RPLP1 promotes tumor metastasis and is associated with a poor prognosis in triple-negative breast cancer patients

Patients and tissue samples

TNBC tissues and adjacent non-tumorous tissues were obtained from 81 TNBC patients who underwent curative resection between 2006 and 2014 at the Affiliated Hospital of Nantong University. All cases were newly diagnosed female patients, who had not yet undergone surgery, radiotherapy, chemotherapy, or biological therapy. Survival data were acquired by periodic interviews with their relatives. Tissue samples were processed immediately following the surgical resection. For histological examination, all tumors and adjacent non-tumor tissues were fixed in formalin and embedded in paraffin blocks. Histological slides stained with hematoxylin and eosin were examined by three pathologists. All studies were approved by the Ethics Committee of Affiliated Hospital of Nantong University.

Immunohistochemistry

TNBC sections were deparaffinized and rehydrated with graded ethanol, soaked in EDTA (1 mmol/L, pH 8.0), and then heated to 121 °C in an autoclave for three min to retrieve the antigen. After natural cooling and rinsing with phosphate-buffered saline (PBS, pH 7.2), 0.3% hydrogen peroxide was applied for 20 min to block endogenous peroxide activity. Thereafter, 10% goat serum was applied for 1 h at room temperature to block any nonspecific reactions. After washing with PBS (pH 7.2), the sections were incubated with a rabbit anti-RPLP1 polyclonal antibody (diluted 1:100; Abcam, ab121190, USA) for 2 h. Negative control slides were processed in parallel using a nonspecific IgG antibody (diluted 1:100; Abcam, USA) at the same concentration as the primary antibody. All sections were processed using the peroxidase-anti-peroxidase kit according to the manufacturer’s instructions (Dako, Germany).

Immunohistochemical evaluation

All the immunostained slides were evaluated by three pathologists who were blinded to sample identity. To assess RPLP1 expression, at least five high-power fields (400×) were chosen for each specimen. More than 500 cells were counted to determine the mean percentage of positively stained cells. Staining results were scored semi-quantitatively. As previous reported [18], the percentage of positive cells was scored as follows: 0 (< 10%), 1 (10–30%), 2 (30–50%), and 3 (50–70%). The staining intensity was scored as follows: 0 (negative), 1 (moderate), 2 (positive), or 3 (strongly positive). The immunostaining score, which value ranged from 0 to 9, was calculated as the level of RPLP1 expression. For statistical analysis, 0–4 is defined as low expression, while 5–9 is defined as high expression.

Western blotting

Cells were promptly homogenized in lysis buffer and then centrifuged at 13,000g for 20 min at 4 °C. The supernatant was diluted twofold in SDS loading buffer and denatured at 100 °C for 15 min. An equivalent amount of protein from each sample was loaded onto a 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). The membranes were incubated overnight at 4 °C with the primary antibodies. The antibodies were as follows: anti-RPLP1 (1:500, ab121190, Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (1:1000, ab1416, Abcam), anti-vimentin antibody (1:1000, ab92547, Abcam), anti-N-cadherin antibody (1:1000, ab18203, Abcam), anti-Snail antibody (1:1000, ab180714), and anti-β-actin (1:5000; Abcam). After washing three times with tris-buffered saline with 0.1% tween-20 (TBST) for 5 min each time, the membranes were then incubated with horseradish peroxidase-conjugated secondary human anti-mouse or anti-rabbit antibodies (1:2000; Abcam) for 2 h at room temperature. The bands were then detected using an enhanced chemiluminescence detection system (Bio-Rad, USA).

Real-time quantitative PCR

The mRNA expression levels of RPLP1 in tissues were assessed by the Real-time quantitative PCR method. The total RNA of tissues was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Carlsbad, CA) according to the manufacturers’ instructions. cDNA for mRNA was synthesized using a Omniscript Reverse Transcription kit (Qiagen, Valencia, CA). For detecting the mRNA level of RPLP1, qPCR was conducted on the Mastercycler Ep Realplex (Eppendorf 2S, Hamburg, Germany). β-actin was used as an internal control. The relative mRNA expression levels were evaluated by the 2^−ΔΔCt method [19]. The primer sequences were as follows: For RPLP1: forward, 5′-TGGCCTGGCTTGTTTGC-3′, reverse: 5′-CTCGGATTCTTCTTTCTTTGCTT-3′; For β-actin: forward, 5′-GCGTGACATTAAGGAGAAG-3′, reverse: 5′-GAAGGAAGGCTGGAAGAG-3′.

Cell cultures

TNBC cell lines MDA-MB-231, MDA-MB-436, MDA-MB-468, MDA-MB-453, and the normal breast epithelial cell line MCF-10A were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These TNBC cells were cultured in Leibovitz’s L-15 Medium (L15 medium, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in an incubator at 37 °C without CO₂. The MCF-10A cells were cultured in MEGM medium (Lonza/Clonetics, USA) in an incubator at 37 °C with 5% CO₂. All the cells were passaged every 3–5 days. At the time of cell culture, we have tested for Mycoplasma infection, and there is no mycoplasma infection in each culture.

RPLP1 knockdown or overexpression vector construction and transfection

The human RPLP1 shRNA vector, which targets sequence 5′-CATTAAAGCAGCCGGTGTAAATGTTGAGC-3′, was subcloned into the PLKO.1 vector (Invitrogen), and the RPLP1 expression vector, which contains the RPLP1 cDNA sequence, was subcloned into the PLKI.1 vector (Invitrogen). For vector transfection, the cells were seeded the day before transfection using antibiotic-free L15 medium with 10% FBS. Transient transfection of the shRNA vectors or overexpression vector was carried out using Lipofectamine 2000 in OptiMEM media, as suggested by the manufacturer (Thermo-Fisher). Cells were incubated with the vectors and lipofectamine reagent complexes for 4 h at 37 °C. FBS was then added to the cells to achieve a final concentration of 10% in medium. Two days after transfection, puromycin (Sigma-Aldrich, USA) was added to the media at 1 μg/mL for 1 week of selection. The expression levels of the target genes were determined by Western blot analysis.

Cell proliferation assay

The cell proliferation assay was performed with the Brdu assay kit according to the manufacturer’s protocol (Roche, Germany). Generally, cells were incubated with 100 µM Brdu labeling solution for 4 h at 37 °C. After removing the culture media, the cells were fixed, and the DNA was denatured with FixDenat solution. The anti-Brdu-POD working solution and substrate solution were then added, and the absorbances of the samples were measured by an ELISA plate reader at 370 nm.

Colony formation assay

Cells were seeded in a 6-well plate at a density (1 × 10³ cells/well). After cultured for 10 days, cells were fixed with 4% paraformaldehyde and then counted after staining with crystal violet. Three independent experiments were performed.

Cell invasion assay

For the invasion assay, cells were suspended in 500 µL serum-free media and placed in the upper compartment of the invasion chamber coated with Matrigel (BD Biosciences). The lower compartment was imbued with a complete medium as the chemoattractant. The experiment was performed in triplicate.

Cell migration assay

A 500 μL cell suspension was placed in each insert chamber containing medium free of FBS, while the medium in the lower chamber contained 10% FBS. The cells that had migrated and attached to the lower surface of the insert were fixed with 4% formaldehyde and stained with crystal violet. After washing with PBS, the number of cells was counted randomly in five scopes under a microscope (400×).

In vivo mouse model

In this study, all animal experiments were approved by the Animal Ethics committee. Nude mice from each group (n = 6) received a tail vein injection of 1 × 10⁵ cells per week for three consecutive weeks [20], and the number of cells injected into each tail vein each time was 1 × 10⁵. The presence of lung metastases was determined by hematoxylin and eosin (H&E) staining after 10 weeks.

Statistical analysis

All statistical analyses were carried out using SPSS 20.0 (Statistical Product and Service Solutions, USA). The association between RPLP1 expression and clinicopathological features was computed using the Chi square (χ²) test. RPLP1 in TNBC cells was studied using the Spearman rank correlation test, because the data were not normally distributed. For survival analysis, the log rank test and Kaplan–Meier method were used. Multivariate analysis was performed with the Cox’s proportional hazards model. For all cases, P < 0.05 was considered statistically significant.

The expression of RPLP1 in TNBC tumors, adjacent normal tissue, and cell lines

To confirm the results of our prior proteomics data, we used immunohistochemistry staining to profile the expression of RPLP1 in paraffin embedded TNBC tissues. Representative examples of reactivity for RPLP1 are shown in Fig. 1. RPLP1 expression in TNBC was scored as positive when cytoplasmic staining was strong. We demonstrated that RPLP1 expression is low in the adjacent normal tissues, while it is high in TNBC tissues. Furthermore, the expression of RPLP1 was elevated in the metastasis group compared to the metastasis negative group (Fig. 1). To confirm these immunohistochemical results, we analysed RPLP1 protein expression (via Western blotting) in TNBC and normal breast epithelial cell lines. RPLP1 protein expression was significantly higher in TNBC cells than in normal breast cells, and in MDA-MB-231 cells, the expression of RPLP1 was relatively high (Fig. 2a). Furthermore, we used quantitative PCR to verify the same expression pattern of RPLP1 in the mRNA level (Fig. 2b). These data indicate that RPLP1 expression is correlated with and may be involved in the development of TNBC.

Correlation of RPLP1 expression with the clinicopathological parameters of TNBC

Prognostic significance of RPLP1 expression

Since we found that RPLP1 was associated with histologic grades, we further investigated the role of RPLP1 in breast cancer prognosis. We ultimatelydetermined that high levels of RPLP1, histologic grade, axillary lymph node status, and distant metastasis were significantly associated with patient survival states (P = 0.014, P = 0.021, P = 0.011, P = 0.034) (Table 2). Furthermore, using the Cox’s proportional hazards regression model, we determined that RPLP1 expression (P = 0.001), histologic grade (P = 0.002), axillary lymph node status (P = 0.001), and distant metastasis (P = 0.004) were independent prognostic factors in patients with TNBC (Table 3). In univariate analysis, using Kaplan–Meier survival curves, we also found that high RPLP1 expression was statistically correlated with poor overall survival (Fig. 3).

Criteria	No. case	Survival status		P value	χ2
		Alive	Dead
Age
< 50	39	34	5	0.105	3.025
≥ 50	42	30	12
Tumor size
≤ 2	35	27	8	0.059	5.661
2–5	41	35	6
> 5	5	2	3
Grade
I	22	14	8	0.021*	7.725
II	32	30	2
III	27	20	7
Axillary lymph node status
N0	48	43	5	0.011*	7.939
Nx	33	21	12
Distant metastasis
Negative	60	51	9	0.034*	5.003
Positive	21	13	8
RPLP1
Low	46	41	5	0.014*	6.572
High	35	23	12

Statistical analyses were performed by the Pearson χ² test

* P < 0.05 is considered significant

	Hazard ratio	95% confidence interval	P
Age	1.150	0.696–1.902	0.586
Tumor size	1.472	0.887–2.443	0.135
Grade	1.890	1.267–2.819	0.002*
Axillary lymph node status	2.541	1.445–4.467	0.001*
Distant metastasis	2.526	1.337–4.770	0.004*
RPLP1	2.552	1.499–4.344	0.001*

Statistical analyses were performed by the Pearson χ² test

* P < 0.05 is considered significant

RPLP1 promotes cell invasion and migration in TNBC

Due to the links between RPLP1 expression and patient characteristics, we went on to investigate the role of RPLP1 in TNBC cell proliferation and invasion. When MDA-MB-231 cells were transfected with the shRNA and expression vector, we confirmed that the shRNA vector effectively knocked down RPLP1 expression, while the expression vector increased RPLP1 expression in TNBC cells (Fig. 4a). When we monitored the proliferation of these cells after 72 h of transfection, we found that changes in RPLP1 expression did not affect cell growth (Fig. 4b). In addition, the clone formation assay has been carried out and we found that changes in RPLP1 expression did not affect cell clone formation abilities (Fig. 4c). We also observe that inhibition of RPLP1 expression can significantly inhibit cell invasion, and overexpression of RPLP1 increased cell invasion in the MDA-MB-231 and MDA-MB-468 cell line (Fig. 4d). Lastly, the migration potential of cells upon overexpression and knockdown of RPLP1 has been evaluated. We also observed that inhibition of RPLP1 expression can significantly inhibit cell migration, and overexpression of RPLP1 enhanced cell migration in the MDA-MB-231 and MDA-MB-468 cell line (Fig. 5). These data indicate that RPLP1 promotes cancer metastasis in TNBC, but it may not be absolutely required for cell growth or proliferation.

The mechanism that RPLP1 affects metastasis may be through EMT

The epithelial-mesenchymal transition plays an essential role in TNBC metastasis. To investigate the mechanism of RPLP1 in TNBC metastasis, we examined the effect of RPLP1 on the epithelial-mesenchymal transition in MDA-MB-231 cells. When we knocked down RPLP1 expression, the epithelial cell marker E-cadherin was increased, and the mesenchymal cell marker N-cadherin was decreased. However, overexpression promoted expression of the mesenchymal cell marker N-cadherin and decreased expression of epithelial cell marker E-cadherin (Fig. 6a). These data indicate RPLP1 mediates cell invasion and affects the cell epithelial-mesenchymal transition. Histologic analysis confirmed that MDA-MB-231 overexpressed RPLP1 cells had an increased ability to metastasize to the lungs compared to the MDA-MB-231 control. Contrary, inhibition of RPLP1 expression can significantly inhibit metastasis (Fig. 6b).