Serum Nardilysin, a Surrogate Marker for Epithelial-Mesenchymal Transition, Predicts Prognosis of Intrahepatic Cholangiocarcinoma After Surgical Resection

Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title Page

Serum nardilysin, a surrogate marker for epithelial-mesenchymal transition,

predicts prognosis of intrahepatic cholangiocarcinoma after surgical resection

Authors

Tomoaki Yoh1), Etsuro Hatano2), Yosuke Kasai1), Hiroaki Fuji1), Kiyoto Nishi3), Kan Toriguchi2),

Hideaki Sueoka2), Mikiko Ohno4), Satoru Seo1), Keiko Iwaisako5), Kojiro Taura1), Rina

Yamaguchi6), Masato Kurokawa6), Jiro Fujimoto2), Takeshi Kimura3), Shinji Uemoto1), Eiichiro

10 Nishi4)

Affiliations

1) Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

2) Department of Surgery, Hyogo College of Medicine, Nishinomiya, Japan

15 3) Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University,

Kyoto, Japan

4) Department of Pharmacology, Shiga University of Medical Science, Otsu, Japan

5) Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University,

Kyotanabe, Japan

20 6) Sanyo Chemical Industries Ltd., Kyoto, Japan

Corresponding author:

Eiichiro Nishi, MD, PhD

Department of Pharmacology, Shiga University of Medical Science, Seta-Tsukinowa-cho, Otsu,

25 520-2192, Japan

Tel.: +81-77-548-2181, Fax: +81-77-548-2183, E-mail: [email protected]

Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Etsuro Hatano, MD, PhD

Department of Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo,

663-8501, Japan

Tel.: +81-798-45-6582, Fax: +81-798-45-6581, E-mail: [email protected]

Running title:

Nardilysin is a novel prognostic biomarker for ICC

Key words:

10 Intrahepatic cholangiocarcinoma, nardilysin, epithelial-mesenchymal transition, biomarker, surgery

Conflicts of Interest:

The authors declare no conflict of interest.

15 Abbreviations

ICC: intrahepatic cholangiocarcinoma

HCC: hepatocellular carcinoma

OS: overall survival

NRDC: nardilysin (N-arginine dibasic convertase)

20 HB-EGF: heparin-binding epidermal growth factor

TNF-α: tumor necrosis factor-α

ADAM: a disintegrin and metalloproteinase

HDAC3: Histone deacetylase 3

PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha

25 IHC: immunohistochemistry

AJCC: American Joint Committee on Cancer

ELISA: enzyme-linked immunosorbent assay

CM: condition medium

FBS: fetal bovine serum

5 KD: knockdown

NC: negative control

qRT-PCR: quantitative real-time polymerase chain reaction

WST-8: tetrazolium salt-based proliferation assay

CCK8: Cell Counting Kit-8

10 PBS: Phosphate-buffered saline

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

DFS: disease-free survival

ROC: receiver operating characteristic

AUC: area under the curve

15 SD: standard deviation

HC: healthy controls

LN: lymph node

CEA: Carcinoembryonic antigen

CA19-9: carbohydrate antigen 19-9

20 EMT: epithelial-mesenchymal transition

ZEB1: zinc finger E-box binding homeobox 1

EMT-TFs: EMT-inducing transcription factors

SOX2: sex-determining region Y-box 2

CSC: cancer stem cell

25 HIF-1α: hypoxia-inducible factor-1α

IGF-1: insulin-like growth factors-1

TGF-β: transforming growth factor-β

EGF: epidermal growth factor

Abstract

Purpose:

Few studies have investigated prognostic biomarkers in patients with intrahepatic

5 cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has

been suggested to play important roles in inflammation and several cancer types. We herein

examined the clinical significance and biological function of NRDC in ICC.

Experimental Design:

We measured serum NRDC levels in 98 ICC patients who underwent surgical resection in two

10 independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in

cancerous tissue specimens from 43 ICC patients. We investigated the roles of NRDC in cell

proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene

silencing.

Results:

15 High serum NRDC levels were associated with shorter overall survival and disease-free survival in

the primary (n=79) and validation (n=19) cohorts. A correlation was observed between serum

protein levels and cancerous tissue mRNA levels of NRDC (Spearman’s ρ=0.413, p=0.006). The

gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor

growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was

20 also accompanied by significant changes in the expression of several epithelial-mesenchymal

transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of

NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from ICC

patients.

Conclusions:

25 Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the

outcome of ICC after surgical resection.

Translational Relevance Few studies have investigated prognostic biomarkers in intrahepatic cholangiocarcinoma (ICC) that

5 may contribute to establishing adjuvant strategies. Nardilysin (NRDC), a metalloendopeptidase of

the M16 family, has been suggested to play important roles in inflammation and several cancer

types. The present results revealed 1) a correlation between serum NRDC levels and cancerous

tissue mRNA levels of NRDC, 2) that preoperative serum NRDC levels were associated with

survival and recurrence, and 3) strong correlations between the mRNA levels of NRDC and EMT-

10 inducing transcription factors, ZEB1 and SNAI1, in ICC cell lines and cancerous tissue. Based on

the potential relationship between NRDC and EMT, the preoperative evaluation of serum NRDC

has potential as a clinical tool for predicting the postoperative outcomes of ICC patients undergoing

surgical resection.

Introduction

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer following

hepatocellular carcinoma (HCC), accounting for 5-15% of all primary liver cancers [1-3]. There are

marked geographic variations in the incidence of ICC, with a higher incidence in East Asia, while

5 the number of ICC patients has been reported to be increasing worldwide [3, 4]. The survival rate of

ICC patients is poor because of the late presentation of the disease and limited therapies. Although

surgical resection is the only curative treatment, 30-40% of ICC patients have surgical indications

[3]. Moreover, the recurrence rate after surgical resection is 50–60% and the 5-year overall survival

(OS) rate after surgical resection is only 25–31% [3, 5, 6], highlighting the need to optimize

10 adjuvant strategies. Recent evidence has suggested that adjuvant chemotherapy is associated with

prolonged survival, particularly in some advanced cases [7, 8]. However, there are no established

methods to define patient subgroups that need adjuvant strategies. The preoperative measurement of

serum tumor markers may identify high-risk patients; however, few studies have investigated

biomarkers in ICC patients possibly due to the difficulties associated with collecting large numbers

15 of serum samples from ICC patients [9-11].

Nardilysin (N-arginine dibasic convertase, NRDC) is a zinc peptidase of the M16 family

that selectively cleaves dibasic sites [12, 13]. NRDC exhibits widespread expression throughout the

body, and regulates multiple biological processes, such as myelination [14], body temperature

homeostasis [15], and insulin secretion [16]. Although NRDC is a soluble cytosolic protein without

20 an obvious signal peptide or nuclear localization signal, it shuttles between the cytoplasm and

nucleus and is secreted via an as yet unknown mechanism [17]. We identified NRDC as a specific

binding partner of heparin-binding epidermal growth factor-like growth factor (HB-EGF). Our

subsequent studies demonstrated that NRDC enhanced the ectodomain shedding of HB-EGF and

other membrane proteins, such as tumor necrosis factor-alpha (TNF-α), through the activation of

25 disintegrin and metalloproteinase (ADAM) proteases [18,19]. In addition to its extracellular

functions, we recently clarified the nuclear functions of NRDC as a transcriptional co-regulator,

which modulates the transcriptional activity of Histone deacetylase 3 (HDAC3) [20], peroxisome

proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) [15], and islet-1 [16].

Furthermore, NRDC is strongly expressed in several cancer types, promotes tumor growth [19, 21-

23], and is associated with a poor prognosis [19, 22, 23], suggesting important roles for NRDC in

5 tumor biology. Our recent findings also indicated the clinical usefulness of serum NRDC in specific

clinical settings. For example, serum NRDC levels were associated with the survival outcomes of

postoperative HCC patients with hepatitis C [23].

In the present study, we retrospectively investigated serum expression levels of NRDC in

ICC patients who underwent surgical resection to clarify whether serum NRDC has potential as a

10 postoperative prognostic indicator. We also examined NRDC mRNA expression in surgical

specimens resected from patients as well as the pathophysiological role of NRDC in ICC using an

RNA interference method in ICC cell lines.

Materials and Methods

Study design and population

5 This study was designed to investigate whether serum NRDC has the ability to predict the outcomes

of ICC patients after surgical resection. We analyzed serum NRDC levels in two independent

cohorts: a primary cohort at Kyoto University and an external validation cohort at Hyogo College of

Medicine. In the primary cohort, we analyzed 79 consecutive patients with ICC who underwent

surgical resection at Kyoto University Hospital (Kyoto, Japan) between January 2006 and July

10 2013. In the validation cohort, we analyzed 19 consecutive patients with ICC who underwent

surgical resection at Hyogo College of Medicine (Nishinomiya, Japan) between January 2009 and

January 2015. The final diagnosis of ICC was histologically confirmed. The follow-up data of the

primary and validation cohorts were updated in April 2017 and February 2018, respectively. The

surgical procedure in the primary cohort was reported previously [6, 7, 9]. Serum samples from ICC

15 patients (n=98) were obtained preoperatively at the time of admission. Fresh frozen cancer tissues

were obtained from 43 out of 79 ICC patients in the primary cohort, from which mRNA was

isolated. Surgical specimens from 20 out of the 43 ICC patients were also assessed by

immunohistochemistry (IHC).

Clinicopathological and survival data were extracted from a prospectively maintained

20 institutional database. Clinicopathological data, including gender, age, hepatitis virus markers, the

Child-Pugh classification, primary tumor characteristics, and treatment-related variables, were

collected. Tumor characteristics and resection margins were ascertained based on a final

pathological assessment. The tumor stage was assessed by the 7th edition of the American Joint

Committee on Cancer (AJCC) classification [24]. Post-surgical adjuvant chemotherapy was

25 administered using gemcitabine (from 2006) and/or tegafur-gimeracil-oteracil potassium (S-1; from

2007) for tumors classified as stage II–IV according to the AJCC classification (from 2006).

Recurrence was diagnosed based on imaging studies and tumor markers.

Written informed consent for the use of serum and resected tissue samples was obtained

from all patients in accordance with the Declaration of Helsinki, and this study was approved by the

5 institutional review committee of the Graduate School of Medicine, Kyoto University (approval

code: R803-1) and Hyogo College of Medicine (approval code: 201807-010).

Measurement of serum NRDC levels

10 Serum was isolated from whole blood and stored at -80C until analyzed. To quantify serum NRDC

levels, an enzyme-linked immunosorbent assay (ELISA) was performed according to a previously

described method [25]. Briefly, to establish a sandwich ELISA system, all combinations of the 7

monoclonal antibodies for NRDC were tested, and the optimum combination of clone #231 for

coating and #304 for detection was selected. An automated analyzer for the chemiluminescent

15 enzyme immunoassay, SphereLight 180 (Olympus, Tokyo, Japan), was utilized to measure serum

NRDC levels according to the manufacturer’s protocol.

Cell culture and preparation of condition medium (CM)

20 The human ICC cell lines HuCCT-1 and HuH28 were provided by the Japanese Collection of

Research Bioresources Cell Bank (Osaka, Japan) and SSP-25 by the RIKEN Bioresource Center

(Tsukuba, Japan). ICC cells were grown in RPMI 1640 media supplemented with 10% fetal bovine

serum (FBS) and 1% penicillin and streptomycin. The human colorectal cancer cell line HCT116

was provided by the American Type Culture Collection (Rockville, TX, USA) and 293 T cells by

25 the RIKEN Bioresource Center (Tsukuba, Japan). These cells were growth in DMEM medium

supplemented with 10% FBS and antibiotics. Cells were cultured at 37C under 5% CO2 and 95%

relative humidity. These cells were incubated in serum-free medium for 24 h before the initiation of

experiments. To prepare CM, confluent monolayers of ICC cells were incubated in RPMI 1640

medium supplemented with 0.1% bovine serum albumin for 48 h. Cultured media were collected

and centrifuged at 3,000 rpm at 4°C for 10 min, and the supernatant was harvested.

Gene knockdown of NRDC

The gene knockdown (KD) of NRDC in ICC cells was performed by the transfection of lentiviral

vectors expressing miRNA as previously reported [19, 23]. The sequences targeting the NRDC gene

10 were as follows: NRDC-KD1: 5’-CTGATGCAAACAGAAAGGAAA-3’; NRDC-KD2: 5’-

GAGAAATGGTTTGGAACTCAA-3’. We used a control vector that contains a non-targeting

sequence for any vertebrate gene as a negative control (NC). The efficiency of the gene KD of

NRDC was evaluated by Western blotting and quantitative real-time polymerase chain reaction

(qRT- PCR) analyses.

Cell proliferation assay

Cell proliferation was examined by a tetrazolium salt-based proliferation assay (WST-8 assay)

using Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Tokyo, Japan). Briefly, 96-well plates

20 were seeded with cells at a density of 5000-10000 cells per well (HuCCT-1: 1.0 × 104 cells, SSP-

25: 5 x 103cell per well) and cultured for 72 h. Ten microliters of CCK8 solution was added to each

well and incubated at 37°C for 2 h. The cell viability ratio was defined as the ratio of absorbance at

450 nm of KD and NC cells. All assays were performed in quadruplicate and repeated at least three

times.

Migration assay

The migration of ICC cells was examined using a wound healing assay and chemotaxis assay as

previously reported [26]. In the wound healing assay, confluent ICC cells in 24-well plates were

wounded using a sterile 200-μl pipette tip. Cells were then grown for an additional 12 and 24 h with

5 serum-free media. Wound closure was observed with an inverted microscope (Keyence, Osaka,

Japan) at 40×magnification. The cell migration distance was measured using ImageJ software

(National Institute of Health) and compared with baseline measurements. All assays were repeated

at least three times.

A chemotaxis assay was performed in 8-μm pore Transwell® chambers (Corning Costar,

10 Cambridge, MA, USA). Briefly, ICC cells (HuCCT-1: 5.0 ×104 cells, SSP-25: 2.0×104 cells) in

serum-free media were placed in the upper chamber. The lower chamber was filled with 750 ml of

RPMI 1640 supplemented with 10% FBS as a chemoattractant. After the incubation at 37°C for 24

h (HuCCT-1) and 12 h (SSP-25), cells were fixed with 4% PFA at 20 min and stained with

hematoxylin and eosin. Cells that migrated through the pores to the lower surface of the filter were

15 counted under a microscope. A total of 3 random fields were counted in duplicate assays.

Chemosensitivity assay

NC and NRDC-KD ICC cells (HuCCT-1: 1.0×104 cells, SSP-25: 5.0×103 cells per well) were

20 seeded on 96-well plates in normal growth media. Twelve hours after seeding, gemcitabine was

added at the indicated concentrations. After a 72-h treatment, cell viability was examined using the

WST-8 assay. The cell viability ratio was defined as absorbance at 450 nm of the sample divided by

the absorbance of the control for each cell. All assays were performed in quadruplicate and repeated

at least three times.

Subcutaneous tumor xenograft

Seven- to 9-week-old male nude mice were used as recipients for xenotransplantation. A total of

1.0×106 of HuCCT-1-NC, -KD1, or –KD2 cells were suspended in 100 μl phosphate-buffered saline

(PBS) and subcutaneously injected into the right (NC) and left (KD1 or KD2) flank of nude mice

5 (n=7, of each). Tumor sizes were measured every week after the inoculation. Tumor volumes were

calculated using the formula: length× (width) 2× 0.52. Animal experiments were performed in

accordance with the protocols approved by the Institutional Animal Care and Use Committee of

Kyoto University. Animal experiments were performed in accordance with the protocols approved

by the Institutional Animal Care and Use Committee of Kyoto University.

IHC

Four-micrometer-thick sections were incubated with an anti-human NRDC mouse monoclonal

antibody (#102, established in our laboratory) at 4 °C overnight. Envision polymer (DAKO), which

15 is a horseradish peroxidase-labeled polymer conjugated with an anti-mouse IgG antibody, was used

as a secondary antibody according to the manufacturer’s protocol. Color was developed with

diaminobenzidine solution (DAKO), followed by counterstaining with hematoxylin.

Quantitative real-time polymerase chain reaction (qRT- PCR)

Total RNA was isolated from HuCCT-1 and SSP-25 cells and 43 fresh frozen cancer tissues using

TRIzol reagent (Thermo Fisher, Scientific) and cleaned using the DNase set and RNeasy Mini Kit

(Qiagen, Hilden, Germany). cDNA generated by reverse transcription with the Omniscript RT Kit

(Qiagen) was subjected to a real-time polymerase chain reaction assay using the Step One Plus

25 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast SYBR Green Master

Mix (Applied Biosystems) according to the manufacturer’s protocols. The average expression levels

of the target genes were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

using the 2−ΔΔCt method. The ICC cell line (SSP-25) was used as a reference sample (i.e.,

2−ΔΔCt value of SSP-25 cells=1) when analyzing human samples. The primers used in this

experiment are listed in Supplementary Table S1.

Western blotting

Twenty micrograms of cell lysates were electrophoresed on a 10% SDS-polyacrylamide gel (SDS

from Wako; acrylamide from Bio Rad, Hercules, CA, USA) and transferred to a nitrocellulose

10 membrane (GE Healthcare, Buckinghamshire, UK). The primary antibodies are listed

in Supplementary Table S2. The blot was observed with EZ-Capture II (ATTO, Tokyo, Japan)

visualized by ECL Prime (GE Healthcare). β-actin was used as the loading control. When analyzing

protein levels in CM, Ponceau 3R Stain Solution (Wako, Osaka, Japan) was used as the loading

control. In figures, representative images were selected from at least 3 independent experiments, in

15 which similar results were obtained.

Statistical analysis

Data from human clinical samples were expressed as median values (range). Regarding continuous

20 variables, data were expressed as a median (range), and compared using the Mann-Whitney U-

test. Categorical variables were expressed as a number [%] and compared using the χ2 test or

Fisher’s exact test where appropriate. A receiver operating characteristic (ROC) analysis was

performed to evaluate the discriminatory power of predictors. The area under the ROC curve

(AUC) was calculated. DeLong’s test was used to compare the AUC. The cut-off values, sensitivity,

25 and specificity of serum NRDC variables were assessed by the Youden index. Other cut-off values

were evaluated by clinically relevant values [5,6]. Relationships between continuous variables were

evaluated by Spearman’s correlation test (the value was expressed as ρ). To label the strength of the

relationship, for absolute values of ρ, 0-0.19 is regarded as very weak, 0.2-0.39 as weak, 0.40-0.59

as moderate, 0.6-0.79 as strong, and 0.8-1 as very strong [27]. OS was calculated from the day of

surgical resection to the date of death or end of the follow-up period, while disease-free survival

5 (DFS) was calculated using the date of death or recurrence as the time of the terminal event

according to the Kaplan-Meier method. Survival was compared using a generalized Wilcoxon test.

A multivariate analysis was performed by Cox’s regression (Step-wise backward model) for

variables identified as significant in the univariate analysis. When collinearity was encountered, a

choice was made based on the p value and clinical reasoning. All analyses were two-sided, and

10 differences were considered significant when p<0.05. Statistical analyses were performed using

JMP ver. 12.1 software (Cary, NC, USA).

Data from in vitro and in vivo experiments were analyzed using the Student’s t-test and expressed as

means ± standard deviation (SD). All statistical analyses were performed using JMP ver. 12.1

software (SAS Institute, Cary, NC, USA).

Results

Prognostic impact of serum NRDC in ICC patients after surgical resection

In the primary cohort, serum NRDC levels were measured in 79 preoperative ICC patients who

consecutively underwent surgical resection at Kyoto University Hospital between 2006 and 2013.

In this study population, most patients had advanced stage disease (AJCC stage III/IV, n=52

[65.8%]) [24]. R0 resection was performed on 63 patients (79.7%) (Table S3). Serum NRDC levels

10 were significantly higher in ICC patients than in healthy controls (HC) (median; 1627.4 (range:

351.9-11318.7) pg/ml vs. 539.8 (range: 5.9-1184.2) pg/ml, p<0.001; Fig. 1A).

We then examined whether serum NRDC levels had prognostic value in ICC patients after

surgical resection. The median follow-up period was 41.6 months (range: 0.1-127.5 months). The

median OS time was 47.6 months, with 3- and 5-year OS rates of 57.0% and 42.3%, respectively.

15 The cut-off value for serum NRDC was selected as 1627.4 pg/ml based on the highest accuracy in

relation to an outcome (death) using an ROC analysis (AUC values of 0.688). Patients with ICC

were divided into two groups according to this cut-off value: high serum NRDC (n=40) and low

serum NRDC groups (n=39). High serum NRDC levels correlated with the presence of multiple

tumors (p=0.006) (Table 1). OS was significantly shorter in patients with high serum NRDC levels

20 than in those with low serum NRDC levels (p=0.002, Fig. 1B). The median OS and 3- and 5-year

survival rates of high and low serum NRDC groups were 31.0 versus 85.6 months, 57.0 versus

71.8%, and 22.3 versus 42.5%, respectively. Moreover, DFS was significantly shorter in patients

with high serum NRDC levels than in those with low serum NRDC levels (p=0.002, Fig. 1C). The

median DFS and 1- and 3-year DFS rates of the high and low serum NRDC groups were 9.4 versus

25 23.0 months, 42.5 versus 71.8%, and 15.0 versus 43.4%, respectively.

To confirm the prognostic relevance of serum NRDC as a biomarker, we performed

univariate and multivariate analyses by Cox’s hazard model using six potential confounders (Table

2). Among known prognostic factors [4-6], lymph node (LN) metastasis (p=0.001), vascular

invasion (p=0.033), and multiple tumors (p<0.001) were poor prognostic factors for OS. LN

5 metastasis (p=0.042), multiple tumors (p<0.001), and poor differentiation (p=0.026) were poor

prognostic factors for DFS. After the multivariate analysis, serum NRDC levels were maintained as

independent prognostic factors for OS (p=0.019) and DFS (p=0.009).

To validate the prognostic impact of NRDC in ICC patients, we analyzed serum NRDC

levels in an external independent cohort of 19 ICC patients who underwent surgical resection at

10 Hyogo College of Medicine between 2009 and 2015. (Table S4). The median follow-up period was

25.4 months (range: 3.8-70.6 months) and the median OS was 27.9 months, with 3- and 5-year OS

rates of 35.1 and 23.4%, respectively. Serum NRDC levels were significantly higher in the

validation cohort (median; 1330, range: 528-14597 pg/ml) than in HC, but were not significantly

different from those in the primary cohort (Fig. 1A). OS and DFS were significantly stratified

15 according to cut-off values (1295 pg/ml: AUC values of 0.729), which were selected independently

in the validation cohort by an ROC analysis. The clinical backgrounds of the high and low serum

NRDC groups and Kaplan-Meier curves for OS and DFS are shown in accordance with this

independent cut-off value (Table S5, Fig, 1D, E). These results reinforced the predictive value of

serum NRDC in postoperative ICC patients.

Comparison of serum NRDC levels with other tumor markers

Carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) are serum tumor

markers that are commonly measured in ICC patients [3, 5, 6]. A correlation was not observed

25 between serum NRDC and CEA or CA19-9 levels (Fig. S1). A Kaplan-Meier curve analysis

revealed that the elevated serum CEA (≥5 ng/mL) and CA19-9 (≥37 IU/ml) levels in ICC patients

correlated with a poor prognosis in the primary cohort (Fig. S2). An ROC analysis in relation to the

outcome (death) showed that the prognostic ability of preoperative serum NRDC levels (AUC:

0.689) was equivalent to that of serum CEA (0.569) and CA19-9 (0.671) levels (Fig. S3). We

additionally analyzed the prognostic value of the combination of 3 markers (Table S6) and found

5 that the combination of NRDC and CA19-9 provided the highest AUC value (0.756), which had a

significantly stronger prognostic value than CA19-9 alone (Fig. S4).

NRDC mRNA levels in resected cancerous tissue correlated with the prognosis of ICC patients

after surgical resection

The up-regulated expression of NRDC in cancer tissue has been reported to have a poor prognostic

impact in several cancer types [19, 22, 23]. In addition to serum NRDC levels, we examined NRDC

expression in surgical specimens resected from ICC patients. An IHC analysis showed the

membranous, cytosolic, and nuclear expression of NRDC in the cancer epithelium (Fig. 2A), which

15 was consistent with previous findings [12-20]. We quantified the mRNA expression levels of

NRDC in cancerous tissues from 43 ICC patients and analyzed the relationship with matched serum

NRDC levels. As expected, a correlation was observed between mRNA and serum NRDC levels

(ρ=0.413, p=0.006, Fig. 2B), suggesting that ICC tumors are a potential source of serum NRDC.

We also assessed the prognostic value of NRDC mRNA levels in tumors. The cut-off

20 values of mRNA levels were selected based on the highest accuracy in relation to the outcome of

death (cut-off 2-ΔΔCt value: 1.708, AUC value of 0.634) and were used to divide patients into two

groups: low NRDC mRNA (n=11) and high NRDC mRNA (n=32) groups. Using these cut-off

values (patient characteristics are shown in Table S7), OS and time to recurrence were significantly

shorter in patients with high NRDC mRNA levels than in those with low NRDC mRNA levels (Fig.

25 2C, D).

Effects of the gene KD of NRDC on the proliferation, migration, and chemosensitivity of ICC

cells

We examined NRDC expression in 3 ICC cell lines and found that all cell lines strongly expressed

NRDC (Fig. S5). NRDC protein levels in ICC cells were similar to those in colon cancer HCT116

5 cells [28] and higher than those in 293T cells [13]. Therefore, to examine the pathophysiological

roles of NRDC in ICC cells, we performed gene KD experiments using 2 ICC cell lines with high

malignant potential: HuCCT-1 (obtained from metastatic ascites [29]) and SSP-25 (spindle cell-type

with mesenchymal features [30]). The sufficient silencing of NRDC expression in both cells was

confirmed by Western blotting and qRT-PCR analyses (Fig. 3A). Secreted NRDC in CM was also

10 clearly decreased by the gene KD (Fig. 3B). We initially evaluated cell proliferation using the

tetrazolium salt assay and found that the proliferation of HuCCT-1 and SSP-25 cells was

significantly decreased by the gene KD of NRDC (Fig. 3C). To further assess the impact of NRDC

on cell growth, control and NRDC-knocked down HuCCT-1 cells were used in tumor xenograft

experiments. Tumor growth after subcutaneous implantation was markedly less in cells with the

15 gene KD of NRDC than in control cells (Fig. 3D). Since we confirmed the similar effects of 2

different siRNAs on in vitro and in vivo cell proliferation, one (KD2) was selected for further

experiments. In two different assays (wound healing assay and chemotaxis assay), HuCCT-1 cells

in which NRDC was knocked down (NRDC-KD2) showed significantly less migratory potential

than control cells (NC) (Fig. 3E and 3F). The similar inhibitory effect of NRDC KD on cell

20 migration was also confirmed in SSP-25 cells (Fig. S6). Furthermore, the influence of NRDC levels

on the chemosensitivity of HuCCT-1 and SSP-25 cells to gemcitabine was investigated. In both cell

lines, NRDC-KD2 cells exhibited greater sensitivity to gemcitabine than NC cells (Fig. 3G and

Fig. S6).

25 Relationship between mRNA levels of NRDC and epithelial-mesenchymal transition (EMT)-

related genes in tumor tissues from ICC patients

To gain mechanical insights into the tumor-promoting and chemoresistant potential of NRDC in

ICC, we investigated gene profiles in ICC cell lines. An analysis by qRT-PCR revealed that several

EMT- and cancer stem cell (CSC)-related genes were down-regulated by the gene KD of NRDC in

5 HuCCT-1 (Fig. 4A) and SSP-25 cells (Fig. 4B). For example, the mRNA levels of vimentin, a

marker of mesenchymal cells, zinc finger E-box-binding homeobox 1 (ZEB1), an EMT-inducing

transcription factor (EMT-TF), sex-determining region Y-box 2 (SOX2), a marker of CSC, and

hypoxia-inducible factor-1α (HIF-1), a trigger of the EMT pathway, were down-regulated by the

gene KD of NRDC. Other EMT-TF, SNAI1 and TWIST1, were also significantly decreased by

10 NRDC KD in SSP-25 cells (Fig. 4B). The Western blot analysis revealed that the protein expression

levels of these genes were markedly reduced (Fig. 4A, B), while E-cadherin, a marker of epithelial

cells, was increased by NRDC KD in HuCCT-1 cells (Fig. 4A).

We then investigated whether relationships existed between the mRNA levels of NRDC

and EMT-related genes in tumor tissues from ICC patients. Notably, the expression levels of NRDC

15 positively and strongly correlated with two EMT-TFs, ZEB1 (ρ=0.679, p<0.001) and SNAI1

(ρ=0.647, p<0.001), and HIF-1α expression (ρ=0.721, p<0.001) (Fig. 4C). NRDC mRNA levels

correlated with SOX2 expression, but not with TWIST1 expression or the Vimentin/E-cadherin

ratio (Fig. S7). We then examined the relationship between serum NRDC and mRNA levels of

EMT-related genes in tumor tissues. Serum NRDC positively correlated with SNAI1

20 (ρ=0.327, p=0.032) and HIF-1α (ρ=0.301, p=0.050) mRNA levels in surgical specimens. ZEB1

mRNA levels were also positively associated with serum NRDC levels (ρ=0.287, p=0.062) (Fig.

4D). Therefore, serum NRDC has potential as a surrogate marker for EMT in the tumors of ICC

patients.

Discussion

The present study highlights the clinical implications of preoperative serum NRDC measurements

in ICC patients, which may contribute to the identification of patients with a poor prognosis. OS

and DFS were significantly stratified by serum NRDC levels in the primary (development) and

5 validation cohorts. The expression levels of NRDC in sera and cancerous tissues were significantly

linked; therefore, an evaluation of pathophysiological features in resected tissues provided an

insight into the clinical significance of serum NRDC. NRDC mRNA levels correlated with EMT-

related genes in primary tumors. Moreover, direct correlations were observed between serum

NRDC and mRNA levels of major EMT-TF, suggesting that serum NRDC has potential as a

10 surrogate marker for EMT features in primary tumors. This hypothesis was supported by a cell

analysis because the gene silencing of NRDC in ICC cell lines reduced EMT-related gene

expression. Functionally, the gene KD of NRDC was accompanied by attenuated

proliferation/migration and increased chemosensitivity to gemcitabine. Importantly, a correlation

was not noted between serum NRDC and CA19-9 or CEA, currently available prognostic markers

15 for ICC. Furthermore, the combination of NRDC and CA19-9 had stronger prognostic value than

either marker analyzed individually, suggesting that serum NRDC is a unique prognostic marker for

ICC patients after surgical resection.

Based on the prognostic impact of serum NRDC levels in ICC patients after surgical

resection, the source of NRDC in serum needs to be identified. According to the following findings

20 of clinical studies: 1) serum NRDC levels were significantly higher in ICC patients than in HC, 2) a

correlation between serum NRDC and NRDC expression levels in resected cancer tissue, we

speculate that a potential source of serum NRDC may be the ICC tumor itself. Experiments using

ICC cell lines also revealed that NRDC is secreted into CM, the amount of which correlated with

the intracellular expression level of NRDC. Our previous analysis of patients with HCC also

25 suggested that serum NRDC reflected the amount of NRDC in cancer tissues [23]. Another possible

source of serum NRDC is inflammatory cells adjacent to tumors. We recently reported that NRDC

levels were increased in the synovial fluid of patients with rheumatoid arthritis [31]. NRDC in

macrophages regulates arthritis via the control of TNF-α secretion because the macrophage-specific

deletion of NRDC markedly ameliorated arthritis [30]. We also demonstrated that inflammatory

cells infiltrating the infarcted myocardium strongly express NRDC [25]. However, clinical data

5 from this study do not strongly support this hypothesis because pre-operative C-reactive protein

levels in ICC patients did not correlate with serum NRDC levels (data not shown). In any case,

serial measurements of serum NRDC levels after surgical resection are needed to identify the real

source, which will also further clarify the relationship between serum NRDC levels and tumor

recurrence.

10 The significant link between patient prognosis and NRDC mRNA expression levels in

cancerous tissues prompted us to examine the pathophysiological functions of NRDC in ICC cells.

The gene KD of NRDC in two different ICC cell lines attenuated cell proliferation and migration,

indicating important roles for NRDC in ICC progression. Moreover, the gene KD of NRDC in

HuCCT-1 cells resulted in increased sensitivity to gemcitabine. Since the poor prognosis of ICC

15 patients is mainly attributed to its highly metastatic characteristics, EMT in the pathogenesis of ICC

has been attracting increasing attention from researchers [32]. Accumulated evidence also suggests

a relationship between chemoresistance and the acquisition of the EMT phenotype and/or existence

of CSC within the tumor [33]. Among EMT-related genes, the significance of EMT-TF, such as

ZEB1, SNAI1, and TWIST1, has been emphasized [32-36]. An analysis of surgical specimens also

20 demonstrated that the strong expression of EMT-TF in resected tissue is associated with the poor

prognosis of ICC patients after surgical resection [32]. We herein showed that the gene KD of

NRDC was accompanied by marked reductions in several EMT-related genes. Moreover, strong

correlations between NRDC and EMT-related genes (ZEB1, SNAI1, and HIF-1α) were

recapitulated in resected cancerous tissue from ICC patients. Park et al. very recently demonstrated

25 that NRDC regulated EMT-related genes, including SNAI1, in colon cancer cells [37]; NRDC was

responsible for the insulin-like growth factor-1 (IGF-1)-induced regulation of EMT-related genes.

Together with our in vitro and in vivo data from ICC cells, NRDC appears to play important and

general roles in the regulation of EMT.

Several signaling pathways triggered, for example, by transforming growth factor-β (TGF-

β) and epidermal growth factor (EGF) as well as hypoxia may induce EMT [35,36]. These signals

5 lead to the activation of EMT-TF, such as ZEB1, SNAI1, and TWIST1, which directly or indirectly

control key EMT-related genes. Due to the multiple functions of NRDC, there are several

possibilities by which NRDC is involved in EMT. Extracellular NRDC may activate EGF or TNF

receptor signaling by enhancing the ectodomain shedding of EGF receptor ligands or TNF-α,

respectively [12,13,18]. Furthermore, nuclear NRDC may regulate the transcription of EMT-related

10 genes, including EMT-TF [38, 39]. While the underlying mechanisms have not yet been elucidated

in detail, the present results suggest that elevated NRDC levels in cancer tissue are associated with

EMT programs. Based on the positive correlation between serum and tumor NRDC levels, it may

be possible to assess the level of EMT features in primary tumors by measuring serum NRDCs.

In conclusion, this is the first study to demonstrate that serum NRDC has potential as a

15 novel prognostic biomarker for ICC, which may reflect EMT features in primary tumor regions. We

propose that the preoperative evaluation of serum NRDC is a potential clinical tool for predicting

tumor recurrence in and the overall prognosis of patients after curative-intent surgery.

Acknowledgments

We gratefully acknowledge the patients who consented to blood collection for this study. The

detection of serum NRDC was supported by Yoshiyuki Amano (Sanyo Chemical Industries). We

thank Takayuki Kawai, Takahiro Nishio, Masayuki Okuno, Seidai Wada, Asahi Sato, Yoshinobu

5 Ikeno, Yusuke Morita, Shintaro Matsuda, and Hiromi Iwai (Kyoto University) for their excellent

help. This study was financially supported by Grants-in-Aid KAKENHI (17H04048, 17K09575,

17K16147, and 18H04694) and AMED (JP17cm0106608). It was also supported by the Takeda

Science Foundation, SENSHIN Medical Research Foundation, and Suzuken Memorial Foundation.

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Table 1

Clinicopathological characteristics and surgical outcomes according to serum NRDC levels

NRDC low NRDC high Variables p value n = 39 n = 40

Clinical factors

Age (years) 69 (32-84) 67.5 (37-83) 0.677

Gender (Male) 23 (58.9%) 26 (65.0%) 0.581

Hepatitis B † 3 (7.7%) 1 (2.5%) 0.356

Hepatitis C † 7 (17.9%) 3 (7.5%) 0.193

CA19-9 (IU/ml) 35.2 (0-5461.1) 75.4 (0-1788.0) 0.111

CEA (ng/ml) 2.1 (0.4-23.7) 2.95 (0-116.6) 0.310

Histological factors

Tumor diameter (cm) 4.1 (1.0-9.0) 4.6 (1.0-14.0) 0.157

Multiple tumors 3 (7.7%) 13 (32.5%) 0.006*

LN metastasis 9 (23.1%) 12 (30.0%) 0.486

Poor differentiation† 2 (5.1%) 7 (17.5%) 0.154

Vascular invasion 20 (51.3%) 24 (60.0%) 0.314

Biliary invasion 18 (46.2%) 17 (42.5%) 0.744

AJCC T3/T4 22 (56.4%) 21 (52.5%) 0.727

AJCC stage III/IV 25 (64.1%) 27 (67.5%) 0.750

Surgical outcomes

R0 resection 32 (82.1%) 31 (77.5%) 0.615

Major hepatectomy (≥3 segments) 34 (87.2%) 35 (87.5%) 1.000

Adjuvant chemotherapy 16 (41.0%) 23 (59.0%) 0.143

Morbidity 17 (43.6%) 18 (45.0%) 0.900

Mortality (<30 days) † 0 (0%) 1 (2.5%) 1.000

* Significant difference p<0.05 † Fisher’s exact test and the χ2 test were used for all other categorical variables. Abbreviations: NRDC, nardilysin; CA19-9, carbohydrate antigen 19-9; CEA, Carcinoembryonic antigen; LN, lymph node; AJCC, American Joint Committee on Cancer; R0, no residual tumor

Table 2

Univariate and multivariate analyses of factors predicting postoperative prognosis

Univariate analysis Multivariate analysis Variables Hazard ratio p value p value (95% Confidence Interval)

Survival

High serum NRDC (versus low) 0.002* 2.047 (1.126-3.821) 0.019* LN metastasis (versus N0/Nx) 0.001* 1.942 (1.032-3.584) 0.040* Vascular invasion (versus negative) 0.033* - - Multiple tumors (versus solitary) <0.001* 2.548 (1.066-4.226) 0.033*

Poor differentiation (versus well/moderate) 0.176 - -

Tumor size ≥5 cm (versus <5 cm) 0.873 - -

Recurrence

High serum NRDC (versus low) <0.001* 2.017 (1.189-3.470) 0.009*

LN metastasis (versus N0/Nx) 0.042* - -

Vascular invasion (versus negative) 0.619 - -

Multiple tumors (versus solitary) <0.001* 2.397 (1.279-4.289) 0.008*

Poor differentiation (versus well/moderate) 0.026* - -

Tumor size ≥5 cm (versus <5 cm) 0.617 - -

* Significant difference p<0.05 Abbreviations: NRDC, nardilysin; LN, lymph node; N0, negative for nodal metastasis; Nx, nodal metastasis status undetermined

Figure Legends

Figure 1

5 Prognostic impact of serum NRDC levels in ICC patients after surgical resection

A. Comparison of serum NRDC levels in HC (n=112) and ICC patients in the primary (n=79) and

validation (n=19) cohorts.

B, C. Kaplan-Meier analyses for OS (B) and DFS (C) in 79 ICC patients in the primary cohort

10 according to serum NRDC levels.

D, E. Kaplan-Meier analyses for OS (B) and DFS (C) in 19 ICC patients in the external validation

cohort according to serum NRDC levels.

NRDC, nardilysin; ICC, intrahepatic cholangiocarcinoma; HC, healthy controls; OS, overall

15 survival; DFS, disease-free survival.

Figure 2

NRDC mRNA levels in resected cancerous tissue correlate with the prognosis of ICC patients

after surgical resection

A. Immunohistochemical staining of NRDC in surgical specimens of ICC. The scale bar represents

100 µm.

B. Relationship between serum NRDC and NRDC mRNA expression in cancer tissue.

C, D. Kaplan-Meier analyses for OS (C) and DFS (D) in 43 ICC patients according to NRDC

10 mRNA levels in resected cancerous tissue.

NRDC, nardilysin; ICC, intrahepatic cholangiocarcinoma; OS, overall survival, DFS, disease-free

survival

Figure 3

Effects of the gene knockdown of NRDC on the proliferation, migration, and chemosensitivity of

ICC cells

A. Stable NRDC-KD ICC cells were established. qRT-PCR and Western blotting were then

performed to confirm the expression of NRDC.

B. NRDC secreted in CM was decreased by the gene KD of NRDC in HuCCT-1 and SSP-25 cells.

C, D. Silencing NRDC attenuated cell proliferation in vitro and tumor growth in vivo (D).

10 E, F. Wound healing assays (E) and chemotaxis assays (F) demonstrated that the migratory ability

of HuCCT-1 cells was decreased by the gene KD of NRDC.

G. Effects of gemcitabine concentrations on the viability of HuCCT-1 cells. Silencing NRDC

increased chemosensitivity.

Data represent the mean ± SD of at least three independent experiments; *p<0.05. **p<0.01.

NRDC, nardilysin; ICC, intrahepatic cholangiocarcinoma; KD, knockdown; CM, condition medium

Figure 4

Relationship between NRDC and epithelial-mesenchymal transition (EMT)-related genes in ICC

A, B. qRT-PCR and Western blot analyses were performed to assess the mRNA and protein levels

of EMT-related genes in NC versus NRDC KD2 in HuCCT-1 cells (A) and SSP-25 cells (B). Data

represent the mean ± SD of at least three independent experiments; *p<0.05. **p<0.01. In Western

blotting, representative images are selected from 3 independent experiments, in which similar

10 results were obtained.

C. Correlation analysis between the mRNA levels of NRDC and EMT-related genes (ZEB1,

SNAI1, and HIF-1α) in surgical specimens from ICC patients.

D. Correlation analysis between the serum NRDC and mRNA levels of EMT-related genes (ZEB1,

SNAI1, and HIF-1α) in surgical specimens from ICC patients.

NRDC, nardilysin; EMT, epithelial-mesenchymal transition; ICC, intrahepatic cholangiocarcinoma;

NC, negative control; KD, knockdown; SD, standard deviation

Serum nardilysin, a surrogate marker for epithelial-mesenchymal transition, predicts prognosis of intrahepatic cholangiocarcinoma after surgical resection

Tomoaki Yoh, Etsuro Hatano, Yosuke Kasai, et al.

Clin Cancer Res Published OnlineFirst October 23, 2018.

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