This article is mentioned in:

Introduction

Breast cancer is classified into several intrinsic subtypes based on gene expression profiles (1). Different subtypes, including luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) and basal-like breast cancer, have been indicated to express different biological characteristics (2). Pathological examination of breast cancer samples is used to detect the expression of estrogen receptor (ER), progesterone receptor (PgR), HER2 and Ki67, in order to select suitable therapeutic strategies in the clinic (3).

Triple-negative breast cancer (TNBC), which is character-ized by the absence of ER, PgR, and HER2, exhibits a relatively aggressive phenotype, therapeutic resistance and is associated with a poor prognosis (4). TNBC is associated with phenotypes of cancer stem cells (CSCs) (5,6) and epithelial-mesenchymal transition (EMT), which is characterized by the downregulation of epithelial markers and mesenchymal phenotypes with high migration ability (7).

Carboxypeptidase A4 (CPA4) catalyzes the release of carboxy-terminal amino acids (8), and its overexpression has been associated with cancer progression in several types of cancer (9-14). Furthermore, CPA4 has been indicated to be secreted in higher amounts from breast cancer cells compared with noncancerous mammary epithelial cells (15). However, few studies have addressed the association between CPA4 expression and clinicopathological factors in patients with breast cancer, including TNBC.

The purpose of the present study was to clarify the significance of CPA4 expression and function in breast cancer. To this end, immunohistochemical analysis was performed to evaluate the association between CPA4 expression and clinicopathological markers, including ER, PgR, HER2, CD44, CD24, aldehyde dehydrogenase 1 (ALDH1), E-cadherin, EGFR, cytokeratin 5/6 (CK5/6), claudins, vimentin and androgen receptor (AR) in 221 breast cancer cases. In addition, the in vitro effects of small interfering RNA (siRNA)-mediated CPA4 knockdown on the viability and migration ability of human TNBC cell lines was examined.

Materials and methods

Cell lines

In the present study, cell lines representative of cancer types were selected as described previously (16): MCF7 and T47D for luminal A type; BT474 for luminal B type; and MDA-MB468, MDA-MB231, HCC70 and HCC1143 for TNBC. The human breast cancer cell lines were obtained from the Riken Cell Bank (MCF7; Riken BioResource Research Center, Tsukuba, Japan) and from the American Type Culture Collection (T47D, BT474, MDA-MB468, MDA-MB231, HCC70 and HCC1143; Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 100 U/ml of penicillin, 100 U/ml of streptomycin and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere containing 5% CO2 at 37°C.

Data mining for new molecular target therapy candidates in TNBC

Four genes were identified as candidates for molecular target therapy in TNBC using whole transcriptome analysis and a lentiviral shRNA-screening library. For transcriptome analysis, total RNA was prepared from cell lines (MCF7, BT474, T47D, MDA-MB468, HCC70 and HCC1143) using NucleoSpin RNA (Takara Bio, Inc., Shiga, Japan). The quality of the RNA was assessed using the RNA integrity number (RIN) obtained by the Agilent RNA6000 Pico Kit and the Agilent 2100 Bioanalyzer (both Agilent Technologies, Santa Clara, CA, USA). Samples used for transcriptome analysis had, on average, an RIN value of 9.4 and a minimum RIN value of 8.9. The library was prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, Inc., San Diego, CA, USA) from 1 µg of total RNA according to the manufacturer's protocol. The resulting libraries were subjected to single-end sequencing of 76-bp reads using the NextSeq 500 High Output v1 Kit on the Illumina NextSeq 500 system (both from Illumina, Inc.). Data processing and analyses were performed using the TopHat version 2 alignment, cufflinks assembly and differential expression apps (all from Illumina, Inc.). Briefly, the reads were filtered, trimmed and aligned in the UCSC reference human genome 19 (hg19) using the Tophat2 (v2.0.7) and Bowtie1 (0.12.9) pipelines. The transcripts were assembled using Cufflinks 2.1.1, and differentially expressed transcripts were detected using Cuffdiff 2.1.1. Genes with a false discovery rate-adjusted q-value of <0.05 and log2-fold change (TNBC/non-TNBC) of >5 were defined as significantly upregulated genes in TNBC cells.

MISSION LentiPlex Human Pooled shRNA Library (SHPH01-1SET; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) is a genome-wide shRNA library that covers ~15,000 genes, with 80,000 shRNA clones. In order to identify shRNAs that targeted genes able to specifically destroy TNBC cell lines compared with non-TNBC cell lines, a mixture of lentiviral particles (SHPH01-1SET; Sigma-Aldrich; Merck KGaA) was introduced to the breast cancer cell lines of non-TNBC (MCF7, T47D and BT474) and TNBC (MDA-MB468, HCC70 and HCC1143) subtypes at multiplicity of infections that yielded 30-50% infected cells according to the manufacturer's protocol. The infected cells were subsequently selected using puromycin for 7 days, and genomic DNA with integrated shRNA was isolated from the cell lines. Thirty cycles of polymerase chain reaction (PCR) was performed using KAPA HiFi HotStart ReadyMix (2X) (KAPA Biosystems; Roche Diagnostics, Indianapolis, IN, USA). The following primers were used for the PCR of the shRNA vector: 5′-ATTTCTTGGCTTTATATATCTTGTGGAAAG-3′ (sense) and reverse primer, 5′-TGTGGATGAATACTGCCATTTGT CTC-3′ (antisense). The PCR were performed on 3-µg genomic DNAs. PCR conditions consisted of initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 98°C for 20 sec, annealing at 60°C for 15 sec and elongation at 72°C for 30 sec. Sequencing adaptors (BIOO Scientific, Austin, TX, USA) were ligated to the PCR amplicons with 8 cycles of PCR and Illumina sequencing was performed for 150 cycles on an Illumina NextSeq500 sequencer with 10% PhiX control DNA (Illumina, Inc.). Data processing was performed in accordance with the manufacturer's protocol. Briefly, FASTQ files were used to trim and align the adapter sequences to the reference shRNA sequences using Bowtie2. The Bowtie output files were converted to count files through a python script 'countBowtieHits.py' and 'RTable.py', which were obtained from the manufacturer's protocol. TCC-edgeR was used for normalization and statistical analysis as described previously (17).

The Reference Expression dataset (RefEx: http://refex.dbcls.jp) was used to examine the expression levels of CPA4 in several noncancerous tissues using RNA sequencing methods (http://refex.dbcls.jp). Specific low-expression genes in 40 normal organs were picked up as maximum fragments per kilobase of transcript per million mapped reads of <1.0. The present transcriptome data were submitted to a public repository, the Gene Expression Omnibus (accession no. GSE113653).

The Cancer Genome Atlas database (cBioPortal Breast Cancer: METABRIC, Nature 2012 and Nat Commun 2016: http://www.cbioportal.org) was used to validate the prognostic significance of CPA4 in patients with breast cancer.

Patients and samples

Tumor specimens from 221 patients with primary breast cancer who underwent surgery for excision of a primary tumor between January 1999 and October 2010 at the Gunma University Hospital (Maebashi, Japan) were retrospectively analyzed. The median follow-up period for survivors was 118 months. Some data from patients with ductal carcinoma in situ, preoperative chemotherapy, preoperative hormone therapy and male breast carcinoma from the present tissue microarray preparations were excluded. Tumor staging was based on the Union for International Cancer Control TNM classification (seventh edition) (18). Nuclear grades (NGs) were defined as the sum of scores for the nuclear atypia, as described previously (19). The present research conformed to the tenets of the Declaration of Helsinki and to the guidelines of the Gunma University Ethical Review Board for Medical Research Involving Human Subjects (approval no. 1297).

Immunohistochemistry (IHC)

For tissue microarray, clinical formalin-fixed paraffin-embedded samples were stored in the archives of the Clinical Department of Pathology, Gunma University Hospital (Maebashi, Japan). For each patient, one paraffin block containing representative non-necrotic tumor areas was selected. Breast cancer tissue cores (2.0-mm diameter per tumor) were punched out from the representative areas near the invasive front and transferred into the paired recipient paraffin block using a tissue array instrument (Beecher Instruments, Inc., Silver Spring, MD, USA).

For IHC, a 4-µm section was cut from the sample paraffin blocks. Each section was mounted on a silane-coated glass slide, deparaffinized and soaked for 30 min at room temperature in 0.3% H2O2/methanol to block endogenous peroxidases. The sections were subsequently heated in boiled 10 mM citrate buffer (pH 6.0) at 98°C for 30 min. Non-specific binding sites were blocked by incubating with 0.25% Casein/1% bovine serum albumin (Code 81-003-3; EMD Millipore, Kankakee, IL, USA) for 30 min at room temperature. A rabbit polyclonal anti-CPA4 antibody (HPA021030; Sigma-Aldrich; Merck KGaA) was applied at a dilution of 1:400 for 24 h at 4°C. A MAX-PO secondary antibody from the Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei Corporation, Tokyo, Japan) was used for 30 min at room temperature according to the manufacturer's instructions. The chromogen 3,3-diaminobenzidine tetrahydrochloride (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was applied as a 0.02% solution containing 0.005% H2O2 in 50 mM Tris-HCl buffer (pH 7.6). The sections were lightly counterstained with Mayer's hematoxylin and mounted. Negative controls were established by omitting the primary antibody. Other IHC was performed using the following primary antibodies: Anti-ER (ready to use; SP1), anti-PgR (ready to use; 1E2), anti-HER2 (ready to use; 4B5), anti-Ki67 (ready to use; 30-9) (Ventana Medical Systems, Inc., Tucson, AZ, USA), anti-EGFR (ready to use; 31G7; Nichirei Corporation), anti-CK5/6 (1:50; 5/16 B4; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), anti-E-cadherin (ready to use; 36; Ventana Medical Systems, Inc.) anti-ALDH1 (1:400; 46/ALDH; BD Biosciences, San Jose, CA, USA), anti-CD44 (1:50; DF1485; Dako; Agilent Technologies, Inc.), anti-CD24 (1:40; SN3b; Thermo Fisher Scientific, Inc.), AR (1:200; AR441; Thermo Fisher Scientific, Inc.), Claudin1 (1:50; 1449527A; Invitrogen; Thermo Fisher Scientific, Inc.), Claudin3 (1:100; GR253635-1; Abcam, Cambridge, UK), Claudin4 (1:50; 3E2C1; Invitrogen; Thermo Fisher Scientific, Inc.), Claudin7 (1:50; GR212894-5; Abcam) and Vimentin (ready to use; V9; Dako; Agilent Technologies, Inc.). A Ventana BenchMark XT system (Roche Diagnostics, Basel, Switzerland) was used for ER, PgR, HER2, Ki67 and E-cadherin. All sections were examined under a BX43 light microscope (Olympus Corporation, Tokyo, Japan).

Immunohistochemical evaluation and intrinsic subtype

CPA4 expression was deemed positive in cells with stained cytoplasms. In addition, the cutoff value for CPA4 positivity was 20% (14). The cutoff value for ER and PgR positivity was 1%. HER2 expression was scored according to the American Society of Clinical Oncology/College of American Pathologists guideline (20). Ki67 labeling index was used to calculate the percentage of cells with high nuclear expression in ~1,000 cells per sample, as described previously (21). EGFR expression was scored in the same manner as HER2 expression; scores of 0 and 1+ were considered as negative, and 2+ and 3+ as positive. A cutoff value of 10% for CK5/6, ALDH1, CD44, CD24, claudin and AR was used (19,22). The cutoff value for high E-cadherin expression was set as >70% (23).

Based on the IHC results, the breast cancer subtypes were defined as follows: Luminal A-like (ER or PgR+ and HER2 0/1+/2+), luminal B-like (ER or PgR+ and HER2 3+), HER2-like (ER-, PgR- and HER2 3+) and TNBC-like (ER-, PgR- and HER2 0/1+/2+).

Fluorescent IHC

The sections were prepared, and endogenous peroxidase was blocked as described above. Antigen retrieval was performed by heating samples in boiled Immunosaver Antigen Activator (Nisshin EM Co., Ltd., Tokyo, Japan) at 98°C for 45 min and stripping was performed by heating in boiled 10 mM citrate buffer (pH 6.0) at 98°C 15 min in a microwave. Nonspecific binding sites were blocked as described above (19). The sections were incubated with primary antibodies CPA4 (1:400) and E-cadherin (1:500) overnight at 4°C or for 1 h at room temperature, respectively. The secondary antibody was used as described for the protocol for IHC. Multiplex covalent labeling was performed (CPA4; Fluorescein, E-cadherin; Cyanine 3) with tyramide signal amplification (Opal 3-Plex Kit; PerkinElmer, Inc., Waltham, MA, USA) according to the manufacturer's protocol. All sections were counterstained with 4′,6-diamidino-2-phenylin-dole at room temperature (Opal 3-Plex Kit) and examined under an All-in-One BZ-X710 fluorescence microscope (Keyence Corporation, Osaka, Japan).

CPA4 small interfering RNA (siRNA) transfection

CPA4-specific siRNAs #1 (5′-CCAAAGAACAUCUGAGAUGtt-3′) and siRNA #2 (5′-CAGCAAAUCUUGUAGGGAUtt-3′) and negative-control siRNA were purchased from GeneDesign, Inc. (Osaka, Japan) and Bonac Corporation (Fukuoka, Japan). MDA-MB231 and MDA-MB468 at a density of 1×106 cells/well were seeded in 100 µl of Opti MEM Reduced Serum Medium (Invitrogen; Thermo Fisher Scientific, Inc.). In total, 20 nM of CPA4-specific siRNAs 1 and 2, and scrambled siRNA (as a negative control) were used to treat cells; siRNAs was transfected using an electroporator CUY-21 EDIT II (Bex Co., Ltd., Tokyo, Japan) according to the manufacturer′s instructions. The subsequent experiments were performed following 24 h of transfection.

Western blot analysis

The proteins were extracted using lysis buffer [10 mM Tris-HCl (pH 7.5), 1 mM ethylene-diaminetetraacetic acid, 10% glycerol, 0.5% NP-40 detergent, 400 mM NaCl, 4 µg/ml of aprotinin, phenylmethylsulfonyl fluoride and dithiothreitol]. The BCA protein assay was used for the protein determination. Total protein (10 µg loaded per lane) was separated using SDS-PAGE (on a 10% polyacrylamide gel), at 300 mA for 90 min and transferred on a nitrocellulose membrane (Invitrogen; Thermo Fisher Scientific, Inc.). Blocking of the membrane was performed using 4% skimmed milk for 60 min at room temperature. The protein expression levels of CPA4, E-cadherin and β-actin were assessed using western blot analysis. These proteins were detected using specific antibodies to CPA4 (1:200; HPA021030; Sigma-Aldrich; Merck KGaA), E-cadherin (1:1,000; M106; Takara Bio, Inc.), and β-actin (1:1,000; #3700; Cell Signaling Technology, Inc.). The membrane was incubated in primary antibodies for overnight at 4°C. β-actin served as a loading control. ECL anti-mouse or anti-rabbit IgG, peroxidase-linked whole antibody was used for secondary antibody (GE Healthcare Life Sciences, Little Chalfont, UK). The signals were detected using the ECL Western Blotting Detection System and an Image Quant LAS 4000 machine (GE Healthcare Life Sciences).

Proliferation assay

Cell proliferation analyses were performed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Breast cancer cell line cells were plated onto 96-well plates in 100 µl of RPMI-1640 medium containing 10% FBS, at ~3,000 cells per well following siRNA transfection. MDA-MB231 and MDA-MB468 cells were evaluated at 48 and 72 h post-CPA4 siRNA treatment, respectively. For quantifying cell proliferation, 10 µl of CCK-8 solution was added to each well and incubated at 37°C for 2 h. Following this, the absorbance of each well at 450 nm was detected using a plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Migration assay

Cell migration was examined using MDA-MB231 and MDA-MB468 cells transfected with a negative control or CPA4 siRNAs. The transfected breast cancer cells were grown in 6-well plates until 100% confluence, and a uniform straight wound was produced in the monolayer in each well using a pipette tip. Wells were washed with phosphate-buffered saline to remove all cell debris Cells were cultured in an atmosphere containing 5% CO2 at 37°C. Closure or filling of the wound was evaluated at 3 and 24 h in MDA-MB231 and MDA-MB468 plates, respectively.

Statistical analysis

Statistical analyses were performed using the Wilcoxon test for continuous variables, and the χ2 test and one-way analysis of variance for categorical variables. Survival curves were generated according to the Kaplan-Meier method. The differences between survival curves were examined using the log-rank test. Cox proportional hazard regression was used to examine the independent prognostic contribution of standard prognostic variables, including CPA4 expression, age, tumor factor, NG, lymph node metastasis (LNM), venous invasion and adjuvant therapy. P<0.05 was considered to indicate a statistically significant difference. JMP software version 12 (SAS Institute, Cary, NC, USA) was used to analyze all statistical analyses.

Results

Identification of CPA4 as a novel therapeutic target according to the shRNA library and transcriptome analysis in breast cancer cells

Among 23,609 genes, 171 that were highly expressed in TNBC compared with their expression in non-TNBC cell lines were selected for transcriptome analysis (Fig. 1A). Following this, 86 of the 171 genes, in which suppression was indicated to have inhibited the viability of TNBC cell lines according to the shRNA library, were selected (Fig. 1B). Finally, 4 genes that were not expressed in normal organs according to a public database were selected (Fig. 1C). It was deduced that these 4 candidates were associated with TNBC viability because they were highly expressed in the TNBC cells compared with non-TNBC cells and normal organs. In the present study, CPA4 was investigated in order to discover a novel molecular target against TNBC.

Figure 1 Identification of new candidate genes that can target TNBC. The new molecular candidates targeting TNBC were selected from 23,609 genes using (A) transcriptome analysis in breast cancer cell lines, (B) shRNA library targeting the TNBC cell lines and (C) a public database of transcriptome analysis in normal tissues. The four candidates selected were associated with TNBC viability and expressed highly in TNBC cells compared with their expression in non-TNBC cells and in normal tissues. TNBC, triple-negative breast cancer; CPA4, carboxypeptidase A4; MUC4, mucin 4; PROM1, prom-inin 1; CD109, cluster of differentiation 109.

Immunohistochemical analysis of CPA4 expression in breast cancer

Cytoplasmic expression of CPA4 in normal breast tissues was decreased compared with breast cancer tissues (Fig. 2). Out of the 221 breast cancer samples, 153 specimens (69.2%) were assigned to the low CPA4 expression group (Fig. 2B) and 68 specimens (30.8%) were assigned to the high CPA4 expression group (Fig. 2C and D).

Figure 2 Immunohistochemical CPA4 and E-cadherin analyses in representative breast tissue samples. (A) Low CPA4 expression in normal breast tissue. Scale bar, 100 µm. (B) Low CPA4 expression in breast cancer tissue. Scale bar, 100 µm. (C) High CPA4 expression in breast cancer tissue (low-power field). Scale bar, 500 µm. (D) High CPA4 expression in breast cancer tissue (high-power field). Scale bar, 100 µm. (E) High CPA4 expression (left panel) and low E-cadherin expression (right panel) in serial TNBC sections using immunohistochemical analysis. Scale bar, 100 µm. (F) Fluorescent immunohistochemical analyses of CPA4 and E-cadherin expression in the representative TNBC tissue. TNBC tissues were immunostained with antibodies against CPA4 (green) and E-cadherin (red). This section was counterstained with DAPI (blue). Scale bar, 100 µm. TNBC, triple-negative breast cancer; CPA4, carboxypeptidase A4; DAPI, 4′,6-diamidino-2-phenylindole.

Association between CPA4 expression and breast cancer clinicopathological features

The association between CPA4 expression and clinicopathological characteristics of the patients were indicated (Table I). Notably, no association was identified between CPA4 expression and clinicopathological characteristics in the 221 breast cancer cases. Furthermore, the association between CPA4 expression levels with breast CSCs markers (ALDH1, CD44 and CD24), EMT markers (vimentin and E-cadherin) and the AR was examined using immunohistochemical staining. An association between high CPA4 expression and high ALDH1 expression was noted (Table II, P=0.027). However, only a few cases exhibited high ALDH1 expression.

Table I Patient characteristics and CPA4 expression in 221 breast cancer cases.

Table I

Patient characteristics and CPA4 expression in 221 breast cancer cases.

Clinical factors	Total breast cancer cohort (n=221)		P-value
	Low CPA4 (n=153)	High CPA4 (n=68)
Age (years)	55.0±12.49	56.7±12.52	0.371
Subtype
Luminal A	89	37	0.435
Luminal B	4	0
HER2	14	9
TNBC	46	22
ER
Negative	62	31	0.482
Positive	91	37
PgR
Negative	80	44	0.086
Positive	73	24
HER2
Score 0, 1+, 2+	135	59	0.758
Score 3+	18	9
EGFR
Score 0, 1+	139	64	0.412
Score 2+, 3+	14	4
CK5/6
Negative	145	66	0.45
Positive	8	2
Ki67 labeling index	21.2±24.22	13.8±15.50	0.068
Tumor size
<2 cm	63	29	0.838
≥2 cm	90	39
Tumor stage
1	70	35	0.881
2	74	29
3	7	3
4	2	1
Lymph node metastasis
Negative	89	48	0.079
Positive	64	20
Metastasis
Negative	152	68	0.504
Positive	1	0
Stage (UICC, 7th Ed)
I	49	28	0.353
II	73	32
III	30	8
IV	1	0
Lymphatic invasion
Negative	46	25	0.325
Positive	107	43
Venous invasion
Negative	103	51	0.252
Positive	50	17
NG
NG1	33	12	0.428
NG2	39	23
NG3	81	33

[i] NG, nuclear grade; CK5/6, cytokeratin; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; PgR, progesterone receptor; ER, estrogen receptor; CPA4, carboxypeptidase A4; TNBC, triple-negative breast cancer; UICC, Union for International Cancer Control.

Table II CPA4 expression in stem cell markers, epithelial-mesenchymal transition markers and AR of 221 breast cancer cases.

Table II

CPA4 expression in stem cell markers, epithelial-mesenchymal transition markers and AR of 221 breast cancer cases.

Clinical factors	Total breast cancer cohort (n=221)		P-value
	Low CPA4 (n=153)	High CPA4 (n=68)
ALDH1
Low	147	60	0.027a
High	6	8
CD44/CD24
High/low	83	43	0.213
Others	70	25
Claudin1
Negative	107	40	0.106
Positive	46	28
Claudin3
Negative	112	46	0.206
Positive	37	22
Unknown	4
Claudin4
Negative	73	23	0.055
Positive	80	45
Claudin7
Negative	82	33	0.511
Positive	68	32
Unknown	3	3
E-cadherin
Low	95	41	0.800
High	58	27
Vimentin
Negative	121	55	0.463
Positive	29	10
Unknown	3	3
AR
Negative	66	26	0.755
Positive	82	39
Unknown	5	3

a P<0.05. ALDH1, aldehyde dehydrogenase 1; CD, cluster of differentiation; AR, androgen receptor; CPA4, carboxypeptidase A4.

Clinicopathological factors were significantly different in 22/68 TNBC cases in the high CPA4 expression group (Tables III and IV). TNBC with high CPA4 expression was associated with low Ki67 expression and the expression of CD44/CD24 (Table III, P=0.011; and Table IV, P=0.016, respectively). The association between CPA4 and E-cadherin, a representative epithelial marker (7), was also assessed. High CPA4 expression was associated with low E-cadherin expression (Table IV, P=0.016). The association between high CPA4 and low E-cadherin expression was validated in representative TNBC sections using IHC (Fig. 2E and F). No other significant differences in the other evaluated factors were indicated.

Table III Patient characteristics and CPA4 expression in 68 TNBC cases.

Table III

Patient characteristics and CPA4 expression in 68 TNBC cases.

Clinical factors	Total TNBC cohort (n=68)		P-value
	Low CPA4 (n=46)	High CPA4 (n=22)
Age (years)	56.3±11.4	61.5±14.9	0.119
Basal-like type
Basal	15	5	0.403
Non-basal	31	17
Ki67 labeling index	41.5±30.35	22.4±22.6	0.011a
Tumor size
<2 cm	21	8	0.469
≥2 cm	25	14
Tumor stage
1	23	10	0.532
2	21	12
3	2	0
4	0	0
Lymph node metastasis
Negative	28	17	0.181
Positive	18	5
Metastasis
Negative	46	22	N.D.
Positive	0	0
Stage (UICC, 7th Ed)
I	17	8	0.302
II	21	13
III	8	1
IV	0	0
Lymphatic invasion
Negative	16	11	0.230
Positive	30	11
Venous invasion
Negative	30	15	0.809
Positive	16	7
NG
NG1	3	3	0.567
NG2	5	3
NG3	38	16

a P<0.05. NG, nuclear grade, TNBC, triple-negative breast cancer; CPA4, car-boxypeptidase A4; UICC, Union for International Cancer Control.

Table IV CPA4 expression in stem cell markers, epithelial-mesenchymal transition markers and AR of 68 TNBC cases.

Table IV

CPA4 expression in stem cell markers, epithelial-mesenchymal transition markers and AR of 68 TNBC cases.

Clinical factors	Total TNBC cohort (n=68)		P-value
	Low CPA4 (n=46)	High CPA4 (n=22)
ALDH1
Low	41	16	0.086
High	5	6
CD44/CD24
High/low	15	14	0.016a
Others	31	8
Claudin1
Negative	18	8	0.826
Positive	28	14
Claudin3
Negative	26	12	0.760
Positive	19	10
Unknown	1
Claudin4
Negative	7	6	0.237
Positive	39	16
Claudin7
Negative	20	10	0.782
Positive	25	12
Unknown	1
E-cadherin
Low	17	15	0.016a
High	29	7
Vimentin
Negative	26	14	0.256
Positive	20	7
Unknown		1
AR
Negative	33	14	0.741
Positive	12	7
Unknown	1	1

a P<0.05. ALDH1, aldehyde dehydrogenase 1; CD, cluster of differentiation; AR, androgen receptor; CPA4, carboxypeptidase A4; TNBC, triple-negative breast cancer.

Prognostic significance of CPA4 expression in patients with breast cancer

The overall and disease-free survival were not significantly associated with CPA4 expression (Fig. 3A and B; P=0.19 and P=0.49, respectively). However, the overall and disease-free survival intervals of high-CPA4-expressing TNBC cells were worse compared with those of low-CPA4-expressing TNBC cells (Fig. 3C and D; P=0.004 and P=0.017, respectively).

Figure 3 Kaplan-Meier analysis of CPA4 expression in the present breast cancer cohort. (A) Overall survival curves according to CPA4 expression in breast cancer (n=221, P=0.19). (B) Disease-free survival curves according to CPA4 expression in patients with breast cancer (n=221, P=0.49). (C) Overall survival curves according to CPA4 expression in patients with TNBC (n=68, P=0.0042). (D) Disease-free survival curves according to CPA4 expression in patients with TNBC (n=68, P=0.017). TNBC, triple-negative breast cancer; CPA4, carboxypeptidase A4.

Using a public database cohort, the prognostic significance of CPA4 expression on overall survival was validated (Fig. 4). As expected, the patients with TNBC and high CPA4 expression exhibited poorer prognoses compared with those with low CPA4 expression; however, the differences were not significant (Fig. 4A and B; P=0.720 and P=0.078, respectively).

Figure 4 Kaplan-Meier analysis of CPA4 expression in the public database cohort. (A) Overall survival curves according to CPA4 expression in patients with breast cancer from TCGA (cBioPortal Breast Cancer: METABRIC, Nature 2012 and Nat Commun 2016: http://www.cbioportal.org) (n=1,904, P=0.72).(B) Overall survival curves according to CPA4 expression in patients with TNBC from TCGA (n=299, P=0.078). CPA4, carboxypeptidase A4; TCGA, The Cancer Genome Atlas.

The association between CPA4 expression and other factors was also explored, including the NG and LNM (Fig. 5). Among NG1-2 and LNM-negative cases, the overall and disease-free survival were not significantly associated with CPA4 expression (Fig. 5A, B, E and F; P=0.39, P=0.66, P=0.29 and P=0.16, respectively). However, among NG3 cases and LNM-positive cases, the overall and disease-free survival intervals of highly expressed CPA4 TNBC cells were worse than those of low CPA4 TNBC cells (Fig. 5C, D, G and 5H; P=0.0002, P=0.019, P=0.0004 and P=0.015, respectively). To clarify the prognostic significance of CPA4, multivariate analysis was performed for survival, and results indicated that high CPA4 expression was an independent prognostic factor for poor survival (Table V; P=0.001).

Figure 5 Kaplan-Meier curves analysis of CPA4 expression in 68 patients with TNBC, with or without progression of NG and LNM. (A) Overall survival in patients with NG1-2 and high or low CPA4 expression (n=14, P=0.39). (B) Disease-free survival in patients with NG1-2 and high or low CPA4 expression (n=14, P=0.66). (C) Overall survival in patients with NG3 and high or low CPA4 expression (n=54, P=0.0002). (D) Disease-free survival in patients with NG3 and high or low CPA4 expression (n=54, P=0.019). (E) Overall survival in LNM-negative patients with high or low CPA4 expression (n=45, P=0.29).(F) Disease-free survival in LNM-negative patients with high or low CPA4 expression (n=45, P=0.16). (G) Overall survival in LNM-positive patients with high or low CPA4 expression (n=23, P=0.0004). (H) Disease-free survival in LNM-positive patients with high or low CPA4 expression (n=23, P=0.015). NG, nuclear grade; LNM, lymph node metastasis; CPA4, carboxypeptidase A4.

Table V Cox univariate/multivariate regression analysis of variables associated with overall survival in patients with TNBC.

Table V

Cox univariate/multivariate regression analysis of variables associated with overall survival in patients with TNBC.

Clinicopathologic variables	Univariate analysis			Multivariate analysis
	RR	95% Cl	P-value	RR	95% a	P-value
CPA4 expression (Low vs. high)	3.43	1.38-15.02	0.007a	30.38	3.44-1071.53	0.001a
Age (<58 vs. 58≤)	1.63	0.71-4.43	0.250	155	0.09-29.95	0.750
Tumor factor CT1 vs.T2-T3)	1.4	0.62-3.76	0.420	2.99	0.23-103.06	0.430
Nuclear grade (NG1-2 vs.NG3)	1.02	0.41-4.48	0.970	0.37	0.01-10.92	0520
Lymph node metastasis (Absent vs. present)	1.32	0.56-3.08	0.510	46.02	152-18844.18	0.020a
Lymphatic invasion (Absent vs. present)	0.81	0.35-1.89	0.610	_	_	_
Venous invasion (Absent vs. present)	1.03	0.38-2.33	0.950	053	0.02-10.29	0.690
Adjuvant therapy (Absent vs. present)	051	0.22-1.37	0.160	0.04	0.0006-0.77	0.030a

a P<0.05. RR, relative risk, CI, confidence interval; CPA4, carboxypeptidase A4; TNBC, triple

Cell viability and migration inhibition in CPA4-knockdown TNBC cells

The role of CPA4 on cell viability and migration ability was assessed using RNA interference. Western blot analysis was performed to validate the CPA4 knockdown experiments in TNBC cell lines treated with specific CPA4 siRNAs (Fig. 6A and B). In addition, it was identified that cell viability in the CPA4 siRNA group was significantly inhibited compared with that in the negative-control group (Fig. 6C, P<0.05). Furthermore, CPA4 knockdown suppressed cell migration in comparison with the negative-control cells (Fig. 6D, P<0.05).

Figure 6 Functional analysis of CPA4 by RNA interference. (A and B) Expression of CPA4 and E-cadherin in MDA-MB231 and MDA-MB468 cells trans-fected with CPA4 siRNA using western blotting. (C) Proliferation potency in MDA-MB231 and MDA-MB468 cells transfected with CPA4 siRNA was assessed using the Cell Counting Kit-8 assay. (D) Migration assay in MDA-MB231 and MDA-MB468 cells transfected with CPA4 siRNA assessed by the wound-healing assay (Original magnification, ×20). CPA4, carboxypeptidase A4; siRNA, small interfering RNA; NC, negative control.

Suppression of E-cadherin in CPA4-suppressed TNBC cells

The association between CPA4 and the expression of E-cadherin was examined. The expression of E-cadherin was increased in CPA4 siRNA groups (Fig. 6A and B). These data were consistent with the inverse association identified between CPA4 and E-cadherin expression in clinical TNBC samples.

Discussion

Lehmann et al (24) indicated that TNBC can be classified according to the gene expression profiles. The subtypes include basal-like1, basal-like2, immunomodulatory, mesen-chymal-like, mesenchymal stem-like, and luminal androgen receptor (LAR) types. Among these subtypes, the present analyses indicated that high CPA4 expression in TNBC was independent of the following characteristics: Basal-like subtypes with high proliferation ability and LAR subtypes with LAR accumulation. Furthermore, the present results indicated E-cadherin upregulation and reduced-migration ability following CPA4 knockdown. The CPA4 accumulation in TNBC tissues were identified to be associated with low E-cadherin expression, high CSC marker expression and a poor prognosis. Furthermore, the data indicated that CPA4 may be a regulator of EMT and CSCs in TNBC cells. Therefore, it was suggested that CPA4 in TNBC may be associated with the mesenchymal-like or mesenchymal stem-like subtypes, with CSC and/or EMT phenotypes. Tanco et al (8) reported that proneurotensin, a precursor of neurotensin, is a substrate of CPA4 using kinetic analysis. Their study revealed that neuro-tensin production was regulated by CPA4 enzyme activity. Notably, neurotensin has also been reported to be associated with EMT induction (25). From these observations, it was suggested that CPA4-mediated EMT may be induced via neurotensin activation via CPA4 enzymes.

The present study was performed to identify therapeutic targets for TNBC, as patients with TNBC lack treatment options available that are readily available options for patients with other types of breast cancer, including hormone therapy and molecularly targeted therapies (26). From the present data, CPA4 expression in normal tissues, including mammary glands and non-TNBC tissues, was significantly decreased compared with that in TNBC tissues. Furthermore, in the present cohort, CPA4 expression in patients with breast cancer was not significantly associated with patient prognosis. However, patients with TNBC and high CPA4 expression had a poorer prognosis compared with those with low CPA4 expression. To the best of our knowledge, the present study is the first to report that high expression of CPA4 may be a specific predictor of poor prognosis for patients with TNBC.

Suppression of CPA4 significantly reduced the cell viability in TNBC cell lines the present study, and previous data has also suggested that CPA4 inhibitors may successfully inhibit cancer cell viability in patients with TNBC (27). The carboxypeptidase inhibitor Sabellastarte magnifica (SmCI) has been reported to suppress the metallo-carboxypeptidase activity of CPA4 by forming a complex with CPA4 (27). SmCI inhibits metallo-carboxypeptidases and serine proteases, such as trypsin and elastase (28). Serine protease is a known promising molecular target in TNBC cells (29). Thus, these findings suggest that targeting CPA4 using inhibitors, such as SmCI, in patients with TBNC may be effective in reducing cancer cell viability via the inhibition of metallo-carboxypeptidases and serine proteases. Therefore, further studies are required before SmCI or other candidate drug trials can be implemented for future clinical applications.

In conclusion, the present results indicated that high CPA4 expression is a powerful marker for poor prognosis and aggressive phenotypes, such as EMT, in TNBC. The present results suggest that targeting CPA4 in TNBC may be a promising therapeutic strategy for controlling aggressive phenotypes in refractory TNBC.

Funding

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS; grant nos. 15K08339, 15K10085, 17K19893 and 18K07665).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

TH, AK, TY, HK and TO contributed to the study design and wrote the manuscript. AY, TF, SO, SK, RKI, NG, MN, TA and KS performed sample collection and data analysis. RKI performed transcriptome analysis.

Ethics approval and consent to participate

This research conformed to the tenets of the Declaration of Helsinki and to the guidelines of the Gunma University Ethical Review Board for Medical Research Involving Human Subjects (approval no. 1297). Patient consent was obtained.

Patient consent for publication

Patient consent was obtained.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

References