Sections of all breast tumor samples (fixed with 4% paraformaldehyde in PBS at 4°C overnight and embedded in paraffin) were obtained from the Department of Pathology of The First Affiliated Hospital of Jinan University (Guangzhou, China) from January 2010 to December 2015. A total of 160 primary ductal breast cancer cases, confirmed by pathology, were selected. The clinical references including ages of patients, tumor type and steroid receptor expression levels were provided by the Department of Pathology.

Dulbecco's modified Eagle's medium (DMEM), PBS and fetal bovine serum (FBS) used in tissue culture were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Additionally, 3,3-diaminobenzidine tetrachloride, 1,4-dithiothreitol (DTT), dimethylsulfoxide, mouse monoclonal anti-hemagglutinin (anti-HA; cat. no. H3663; 1:1,000), protease inhibitor cocktail, penicillin and streptomycin were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Polyethyleneimine (PEI) was purchased from Polyplus-transfection SA (Illkirch, France). L-803087 and Protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Blue Range™ Prestained Protein Molecular Marker, used for SDS-PAGE, was purchased from Pierce; Thermo Fisher Scientific, Inc. Rabbit monoclonal anti-SSTR1 (cat. no. ab137083; 1:500), rabbit monoclonal anti-SSTR2 (cat. no. ab134152; 1:1,000), rabbit monoclonal anti-SSTR3 (cat. no. ab137026; 1:10,000), rabbit polyclonal anti-SSTR4 (cat. no. ab28578; 1:1,000) and rabbit monoclonal anti-SSTR5 (cat. no. ab109495; 1:1,000) were purchased from Abcam (Cambridge, MA, USA). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (cat. no. TA140003; 1:10,000), HRP-conjugated goat anti-mouse IgG (cat. no. TA130004; 1:20,000) used for western blot analysis were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). The tetramethylrhodamine-conjugated goat anti-rabbit IgG (cat. no. 111-025-003; 1:100) and the fluorescein isothiocyanate-conjugated mouse anti-rabbit IgG (cat. no. 111-095-003; 1:100) used in immunofluorescence were purchased from Jacksons ImmunoResearch Europe, Ltd. (Newmarket, UK). PrimeSTAR DNA Polymerase, EasyTaq DNA Polymerase, EcoRI, XhoI and T4 DNA ligase were purchased from Takara Bio, Inc. (Otsu, Japan). Ethanol, xylene and KCl were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). EDTA, 0.1% Triton X-100, hematoxylin and glycerol were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). MDA-MB-435S cells were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in our laboratory according to the instruction.

Total mRNA was extracted from cultured HeLa cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) reagent, according to the manufacturer's protocol. A total of 2 µg total RNA in each reaction was reverse-transcribed into cDNA with oligo(dT) primers using Moloney Murine Leukemia Virus Reverse Transcriptase kit (Promega Corporation, Madison, WI, USA). The cDNA of hSSTR1 and hSSTR4 was amplified with PrimeSTAR using the polymerase chain reaction (PCR) with the following primers: hSSTR1 (1,176 bp), sense, 5′-CCGGAATTCGCCACCATGTTCCCCAATGGCACCG-3′, and antisense, 5′-CCGCTCGAGTCAGAGCGTCGTGATCCGG-3′; and hSSTR4 (1,167 bp), sense, 5′-CCGGAATTCGCCACCATGAGCGCCCCCTCGACG-3′, and antisense, 5′-CCGCTCGAGTCAGAAGGTGGTGGTCCTGG-3′. PCR was performed in a Peltier Thermal Cycler 200 programmed with an initial denaturation at 98°C for 5 min followed by 35 cycles of denaturation at 98°C for 30 sec, annealing at 55–58°C for 30 sec and extension at 72°C for 90 sec. The PCR products were verified by electrophoresis on 1% agarose gel and visualized by ethidium bromide staining under ultraviolet light. The purified PCR products were then digested with EcoRI/XhoI and cloned using T4 ligase into pcDNA6-Myc/His (Invitrogen; Thermo Fisher Scientific, Inc.) and pCMV-HA (Clontech Laboratories, Inc., Mountainview, CA, USA). The constructs were screened using PCR with Taq polymerase and the sequences of the constructs were verified by DNA sequencing (RuibioBiotech, Beijing, China).

Human cancer cell line MDA-MB-435S was maintained in DMEM, supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in an atmosphere containing 5% CO₂ at 37°C. Cells were transfected with the indicated constructs using PEI according to the manufacturer's protocol.

A total of five 6 µm paraffin sections were prepared for each sample. The paraffin sections of breast tumor tissues were baked in an oven at 65°C for 4 h prior to being deparaffinized in xylene for 20 min twice at room temperature. The samples were subsequently rehydrated by incubating at room temperature in an ethanol gradient series for 3 min at each step (100, 95, 80, 70 and 50%, and distilled water). The tissue sections were then washed with PBS three times prior to being boiled in a microwave in 0.01 M citric acid (pH 6.0) for 20 min. The samples were then washed once with PBS.

For immunostaining, the sections were treated with 3% H₂O₂ for 10 min at room temperature and blocked with 1% bovine serum albumin (Thermo Fisher Scientific, Inc.) plus 0.1% Triton X-100 in PBS for 30 min at room temperature. The sections were incubated with the indicated primary antibodies (with PBS in blank control) at 4°C overnight and then washed with 3X PBS. The slides were probed with HRP-conjugated secondary antibodies for 1 h at room temperature and the excess antibody was washed off with PBS. Subsequently, 250 µl DAB per section was then added for color reaction. Following an intensive wash with PBS, the slides were counterstained with hematoxylin for 8 min at room temperature. The expression levels of SSTRs were scored according to the color and number of positive cells (positive cells <5%, 0; positive cells 5–25%, 1; positive cells >25–50%, 2; positive cells >50%, 3; light tea color, 1; yellow-brownish, 2; brown, 3). An overall score of 0–1 represented negative, a score of 2–3 points represented +, a score of 4–6 represented ++ and a score of >6 represented +++. The incidences of SSTR expressions were presented in percentages. The association between SSTR expression levels and other clinical indexes were analyzed using a χ² test and P<0.05 was used as statistical significance. A colon cancer tissue sample that was confirmed to exhibit positive SSTR1-5 expression was stored in our lab and included in every experiment as a positive control.

Cultured cells were collected and resuspended in lysis buffer containing 50 mM Tris/HCl (pH 7.4) (Invitrogen; Thermo Fisher Scientific, Inc.), 100 mM KCl, 10% glycerol, 1 mM EDTA, 1% Triton X-100 and 1 mM DTT and protease inhibitor cocktail. The supernatants were collected by centrifugation at 12,000 × g for 15 min at 4°C. Protein A/G PLUS-Agarose was used for immunoprecipitation, which was conducted according to the Current Protocols in Cell Biology (28). Aliquots (15 µl) of protein samples were then separated by SDS-PAGE (12% gel) and transferred onto a polyvinylidene fluoride membrane (Whatman; GE Healthcare Life Sciences, Little Chalfont, UK). The membrane was then blocked in 5% skimmed milk in PBS plus 1% Triton X-100 for 1 h at room temperature. Specific primary antibodies and HRP-conjugated secondary antibodies were then used for probing at room temperature for 1 h. The immunoblots were visualized by chemiluminescence with an enhanced chemiluminescent kit (GE Healthcare Life Sciences) and the results were further analyzed with AlphaEase FC software 4.1.0 (Protein Simple, San Jose, CA, USA). Densitometry was performed using a FluorChem™ system (Protein Simple).

The cultured MDA-MB-435S cells were plated on coverslips and transfected using 16 µg PEI per 4 µg of pCMV-HA-SSTR1 and/or pcDNA-MYC-SSTR4. At 24 h post-transfection, the SSTR1 and SSTR4 subtype-specific SSA L-803087 was added into the culture medium to a final concentration of 10 nM (DMEM was added as mock treatment in control cells). The cells were maintained for an additional 24 h prior to being analyzed. The cells were rinsed twice with PBS and fixed with 3.5% paraformaldehyde in PBS for 15 min at room temperature. The cells were subsequently permeabilized or not with 0.2% Nonidet-P40 in PBS, followed by staining for 1 h with the appropriate dilutions of the indicated primary antibodies at room temperature, according to the manufacturer's protocol. Excess primary antibodies were washed off with PBS and attached antibodies were then detected with appropriate dilutions of HRP-conjugated secondary antibodies for 1 h at room temperature, according to the manufacturer's protocol. The coverslips were inverted and mounted onto glass slides for visualization under the 505, 595 and 400 nm wavelengths on a Zeiss LSM710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). The images were obtained using a Zeiss LSM 510 EMTA camera (Carl Zeiss AG) and processed with Zeiss LSM Image Browser 4.0 (Zeiss GmbH, Jena, Germany). A total of five different views of co-transfected cells were randomly selected and the dimerization between SSTRs was calculated with Pearson's correlation using Volocity 6.11 (PerkinElmer, Inc., Waltham, MA, USA). Results are presented as the mean ± standard deviation (SD) and the P-values were determined with Student's t-test.

In total, 1×10⁵ MDA-MB-435S cells were used to inoculate 1 ml culture medium per well in 24-well Petri dishes. The cells were transfected with pCMV-HA-SSTR1 and/or pcDNA-MYC-SSTR4, and at 24 h post-transfection, the SSTR subtype-specific SSA, L-803087, was added into the culture medium, whereas PBS was added in the mock treatment. The cells were maintained in the aforementioned cell culture conditions for an additional 24 h prior to further analysis. The cells were then trypsinized and resuspended in fresh medium followed by flow cytometry analysis. For the cell cycle assay, cells were fixed with 70% ethanol on ice for 30 min. The cells were then suspended in PBS and treated with RNase A (final concentration 100 µg/ml; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37°C for 30 min. Following removal of RNase A by washing once with PBS, the cells were stained with propidium iodide (PI) at 5 µg/ml for 15 min at room temperature and the cell cycle phases were determined with flow cytometry using a FACSAria I instrument (BD Biosciences, Franklin Lakes, NJ, USA). The data were further analyzed using Modfit LT 4.1 (Verity Software House, Inc., Topsham, ME, USA). The results were obtained from three replicas for each experiment and are presented as the mean ± SD. P-values were determined with Student's t-test.

Paraffin sections of 160 surgically removed human breast-infiltrating ductal carcinoma tumor tissues were collected from the Department of Pathology of The First Affiliated Hospital of Jinan University. The clinical information, including the patient age, cell differentiation and the expression levels of the relevant molecular markers, was provided by the Department of Pathology of The First Affiliated Hospital of Jinan University. Among these 160 breast cancer tissue samples, the cancer cells were determined to be poorly differentiated in 83 samples, moderately differentiated in 54 samples and well-differentiated in 23 samples, according to the Scarff-Bloom-Richardson system recommended by World Health Organization (29). Histological information of hormone receptor [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2] expression levels was provided by the Department of Pathology of The First Affiliated Hospital of Jinan University and was available for 46 samples. The expression levels of the five SSTRs were determined for each sample using immunohistology and their associations with the clinical indexed were further analyzed (data not shown).

All five SSTRs were determined to be expressed at variable levels in breast cancer tissues (Fig. 1), and the expression levels of SSTR1-5 were detected in 90.0, 34.4, 41.9, 71.3 and 44.4% of cancer tissues, respectively (Table I). The expression levels of these SSTRs were determined to not be associated with the ages of patients. Different subtypes of SSTRs were frequently determined to be co-expressed in tumor tissues. Only 5 samples were observed to be negative for all five subtypes of SSTR expression. A total of 19 cases expressed just one subtype of SSTR, and, among them, 18 were SSTR1-positive and only 1 case expressed SSTR5 alone. The positive expression instances of all five SSTRs was negatively associated with cancer tissue differentiation, and the statistical significance (P<0.05) in these differences was revealed for the expression levels of SSTR1 and SSTR4 in tumor tissues of different differentiations (Table I). Furthermore, high expression levels of SSTR2 and SSTR3 were only observed in poorly differentiated tumor tissues (Fig. 2). No associations of hormone receptor (estrogen, progesterone and erB-2) expression levels with SSTR expression levels were identified (Table II). However, the expression levels of SSTR2, SSTR3, SSTR4 and SSTR5 exhibited an increased frequency of observation in ER/PR-negative tumor cases.

Owing to the common features of ligand-induced dimerization for G-protein-coupled receptors, the potential dimerization of the two most commonly expressed SSTRs, SSTR1 and SSTR4, were investigated further. The full-length coding sequences of SSTR1 and SSTR4 were cloned into pCMV-HA and pcDNA6-Myc/His, respectively. Exogenous HA-SSTR1 and MYC-SSTR4 were then co-expressed in cultured MDA-MB-435S cells. No endogenous expression of SSTRs was observed, which was verified using immunoblotting with specific SSTR antibodies (data not shown). The cells were permeabilized or not permeabilized prior to the immunofluorescence assay to primarily display the cytoplasmic or plasma membrane-bound proteins. The target proteins were visualized using confocal microscopy and the potential protein dimerizations were calculated with Pearson's correlation. The heterodimerizations of SSTR1/SSTR4 natively existed on the plasma membrane as well as in the cytoplasm of cells co-expressing HA-SSTR1 and MYC-SSTR4. The Pearson's correlation value of SSTR1 and SSTR4 was significantly increased in permeabilized cells (from 0.74±0.07 to 0.89±0.02) as well as in non-permeabilized cells (from 0.63±0.04 to 0.83±0.03) upon subtype-specific SSA L-803087 activation, compared with non-activated cells. Overexpressed HA-SSTR1 and MYC-SSTR4 were predominantly observed on cytoplasmic membranes prior to activation whereas translocation of activated receptors to the cytoplasm was observed upon ligand induction (Fig. 2).

The dimerization of SSTR1 and SSTR4 was also observed using co-immunoprecipitation. The cell lysates of MDA-MB-435S cells overexpressing HA-SSTR1 and MYC-SSTR4 were immunoprecipitated with an anti-HA antibody. Western blot analysis of the precipitated proteins using an anti-MYC antibody revealed the association between SSTR1 and SSTR4 (Fig. 3A). The heterodimerization of SSTR1 and SSTR4 was significantly increased upon receptor activation using L-803087 (Fig. 3B).

The influence of SSTR expression and receptor activation on cell proliferation was further investigated in cultured MDA-MB-435S cells using flow cytometry. The cells were transfected with pCMV-HA-SSTR1 and/or pcDNA-MYC-SSTR4, followed by receptor activation using 10 nM L-803087. The control cells were transfected with empty vectors and the mock cells were treated with buffer only. The cell cycles of these cells were subsequently analyzed using flow cytometry. Compared with the control (39.45±0.63% cells in S-phase), the proportion of cells overexpressing SSTR1 or SSTR4 was slightly decreased in S-phase (38.36±2.71%; P>0.05; Fig. 4). However, the proportion of S-phase cells overexpressing SSTR1 and SSTR4 was significantly decreased when treated with 10 nM L-803087 (26.06±2.79%; P<0.05). The expression levels of the relevant SSTRs in these cells were verified using western blot analysis (data not shown). The results indicated that the inhibition of cell proliferation was mediated via receptor activation and was subtype-specific.