surgery,

Peking Union Medical College Hospital, Chinese Academy of Medical

Sciences, Peking Union Medical College, Beijing 100730, PR China. Phone:

86-10-69156007, Fax: 86-10-65124875, E-mail: [email protected].

Changzheng Liu, Department of Biochemistry and Molecular Biology, State

Key Laboratory of Medical Molecular Biology, Institute of Basic Medical

Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine,

Peking Union Medical College, Beijing 100005, PR China. Phone:

86-10-69156424, Fax: 86-10-65253005, E-mail: [email protected]

Disclosure of Potential Conflicts of Interest:

No potential conflicts of interest were disclosed by the authors.

Word count for the abstract: 190

Word count for the text: 5000

Total Figures: 7

ABSTRACT

Mutant p53 (mutp53) proteins promote tumor invasion and metastasis in

pancreatic ductal adenocarcinoma (PDAC). However, the mechanism

underlying sustained activation of mutp53 oncogenic signaling is currently

unclear. In this study, we report that NOP14 nucleolar protein (NOP14)

expression is upregulated in PDAC tumors and metastatic tissue specimens.

NOP14 overexpression promoted cell motility, whereas NOP14 inhibition

decreased invasive capacity of PDAC cells. In vivo invasion assays conducted

on established subcutaneously, orthotopically, and intravenously injected

tumor mouse models also indicated NOP14 as a promoter of PDAC

metastasis. Mechanistically, mutp53 was validated as a functional target of

NOP14; NOP14 primed tumor invasion and metastasis by increasing the

stability of mutp53 mRNA. The NOP14/mutp53 axis suppressed p21

expression at both the transcriptional and post-transcriptional levels via

induction of microRNA-17-5p in PDAC cells. In vivo, high NOP14 expression in

PDAC patient tumors correlated with local metastasis and lymph invasion.

Overall, our findings define a novel mechanism for understanding the function

of NOP14 in the metastatic cascade of PDAC. Targeting NOP14 allows for

effective suppression of tumor invasion in a mutp53-dependent manner,

implicating NOP14 inhibition as a potential approach for attenuating

metastasis in p53 mutant tumors.

INTRODUCTION

The concept that gain-of-function of mutant p53 (mutp53) proteins led to

cancer progression was established over two decades ago, and a number of

hotspots have been identified in diverse cancers, including pancreatic ductal

adenocarcinoma (PDAC).(1) In human PDAC, p53 accumulation has been

correlated with lymph node metastasis, and knock-in Trp53 mutations result in

the acquisition of critical functions for overcoming growth arrest/senescence

and driving metastasis.(2) Another study has revealed that sustained mutp53

expression is required to maintain the prometastatic phenotype and that

platelet-derived growth factor receptor b (PDGFRb) is involved in

mutp53-driven PDAC metastasis.(3) These findings indicate that mutp53 is a

potential antimetastatic target for PDAC prevention. However, mutp53 proteins

have been proven to be undruggable to date.(4-5) Efforts have been focused

on searching for potential mediators of mutp53 activity in cancer progression,

and targeting of these related proteins might facilitate the suppression of

metastasis driven by mutp53.

NOP14 has been reported to be a nucleolar protein required for maturation of

18S rRNA and for 40S ribosome production.(6) Our preliminary data have

revealed that NOP14 causes increased PDAC cell growth and invasion and we

have also detected NOP14 mutations in primary and metastatic PDAC

tissues,(7-8) indicating a potential association between NOP14 and cancer

invasion. However, the correlation between dysregulated NOP14 and PDAC

progression remains unclear, and little else is known about the mechanisms

underlying the function of NOP14 in the metastasis of p53 mutant tumors.

Herein, we demonstrated that PDAC cells with increased NOP14 expression

possess an enhanced invasive capacity. We also showed that NOP14 drives

PDAC metastasis by stabilizing mutp53 mRNA, thereby affecting its functional

targets. Overall, our data define a mechanism of the NOP14/mutp53

regulatory axis in suppressing P21 expression at both the transcriptional and

post-transcriptional levels by induction of microRNA-17-5p (miR-17-5p) in

PDAC cells.

MATERIALS and METHODS

Clinical specimens and cell lines

Tissues were collected as previously described.(9) The patient characteristics

are provided in Table S1. MIA PaCa-2, Su.86.86, T3M4, PANC-1, SW1990,

BxPC-3, and AsPC-1 cell lines were obtained from the American Type Culture

Collection (ATCC) and grown in DMEM or RPMI1640 with 10% FBS (HyClone)

at 37ºC in 5% CO2 cell culture incubator. Cell lines were tested and

authenticated 1 month before the experiment according to microscopic

morphology, growth curve analysis, and mycoplasma detection according to

the ATCC cell line verification test recommendations.

Quantification of RNAs and proteins

Quantitative real-time PCR (qRT-PCR) analysis was performed using SYBR

Green PCR Master Mix with a Bio-Rad IQ5 qRT-PCR system. To determine

the miRNA levels, qRT-PCR was conducted using Taqman probes (Invitrogen,

Carlsbad, CA, USA) according to the manufacturer′s instructions. The primers

are shown in Table S2 and S3. Immunoblotting was performed as described

previously.(9) The antibodies used included those against NOP14, p53,

PDGFRb, EGFR, TGFB1, SMAD2, ZEB1, P21, and β-actin (p53, ZEB1, EGFR,

P21, and β-actin were purchased from CST; and NOP14, PDGFRb, SMAD2,

and TGFB1 were obtained from Abcam).

RNA sequencing and data analysis

Directional (stranded) libraries for the paired-end sequencing of PANC-1 cells

were generated with an Illumina platform. Differential expression analysis to

obtain sequence count data (fragments per kilobase of transcript per million

mapped reads [FPKM] values) was conducted using Cuffdiff.

Actinomycin D (ActD) treatment

PDAC cells were treated with 10 μg/ml ActD (Sigma-Aldrich) as described

previously,(10) and cells were collected various time intervals. The mutp53

mRNA and pri-miR-17-5p levels were determined by qRT-PCR.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed as previously described.(11) Briefly, protein A/G

Sepharose beads conjugated to anti-p53 antibody (Thermo Scientific) were

used to immunoprecipitate p53 form whole-cell lysates. Quantitative ChIP was

performed with an ABI StepOne Plus using SYBR green dye.

PDAC cell-engrafted tumor mouse models

All experimental procedures involving animals were performed according to

the institutional ethical guidelines for animal experiments of Peking Union

Medical College.

Immunohistochemistry (IHC)

IHC was conducted to measure NOP14 and P21 expression using NOP14

(Sigma-Aldrich) and P21 (CST) antibody as previously described.(9)

Statistics

Each experiment was repeated at least three times. Student's t test (two-tailed)

was performed and three-group data were analyzed using one-way analysis of

variance. All statistical analyses were performed using SPSS 16.0 software

(SPSS Inc., Chicago, IL, USA). P-values <0.05 were considered statistically

significant.

Additional details are provided in online supplementary materials and

methods

RESULTS

Increased NOP14 expression is correlated with enhanced PDAC

metastasis.

NOP14 mutations have been detected in the metastatic nodes of PDAC,

suggesting a potential role of NOP14 in PDAC invasion.(8) Here, we examined

the function of NOP14 in PDAC metastasis. Thus, IHC analysis was performed

to evaluate NOP14 expression in PDAC tissues from 20 patients, which

revealed that NOP14 expression was upregulated in the PDAC samples

compared with the matched normal controls (Fig.1A, B). Additionally, NOP14

overexpression was significantly increased in 7 metastases, including 4

hepatic, 2 peritoneum metastases, and 1 lymph node, as shown by IHC

analysis (Fig.1C, D; Table S4). These data indicated that NOP14 expression

was positively correlated with PDAC metastasis. To further confirm these

findings, NOP14 expression was measured in several PDAC cell lines as

shown in Fig.S1A. Further, the invasive capacity of these cells was evaluated

by in vitro invasion assays, which showed that PDAC cells with higher NOP14

levels exhibited increased invasiveness (Fig.S1B, C).

Altogether, these data demonstrate that increased NOP14 expression is

associated with enhanced PDAC invasion and metastasis.

NOP14 promotes PDAC cell migration and invasion.

Given the parallels between NOP14 and metastatic potential, we next

investigated the role of NOP14 in PDAC cell movement. Thus, we transfected

PANC-1 cells with a construct to achieve NOP14 overexpression (Fig.S2A, B).

Then, wound-healing assay was performed, which revealed that NOP14

overexpression led to increased cell migration compared with vector-treated

PANC-1 cells (Fig.1E). We next inhibited NOP14 expression in PANC-1 cells

using a specific siRNA (si-NOP14) (Fig.S2C, D) and performed wound-healing

assay, which revealed that NOP14 inhibition resulted in reduced wound

closure compared with si-Con-treated cells (Fig.1E). Similar results were

observed in MIA PaCa-2 cells (Fig.S3A; Fig.S4). In vitro transwell and invasion

assays were employed to further evaluate the impact of NOP14 on PDAC cell

movement, which showed that cell migration and invasion was increased by

NOP14 overexpression and suppressed by NOP14 inhibition in PANC-1 cells

(Fig.1F). Similar results were observed in MIA PaCa-2 cells (Fig.S3B).

Moreover, the invasive capacity of PDAC cells into collagen gels was

determined in inverted invasion assay. We found that NOP14 overexpression

enhanced cell invasion and NOP14 inhibition led to decreased invasiveness

both in PANC-1 and MIA PaCa-2 cells (Fig.1G).

Altogether, our data demonstrate that NOP14 overexpression increases PDAC

cell migration and invasion.

NOP14 inhibition deceases the invasion of PDAC cell-engrafted tumors.

We next examined the effect of NOP14 on tumor invasion using transplanted

tumor mouse models. To this aim, we inhibited NOP14 in PANC-1 cells

expressing increased levels of NOP14 and with strong invasiveness using a

Lentivirus system expressing siRNA (Lenti-shNOP14) (Fig.S5A, B). Next,

Lenti-shNOP14- or Lenti-shCon-infected PANC-1 cells were subcutaneously

inoculated into the posterior flanks of nude mice. 4 weeks after tumor

formation, we monitored the growth of transplanted tumors by

bioluminescence imaging (BLI) and observed that NOP14 inhibition

suppressed the growth of PANC-1-engrafted tumors (Fig. 2A). In agreement

with the tumor volumes, the weights of those that were treated with

Lenti-shNOP14 were significantly lower than the control tumors (Fig.2B).

Moreover, we performed H&E and NOP14 staining of randomly selected

mouse tumors and observed that NOP14 inhibition resulted in decreased

PDAC invasion to adjacent muscle tissues and downregulation of NOP14

expression (Fig.2C; Fig.S5C). Next, PANC-1 cells infected with

Lenti-shNOP14 or Lenti-shCon were orthotopically injected into the pancreas

of nude mice. NOP14 inhibition was found to significantly retard the growth of

transplanted PANC-1 cells (Fig.2D). Further, the Lenti-shCon-PANC-1 cells

exhibited extensive metastasis, as demonstrated by H&E staining in the

dissected tissue samples, whereas the Lenti-shNOP14-PANC-1 cells failed to

metastasize to the majority of the examined tissues (Fig.2E, F).

Altogether, these data indicate that NOP14 inhibition suppresses the invasion

of PANC-1-engrafted tumors.

NOP14 inhibition contributes to reduced metastasis of PDAC cells in

vivo.

To confirm the above findings, we further conducted in vivo invasion assays.

Thus, 5×105 viable PANC-1 cells infected with Lenti-shNOP14 or Lenti-shCon

were resuspended in 0.1 ml PBS and injected into the lateral tail veins of SCID

mice. Eight weeks after injection, the animals were sacrificed, and metastasis

progression was monitored. The metastatic capacity of the

Lenti-shNOP14-infected PANC-1 cells was found to be decreased compared

with that of the Lenti-shCon-infected cells (Fig.2G). Next, the lungs and livers

were dissected for microscopic histological analysis, which revealed extensive

metastasis to the lungs in the SCID mice infected with Lenti-shCon-PANC-1

cells (4/5); in addition, NOP14 inhibition significantly decreased the frequency

of lung metastasis (1/5), and the lung weights dissected from the

Lenti-shNOP14-infected mice were significantly decreased compared with

those from the Lenti-shCon-infected mice (Fig.2H). Further, NOP14

expression was decreased in the examined lung tissues, as demonstrated by

IHC analysis (Fig.2I; Fig.S5D). No significant changes were observed in the

outward appearances of the livers (data not shown).

Together, our findings indicate that NOP14 inhibition decreases the metastatic

capacity of PDAC cells.

Identification of a gene expression signature of NOP14 induction in

PDAC cells.

To identify a genetic signature that may be associated with the

NOP14-mediated PDAC metastasis, we performed whole transcriptome

RNA-sequencing (RNA-seq) analysis using PANC-1 cells with NOP14

overexpression or inhibition. The expression levels of 144 and 100 genes were

found to be positively and negatively correlated with that of NOP14,

respectively (Fig.3A; Table S5). Among these 244 genes, most were involved

in physiological regulation (45%) and cellular metabolism (31%), and a few

(12%) were involved in cell growth (Fig.3B). To further narrow down the genes

related to PDAC metastasis, those genes from the RNA-seq dataset with at

least a 1.5-fold increase or decrease in their expression in PANC-1 cells with

NOP14 overexpression or inhibition were selected. A set of 22 genes was

validated by qRT-PCR, which indicated that the expression of a number of

oncogenes, including PDGFRb, SLUG, ZEB1, EGFR, SMAD2, TWIST1,

S100A4, PIM1, and E2F1, was positively regulated by NOP14 and that the

expression of several tumor suppressor genes, including TP63, P21, and

TP53I3, was negatively regulated by NOP14 in PANC-1 and MIA PaCa-2 cells

(Fig.3C). PANC-1 and MIA PaCa-2 cells carry mutations in p53, and we

observed that mutp53 expression was upregulated both in these two cells with

NOP14 overexpression, whereas it was downregulated with NOP14 inhibition,

as shown by qRT-PCR and immunoblotting analyses (Fig.3D).

Together, these data indicate that a number of tumor-related genes are

involved in NOP14-driven PDAC progression.

NOP14 regulates PDAC metastasis by stabilizing mutp53 mRNA.

Because an alteration in the steady-state level of mutp53 mRNA could be

attributed to the induction of either transcription or mRNA degradation by

NOP14, we sought to directly explore whether NOP14 inhibition affects the

stability of mutp53 mRNA. To this aim, PANC-1 and MIA PaCa-2 cells were

treated with ActD to inhibit transcription, which resulted in a decreased mutp53

mRNA half-life in cells with NOP14 inhibition compared with si-Con-treated

cells (Fig.3E). These data indicated that NOP14 enhanced the stability of

mutp53 mRNA in PDAC cells. Interestingly, several of the above-mentioned

genes have been reported to be direct or indirect effectors of p53 in diverse

cancers.(2-3, 12-14) To determine whether these genes are regulated by

mutp53 in PDAC cells, qRT-PCR and immunoblotting analyses were

performed and we found that mutp53 inhibition led to increased expression of

P21 and decreased expression of PDGFRb, TGFB1, ZEB1, EGFR, and

SMAD2 in PANC-1 and MIA PaCa-2 cells (Fig.3F; Fig.S6). Further, the

expression of EGFR, TGFB1, ZEB1, and SMAD2 were upregulated and

downregulated in PANC-1 and MIA PaCa-2 cells with NOP14 overexpression

and inhibition, respectively, and P21 expression was negatively regulated by

NOP14 in these two cells, as shown by immunoblotting analysis (Fig.3G).

Although NOP14 overexpression promoted cell invasion in both PANC-1 and

MIA PaCa-2 cells, the target genes were not the same, i.e. PDGFRb was

regulated by the NOP14/mutp53 axis in MIA PaCa-2 cells but not in PANC-1

cells (Fig.3G). These discrepancies might be attributed to different p53

mutation sites.

Together, these data demonstrate that NOP14 affects the expression of

mutp53 targets by regulating the stability of mutp53 mRNA (Fig.3H).

NOP14 promotes PDAC metastasis by regulating miR-17-5p expression

We further identified the miRNAs induced by NOP14 in PANC-1 cells by

miRNA sequencing (miRNA-seq). Analysis of the miRNA-seq data revealed

that the expression of 46 miRNAs was induced by NOP14 whereas that of 42

miRNAs was inhibited by NOP14 in PANC-1 cells (Fig.4A). Among these 88

miRNAs, most were found to be involved in tumorigenesis and progression

(55%), cellular metabolism (3%), and immune regulation (1%) (Fig.4B). Among

these cancer-related miRNAs, the expression of miR-17-92 family, miR-31,

miR-1244, and miR-4517, was significantly induced by NOP14. To confirm

these findings, we performed qRT-PCR using PANC-1 and MIA PaCa-2 cells

with NOP14 overexpression or inhibition and found similar alterations in

miR-17-5p expression (Fig.4C, D). Because previous studies have indicated

that miR-17-5p promotes cancer metastasis,(15-16) we next examined

whether NOP14 enhances the invasive capacity by inducing miR-17-5p

expression in PDAC cells. Thus, we conducted a series of rescue experiments.

NOP14 was overexpressed in PANC-1 cells, thereby resulting in increased

miR-17-5p expression. The upregulation of miR-17-5p was suppressed by a

specific inhibitor (Anti-17-5p), as demonstrated by qRT-PCR (Fig.4E).

Subsequent in vitro transwell and invasion assays revealed that miR-17-5p

inhibition to prevent the induction of NOP14 caused decreased cell migration

and invasion (Fig.4F). Further, PANC-1 cells were transfected with miR-17-5p

mimic to rescue the downregulated expression of endogenous miR-17-5p

induced by NOP14 inhibition (Fig.4G). Subsequent in vitro transwell and

invasion assays indicated that miR-17-5p overexpression to prevent NOP14

inhibition led to increased cell migration and invasion (Fig.4H). Similar results

were observed in MIA PaCa-2 cells subjected to the same treatment (Fig.S7).

Taken together, these data indicate that NOP14 promotes PDAC metastasis at

least partly by inducing miR-17-5p expression.

P21 is a direct target of miR-17-5p in PDAC cells

P21 has been reported to be a target of miR-17-5p in diverse cancer cells and

in pancreatic cancer stem cells.(17-18) We further investigated the correlation

between miR-17-5p and P21 in PDAC cells. Thus, PANC-1 and MIA PaCa-2

cells were transfected with a p21-3´UTR-wild-type or p21-3´UTR-mutant

luciferase reporter, and luciferase activity was assessed, which demonstrated

that endogenous miR-17-5p repressed the luciferase activity of the wild-type

reporter, whereas mutation of the miR-17-5p sites abrogated this reduction

(Fig.5A, B; Fig.S8). Further, miR-17-5p overexpression resulted in reduced

P21 expression, and miR-17-5p inhibition led to upregulation of P21 in PANC-1

and MIA Pa2Ca-2 cells (Fig.5C). These data indicate that miR-17-5p regulates

P21 expression by binding to its complementary sites. NOP14 enhanced

miR-17-5p expression in PDAC cells, suggesting that P21 expression might be

affected by NOP14. This notion was validated by the above-mentioned

findings. Additionally, immunostaining analysis was performed to evaluate P21

expression in PANC-1 cells with miR-17-5p overexpression or inhibition,

revealing that miR-17-5p repressed its expression (Fig.5D). Similar results

were observed in PANC-1 cells with NOP14 overexpression or inhibition

(Fig.5E), indicating a positive regulatory association between NOP14 and

miR-17-5p.

We also investigated whether P21 was expressed at lower levels in PDAC

tissues with upregulated miR-17-5p expression. qRT-PCR was conducted to

assess 20 paired PDAC tissues, demonstrating that the miR-17-5p levels were

increased in these samples (Fig.5F). Moreover, P21 expression was

significantly downregulated in these specimens, as shown by IHC analysis

(Fig.5G).

Altogether, these findings indicate P21 is a direct target of miR-17-5p in PDAC

cells.

NOP14 induces miR-17-5p expression by stabilizing mutp53 mRNA

To explore the mechanism underlying the impact of NOP14 on the

upregulation of miR-17-5p in PDAC cells, we first evaluated the RNA stability

of primary miR-17-5p (pri-miR-17-5p) in PANC-1 and MIA PaCa-2 cells. We

found that NOP14 inhibition did not affect the stability of pri-miR-17-5p in these

two cells (Fig.S9). We next assessed whether NOP14 regulates miR-17-5p

expression at the transcriptional level. miR-17-92 family has been

demonstrated to be a transcriptional target of p53 in colorectal carcinoma and

B cell chronic lymphocytic leukemia.(11, 19) We showed that NOP14

enhanced the stability of mutp53 mRNA in PDAC cells, as stated above. Thus,

we hypothesized that NOP14 induces miR-17-5p expression by stabilizing

mutp53 mRNA in PDAC cells. To test this idea, we measured miR-17-5p

expression in PANC-1 and MIA PaCa-2 cells with NOP14 inhibition and found

that si-NOP14 treatment led to decreased expression of pri-miR-17-5p and

miR-17-5p precursor (pre-miR-17-5p), as shown by qRT-PCR (Fig.6A). We

next performed qRT-PCR to evaluate miR-17-5p expression in PANC-1 and

MIA PaCa-2 cells with mutp53 inhibition and observed that si-p53 treatment

reduced the levels of pri-miR-17-5p and pre-miR-17-5p in these two cells

(Fig.6B). The direct binding of mutp53 to the promoter of miR-17-5p in PANC-1

and MIA PaCa-2 cells was validated by luciferase reporter gene assay and

ChIP analysis (Fig.6C, D). We further transfected NOP14-overexpressing

PANC-1 and MIA PaCa-2 cells with si-p53 and found that si-p53 treatment to

prevent the induction of NOP14 led to decreased miR-17-5p expression, as

shown by qRT-PCR (Fig.6E). The following rescue assay revealed that

miR-17-5p overexpression to prevent the suppression of si-p53 on P21

expression led to increased cell invasion (Fig.6F, G).

Altogether, our data indicate that NOP14-induced PDAC metastasis is at least

partly due to mutp53-regulated miR-17-5p/P21 signaling (Fig.6H).

NOP14 expression and PDAC progression

To determine the physiological relevance of NOP14 in PDAC, we performed

IHC analysis on sections of PDAC and adjacent normal tissues (Table S6).

Representative NOP14-staining on tissue microarrays (TMAs) containing 90

core pancreatic cancer tissue specimens, including 82 paired PDAC samples,

is shown on Fig.7A. We scored NOP14 expression in the core specimens on a

scale from 0 (no expression) to 2 (high expression) based on the IHC scores of

NOP14 and observed that NOP14 expression was increased in the PDAC

samples (Fig.7B). These findings are consistent with those obtained for the

above-mentioned 20 PDAC tissues. The correlation between NOP14

expression and PDAC progression was further explored, which revealed that

PDAC patients with increased NOP14 levels in tumors tended to have tumor

invasion and more aggressive tumor phenotypes (Fig.7C, D, E; Table S6).

Additionally, a higher NOP14 level was found to be correlated with shorter OS,

as demonstrated by Kaplan-Meier analysis (P=0.008) (Fig.7F).

Taken together, these data indicate that increased NOP14 expression is

correlated with PDAC development.

Discussion

Cancer cells expressing mutp53 possess the oncogenic capacity to promote

cell proliferation, survival, and metastasis, indicating that targeting mutp53

may be a potential therapeutic strategy for cancer prevention. Because no

effective strategies have been validated for the targeting of mutp53, recent

studies have focused on elucidating the mechanisms underlying the regulation

of mutp53-induced oncogenic signaling. A number of molecules have been

identified as potential mediators of mutp53 function in cancer progression,

such as PDGFRb, proteins involved in integrin recycling, mevalonate pathway,

and miRNA biogenesis.(3, 20-22) Here, we showed that NOP14 promoted

PDAC metastasis by stabilizing mutp53 mRNA in PANC-1 (Trp53R273H) and

MIA PaCa-2 (Trp53 R248W) cells. These data suggest that NOP14 acts as a

specific regulator of mutp53 and define a novel mechanism for understanding

the oncogenic role of mutp53 in tumorigenesis.

Given that NOP14 increases the mutp53 protein levels by stabilizing mutp53

mRNA, we proposed that NOP14 might affect mutp53-induced oncogenic

signaling. We performed RNA-seq to identify the functional targets of NOP14

involved in PDAC metastasis. A number of protein-encoding genes were

verified to be regulated by NOP14. Among them, P21, EGFR, SMAD2,

TWIST1, and TP63 are downstream mediators of p53 and these proteins are

regulated by mutp53 in PDAC cells, suggesting that NOP14 promotes invasion

and metastasis by regulating the stability of mutp53 mRNA, thereby affecting

the associated signaling pathways.

miRNA-seq data revealed that miR-17-5p expression was significantly

regulated by NOP14 in PDAC cells. miR-17-5p has been previously shown to

be induced by c-Myc.(23) However, we found that NOP14 did not affect the

c-Myc levels in PDAC cells (Fig.S10), indicating that NOP14 regulated

miR-17-5p expression through other proteins. Another study has demonstrated

that miR-17-5p is negatively regulated by wild-type p53 in HCT116 colon

cancer cells.(11) Our data confirmed this finding in PANC-1 and MIA PaCa-2

cells SW1990 cells and revealed that miR-17-5p was regulated by mutp53.

Taken together, our results indicate that NOP14 promotes mutp53-driven

PDAC metastasis, at least partly through miR-17-5p-mediated P21 signaling.

More interestingly, P21 was not only validated as a direct transcriptional target

of mutp53, but it was also found to be inhibited by miR-17-5p, demonstrating

that the NOP14/mutp53 regulatory axis affects P21 expression at both the

transcriptional and post-transcriptional levels. Further analysis of the RNA-seq

and miRNA-seq data validated that several tumor-related genes, including

TP63 (target of miR-20a and let-7i), BCL2 (target of miR-195 and miR-34), and

PDGFRb (target of miR-195), were transcriptional targets of p53 and that

these miRNAs were also directly regulated by p53.(3-4, 24-26) These results

revealed a global mechanism that increased understanding of the influences of

the NOP14/mutp53 regulatory axis on its downstream molecules, i.e. the

expression of the p53 transcriptional effectors was also found to be regulated

by p53-induced miRNAs at the post-transcriptional level (Fig.6H).

In summary, our data define the function and mechanism of NOP14 in

mutp53-driven PDAC metastasis. Thus, targeting of NOP14 may represent a

realistic approach for the prevention of PDAC with mutp53.

Acknowledgements

We thank Shuangni Yu, Hui Zhang, and Hongkai Zhang for providing

assistance with IHC analysis and Bo Rong for providing assistance with ChIP

assay.

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Figure legends

Fig.1. NOP14 promotes PDAC cell migration and invasion.

(A) Representative images of NOP14-staining of the PDAC and matched normal tissues

(NC). Magnification: 40×. Bars: 500 μm. Images shown in the top-right panels represent

the magnified views. (B) Evaluation of NOP14 expression in PDAC tissues is shown. (C)

Representative images of NOP14-staining of in 7 metastases. Magnification: 40×. Bars:

500 μm. The boxes show the enlarged areas. (D) Evaluation of NOP14 expression in the

metastases is shown. (E) Wound-healing assays were conducted in PANC-1 cells with

NOP14 overexpression or inhibition. Wounds were photographed, and the wound closure

percentage from a representative experiment (n=3) was measured using AxioVision

software. Bars: 500 μm. (F) In vitro transwell and invasion assays were performed in

PANC-1 cells with NOP14 overexpression or inhibition. Representative images are shown.

Magnification: 100×. Bar: 200 μm. Normalized ratios of migrated or invasive cells are

shown in the bottom panels. (G) PANC-1 and MIA PaCa-2 cells with NOP14

overexpression or inhibition were allowed to invade into collagen for 24 h before

quantification. Representative three-dimensional (3D) reconstructions of each condition

selected from three repeated experiments are shown in the top panels. Cells were stained

for F-actin (red) and DAPI (blue). The dashed line indicates the approximate position of

the transwell membrane, and the arrow indicates the direction of cell movement. The

average numbers of invaded cells for six replicates ± SD are shown in the bottom panels.

Fig.2. NOP14 inhibition suppresses PDAC cell invasion in vivo.

(A) NOP14 inhibition decreased the tumor formation capacity of PANC-1 cells as

observed in xenograft models. Representative IVIS images of nude mice are shown, and

there were 6 mice in each group. The right panel shows the tumor volumes on the

indicated days. (B) Photographs of dissected tumors from nude mice and the tumor

weights calculated at the end of the experiment are shown in the right panel. (C)

Pathological analysis of tissue sections from recipient mice at 8 weeks post-injection.

H&E- and NOP14-staining were performed. Bars: 500 μm. The boxes show the enlarged

areas. The arrows indicate the muscle tissues invaded by PDAC cells. Normalization of

NOP14 expression is shown in the right panel. (D) Representative IVIS images of a

mouse orthotopic pancreatic tumor model are shown, and there were 5 mice in each

group. (E) The primary pancreas tissues, lungs, livers, and kidneys were dissected for

microscopic histological analysis. The numbers of metastatic nodes were calculated and

shown in the right panel. (F) Representative images obtained in histological analysis,

which was conducted on sections from the primary pancreas tissues, liver, lung, spleen,

mesentery, appendix, diaphragm, and colon tissues. The images shown in the top-left or

bottom-right panels represent magnified views of the boxed regions. (G) SCID mice were

injected with Lenti-shNOP14- or Lenti-shCon-infected PANC-1 cells through the lateral tail

vein. (H) The lungs were dissected for microscopic histological analysis, and the wet

weights of the lungs are shown. (I) Representative images of H&E and NOP14 stained

lung tissues are shown in the left panel, and the NOP14 expression scores are shown in

the right panel.

Fig.3. Genome-wide target identification indicates mutp53 as a functional effector

of NOP14 in PDAC cells.

(A) Cluster heat map of mRNA expression profiles in PANC-1 cells with NOP14

overexpression or inhibition. (B) Analysis of the gene expression profiles revealed that the

expression of 144 genes was positively regulated by NOP14 whereas 100 genes was

negatively correlated with the NOP14 level in PANC-1 cells. (C) The expression of 22

genes was validated in PANC-1 and MIA PaCa-2 cells by qRT-PCR. GAPDH served as a

loading control. (D) The expression of mutp53 was determined in PANC-1 and MIA

PaCa-2 cells with NOP14 overexpression or inhibition by qRT-PCR and immunoblotting

analyses. GAPDH and β-actin were used as a loading control, respectively. (E) PANC-1

and MIA PaCa-2 cells were transfected with si-NOP14, and total RNA was isolated at

different time points after ActD addition. The stability of mutp53 mRNA was determined by

qRT-PCR. (F, G) The expression of p53 targets was validated in PANC-1 and MIA PaCa-2

cells with si-p53 transfection by qRT-PCR (F), and immunoblotting analysis (G). β-actin

served as a loading control. (H) Schematic representation of NOP14-induced signaling

molecules involved in regulating PDAC metastasis.

Fig. 4. NOP14 promotes PDAC metastasis by regulating miR-17-5p expression.

(A) Cluster heat map of miRNA expression profiles in PANC-1 cells with NOP14

overexpression or inhibition. (B) Analysis of the miRNA expression profiles showed that

the expression of 46 miRNAs was positively regulated by NOP14 whereas 42 miRNAs

was negatively correlated with the NOP14 level in PANC-1 cells. (C, D) The expression of

several miRNAs was validated in PANC-1 and MIA PaCa-2 cells (C). miR-17-5p

expression depicted similar alterations in these two cells with NOP14 overexpression or

inhibition as shown by qRT-PCR. U6 snRNA served as a loading control (D). (E) The

expression of miR-17-5p was evaluated in PANC-1 cells with miR-17-5p inhibition

following transfection with an NOP14 construct. U6 snRNA served as a loading control. (F)

In vitro transwell, and invasion assays were conducted in the rescue experiments.

Magnification: 100×. The normalized ratios of migrated or invasive PANC-1 cells are

shown in the right panels. (G) The expression of miR-17-5p was evaluated in PANC-1

cells with miR-17-5p overexpression upon transfection with si-NOP14. U6 snRNA served

as a loading control. (H) In vitro transwell, and invasion assays were performed in the

rescue experiments. Magnification: 100×. The normalized ratios of migrated or invasive

PANC-1 cells are shown in the right panels.

Fig.5. P21 is a direct target of miR-17-5p in PDAC cells.

(A) Sequence in the 3′-UTR of P21 targeted by miR-17-5p. The sequences shaded in gray

represent mutants of the miR-17-5p-matched seed sequence. (B) Endogenous miR-17-5p

expression decreased luciferase activity when linked to the segment containing the target

sequence within the 3′-UTR of P21 mRNA in PANC-1 cells. Mutation of this sequence

abolished miR-17-5p-dependent repression. (C) miR-17-5p overexpression decreased

the P21 protein levels, and miR-17-5p inhibition increased P21 expression in PANC-1 and

MIA PaCa-2 cells, as demonstrated by immunoblotting analysis. β-actin served as a

loading control. The numbers below the panels represent the normalized protein

expression levels. (D) Immunofluorescence microscopy of PANC-1 cells stained with

anti-P21, F-actin, and DAPI (nuclei), showing decreased P21 staining after a miR-17-5p

mimic treatment and increased P21 staining following Anti-17-5p treatment. Bars: 20 μm.

(E) Immunofluorescence staining revealed decreased P21 expression in PANC-1 cells

with NOP14 overexpression and increased P21 expression in these cells with NOP14

inhibition. Bars: 20 μm. (F) miR-17-5p expression was upregulated in 20 PDAC tissues,

as shown by qRT-PCR. U6 snRNA served as a loading control. NC indicates the matched

normal control. (G) Representative images from P21 staining of the same samples

described in panel F. Magnification: 200×. Bars: 50 μm. The P21 expression scores are

shown in the bottom panel.

Fig.6. NOP14 inhibition results in decreased miR-17-5p levels by stabilizing mutp53

mRNA.

(A) The pri- and pre-miR-17-5p levels were determined in PANC-1 and MIA PaCa-2 cells

with NOP14 inhibition by qRT-PCR. GAPDH served as a loading control. (B) The

expression of pri-, pre- and mature miR-17-5p was evaluated in PANC-1 and MIA PaCa-2

cells with p53 inhibition by qRT-PCR. GAPDH (pre-miR-17-5p and pri-miR-17-5p) and U6

snRNA (mature miR-17-5p) served as loading controls. (C) Mutp53 inhibition in PANC-1

and MIA PaCa-2 cells resulted in decreased luciferase activity when linked to the segment

containing the target sequence within the promoter of miR-17-92 family. Mutation of the

target sequence abolished mutp53-dependent activation. (D) ChIP assay using p53

antibody on PANC-1 and MIA PaCa-2 cells. The values are the mean ± SD. (E) Rescue

assays were conducted on PANC-1 and MIA PaCa-2 cells, showing that NOP14

increased the expression of miR-17-5p by stabilizing mutp53 mRNA. (F) In vitro invasion

assay was performed in the rescue assay. Representative images are shown

(Magnification: 100×). The normalized ratio of invasive cells is shown in the bottom panels.

(G) The p53 and P21 protein levels were assessed in PDAC cells conducted rescue assay.

β-actin served as a loading control. The numbers below the panels represent the

normalized protein expression levels. (H) Schematic representation of NOP14 function in

mutp53-driven PDAC metastasis.

Fig.7. Increased NOP14 expression is correlated with PDAC progression.

(A) Representative images of NOP14-stained paired PDAC tissues. Magnification: 20×.

Bars: 1000 μm. The boxes show the enlarged areas. (B) Quantification of NOP14

expression, as determined by IHC analysis of a TMA containing 82 matched PDAC and

normal tissues. (C) NOP14 expression was correlated with PDAC progression. (D, E)

NOP14 expression levels were scored from 0 (negative) to 2 (high expression). The

numbers of paired PDAC tissues are indicated on the tops of the columns. Significance

was assessed using the Mann-Whitney test. ⎯X = mean. Patients with high tumor NOP14

expression tended to have local invasion (D) and lymph invasion (E). (F) Comparison of

the OS of patients with different NOP14 levels using the Kaplan-Meier method. P=0.0080.

Pancreatic cancer progression relies upon mutant p53-induced oncogenic signaling mediated by NOP14

Yongxing Du, Ziwen Liu, Lei You, et al.

Cancer Res Published OnlineFirst March 9, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-2339

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