Edited by: San-Gang Wu, First Affiliated Hospital of Xiamen University, China
Reviewed by: Krishna B. Singh, University of Pittsburgh, United States; Umberto Malapelle, University of Naples Federico II, Italy
This article was submitted to Women's Cancer, a section of the journal Frontiers in Oncology
†These authors have contributed equally to this work
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Breast cancer is the most common female malignancy worldwide, however its molecular pathogenesis still needs in-depth investigation. Here we first revealed that the olfactory receptor family 2, subfamily T, member 6 (OR2T6) was significantly over-expressed in breast cancer tissues compared with normal breast tissues. OR2T6 expression was tightly correlated with higher TNM staging, positive lymph node metastasis, and associated with poorer patients' overall and disease-free survival. And OR2T6 enhanced the proliferation, invasion, and migration ability of breast cancer cell lines
Breast cancer is the most common cancer and the fifth leading cause of cancer death among women worldwide. More than 1,000,000 new cases are diagnosed annually and ~373,000 result in death each year (
Recently, we used a gene microarray method to analyze the differential expression between cancers with and without metastasis (GEO accession number:
The olfactory receptor (OR) family is a key member of the G-protein-coupled receptor (GPCR) family in humans (
In the present study, to verify the possible role of OR2T6 in breast cancer, we tested the mRNA and protein levels of OR2T6 in breast cancer tissues
This study was approved by the ethics committee of the Medical School of Shandong University (approval code: 2012028). Written informed consent was obtained from all patients before the use of the materials. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
We collected 102 samples of breast cancer tissue from patients who underwent modified radical mastectomy because of breast cancer and 60 cases of normal breast tissue from patients who underwent local resection because of fibrocystic disease of the breast at Qilu Hospital of Shandong University between July 2005 and July 2012. None of the patients had received radiotherapy or chemotherapy prior to surgery. Hematoxylin and eosin (HE)-stained sections and paraffin-embedded tissue sections were obtained from the Department of Pathology, and patient information was obtained from the patient medical record room at Qilu Hospital. All of the HE sections were reviewed by two experienced pathologists, and the diagnoses were classed according to the World Health Organization Classification of Tumors.
Patients were followed up every 3 months until death or July 2017. Survival time, disease-free interval, and development of metastases were recorded as survival data. The interval between the dates of surgery and death was used as the overall survival (OS) time. The interval between the date of the operation and recurrence or death was recorded as the disease-free survival (DFS) time. Patients still alive at the last follow-up were classified as censored observations.
The cBioPortal for cancer genomics (
The human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from the American Type Culture Collection (Manassas, VA, USA) (last authenticated in April 2018 using STR profile analysis). Cells were cultured per routine in Dulbecco's modified Eagle's medium (MCF-7) or Leibovitz's L15 medium (MDA-MB-231) with 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO2.
Total RNA was extracted using Trizol (Ambion, cat. 15596-026) and synthesized into cDNA with the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, cat. K1621). The detailed primer sequences were listed in
For plasmid construction, the whole OR2T6 sequence was synthesized and inserted into the pcDNA3.1 vector at the Nhel/Xhol restriction enzyme site for the construction of the pcDNA3.1-OR2T6 plasmid (Boshang Biological Technology, Shanghai, China). The pcDNA3.1 vector was used as the control. The siRNA sequences were as follows: OR2T6, 5′-GCUCUUCACUCACAAUAAAdTdT-3′ and NControl_05815 (siN05815122147) (Ribobio, Guangzhou, China). MCF-7 and MDA-MB-231 cells were seeded in 6-well culture plates at 2 × 105 cells per well and cultured in medium without antibiotics for 24 h before transfection. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the siRNAs or plasmids into cells.
Total protein was extracted from cells using CelLyticTM MT Cell Lysis Reagent (Sigma, cat. C3228) and qualified using the BCA reagent kit (Beyotime, cat. P0011). Protein was loaded onto 10% gels for immunoblotting with antibodies against OR2T6, E-cadherin, N-cadherin, β-catenin, vimentin, H-RAS, c-Myc, ERK, c-fos, or β-actin separately. β-actin was used as the loading control. Detailed information about the antibodies was listed in
Primary antibodies used in this study.
OR2T6 | WB, IHC-P | WB:1:1000, IHC-P:1:150 | Santa Cruz | Goat | Polyclonal | sc-104547 |
H-RAS | WB | 1:300 | Sangon Biotech | Rabbit | Polyclonal | D260079 |
c-Myc | WB | 1:1000 | Sangon Biotech | Rabbit | Polyclonal | D110006 |
ERK | WB | 1:2000 | CST | Rabbit | Monoclonal | 4370S |
c-fos | WB | 1:500 | Sangon Biotech | Rabbit | Polyclonal | D120415 |
E-cadherin | WB | 1:1000 | CST | Rabbit | Monoclonal | mAb 3195 |
N-cadherin | WB | 1:1000 | CST | Rabbit | Monoclonal | mAb13116 |
β-catenin | WB | 1:1000 | CST | Rabbit | Monoclonal | mAb 8480 |
Vimentin | WB | 1:1000 | CST | Rabbit | Monoclonal | mAb 5741 |
Cell proliferation was measured using Cell-Light™ EdU DNA Cell Proliferation (EdU) assays (Ribobio, cat. C10310). Briefly, cells were seeded onto 96-well plates at a density of 5 × 103 cells per well at 24 h after transfection. After EdU labeling, cells were treated with 100 μL of 1× Apollo reaction cocktail, stained with 100 μL of Hoechst 33342 (5 μg/mL), and observed under a fluorescence microscope (Olympus, Japan). The proliferation rate was determined by calculation of the percentage of EdU-positive cells.
Cells were collected at 72 h after transfection and stained with the Annexin V-FITC/PI Apoptosis Detection Kit (BestBio, cat. BB4101), according to the manufacturer's instructions. Cells with negative PI and positive FITC staining were defined as early apoptotic cells, while those with positive PI and FITC staining were defined as late apoptotic cells. The total apoptotic rate was calculated as the early apoptotic rate plus the late apoptotic rate.
Migration assays were conducted using Transwell inserts (8.0 μm, 24-well format; Corning, NY, USA). For the invasion assay, the inserts were pre-coated with a Matrigel matrix (BD Science, Sparks, MD, USA). Cell migration and invasion assays were performed as described previously (
MCF-7 cells were transfected with OR2T6 siRNA or negative control and then subjected to human gene expression microarray (KangCheng Inc., Shanghai, China) assay. Genes with a fold-change above two after transfection were defined as differentially expressed genes (DEGs). Bioinformatic analyses, namely, DEGs gene ontology (GO) enrichment analysis, together with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online tool (
An immunohistochemical method was used to detect the protein expression in 162 tissue samples. Briefly, slides were baked at 60°C, deparaffinized, and rehydrated. After antigenic retrieval, the sections were treated with 3% hydrogen peroxide and incubated with normal serum. Then, slides were further incubated with antibodies for OR2T6 overnight at 4°C. On the second day, the slides were treated with reagent 1 containing the secondary antibody and reagent 2 containing streptavidin-horseradish peroxidase complex (PV-9000 kit, Zhongshan Biotechnology Company, Beijing, China) for 30 min at room temperature separately. After diaminobenzidine (DAB) staining, the sections were counterstained with hematoxylin. PBS buffer was used as a negative control to replace OR2T6 antibody.
The staining results were independently judged by two pathologists blinded to the patients' information. Staining index (SI) was used to determine the results by combining the staining intensity and the proportion of positive cells. In brief, staining intensity was graded as follows: 0 (no staining), 1 (light yellow), 2 (yellow brown), and 3 (strong brown). The positive proportion of tumor cells was evaluated as follows: 0 (positive cells <5%); 1 (positive cells: 5–25%); 2 (positive cells: 26–50%); 3 (positive cells: 51–75%); and 4 (positive cells >75%). Then, the final SI scores were graded as 0, (-); ≤ 5, (+); 6–8, (++); and ≥ 9, (+++). Tumor samples scored (-) to (+) were considered negative, and others were considered positive.
SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The chi-square test was used to evaluate the difference in OR2T6 expression between normal breast and breast cancer tissues. Student's
We first examined the mRNA levels of OR2T6 in normal breast tissues (
OR2T6 expression in breast cancer tissues and its association with patients' outcome.
We next investigated the correlation of OR2T6 expression with the clinicopathological characteristics of breast cancer patients. Statistical analysis showed that the positive expression of OR2T6 was tightly associated with lymph node metastasis (
Association of clinicopathological parameters with OR2T6 protein expression.
0.821 | ||||
<50 | 40 | 10 | 30 | |
≥50 | 62 | 18 | 44 | |
No | 48 | 6 | 42 | |
Yes | 54 | 22 | 32 | |
0.503 | ||||
≤2 cm | 43 | 10 | 33 | |
>2 cm | 59 | 18 | 41 | |
I | 24 | 4 | 20 | |
II | 55 | 13 | 42 | |
III | 14 | 5 | 9 | |
IV | 9 | 6 | 3 | |
0.095 | ||||
I | 7 | 0 | 7 | |
II | 76 | 20 | 56 | |
III | 19 | 8 | 11 |
Univariate and multivariate Cox proportional hazards regression analysis of survival in 102 patients with breast cancer.
OR2T6 expression | 2.831 | 1.246–6.431 | 1.362 | 0.490–3.786 | 0.554 | |
Histological Grade | 2.006 | 0.899–4.475 | 0.089 | 2.716 | 1.081–6.824 | |
LN metastasis | 4.759 | 1.615–14.027 | 1.781 | 0.513–6.182 | 0.363 | |
TNM staging | 5.035 | 2.978–8.513 | 5.728 | 3.254–10.084 | ||
Tumor size | 2.348 | 0.925–5.958 | 0.072 | 1.209 | 0.394–3.708 | 0.741 |
Age | 2.079 | 0.910–4.750 | 0.082 | 2.458 | 1.050–5.751 | |
OR2T6 expression | 2.691 | 1.186–6.102 | 1.275 | 0.480–3.388 | 0.626 | |
Histological grade | 1.852 | 0.841–4.078 | 0.126 | 2.039 | 0.832–4.994 | 0.119 |
LN metastasis | 5.109 | 1.736–15.038 | 4.228 | 1.403–12.745 | ||
TNM staging | 5.322 | 3.115–9.091 | 2.265 | 0.820–6.256 | 0.115 | |
Tumor size | 2.395 | 0.944–6.078 | 0.066 | 1.213 | 0.386–3.809 | 0.741 |
Age | 2.140 | 0.938–4.883 | 0.071 | 1.566 | 0.655–3.742 | 0.313 |
We used cBioPortal to study the genetic changes of OR2T6 gene in 10 breast cancer studies. In 8,023 cases of breast cancer, the genetic change rate of OR2T6 ranged from 0 to 22.23%, with an average of 9.21%. Of 743 cases of genetically altered breast cancer, 734 cases had OR2T6 amplification, 5 cases had mutation, and 4 cases had deep deletion (
To explore the role of OR2T6 in the proliferation and apoptosis of breast cancer cells, we performed EdU staining and flow cytometry assays in MCF-7 and MDA-MB-231 cells. The results showed that the overexpression of OR2T6 in MCF-7 cells significantly increased the percentage of EdU-positive cells, which are the proliferating cells (
OR2T6 promotes the proliferation of breast cancer cell lines MCF-7 and MDA-MB-231. The percentage of EdU positive cells was recorded as the proliferation rate. Student's
For the apoptosis assay, when we increased OR2T6 expression in MCF-7 cells, the percentage of apoptotic cells was reduced from 29.0 to 16.5% (
OR2T6 inhibited apoptosis of breast cancer cell lines. Cells were collected at 72 h after transfection, stained with Annexin V-FITC and PI and then analyzed by flow cytometry. Apoptosis of MCF-7 and MDA-MB-231 cells was inhibited by overexpression of OR2T6
To assess the effects of OR2T6 on the invasion and migration capability of breast cancer, we first transfected breast cancer cells with pcDNA3.1-OR2T6 plasmid. The results showed that the overexpression of OR2T6 significantly enhanced the migration and invasion of MCF-7 cells (
OR2T6 enhances cell migration and invasion of breast cancer cell lines. Cells (1 × 105) were seeded in 12-well plates the day before transfection. After 24 h of transfection, the cells (1 × 104) were resuspended in serum-free medium and seeded to the upper chamber of a Boyden chamber Transwell. Culture medium containing 10% fetal bovine serum was added to the lower chamber. Then, 36 h later, any cells that had migrated were fixed, stained, and counted. For the invasion assay, an experiment was done using membranes coated with a Matrigel matrix. The migration and invasion of MCF-7 and MDA-MB-231 cells were increased by overexpression of OR2T6
To clarify the underlying mechanism responsible for the effects of OR2T6 on cell migration and invasion, we investigated the role of OR2T6 in the EMT process. When we transfected the pcDNA3.1-OR2T6 plasmid into MCF-7 cells, the protein expression of OR2T6 increased significantly, indicating the specificity of the vector (
OR2T6 plays a role in the regulation of the EMT process and the MAPK/ERK pathway in breast cancer cells.
We next investigated the molecular signaling pathways via which OR2T6 functions in breast cancer. To achieve this, we transfected MCF-7 cells with OR2T6 siRNA and subjected them to human gene expression microarray assays. The mRNA levels of 28,186 genes were compared between the cells transfected with siOR2T6 and negative control. Bioinformatic analysis revealed that 13,452 genes were up-regulated, while 14,734 genes were down-regulated in the siOR2T6 group compared with the control group (data not shown). Furthermore, 385 differentially expressed genes (DEGs) were identified, among which 213 genes were up-regulated, and 172 genes were down-regulated after the interference with OR2T6 gene expression (
To obtain a more comprehensive knowledge of the DEGs, GO, and KEGG pathway enrichment analysis were performed. GO analysis showed that the DEGs were mainly enriched in regulation of biological process, single-organism process, cellular response to chemical stimulus, and biological adhesion. KEGG analysis suggested that the DEGs were enriched in cAMP, calcium signaling pathway and pathways in cancer including Rap1, PI3K-AKT, and Ras signaling pathways. To verify the microarray data, real-time quantitative PCR was conducted in MCF-7 cells to validate the related molecules in Rap1 signaling pathway (Rap1A, Rap1B, MLLT4, CTNND1)and PI3K-AKT pathway (PIK3R5, AKT2, AKT3, BAD, and Bcl-2). However, results showed that the expression of these genes were not changed obviously (
Although different ORs have different copy numbers and different functional alleles, highly related ORs often cluster at the same locus, indicating their specific functions in humans (
It is well-known that the truncating mutation 1100delC of checkpoint kinase 2
The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesions and gain migratory and invasive properties (
The MAPK/ERK pathway is one of the most important for tumor initiation. Once the pathway is activated, tumor cell proliferation is greatly enhanced, and apoptosis is inhibited (
Recent reports have shown three patterns of subcellular protein localization of ORs. OR51E1 protein was observed in the membrane and cytoplasm of epithelial cells (
OR2T6 belongs to GPCRs, the largest family of cell-surface receptors. GPCRs have been proved to participate in the development and progression of many tumors including breast cancer (
In the present study, we identified that OR2T6, as a new oncogene, might be involved in the initiation and progression of breast cancer. Its over-expression was associated with higher TNM staging and lymph node metastasis. Up-regulation of OR2T6 expression in breast cancer may enhance migration and invasion by initiating the EMT, and promote proliferation and inhibit apoptosis via activation of the MAPK/ERK pathway (
The datasets generated for this study can be found in the GEO database (accession number
The studies involving human participants were reviewed and approved by the ethics committee of the Medical School of Shandong University (approval code: 2012028). The patients/participants provided their written informed consent to participate in this study.
PG conceived the experiments and wrote the paper. ML and XW carried out the
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank LetPub (
The Supplementary Material for this article can be found online at: