Frequent downregulation of LRRC26 by epigenetic alterations is involved in the malignant progression of triple-negative breast cancer
- Authors:
- Published online on: March 5, 2018 https://doi.org/10.3892/ijo.2018.4301
- Pages: 1539-1558
Abstract
Introduction
Triple-negative breast cancer (TNBC), defined as a clinical breast cancer subtype that is negative for estrogen receptor (ER) and progesterone receptor (PgR) expression and human epidermal growth factor receptor 2 (HER2) gene amplification, accounts for 15% of all invasive breast cancer cases. TNBC comprises a heterogeneous group of tumors with different histological and molecular characteristics. Clinically, as TNBC patients do not benefit from endocrine therapies (selective estrogen receptor modulators, selective estrogen receptor downregulators and aromatase inhibitors) (1–3) or anti-HER2 drugs (trastuzumab, lapatinib, pertuzumab and trastuzumab emtansine) (4) due to a lack of targeted therapeutic receptors (5,6), these therapies are not recommended in National Comprehensive Cancer Network guidelines (7). Therefore, chemotherapy using conventional cytotoxic agents, including anthracyclines, cyclophosphamides, taxanes, platinum agents and eribulin (8,9), is currently the mainstay of systemic treatment, although patients with TNBC have worse outcomes following chemotherapy than patients with breast cancer of other subtypes, including hormone receptor-positive and HER2-positive breast cancer (5,6,10). Thus, since there is no optimized standard chemotherapy protocol for TNBC, detailed subtyping of TNBC is necessary to identify more effective molecular-targeted therapies.
Current omics-based studies, such as genomics and transcriptomics, can provide data that can be used to categorize TNBC. Recent gene expression profiling-based cluster analysis using a dataset consisting of 587 TNBC cases from 21 independent datasets identified six subtypes, including two basal-like subtypes, as well as an immunomodulatory, a mesenchymal, a mesenchymal stem-like and a luminal androgen receptor subtype (11). Moreover, next generation sequencing (NGS)-based studies detected tumor protein p53 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α mutations in 80 and 9% of TNBC cases, respectively (12), highlighting these gene mutations as key genetic events in this subtype. Notably, additional NGS studies led to the identification of actionable molecular characteristics, including 'BRCAness', defined as a defect in homologous recombination repair due to BRCA1 DNA repair associated (BRCA1) or BRCA2 mutation, in TNBC (13,14). Recently, poly(ADP-ribose) polymerase inhibitors were shown to take advantage of synthetic lethality in targeting TNBC with BRCAness (15,16). Furthermore, the nuclear factor-κB (NF-κB) signaling pathway is constitutively activated in the basal-like subtype of breast cancer cells, ~80% of which include TNBC (17). However, the underlying causes of NF-κB signaling activation have remained poorly understood.
To characterize the molecular features of TNBC, RNA-sequencing (RNA-seq) analysis was performed in the present study and it was found that leucine-rich repeat (LRR)-containing 26 (LRRC26), a member of the LRR super-family, was frequently downregulated in patients with TNBC. LRRC26, also known as CAPC, is reported to act as a negative regulator of NF-κB activity, and to inhibit cancer cell proliferation and migration (18). However, the pathophysiological role of LRRC26 in carcinogenesis and TNBC progression, particularly the mechanisms regulating LRRC26 expression, has not yet been elucidated.
The present study aimed to characterize the molecular mechanism of LRRC26 downregulation in TNBC.
Materials and methods
Cell lines and clinical specimens
The human breast cancer cell lines, MDA-MB-231, HCC1143, BT-20, BT-549, HCC1395, HCC1937, MDA-MB-157, HCC38, HCC70, MDA-MB-468, BT-474, SK-BR-3, T-47D and ZR-75-1, were purchased from the American Type Culture Collection (Rockville, MD, USA). MCF-7 cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). 293T cells and MDA-MB-453 cells were provided by the RIKEN BioResource Center (Tsukuba, Japan). KPL-3C cells were kindly provided by Dr Junichi Kurebayashi (Kawasaki Medical School, Kurashiki, Okayama, Japan) (19). Each cell line was cultured according to optimal conditions recommended by their respective depositors (Table I). No Mycoplasma contamination was detected in any of the cultures using a Mycoplasma Detection kit (Takara Bio, Inc., Otsu, Japan). A total of 26 TNBC samples and 26 normal mammary tissues were surgically resected with informed consent from patients who were treated at the Tokushima Breast Care Clinic (Tokushima, Japan), as previously described (20) (Table II). Samples were immediately embedded in TissueTek OCT compound (Sakura, Tokyo, Japan), frozen and stored at −80°C.
 | Table IIClinical and pathological features of 26 triple-negative breast cancer cases enrolled in RNA-seq and qPCR analyses. |
The present study, as well as the use of all clinical materials aforementioned, was approved by the Ethics Committee of Tokushima University (Tokushima, Japan).
RNA-seq analysis
Total RNA was extracted from frozen tumors and adjacent normal breast tissues from the 15 out of 26 TNBC samples that could produce a large enough amount of RNA for RNA seq analysis using the Nucleospin RNA II system (Takara Bio, Inc.) according to the manufacturer's protocols. Whole-transcriptome RNAs seq analysis was performed using the SureSelect strand-specific RNA library preparation kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocols, followed by paired-end 100-bp sequencing on an Illumina HiSeq 1500 platform (Illumina, Inc., San Diego, CA, USA). All primary sequence data files are deposited in the DNA Data Bank of Japan (accession no. JGAS00000000116; http://www.ddbj.nig.ac.jp/). Data were analyzed using CLC Biomedical Genomics Workbench version 4.0 (Qiagen GmbH, Hilden, Germany) with default parameters. Transcript abundance was calculated as transcript per million.
Quantitative PCR (qPCR) and semi-quantitative PCR
Total RNA extracted from clinical breast cancer samples and breast cancer cell lines using the NucleoSpin RNA II system (Takara Bio, Inc.) and the poly A-RNAs of a normal human mammary gland (Clontech Laboratories, Inc., Mountainview, CA, USA) were reverse transcribed using SuperScript II (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). qPCR analysis using the ABI PRISM 7500 Real-Time PCR system was performed with SYBR® Premix Ex Taq (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were 10 min at 94°C, followed by 45 cycles of denaturation at 94°C for 15 sec, 1 min annealing and extension at 60°C, and reading of the fluorescence. Following threshold-dependent cycling, melting curve analysis was performed from 60 to 94°C. The quantitative calculation was analyzed using 2−ΔΔCq (21). Semi-quantitative PCR analysis was performed as described previously (22,23). Gene-specific primers used for qPCR and semi-quantitative PCR were as follows: LRRC26 forward, 5′-CTGCTGCTGGACCACAACC-3′ and reverse, 5′-AGAAGGCTCGCACATGCAC-3′; interleukin-6 (IL-6) forward, 5′-GGGCATTCCTTCTTCTGG-3′ and reverse, 5′-ACTGCATAGCCACTTTCCA-3′; IL-8 forward, 5′-GCAAAATTGAGGCCAAGG-3′ and reverse, 5′-GGCACAGTGGAACAAGGA-3′; and C-X-C motif chemokine ligand-1 (CXCL-1) forward, 5′-GCTCTTCCGCTCCTCTCACA-3′ and reverse, 5′-GCTTCCTCCTCCCTTCTGGT-3′. The β-actin primer sequences used as quantitative controls were as follows: Forward, 5′-ATTGCCGACAGGATGCAG-3′ and reverse, 5′-CTCAGGAGGAGCAATGATCTT-3′ for qPCR; and forward, 5′-GAGGTGATAGCATTGCTTTCG-3′ and reverse, 5′-CAAGTCAGTGTACAGGTAAGC-3′ for semi-quantitative PCR.
Methylation analysis using bisulfite pyrosequencing and bisulfite sequencing
Genomic DNA (1 µg) extracted from clinical samples and cell lines was modified with sodium bisulfite using an EpiTect Bisulfite kit (Qiagen GmbH), subsequent to which bisulfite pyrosequencing was performed as previously described (24). The methylation ratio (%) of LRRC26 in each sample was analyzed using PyroMark Q96 software version 2.5.8 (Qiagen GmbH). For bisulfite sequencing, thermocycling conditions were 10 min at 94°C, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 10 min. Amplified PCR products were cloned into the pCR2.1-TOPO vector (Thermo Fisher Scientific, Inc.), and 11 clones from HCC1937, 12 clones from BT-20 and 15 clones from HCC70 were sequenced using an ABI3130x automated sequencer (Thermo Fisher Scientific, Inc.). Primer sequences and PCR product sizes are listed in Table III.
5′-aza-dC treatment
To restore epigenetically silenced LRRC26 gene expression, HCC1937 cells were plated onto 6-well dishes at a density of 3.5×105 cells/well and treated with several concentrations (0, 1, 2.5, 5 and 10 µM) of 5′-aza-dC (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 5 days, replacing the reagent and RPMI-1640 (Sigma-Aldrich; Merck KGaA) medium every 24 h. LRRC26 gene expression following treatment with 5′-aza-dC was monitored by semi-quantitative PCR as aforementioned.
Small interfering RNA (siRNA)-mediated gene silencing
ON-TARGETplus siRNA-SMARTpool (4 types of siRNA; catalog no. L-029447-01-0005; GE Healthcare Dharmacon, Inc., Lafayette, CO, USA) was used to knockdown LRRC26 expression in the HCC70 cells, with siRNA against enhanced green fluorescent protein (si-EGFP) used as a control. HCC70 cells were plated in 6-well dishes at 1.0×105 cells/well. Transfection of 10 nM ON-TARGETplus siRNAs was performed using Lipofectamine RNAiMax (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Knockdown efficiency was evaluated by qPCR using the aforementioned protocol at 48, 72, 96 and 120 h post-transfection. Cell proliferation assays were performed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) as previously described (20). All experiments were performed in triplicate.
Soft agar colony formation assay
Anchorage-independent growth was assessed by the soft agar assay. HCC70 cells were transfected with 10 nM ON-TARGETplus siRNAs for 36 h, and 2.5×103 cells were plated in triplicate in 5 ml RPMI-1640 medium containing 0.4% soft agarose, overlaid on 5 ml 0.72% agarose in 6-cm dishes and cultured at 37°C for 11 days. Colonies per well were counted on a Power IX71 microscope (Olympus Corporation, Tokyo, Japan).
Migration assay
BD Falcon Cell Culture Inserts (24 wells, 8-µm pore size; BD Biosciences, Franklin Lakes, NJ, USA) were rehydrated with serum-free RPMI-1640 at 37°C in a 5% CO2 incubator for 2 h. Following rehydration, the medium was removed and 0.5 ml RPMI-1640 was added to the upper chamber, and 0.5 ml RPMI-1640 with 10% fetal bovine serum (FBS) was added to the lower chamber. RPMI/FBS medium (0.5 ml) containing 2.5×104 HCC70 cells previously transfected with siRNA for 36 h was added to each insert. The HCC70 cells in the migration chamber underwent incubation at 37°C for 48 h. Following incubation, the chamber inserts were fixed (4% paraformaldehyde for 10 min) prior to being stained with 1% Giemsa at room temperature for 2 days. The total number of migrated cells for each treatment was calculated from five random fields for each insert counted on a Power IX71 microscope (Olympus Corporation) at ×40 magnification. The number of migrated cells per well for each treatment was averaged from duplicate samples and expressed as the mean ± standard deviation.
Invasion assay
Matrigel invasion chambers (24 wells, 8-µm pore size; Corning Inc., Corning, NY, USA) were rehydrated with serum-free RPMI at 37°C in a 5% CO2 incubator for 2 h. Following rehydration, the medium was removed and 0.5 ml RPMI-1640 was added to the upper chamber, and 0.5 ml RPMI-1640 plus 10% FBS was added to the lower chamber. RPMI/FBS medium (0.5 ml) containing 2.5×104 HCC70 cells previously transfected with siRNA for 36 h was added to each insert. The HCC70 cells in the invasion chamber were incubated at 37°C for 48 h. Following incubation, chamber inserts were fixed with 4% paraformaldehyde for 10 min prior to being stained with 1% Giemsa at room temperature for 2 days. The total number of migrated cells for each treatment was calculated from five random fields for each insert counted on a Power IX71 microscope (Olympus Corporation) at ×40 magnification. The number of invaded cells per well for each treatment was averaged from duplicate samples and expressed as the mean ± standard deviation.
Construction of expression vectors
pCAGGSSnHC and pCAGGSn3FC, in which an HA-tag and a 3xFLAG-tag, respectively, are inserted in the C-terminus of the cloning sites of pCAGGS vector, were constructed and gifted by Dr Yusuke Nakamura (University of Tokyo, Tokyo, Japan). Briefly, to construct the LRRC26 and B cell receptor associated protein 31 (BAP31) expression vectors, the entire coding sequences of LRRC26 and BAP31 cDNAs were amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) with the following primers: LRRC26 forward, 5′-GGAATTCATGCGGGGCCCTTCCTGGTCG-3′ and reverse, 5′-CCGCTCGAGGGCTTGGGCGGCAGCGGCGG-3′; and BAP31 forward, 5′-CGGAATTCACCATGGGTGCCGAGGCGTC CTC-3′ and reverse, 5′-CCGCTCGAGCTCTTCCTTCTTGTCCATGGGAC-3′ (bold indicates the restriction enzyme sites). The thermocycling conditions were as follows: For LRRC26, an initial denaturation of 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 15 sec, annealing at 62°C for 30 sec and elongation at 68°C for 80 sec; for BAP31, an initial denaturation of 2 min at 94°C, followed by 30 cycles of denaturation at 94°C for 15 sec, annealing at 52°C for 30 sec and elongation at 68°C for 1 min. BAP31 is an a transmembrane protein associated with the endoplasmic reticulum (ER) and ER-Golgi intermediates, and has been involved in apoptosis and protein trafficking (25,26). Each PCR product was inserted into the EcoRI and XhoI sites of pCAGGSnHA in frame with the C-terminal HA-tag (pCAGGSSnHC-LRRC26) and pCAGGSn3FC in frame with the C-terminal FLAG-tag (pCAGGSSn3FC-BAP31), respectively. To construct the pGL3-NF-κB expression vector for the luciferase reporter assay as described next, the oligonucleotides for NF-κB-U (5′-CTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTATATAC-3′) and NF-κB-L (5′-TCGAGTATATAGCGGAAAGTCCCCAGCGGAAAGTCCCCAGCGGAAAGTCCCCAGCGGAAAGTCCCCAGGTAC-3′) (bold font indicates restriction enzyme sites and underlining indicates 4× NF-κB binding sites) was annealed, and cloned into the KpnI and XhoI sites of pGL3-basic (Promega Corporation, Madison, WI, USA). The DNA sequence of all constructs were confirmed by DNA sequencing (ABI3500xL; Thermo Fisher Scientific, Inc.).
Luciferase reporter assay
293T cells (2.0×105 cells/well in 6-well plates) were co-transfected with 200 ng pGL3-NF-κB in combination with 1.0 µg pCAGGSnHC-LRRC26 or the mock vector using Fugene 6 (Promega Corporation). pRL-TK (Promega Corporation) was used as an internal control. After 24 h, the cells were treated with TNF-α (40 ng/ml; Sigma-Aldrich; Merck KGaA) for 0, 4, 8 and 12 h. Next, the cells were harvested and analyzed for Firefly luciferase and Renilla luciferase activity using the PicaGene Dual Sea Pansy Luminescence kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer's protocols. Data are expressed as the fold increase over mock-transfected cells (set at 1.0) and represented as the mean ± standard error of two independent experiments.
Analysis of public database of RNA expression and methylation in breast cancer
Publicly available gene expression and methylation data from The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/) were downloaded. Differential expression (by fold-change value) between breast cancer and the adjacent normal breast tissues was calculated according to the normalized gene expression value of each sample.
Microarray analysis
HCC70 cells were seeded at a density of 2×105 cells/well onto 6-well plates, followed by transfection with 10 nM siEGFP (Sigma-Aldrich; Merck KGaA) or siLRRC26 (GE Heathcare Dharmacon, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. After 48 and 72 h of siRNA transfection, total RNA was extracted using a NucleoSpin RNA kit (Takara Bio, Inc.) according to the manufacturer's protocols. A total of 100 ng total RNA from each sample was amplified and Cy3-labeled. Next, 0.825 µg Cy3-labeled cRNA was fragmented, hybridized onto the Agilent SurePrint G3 Hmn GE 8×60K Ver 2.0 platform (Agilent Technologies, Inc.) and then incubated with rotation at 65°C for 17 h. Data were analyzed using GeneSpring software version 13.0 (Agilent Technologies, Inc.) as previously described (20). To identify genes that were significantly altered between siLRRC26-treated cells and siEGFP-treated cells, the mean signal intensity values in each analysis were compared. The extent and direction of the differential expression between 48 and 72 h were determined by calculating the fold-change value. Data from this microarray analysis have been submitted to the NCBI Gene Expression Omnibus archive as series GSE90582.
Functional gene annotation clustering
The Database for Annotation, Visualization and Integrated Discovery (DAVID 6.8) was approved to detect functional gene annotation clusters based on gene expression profiling by gene annotation enrichment analysis (http://david.abcc.ncifcrf.gov/) (27,28). The clusters from the gene annotation enrichment analysis were selected in this study based on a previous study (29).
Scratch wound cell migration and invasion assays by IncuCyte
BT20 cells (2.0×105 cells/well in 6-well plates) were transfected with 2.0 µg pCAGGSnHC-LRRC26 or the mock vector. For migration assay, after 48 h, cells (2.5×104 cells/well) were re-seeded onto the 96-well ImageLock™ plate (Essen BioScience, Ann Arbor, MI, USA), which was coated with Matrigel (100 µg/ml) prior to seeding the cells. After 4 h, the wound area was created in the cell monolayer with a 96-well WoundMaker™ (Essen BioScience). The plate was washed with phosphate-buffered saline (PBS) (−) to remove detached cells, and 100 µl fresh medium was added. For the invasion assay, the cells were covered with 50 µl Matrigel solution (8 mg/ml) and incubated for 30 min. Next, 100 µl of additional culture media was added to each well and the plates were placed into the IncuCyte ZOOM (Essen Bioscience). The two plate types were scanned every 2 h for 72 h. Data were analyzed using ZOOM software version 2016B (Essen BioScience) according to the manufacturer's protocols. Cell migration and invasion were expressed as relative wound density.
Immunocytochemical staining analysis
To examine the subcellular localization of the LRRC26 protein in TNBC cells, BT20 cells (2.0×105 cells/well in 6-well plates) were transfected with 2.0 µg pCAGGSnHC-LRRC26 or the mock vector using FuGENE HD transfection regent (Roche Diagnostics GmbH, Mannheim, Germany). In addition, BT-20 cells were co-transfected with 2.0 µg pCAGGSnHC-LRRC26 and pCAGGSn3FC-BAP31 or the empty vector (Mock). At 48 h post-transfection, BT-20 cells (2.0×104 cells/well) were plated onto an 8-well glass slide chamber (Thermo Fisher Scientific, Inc.). After 24 h of incubation, the cells were fixed with 4% paraformaldehyde for 30 min at 4°C and then peameabilized with 0.1% Triton X-100 for 2 min at room temperature. Next, the cells were covered with 3% bovine serum albumin for 1 h at room temperature and then incubated with anti-HA antibody (1:1,000 dilution; cat. no. 1867423; Roche Diagnostics GmbH) and anti-FLAG M2 (1:1,000 dilution; cat. no. F-3165; Sigma-Aldrich; Merck KGaA) or anti-78-kDa glucose-regulated protein (GRP78; 1:200 dilution; cat. no. ab108615; Abcam, Cambridge, UK) antibodies overnight at 4°C. Subsequent to washing with PBS (−), the cells were stained with Alexa 488-conjugated anti-rat (cat. no. A-21210) and Alexa 594-conjugated anti-mouse (cat. no. A-11032) or anti-rabbit (cat. no. A-11037) antibodies (1:1,000 dilution; Molecular Probes; Thermo Fisher Scientific, Inc.) at room temperature for 1 h, respectively. The nuclei were stained with 4′,6′-diamidine-2′-phenylindole dihydrochloride. Fluorescence was observed was obtained using an IX71 microscope (Olympus Corporation). Scale bars indicate 20 µm.
Immunoblotting analyses
Immunoblotting analyses were conducted as previously described (30). Briefly, cell lysates were prepared with lysis buffer at 48 h post-transfection. The lysates were electrophoresed, transferred to a nitrocellulose membrane and blocked with 4% BlockAce solution (Dainippon Sumimoto Pharma Co., Ltd., Osaka, Japan) for 1 h. Subsequently, the membrane were incubated with anti-HA (1:3,000 dilution; cat. no. 1867423) and anti-α/β-Tubulin antibodies (1:1,000 dilution; cat. no. 2148) (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight, respectively. Following incubation with a horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; catalog no. sc-2006 for anti-rat antibody; catalog no. sc-2004 for anti-rabbit antibody; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h, the membranes were developed with an enhanced chemiluminescence system (GE Healthcare Life Sciences, Little Chalfont, UK) and were scanned using an Image Reader LAS-3000 mini (Fujifilm, Tokyo, Japan).
Statistical analysis
Statistical significance was calculated using the Kruskal-Wallis test and Dunnett's post-hoc test with SPSS version 20.0 software (IBM, Armonk, NY, USA) for the comparison between the gene expression and methylation levels from the TCGA dataset. For multiple comparisons of Figs. 1D and 2B, a one-way analysis of variance with Dunnett's and Tukey's post hoc tests, respectively, were performed. Wilcoxon signed rank test was performed using JMP 12.1.0 (SAS Institute Japan, Ltd., Tokyo, Japan) to assess methylation levels between tumor and paired normal tissues. The χ2 test was performed using Microsoft® Excel 2016 to assess the associations between the expression of the LRRC26 gene and patient characteristics. Student's two-sided t-test was performed using Microsoft® Excel 2016 to calculate statistical significance in experiments for the gene expression, methylation status, colony formation, migration, invasion and luciferase activity. P<0.05 was considered to indicate a statistically significant difference.
Results
LRRC26 is specifically downregulated in TNBC clinical specimens and cell lines due to methylation of CpG islands
To characterize the molecular features of TNBC, RNA-seq analysis was first performed and it was found that LRRC26 expression was downregulated in 10 out of 15 patients with TNBC, but upregulated in 4 (no regulation change in 1 patient) (Fig. 1A). Subsequent qPCR confirmed that LRRC26 was significantly downregulated in 16 out of the 26 patients with TNBC (6 of the 15 patients who also underwent RNA-Seq) compared with that in paired normal breast tissues (Fig. 1B), which was consistent with the RNA-seq results. Subsequently, statistical analysis of the association between LRRC26 expression level and clinicopathological features, including the tumor stage or grade of TNBC cases used for qPCR, was performed, and it was found that LRRC26 downregulation was significantly associated with increased histοlogical grade in patients with TNBC (P=0.017; χ2 test) (Table IV). Furthermore, the expression of the LRRC26 gene was examined by the analysis of TCGA dataset, including a much larger number of TNBC cases and normal controls, and it was found that the LRRC26 gene was significantly downregulated in 123 TNBC cases compared with that in 113 normal controls (Fig. 1C). These results suggested the possibility that the downregulation of LRRC26 may be associated with the carcinogenesis of TNBC. Accordingly, the present study focused on understanding the mechanism of LRRC26 downregulation in TNBC, although further analysis of the mechanism of LRRC26 upregulation in breast cancer will be necessary. Next, to determine whether LRRC26 downregulation is associated with CpG hypermethylation in its promoter region, bisulfite pyrosequencing analysis was performed using a set of 12 TNBC clinical tissues and adjacent normal breast tissues. LRRC26 methylation levels were quantitatively measured at two CpG islands containing 'Seq.1' and 'Seq.2′, located 206 and 158 bp upstream of the transcription start site, respectively (Fig. 1D). The average methylation levels of 'Seq.1' in the tumor and normal tissues was 43.63% (range, 18.37–62.89%) and 26.55% (range, 18.47–40.09%), respectively, and that of 'Seq.2′ in the tumor and normal tissues was 18.79% (range, 8.52–23.15%) and 11.63% (range, 8.86–15.32%), respectively (Fig. 1D). Accordingly, the methylation levels of the two CpG islands were significantly higher in 11 out of 12 tumor tissues compared with those in adjacent normal mammary gland tissues (Fig. 1D; Wilcoxon signed-rank test: Seq.1, P=0.0015 and Seq.2, P=0.0024, respectively) (Fig. 1D). To further validate this result, LRRC26 expression levels were analyzed in breast cancer cell lines by qPCR. LRRC26 expression was significantly downregulated in 8 out of 10 TNBC cell lines compared with that in normal mammary glands (Fig. 1E), whereas its expression level was extremely high in all HER2-positive and ER-positive/HER2-negative cell lines (Fig. 1E). Furthermore, LRRC26 methylation status was analyzed in TNBC cell lines, and it was observed that high levels of methylation at the two CpG island sites in LRRC26 were in agreement with LRRC26 expression levels in 4 out of 6 TNBC cell lines (Fig. 1E and F). Low levels of methylation at the two CpG island sites were in agreement with high levels of LRRC26 expression in the two HER2-positive cell lines, BT-474 and MDA-MB453. Of the remaining TNBC cell lines, LRRC26 was expressed in HCC70, whereas high DNA methylation levels (>60%) were detected at Seq.1 and low levels at Seq.2. Conversely, LRRC26 was downregulated in MDA-MB-231, which displayed high DNA methylation levels at Seq.1 and moderate levels at Seq.2 (Fig. 1E and F). These results indicated that a different mechanism, such as histone modification or the existence of other critical CpG sites in the LRRC26 promoter region, may underlie LRRC26 silencing in these cells.
| Table IVAssociation between leucine-rich repeat-containing 26 downregulation in triple-negative breast cancer cases and patient characteristics. |
LRRC26 downregulation and hypermethylation occur exclusively in TNBC
To clarify the detailed methylation status, bisulfite sequencing was performed using TNBC cell lines HCC1937, BT20 and HCC70. It was found that the 200 bp upstream of the transcription initiation site, including Seq.1 and Seq.2 CpG islands, in LRRC26 were densely methylated in HCC1937, but not in HCC70, although the Seq.1 CpG island was methylated (Fig. 2A). Moreover, the same regions in LRRC26 were partially methylated in BT20, indicating that the methylation levels at the LRRC26 promoter region were in agreement with the level of LRRC26 expression in all three cell lines. Notably, LRRC26 expression was restored in a dose-dependent manner following treatment with 5′-aza-dC in HCC1937 (Fig. 2B). Moreover, the methylation level of LRRC26 was also significantly reduced in HCC1937 cells following treatment with 5′-aza-dC (Fig. 2B).
Subsequently, RNA-seq analysis of 919 breast cancer cases from TCGA dataset revealed that LRRC26 gene expression in TNBC cases was significantly lower than that in other subtypes, such as ER(+)/HER2(−), ER(±)/HER2(+) (Fig. 2C), whereas LRRC26 gene methylation analysis of 625 cases on the same dataset was significantly higher in TNBC cases than in other subtypes (Fig. 2D). Furthermore, bisulfite pyrosequencing analysis was performed using a set of 12 TNBC clinical tissues and adjacent normal breast tissues to analyze the methylation level of LRRC26 at seqTCGA located 373 bp upstream of the transcriptional start site (Fig. 2E). The results showed that the methylation level of the seqTCGA CpG island was significantly higher in all tumor tissues than that in the adjacent normal mammary gland tissues (Wilcoxon signed-rank test: P=0.002) (Fig. 2E). These data strongly suggested that low LRRC26 expression due to hypermethylation in its promoter region is exclusive to TNBC cases.
LRRC26 as a tumor suppressor in TNBC cells
To investigate the impact of LRRC26 on cell growth in HCC70 TNBC cells, standard cell proliferation assays were performed. First, the knockdown of LRRC26 expression was confirmed in HCC70 cells, in which LRRC26 was highly expressed, as determined by qPCR (Fig. 3A), and it was found that LRRC26 silencing did not affect cell proliferation (Fig. 3A), consistent with the findings of a previous study using LNCaP prostate cancer cells (18). Next, to further assess the tumor suppressive function of LRRC26 on the development and progression of TNBC cells, soft agar colony formation, invasion and migration assays were performed to evaluate metastatic properties. Knocking down LRRC26 expression significantly increased the number of colonies in the soft agar (Fig. 3B), suggesting a critical role for LRRC26 in anchorage-independent growth. Subsequent Matrigel invasion and migration assays also revealed that siRNA-mediated depletion of LRRC26 expression led to significant facilitation of HCC70 cell invasion and migration compared with that in siEGFP-transfected cells (Fig. 3C and D). Furthermore, the effects of LRRC26 overexpression on migration, invasion and cell proliferation in BT-20 cells, which express LRRC26 at a low level, were examined. The results showed that LRRC26 overexpression led to a reduction in the migration and invasion and abilities (Fig. 3F and G) of the cells, but did not effect cell viability (Fig. 3H). Taken together, these findings strongly suggest that LRRC26 suppresses the aggressive behavior of TNBC cells.
LRRC26 expression negatively regulates the TNF-α-induced NF-κB pathway
LRRC26 has been reported to negatively regulate NF-κB signaling in prostate cancer LNCaP cells (18). Therefore, the effect of LRRC26 on NF-κB activity was examined in the present study using a luciferase reporter assay and qPCR analysis. A significant time-dependent decrease in luciferase activity was observed in the presence of TNF-α stimulation with ectopic LRRC26 expression compared with that in the mock control vector in 293T cells (Fig. 4A). qPCR analysis showed that siRNA-mediated LRRC26-knockdown significantly increased the TNF-α-induced expression of the NF-κB target genes IL-6, IL-8 and CXCL1 in HCC70 cells (Fig. 4B). These results suggested that LRRC26 negatively regulates TNF-α-induced NF-κB activity.
Functional gene annotation clustering analysis
To further clarify the biological role of LRRC26 in the progression or development of TNBC cells, the present study attempted to identify the processes or pathways associated with LRRC26 in TNBC cells. siLRRC26, or siEGFP as a control, was transfected into HCC70 cells, which highly express LRRC26, and alterations in gene expression were measured using DNA microarray analysis. To identify genes associated with LRRC26, genes were selected using the following criterion: Expression level was decreased or increased by >3-fold in siLRRC26-transfected cells compared with that in siEGFP-transfected cells. This approach identified 230 genes that were altered upon LRRC26-knockdown (Table V). Among them, the downregulation of olfactory receptor family 5 subfamily M member 1 (OR5M1) and OR5T1 genes, which encode members of olfactory GPCR protein, was verified in LRRC26-depleted cells, as determined by qPCR (Fig. 5A). Notably, OR5T1 protein is predicted to be N-glycosylated at Asn17, suggesting the possibility that LRRC26 downregulation may be important for ORT51 glycosylation. To further identify significantly overrepresented Gene Ontology terms affected by LRRC26 expression in HCC70 cells, these upregulated or downregulated genes were analyzed using the DAVID algorithm (27,31). The most prominent cluster (annotation cluster 1; gene enrichment score, 3.12) was identified, which contained features related to 'glycoprotein', 'signal peptide', 'secreted' and 'N-linked glycoprotein (GlcNAc)' (Table VI). Furthermore, the immunocytochemical staining experiments showed that exogenous HA-LRRC26 was found to be partially co-localized with endogenous GRP78, a molecular chaperone localized in the endoplasmic reticulum (Fig. 5B), and exogenous FLAG-tagged BAP31, an integral membrane protein of the endoplasmic reticulum, in BT20 cells (Fig. 5C), indicating the possibility that LRRC26 may function in the endoplasmic reticulum of TNBC cells. These findings suggested the possibility that LRRC26 downregulation may affect the secretory pathway from the Golgi apparatus to the cell surface or vesicle transport from the endoplasmic reticulum to the Golgi apparatus in TNBC cells.
| Table VGenes that were altered upon leucine-rich repeat-containing 26-knockdown according to DNA microarray analysis (n=230). |
| Table VIFunctional gene annotation clustering analysis of annotation cluster one (enrichment score: 3.12) based on the Database for Annotation, Visualization and Integrated Discovery algorithma. |
Discussion
Genetic and epigenetic inactivation involving DNA methylation and histone modifications of tumor suppressor genes serve a crucial role in the progression and development of breast cancer (20,32). Notably, aberrant promoter hypermethylation of tumor suppressor genes is commonly observed in breast cancer and is the predominant mechanism for loss of function. In the present study, it was demonstrated that LRRC26 downregulation contributes to the progression and development of TNBC, although further analysis of understanding of the mechanism of LRRC26 upregulation in breast cancer, particularly ER- and HER2-positive breast cancer, will be necessary. Although RNA-seq and public TCGA database analyses identified no somatic mutations of the LRRC26 mRNA in the TNBC tissues and cell lines, the frequent downregulation of LRRC26 due to promoter hypermethylation at Seq.1 and Seq.2 in TNBC tissues and cell lines was observed (Fig. 1B–E). Notably, LRRC26 was highly expressed in HCC70 cells, but not in MDA-MB-231 cells, whereas LRRC26 methylation levels at the two CpG sites were similar between HCC70 and MDA-MB-231 cells (Fig. 1D and E). This discrepancy implies the existence of other critical CpG sites in the LRRC26 promoter region that may be responsible for gene silencing. In fact, the methylation level of the seqTCGA CpG island, located 373 bp upstream of the transcriptional start site, was significantly higher in all tumor tissues compared with that in adjacent normal mammary gland tissues (Fig. 2E). By contrast, LRRC26 was also not methylated in other CpG islands within 200 bp of the transcription initiation site, particularly around the transcription initiation site of the LRRC26 gene in HCC70 cells. Notably, miRDB, an online database for miRNA target prediction (33), predicted LRRC26 as a target of hsa-miR-1275. hsa-miR-1275 is reported to be upregulated in young women (<35 years old) with breast cancer compared with that in older women (>65 and 45–65 years old) (34), as well as in MDA-MB-231 TNBC cells (35), suggesting the possibility that epigenetic and miRNA-mediated inactivation may contribute to LRRC26 downregulation.
By analyzing TCGA RNA-seq public databases in the present study, it was found that LRRC26 gene expression was significantly lower in TNBC cases than in ER-positive/HER2-negative and ER-negative/HER2-positive breast cancer cases (Fig. 2C and D), suggesting that hypermethylation-mediated LRRC26 inactivation may be a TNBC-specific event in the progression and development of cancer cells. Further statistical analysis demonstrated that LRRC26 downregulation was significantly associated with increased histological grade in patients with TNBC (P=0.017; χ2 test) (Table IV), whereas Kaplan-Meier analysis revealed no significant association between LRRC26 downregulation and the overall survival of patients with TNBC (data not shown). These findings suggested that LRRC26 downregulation may be involved in the aggressiveness of TNBC, but that in addition to LRRC26 downregulation, other factors may also be necessary to drive malignacy. Further analysis of the association between LRRC26 downregulation and prognosis using a large number of TNBC samples will also be necessary. Furthermore, LRRC26 has been reported to suppress tumor growth by negatively regulating NF-κB signaling in LNCaP and MDA-MB-231 cells (18). However, in the present study, the upregulation of NF-κB-target genes in the absence of TNF-α stimulation was not observed in LRRC26-depleted HCC70 cells (data not shown), although the upregulation of NF-κB-target genes in the presence of TNF-α stimulation was observed in these cells (Fig. 4B). These results suggest that LRRC26 downregulation may be important for TNF-α-mediated NF-κB activation in TNBC cells. Furthermore, LRRC26 expression has been reported to promote anchorage-independent growth in MDA-MB231 cells (18). The present study also found that knocking down LRRC26 increased not only anchorage-independent growth, but also invasion and migration in HCC70 cells; however, it did not promote proliferation in the absence of TNF-α stimulation, suggesting that LRRC26 downregulation is critical for the TNF-α-mediated NF-κB-independent progression of TNBC.
To investigate biological roles of LRRC26 distinct from the NF-κB pathway in TNBC cells, DNA microarray analysis was performed using siLRRC26-transfected HCC70 cells. Functional annotation clustering revealed that upregulated and downregulated genes in LRRC26-depleted HCC70 cells are functionally associated with protein secretion and N-linked glycosylation. These findings suggest the possibility that LRRC26 downregulation may affect the secretory pathway from the Golgi apparatus to the cell surface or vesicle transport from the endoplasmic reticulum to the Golgi apparatus.
LRRC26 protein, a member of the LRR superfamily, has been reported to act as a big potassium (BK) channel auxiliary subunit, whereas the regulation of NF-κB activation by LRRC26 is independent of BK channels (36). Notably, LRRC26 is also predicted to be present on the endoplasmic reticulum membrane via its N-terminal LRR domain, as LRRC26 is N-glycosylated at an Asn147 site in the endoplasmic reticulum (36). LRRC26 has been reported to localize in the endoplasmic reticulum (36). In fact, the present study demonstrated that LRRC26 was observed to be co-localized with GRP78 and BAP31 in the endoplasmic reticulum of TNBC cells (Fig. 5B and C). Moreover, N-linked glycoproteins are composed of a polypeptide glycosylated in the endoplasmic reticulum with several carbonate chains via asparagine residues, and have critical roles in cell-cell interaction and cell adhesion for invasive and metastatic behaviors in breast cancer. These results strongly suggest that LRRC26 may serve a role in breast cancer progression (37–40). However, further analyses are required to elucidate the effects of LRRC26 downregulation on N-linked glycosylation in TNBC cells.
In summary, the present study demonstrated that the methylation-mediated inactivation of LRRC26 resulted in enhancement of anchorage-independent growth-, invasion- and migration-associated metastatic behavior. Notably, frequent methylation-mediated inactivation of LRRC26 is a TNBC-specific event that may be a potential diagnostic biomarker.
Acknowledgments
The authors would like to thank Dr Junichi Kurebayashi (Kawasaki Medical School, Kurashiki, Okayama, Japan) for gifting the KPL-3C breast cancer cell line, and Ms. Hinako Koseki and Ms. Hitomi Kawakami (Division of Genome Medicine, Institute for Genome Research, Tokushima University, Tokushima, Japan) for providing excellent technical support.
Notes
[1] Funding
This research was supported by the Tailor-Made Medical Treatment with the BBJ Project (grant no. 13418656) and Practical Research for Innovative Cancer Control (grant no. 12103129) from the Japan Agency for Medical Research and Development.
[2] Availability of data and materials
The assessed TCGA data set was from the TCGA portal (http://cancergenome.nih.gov/). The RNA-Seq data (accession no. JGAS00000000116) used in Fig. 1A were deposit in the DNA DataBank of Japan (http://www.ddbj.nig.ac.jp/). The microarray data (GSE90582) were submitted to the NCBI Gene Expression Omnibus archive (https://www.ncbi.nlm.nih.gov/geo/).
[3] Authors' contributions
YMiyagawa performed all experiments, interpreted all data and prepared the draft of the manuscript. YMatsushita performed the analyses for LRRC26 expression, invasion, migration assay by Incucyte and immunocytochemical staining, interpreted all data and prepared the draft and final version of the manuscript. HS performed the methylation of LRRC26 and analyses for TCGA data sets. MK and TY provided the interpretation of LRRC26 expression and function. RK performed the analyses for the luciferase assay. AY and AT provided the interpretation of the clinical association data. JH and MS prepared the clinical specimens and provided the interpretation of the clinical association data. YMiyoshi discussed the interpretation of all data. TK was involved in the conception and design of all studies, the interpretation of the data, and the preparation of the draft and final version of the manuscript. All authors read and approved the final manuscript.
[4] Ethics approval and consent to participate
The present study, as well as the use of all clinical materials aforementioned, was approved by the Ethics Committee of Tokushima University (permission no. H29-15 for expression profile analysis and permission no. H29-14 for RNA-seq analysis).
[5] Consent for publication
Clinical specimens were obtained with informed consent from patients who were treated at the Tokushima Breast Care Clinic (Tokushima, Japan), as previously described (20), with permission to publish their data.
[6] Competing interests
The authors declare that they have no competing interests.
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