A total of 120 patients with breast cancer who underwent surgical treatment and had complete tumor tissue blocks preserved at the Department of Breast Surgery, Qilu Hospital of Shandong University (Jinan, China) between January 2004 and May 2016 were recruited. All 120 patients were female, and their ages ranged between 26 and 72 years old, with an average age of 46.3±9.9 years old. The inclusion criteria were as follows: Diagnosed with breast cancer based on the post-operative pathological section; completed clinical follow-up survey; >18 years old; and never received pre-operative chemotherapy or radiotherapy. The exclusion criteria were as follows: <18 years old; had incomplete clinical follow-up data or other malignant tumors at the same time; or received pre-operative chemotherapy or radiotherapy. Patients all underwent surgery: 88 had modified radical mastectomy (73%) and 32 had breast-conserving therapy (27%). After surgery, the patients were treated with chemotherapy or radiotherapy. Indications for post-operative chemotherapy, radiotherapy or chemoradiotherapy were patients with various pathological features, such as advanced cancer stage, lymph node positivity, perineural invasion, lymphovascular invasion and receptor status. Patients positive for hormone receptors received adjuvant endocrine therapy for 5 years and patients positive for human epidermal growth factor receptor 2 (HER2) received trastuzumab therapy for 1 year. After finishing treatment, patients were regularly followed-up with clinical examinations and imaging. Patients were scheduled for clinical visits every 4–6 months during the first and second years, every 6 months during the third, fourth and fifth years, and annually thereafter.

Formalin-fixed (10% neutral buffered formalin at 4°C for 12–24 h at room temperature), paraffin-embedded sections (size, 4 µm) from breast cancer and normal breast tissue samples were obtained. The normal tissue samples were obtained from the adjacent tissue of the same patients, and the distance between tumor and normal tissues was ≥0.5 cm (17). The sections were deparaffinized, rehydrated in a descending alcohol series and heated at 120°C for 10 min in 10 mM sodium citrate (pH 6.0) to retrieve antigens. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide at room temperature for 10 min. The sections were then incubated with an anti-SHOC2 antibody (1:400; cat. no. Fnab06912; Wuhan Fine Biotech Co., Ltd.) at room temperature for 1 h, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. 8114; Cell Signaling Technology, Inc.) at room temperature for 15 min. The sections were developed in diaminobenzidine for 5 min and counterstained with hematoxylin for 3 min at room temperature. The primary antibody was omitted for the negative controls. Sections were visualized and photographed using an optical microscope (Leica Microsystems GmbH; magnification, ×50 and ×200). The expression levels of SHOC2 were semi-quantified using a semi-quantitative IHC scoring system, as previously described (18,19). The percentage of positive tumor cells was graded on a scale between 0 and 4, as follows: 0, none; 1, 1–10%; 2, 11–50%; 3, 51–80%; 4, >80%. The intensity of staining was graded on a scale between 0 and 3, as follows: 0, none; 1, weak staining; 2, moderate staining; 3, strong staining. The combination of the extent (E) and intensity (I) of staining was obtained as the product of E and I (EI), which varied between 0 and 12 for each sample. Using the X-tile software program (version 3.6.1; The Rimm Lab, Yale University; http://medicine.yale.edu/lab/rimm/research/software.aspx), a significant cutoff point for SHOC2 was identified in terms of overall survival (OS) in patients with breast cancer, as previously described (20). A cutoff score of 8 was selected; 0–8 was considered low expression, whereas >8 was considered high expression.

MCF-7 and MDA-MB-231 breast cancer cell lines and the 293T cell line were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and maintained by the Center Lab of Shandong University. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin, and grown at 37°C in 5% CO₂. Stable knockdown of SHOC2 in the breast cancer cell line was performed using lentiviruses. Briefly, the target sequence (5′-TGCTTGATTTACGGCATAA-3′) for short hairpin RNA (shRNA)-SHOC2 and the nonspecific control sequence (5′-TTCTCCGAACGTGTCACGT-3′) (21) were inserted into the GV493 shRNA vector (Shanghai GeneChem Co., Ltd.) linearized with AgeI and EcoRI. For GFP expression in this vector, GFP-IRES was cloned in front of the puromycin-resistant marker gene. Virus production was carried out using 293T cells. Briefly, cells were transfected with lentiviral DNA constructs alongside the lentiviral packaging plasmids pHelper 1.0 and pHelper 2.0 (Shanghai GeneChem Co., Ltd.) at a ratio of 4:3:2. The viral supernatants were harvested 48 h post-transfection, filtered using a 0.45-µm pore filter, and used for infection. To establish stable cell lines, cells were seeded in 6-well plates (2×10⁵/well) until they reached 60–70% confluence before being infected with either SHOC2 knockdown or the control lentivirus (MOI=10), and selected with puromycin (2 µg/ml) to obtain single-cell clones. The single-cell clones obtained were cultured for 2 weeks, and amplified for further experiments in culture media containing puromycin (1 µg/ml).

RT-qPCR was performed to detect the mRNA expression levels of SHOC2 in 11 pairs of fresh breast cancer and adjacent noncancerous human breast tissues. Total RNA was isolated from breast cancer and adjacent noncancerous tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Random-primed cDNA synthesis was performed using a QuantiTect^® RT kit (Qiagen GmbH). The RT reaction was performed as follows: Initial incubation at 42°C for 2 min, followed by sequential steps at 42°C for 15 min, and at 95°C for 3 min. RT-qPCR was performed using a PTC-100^® Thermal cycler (MJ Research, Inc.) with a PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocols. For qPCR, the thermocycling conditions were: Pre-denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and of annealing at 60°C for 30–60 sec. For amplification, the following primers were designed: SHOC2, forward 5′-TCAGTGGTGTATAGGCTGGATTCT-3′, reverse 5′-GCTACATCCAGCGTAATGAGGT-3′; GAPDH, forward 5′-CCTCCGGGAAACTGTGGCGTGATGG-3′ and reverse 5′-AGACGGCAGGTCAGGTCCACCACTG-3′. Each sample was examined three times, and the levels of PCR product were adjusted to GAPDH, which served as an internal control. The fold change of mRNA expression was calculated using the formula: 2^−ΔΔCq (22).

Confluent cells were washed twice with ice-cold PBS and lysed in RIPA buffer with 1 mM PMSF (Beyotime Institute of Biotechnology). The concentrations of the total protein extracts were determined by the bicinchoninic acid (BCA) method using a commercial kit from Pierce; Thermo Fisher Scientific, Inc. Equal amounts of protein samples (40 µg) were separated by SDS-PAGE on 10% gels and transferred onto PVDF membranes. After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against SHOC2 (1:1,000; cat. no. Fnab06912; Wuhan Fine Biotech Co., Ltd.), Bcl-2 (1:1,000; cat. no. 15071; Cell Signaling Technology, Inc.), Bax (1:1,000; cat. no. 5023; Cell Signaling Technology, Inc.), poly (ADP-ribose) polymerase (PARP) (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), phosphorylated (p)-AKT (1:1,000; cat. no.4060; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 4691; Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 4376; Cell Signaling Technology, Inc.), ERK (1:1,000; cat. no. 4696; Cell Signaling Technology, Inc.) and GAPDH (1:1,000; cat. no. AP7873a; Abgent, Inc.), followed by incubation with either HRP-conjugated Anti-Rabbit secondary antibodies (1:5,000; cat. no. R4880; Sigma-Aldrich; Merck KGaA) or Anti-Mouse secondary antibodies (1:5,000; cat. no. 7077; Cell Signaling Technology, Inc.) at room temperature for 1 h. Protein bands were detected by ECL western blot substrate (Thermo Fisher Scientific, Inc.). Immunoreactive bands were visualized by ImageQuant LAS 4000 series (GE Healthcare). Image J v.1.43 (National Institutes of Health) software was used to quantify relative protein expression.

Cells (5 ×10³/well) were seeded and incubated in 96-well plates at 37°C in a 5% CO₂ environment for 1, 2, 3, 4 and 5 days. At each time point, 5 mg/ml MTT was added to each well. After incubation at 37°C for 4 h, the supernatants were removed. Subsequently, 100 µl DMSO was added to each well, and the well contents were thoroughly mixed for 5 min. The absorbance was measured at 490 nm using a microplate reader (TECAN Infinite^® 200; Tecan Group, Ltd.).

Cells were cultured to 85% confluence, collected and then rinsed with cold D-Hanks balanced salt solution. After centrifugation at 1,000 × g for 5 mins at room temperature, the supernatants were removed, and the cells were fixed in 75% cold ethanol for 1 h. The fixed cells were rinsed with D-Hanks solution and permeabilized with 0.1% Triton X-100 and 2 mg/ml RNase A in D-Hanks solution for 30 min at 37°C. The cells were then rinsed with D-Hanks solution and stained with 50 mg/ml propidium iodide (Sigma-Aldrich; Merck KGaA) at 4°C for 1 h. The stained cells were analyzed with a Millipore Guava^® easyCyte 5HT flow cytometer (EMD Millipore). A total of 8 days after lentiviral infection, cells (5×10⁵) were collected and rinsed with cold D-Hanks balanced salt solution. After centrifugation at 1,000 × g for 3 mins at room temperature, the supernatants were removed. The cells were resuspended with 200 µl binding buffer and then stained with 10 µl Annexin V-APC (cat. no. 88-8007; eBioscience; Thermo Fisher Scientific, Inc.) at room temperature in the dark for 10–15 min. Subsequently, flow cytometry was performed using the Guava easyCyte 5HT flow cytometer. Data were analyzed using FlowJo software v.10 (FlowJo LLC).

All results were repeated three times and the data are presented as the mean ± standard deviation. The association among SHOC2 expression, proliferation and apoptosis was assessed using unpaired Student's t-test. The mRNA expression was compared between normal and tumor tissues using paired Student's t-test. IHC data were compared between normal and tumor tissues using Wilcoxon signed-rank test. The relationship between SHOC2 expression and clinicopathological characteristics was analyzed by the χ² test. Survival curves were plotted by the Kaplan-Meier method and compared using the log-rank test. Survival data were evaluated using univariate and multivariate Cox regression analyses. All statistical analyses were performed using SPSS version 18.0 (SPSS, Inc.). P<0.05 was considered to be statistically significant.

MCF-7 and MDA-MB-231 breast cancer cells were infected with shRNA-SHOC2 to induce SHOC2 knockdown (Fig. 1A). Subsequently, MTT assays were performed to determine the effects of SHOC2 on breast cancer cell proliferation, which revealed that cell counts were significantly decreased by SHOC2 knockdown. Therefore, SHOC2 knockdown may inhibit the growth of MCF-7 and MDA-MB-231 breast cancer cells (P<0.001; Fig. 1B and C). The mechanism underlying the antiproliferative effects of SHOC2 knockdown was examined by analyzing apoptosis using Annexin V staining. The number of Annexin V-positive cells was significantly higher in shRNA-SHOC2 breast cancer cells than in control cells (P<0.001; Fig. 1F and G). Moreover, cell cycle distribution was analyzed using flow cytometry, which showed that SHOC2 knockdown led to cell arrest in the G1 and G2/M phases (P<0.05; Fig. 1D and E). Next, a series of cellular apoptosis-related proteins were analyzed; the expression levels of the anti-apoptosis marker Bcl-2 were significantly decreased, whereas the levels of the pro-apoptotic markers Bax and cleaved-PARP were increased in shRNA-SHOC2 cells (P<0.001; Fig. 2A). These results indicated that knockdown of SHOC2 significantly inhibited proliferation, and promoted apoptosis and cell cycle arrest in breast cancer cells.

Analysis of RAS-MAPK/PI3K pathway-associated protein expression revealed that after SHOC2 was knocked down in MCF-7 and MDA-MB-231 breast cancer cells, p-ERK and p-AKT expression was decreased. These findings indicated that the Ras-ERK and PI3K-AKT pathways were inhibited by SHOC2 knockdown, as revealed by the decrease in positive signals for p-ERK and p-AKT (P<0.001; Fig. 2B).

To explore the roles of SHOC2 in human breast cancer, SHOC2 mRNA was evaluated in fresh clinical samples by RT-qPCR. SHOC2 expression was significantly higher in human breast cancer tissues than in paired normal breast tissues (P<0.01; Fig. 3A). In the IHC analysis, SHOC2 protein was predominantly found in the cytoplasm of tumor cells, and this demonstrated that 88 of 120 (73.3%) breast cancer patients had high SHOC2 expression. Consistent with the RT-qPCR results, increased SHOC2 expression levels, compared with those in normal breast tissues, were detected in the tumor tissues (P<0.01; Fig. 3B-F).

The associations between SHOC2 expression and the clinicopathological features of breast cancer are summarized in Table I. The results showed that significant associations were observed between high SHOC2 expression and high histopathological grades (P<0.01). Similarly, regarding tumor size, SHOC2 expression scores were higher in T2-T3 (>2 cm) tumors than in T1 (≤2 cm) tumors (P=0.023). In addition, SHOC2 expression was associated with estrogen receptor (ER) status and was higher in patients with ER-negative breast cancer than in patients with ER-positive breast cancer (P=0.028). However, the associations between SHOC2 expression and patient age, progesterone receptor, HER2 or lymph node status were not statistically significant.

The Kaplan-Meier method was applied to explore the significance of SHOC2 expression for the OS rates of the enrolled patients. The results revealed that patients with low SHOC2 expression had improved OS than those with high SHOC2 expression (Fig. 3G). The prognostic value of SHOC2 expression was analyzed in different subgroups of patients. For the patients with different SHOC2 expression statuses, differences in prognosis were more notable in the ER-negative subgroup than in the ER-positive subgroup. The OS was significantly lower for patients with high SHOC2 expression than for patients with low expression (P<0.05; Fig. 3H and I); this result implied that the expression of SHOC2 may be more important for ER-negative patients. The univariate analysis revealed that tumor size (P=0.023), ER status (P=0.015), lymph node status (P<0.001) and SHOC2 expression (P=0.020) were all related to OS (Table II). These data suggested that SHOC2 might have prognostic value for patients with breast cancer. Subsequently, a multivariate analysis was performed using the Cox proportional hazards model and demonstrated that high SHOC2 expression (P<0.001), lymph node status (P<0.001) and negative ER expression status (P=0.010) were independent prognostic factors for survival (Table II).