Low serum gastrin associated with ER⁺ breast cancer development via inactivation of CCKBR/ERK/P65 signaling

All 93 BC cases, female and age ranging from 20 to 70 years (mean = 46.2 years), were collected from Shanghai General Hospital (Shanghai, China) and Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine (Zhuhai, China) in 2016 (detail in Additional file 1: Table S2). All necessary BC patient information was available, including tumor characteristics (grade, lymph node stage, and size). Not all BC patients received chemo-radiotherapy before enrollment. Normal, healthy female volunteers who received a physical examination at Shanghai General Hospital were used as controls for gastrin measurement (N = 20). All patients gave verbal informed consent before inclusion. Approval from Ethics Committee of the Shanghai Jiao Tong University School of Medicine was obtained after they reviewed the study protocol and purpose.

Patients were given a physical examination every 3 months for the first 2 years after surgery and were subsequently examined every 6 months. Disease-free survival (DFS) was calculated as the duration between the date of surgery and the date of first evidence of local recurrence, distant metastasis, or diagnosis of a second primary tumor or cancer-associated mortality. Overall survival (OS) was calculated as the time between the date of surgery and the date of mortality from any cause.

Female athymic BALB/C nude mice (4–6 weeks-old) were purchased from the Shanghai Experimental Animal Center at the Chinese Academy of Science (Shanghai, China). BC-bearing mice were given estrogen subcutaneously 3 d before injecting MCF-7 cells (1 × 10⁷) into the bilateral mammary fat pad. These mice were then randomly divided into control (N = 6) and experimental (N = 6) groups when tumors became outwardly visible (approximately ≥10 mm³). Control and experimental groups received PBS or gastrin treatment (2 mg/kg, twice/d), respectively, by caudal vein injection for 12 d; tumor volume was measured every other day. At the end of the study period, mice were sacrificed by carbon dioxide euthanasia and tumors were removed, measured, weighed, and prepared for Western blot analysis. Tumor volume was calculated according to the formula: V (mm³) = (π x length x width²) / 6. All animal studies were conducted with the approval and guidance of Shanghai Jiao Tong University Medical Animal Ethics Committees.

All human BC cell lines (MCF-7, T-47D, and MDA-MB-231) used in the study were purchased from the Cell Bank of Shanghai Institute of Cell Biology at the Chinese Academy of Science in 2016. The three cell lines were authenticated in Ministry of public security Evidence Identification Center by testing the DNA profiling (STR) entrusted by Shanghai Institute of Cell Biology in 2012 and 2013 respectively. The testing results were consistent with the profiles reported in ATCC. Mycoplasma detection was performed using MycAwayTM–color one step Mycoplasma detection Kit (Shanghai YEASEN Biotechnology Co., Ltd.) according to the manufacturer’s instructions. Cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and incubated in air with 5% CO₂ at 37 °C. In addition, the culture media for MCF-7 and T-47D were not supplemented with estradiol. Antibodies against ERK1/2 (monoclonal, 4695S, 137F5, CST, USA), phoshorylated ERK1/2 (p-ERK1/2) (monoclonal, 4370S, D13.14.4E, CST, USA), P65 (monoclonal, 8242S, D14E12, CST, USA), phoshorylated P65 (p-P65) (monoclonal, 3033S, 93H1, CST, USA), CCKBR (polyclonal, SC33221, Santa Cruz, USA), HER2 (monoclonal, GT210029, sp3, Gene Tech, Shanghai, China), ER (monoclonal, GT210029, sp1, Gene Tech, Shanghai, China), and PR (monoclonal, GT210029, sp2, Gene Tech, Shanghai, China) were used as primary antibodies for Western blot or/or immunohistochemistry (IHC). Gastrin was purchased from China Peptides (Shanghai, China). Lipopolysaccharides (LPS), PD98059, betulinic acid (BA), and parthenolide (PN) were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8; Dojindo, Japan).

Protein was extracted from cells and tissues with RIPA lysis buffer (50 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl, 0.5% DOC, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mmol/l NaF, 1 mmol/l Na₃VO₄, and 1 mM PMSF [Cell Signaling Technologies, Danvers, MA, USA]) purchased from Yeasen Biotech before being centrifuged at 12,000 x g for 20 min at 4 °C. The protein concentrations were determined by a BCA kit (Thermo Fisher Scientific, USA). Protein lysates were applied to Western blot.

Equal amount of proteins were separated on 10% sodium dodecyl sulfate –polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% bovine serum albumin (Amresco, USA) with shaking for 1 h at room temperature, and then washed in 1X Tris-buffered saline-Tween-20 (TBST) buffer thrice for 5 min each. Membranes were incubated overnight at 4 °C with anti-CCKBR (1:200), anti-ERK (1:1000), anti-p-ERK (1:1000), anti-P65 (1:1000), and anti-p-P65 (1:1000) primary antibodies. Then, membranes were washed thrice for 5 min each with 1X TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies (Cell Signaling Technologies, Danvers, MA, USA) for 1 h at room temperature. Membranes were washed thrice for 5 min each with 1X TBST, and antigen-antibody complexes were visualized with a chemiluminescent ECL detection system (Pierce, Rockford, IL, USA). Blots were scanned and analyzed via Image J software.

CCK-8 assay was performed to evaluate cell proliferation. BC cell lines (MCF-7, T-47D, and MDA-MB-231) were seeded onto 96-well plates at a density of 2 × 10³ cells/well, cultured, and treated with gastrin (10^− 7 M), lipopolysaccharides (1 μg/ml), PD98059 (10 μM), betulinic acid (10 μg/ml), or parthenolide (10 μg/ml) for 1, 3, 5, and 7 d, respectively. At the time points indicated above, 90 μL DMEM and 10 μl CCK-8 were added to each well and incubated for an additional 50 min. Then, the media supernatant was removed and the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA). Wells containing 10% CCK-8 (90 μl DMEM and 10 μl CCK-8) were regarded as blanks. The experiment was repeated three times, with four repeated measures of each experimental value.

For tamoxifen treatment, the experiments were performed according to the previously published data [35]. Briefly, 2 × 10³ cells were plated into 96-well plate and culture for 24 h. Then, the cells were washed with 1× PBS, the culture medium was changed with phenol red free DMEM (Gibco, USA) containing 5% charcoal-stripped steroid depleted FBS (Hyclone, USA). Aliquot of gastrin and / or tamoxifen (2 μM) was added to the medium after 24 h culture for 1, 3, 5, and 7 d, respectively. At the time points indicated above, CCK-8 assay was performed to evaluate cell proliferation. The experiment was repeated three times, with four repeated measures of each experimental value.

BC and para-BC tissue samples from 50 patients were embedded in paraffin and sliced into 4-μm-thick sections for IHC. After deparaffinization, the sections were placed into a pressure cooker with 10 mM sodium citrate buffer (pH 6.0) at high power for 3 min and then an oven at 100 °C for 15 min, followed by treatment with 3% H₂O₂ for 15 min at room temperature. Anti-HER2 (1:200), anti-PR (1:400), anti-ER (1:400), Anti-CCKBR (1:50), anti-p-P65 (1:250), and anti-p-ERK (1:250) primary antibodies were incubated on sections overnight at 4 °C. After being placed at room temperature for 30 min, sections were incubated with a secondary antibody for 15 min. DAB and hematoxylin stained images were obtained on a LEICA microscope (CTR6000) equipped with a digital camera (400X magnification). The positive and negative controls were performed in each IHC experiments simultaneously. The positive controls were stained with the slices that were performed with the same antibody in clinic previously while the negative controls were used PBS instead of the primary antibody in each IHC. In addition, IHCs were performed manually. Three researchers who were blinded to patient prognosis evaluated the slides independently, two of them were pathologists. The criteria for “para BC” regions of the slide were that we collected the tissues distance from cancer 3 cm, which do not include fat tissue. The criteria for the cutoff between positive and negative staining as follows: less than 10% of expression was considered to be “loss” (−), and more than 10% of expression was designated (+).

The siRNAs targeting CCKBR and negative control siRNAs were obtained from Shanghai Gene Pharma (Shanghai, China). The siRNA transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, 2 × 10⁵ cells plated in the 6-well plate were transfected. The siRNAs (20 nM, 8 μl) were incubated in OMEM (250 μl) for 5 min while Lipofectamine 3000 reagent (5 μl) was diluted into OMEM (250 μl) gently for 5 min either. Then the above two dilutions were mixed and Incubated for 20 min at room temperature. Lastly DNA-lipid complex was added to cells.

Statistical analysis was performed using the SPSS v. 21. A chi-square test was used to assess associations between categorical data. A Student’s t-test was used for continuous variables using GraphPad Prism v. 6.0. All results are presented as means ± standard error of the means. A P < 0.05 was considered significant for all statistical tests.

In order to explore the role of gastrin in BC, we first measured the serum level of gastrin in 93 BC patients and 20 control subjects. The results showed that gastrin levels were significantly reduced in the majority of BC patients compared to normal controls (Fig. 1A). Importantly, low gastrin level was correlated to clinicopathological characters involving ER subtype and tumor size (Table 1). Particularly, serum gastrin level in ER⁺ BC patients was further decreased indicating a correlation between gastrin and this special subtype of BC. Given that CCKBR served as the receptor of gastrin, it was also determined in BC patients through IHC, and the results showed CCKBR expression was also markedly reduced in this subtype of BC (Fig. 1B), which was increased in MCF-7 cells treated with gastrin (Fig. 1C). According to previous reports that ERK could be activated by downstream CCKBR signal, p-ERK was stimulated in gastrin-treated BC cells as well as p-P65 which was phosphorylated by p-ERK (Fig. 1C). Consistently, p-ERK and p-P65 were decreased in ER⁺ BC subtypes compared to adjacent normal tissues (Fig. 1D). Furthermore, the association of four gene expressions with relapse free survival (RFS) was performed using the KM Plotter Online Tool (http://​www.​kmplot.​com) and the results suggested that the expression level of gastrin/CCKBR/ERK/P65 was found to be correlated with better RFS in BC (Fig. 1E).

To further confirm the association of CCKBR/ERK/P65 and ER positive BC, the levels of CCKBR, p-ERK and p-P65 in fresh tumor and corresponding adjacent normal BC tissues (N = 5) were determined by western blot and IHC (ER, PR and HER2 status was determined in Additional file 2: Figure S1). The results indicated that expressions of CCKBR, p-ERK and p-P65 were all decreased in these examples of ER⁺ subtype, but not in ER⁻ or TNBC BC subtype (Fig. 2A and B). Of note, expression of ERK/P65 was activated in TNBC and ER⁻ BC with or without reduction of CCKBR, suggesting ERK/P65 might be under the regulation of other signaling pathways in these two molecular subtypes of BC (Fig. 2). These results were confirmed by experiments in MCF-7 (ER⁺), T-47D (ER⁺), and MDA-MB-231 (ER⁻) BC cell lines. Moreover, expression of p-ERK/p-P65 was decreased in MCF-7 and T-47D versus MDA-MB-231 cells (Fig. 2C-E).

To explore the role of gastrin in BC, MCF-7, T-47D, and MDA-MB-231 cells were treated with gastrin for 7 days, and the CCK-8 assay was performed at each time point. The results showed that gastrin inhibited growth of MCF-7 and T-47D cells, but not MDA-MB-231 cells (Fig. 3A). To test whether gastrin plays a CCKBR-dependent role, CCKBR was knockdown by the targeted siRNAs (S1, most efficient) in MCF-7 and T-47D cells co-treated with gastrin. As shown in Fig. 3B and C, S1 significantly inhibited expression of CCKBR and gastrin–mediated inhibition on proliferation of BC cells was greatly weakened in these BC cells. In addition, the inhibitory effect of gastrin on ER⁺ BC was further confirmed in mice bearing MCF-7 tumors (Fig. 3D).

To define the role of gastrin in BC through CCKBR-mediated regulation of p-ERK/p-P65, expression of p-ERK/p-P65 in BC cell lines and mice bearing tumors was detected by Western blot. As show in Fig. 4, gastrin treatment led to up-regulation of CCKBR/p-ERK/p-P65 along with growth inhibition of MCF-7 and T-47D cells (Fig. 3A).

To test whether ERK/P65 activator also inhibits growth of ER⁺/CCKBR⁻/ERK⁻/P65⁻ BC cells, MFC-7 and T-47D cells were treated with the activators and the inhibitors of ERK/P65, respectively. As shown in Fig. 5, the ERK/P65 inhibitors significantly inhibited expression of p-ERK/p-P65 without growth inhibition of either cell line (Fig. 5 A, E, C, and G). While the ERK/P65 activators activated the two proteins, they also inhibited proliferation of cells (Fig. 5B, F, D, and H).

Tamoxifen is currently used for the treatment of ER⁺ BC. It is also approved by the US Food and Drug Administration for the prevention of BC in women at high risk for developing the disease. However, the underlying mechanism is still not fully understood. If the inactivation of CCKBR/ERK/P65 is a key event in BC development, then tamoxifen must affect expression of CCKBR/p-ERK/p-P65 in BC cells. To test this, MCF-7 and T-47D cells were separately treated with tamoxifen or gastrin alone or in combination. The results showed that both agents remarkably inhibited growth of the both BC cell lines (Fig. 6). Notably, combination treatment produced a synergistic inhibitory effect (Fig. 6A and B). As with gastrin treatment, tamoxifen also increased expression of CCKBR/p-ERK/p-P65 in both BC cell lines, and it is likely that tamoxifen and gastrin play a cooperative role in down-regulation of CCKBR/p-ERK/p-P65 (Fig. 6C and D).