ClpP regulates breast cancer cell proliferation, invasion and apoptosis by modulating the Src/PI3K/Akt signaling pathway

Tissue specimens

Human BC tissues and corresponding adjacent normal tissues were collected from patients who underwent surgery at the First Affiliated Hospital of Chongqing Medical University from 2014 to 2018, snap-frozen in liquid nitrogen, and then stored at −80 °C. This study was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Chongqing Medical University (approval no. 2019-208) and conducted in accordance with the tenets of the Declaration of Helsinki. Each participant signed an informed consent form prior to study inclusion.

Cell culture

Seven human BC cell lines (T47D, MCF-7, ZR-75-1, SK-BR-3, MDA-MB-231, MDA-MB-468 and BT-549) and two normal mammary epithelial cell lines (MCF-10A and HBL-100) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-10A cells were cultured as described previously (Debnath, Muthuswamy & Brugge, 2003) and all other cell lines were cultured in Roswell Park Memorial Institute 1640 medium (Gibco BRL, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL) at 37 °C under a humidified atmosphere of 5% CO₂/95% air.

Small interfering RNAs (siRNAs), plasmids, and transfection

ClpP-specific and the negative control siRNAs were synthesized by OriGene (OriGene Technologies Inc., Rockville, MD, USA). The following siRNAs sequences were generated: SR305388A-rGrCrUrCrArArGrArArGrCrArGrCrUrCrUrArUrArArCrATC; SR305388B-rGrUrUrUrGrGrCrArUrCrUrUrArGrArCrArArGrGrUrUrCTG; and SR305388C-rGrGrCrCrArUrCrUrArCrGrArCrArCrGrArUrGrCrArGrUAC. The expression vector pCMV6-Myc-DDK-ClpP was produced by OriGene (OriGene Technologies Inc., Rockville, MD, USA); the empty pCMV6-Myc-DDK vector was used as a control. Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA) was used for siRNAs and plasmids transfection, in accordance with the manufacturer’s protocols.

RNA extraction and quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from cells and tissues with TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. RT-qPCR was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the SYBR Green kit (Invitrogen). β-actin was used as an internal control. Each sample was tested in triplicate. The followig primers were used for RT-qPCR analysis: ClpP, forward primer: 5′-GCC AAG CAC ACC AAA CAG A-3′, reverse primer: 5′-GGA CCA GAA CCT TGT CTA AG-3′; β-actin, forward primer: 5′-CCT GTG GCA TCC ACG AAA CT-3′, reverse primer: 5′-GAA GCA TTT GCG GTG GAC GAT- 3′.

Western blot analysis

Total proteins were extracted with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Western blot analysis was conducted as previously described (Li et al., 2018) with the following antibodies: anti-ClpP (OriGene Technologies Inc., TA502075), anti-c-Src (Santa Cruz Biotechnology, sc-130124), anti-p-Src (Santa Cruz Biotechnology, sc-166860), anti-PI3K (Santa Cruz Biotechnology, sc-12930), anti-p-PI3K (Santa Cruz Biotechnology, sc-12929), anti-Akt (Wanleibio, WL0003b), anti-p-Akt (Proteintech Group, 66444-1-ig), anti-caspase 3 (Santa Cruz Biotechnology, sc-271759), anti-cleaved-caspase 9 (Wanleibio, WL01838), anti-cleaved-caspase 8 (Wanleibio, WL0153), anti-cleaved PARP (Cell Signaling Technology, #9541), anti-MMP7 (Santa Cruz Biotechnology, sc-80205), anti-E-cadherin (Abcam, ab40772), anti-vimentin (Santa Cruz Biotechnology, sc-965) and anti- β-actin (Santa Cruz Biotechnology, sc-47778), anti-mouse IgG (Cell Signaling Technology, #7076), anti-rabbit IgG (Cell Signaling Technology, #7074) and anti-goat IgG (Proteintech Group, SA00001-4).

Immunohistochemical (IHC) analysis

All specimens were formalin-fixed, paraffin-embedded and cut into 4 µm-thick sections, which were mounted onto glass slides. IHC analysis was conducted as described previously (Li et al., 2018). Briefly, the slides were incubated with a primary antibody against ClpP overnight at 4 °C. Images were obtained using a Leica microscope equipped with a digital camera (Leica Microsystems, Wetzlar, Germany) and the IHC results were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MA, USA). The grayscale units were converted to optical density, units and the area and integrated optical density (IOD) of the sections were then measured to calculate the mean optical density (MOD) for semi-quantitative statistical analysis, where MOD = total IOD/total area. The mean density was calculated as IOD/area.

Cell apoptosis analysis

Cell apoptosis was detected with the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) in accordance with the manufacturer’s protocol and a FACSCalibur flow cytometer (BD Biosciences).

Colony formation assay

MDA-MB-231 and ZR-75-1 cells were seeded into triplicate wells of 6-well plates at 1,000 cells/well, and cultured for 7 days. The cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution (C0121, Beyotime Institute of Biotechnology, Haimen, China).

Cell migration and invasion assay

Transwell chambers (8-µm pore size; Corning, NY, USA) were used to detect the migratory and invasive capabilities of BC cells. For the transwell migration assays, 200-µlL aliquots of transfected MDA-MB-231, ZR-75-1, MCF-7 and T47D cells (4 * 10⁴ cells/well) were added into the upper transwell chamber, and 800 µL of medium containing 10% FBS were added to the lower chamber. After incubation at 37 °C under an atmosphere of 5% CO₂/95% air for 24 h (MDA-MB-231 and ZR-75-1 cells), or 72 h (MCF-7 and T47D cells), cells that migrated through the membrane pores were fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (DC079; Genview, Beijing, China) for 15 min at room temperature. Matrigel™-coated transwell filters (Matrigel™: serum-free medium = 1:7, 70 µl/chamber) were used to evaluate the invasion capability of the cells. The subsequent procedures were the same as those for the cell migration assay. Cells from six random fields were counted under a microscope. All experiments were repeated three times.

Database analysis

TCGA dataset analyses

Gene expression data of BC tissues were downloaded from the TCGA database (https://tcga-data.nci.nih.gov/tcga/). The study cohort consisted of a total of 1010 BC patients. ClpP expression data of 112 normal breast samples were included to compare differences in ClpP expression levels in BC tissues. Overall survival (OS) and complete clinicopathological data of 990 BC patients, and RFS data of 792 BC patients were screened. ClpP expression levels were ranked from low to high based on median values. The first 50% of patients were considered as the low-expression group and the second 50% as the high-expression group.

Kaplan Meier-plotter dataset analyses

Prognosis based on ClpP levels in BC patients was analyzed using the Kaplan Meier-plotter database (http://kmplot.com/analysis/).

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 22.0 (Armonk, NY, USA) and GraphPad Prism 7.0 software (San Diego, CA, USA). The two-tailed Student’s t-test was used to compare two groups of independent samples. The chi-square test was used to assess the correlation between ClpP expression and the clinicopathological characteristics of BC patients. Kaplan–Meier analysis was performed to plot survival curves, which were compared with the log-rank test. A p-value < 0.05 was regarded as statistically significant.

ClpP is overexpressed in human BC tissues and associated with clinical outcomes

ClpP expression levels in BC tissues were evaluated using the TCGA dataset. The results showed that ClpP expression was significantly up-regulated in BC tissues, as compared with normal tissues (p < 0.001) (Fig. 1A). Receiver operating characteristic (ROC) curve analysis was conducted to estimate the diagnostic value of ClpP. The area under the ROC curve (AUC) was 0.829 (95% confidence interval [CI] [0.79–0.869], p < 0.0001) (Fig. 1B). To verify the results, 18 pairs of BC and adjacent normal tissues were used for RT-qPCR analysis of ClpP expression levels. Consistently, ClpP expression was significantly increased in BC tissues, as compared with normal tissues (p < 0.01) (Fig. 1C). The AUC was 0.713 (95% CI [0.543–0.883], p = 0.029) (Fig. 1D). ClpP protein expression was also assessed by IHC analysis. As compared with normal tissues, cytoplasmic ClpP immunoreactivity was markedly higher in tumor tissues (p < 0.001) (Figs. 1E–1G). These results showed that ClpP expression was significantly increased in BC tissues, suggesting high diagnostic potential. The clinical significance of ClpP in BC was assessed in tissues from 990 BC patients with complete clinicopathological characteristics (Table 1). High ClpP expression was found to be significantly associated with the T stage (p = 0.0154) and ER expression (p = 0.0164). Kaplan–Meier survival curves from TCGA indicated that ClpP was not associated with RFS (p = 0.506) or OS (p = 0.619) (Figs. 2A and 2B). However, within the Kaplan Meier-plotter database, high ClpP expression was associated with poor RFS (p = 0.00071) (Figs. 2C and 2D). These results suggest that upregulation of ClpP was correlated with the T stage, ER expression, and poor RFS, suggesting a potential essential role in BC tumorigenesis.

Silencing of ClpP inhibits proliferation, migration, invasion and induces apoptosis of BC cells

ClpP mRNA and protein expression levels in a panel of BC and normal breast epithelial cell lines were determined. The results showed that ClpP was increased in most malignant cell lines, as compared with normal cells (Figs. 3A and 3B). To further investigate the potential biological functions of ClpP in BC, gain and loss-of-function studies were performed. The results showed that ClpP expression was significantly higher in MDA-MB-231 and ZR-75-1 cells, but lower in MCF-7 and T47D cells. Thus, ClpP was knocked-down in MDA-MB-231 and ZR-75-1 cells, and overexpressed in MCF-7 and T47D cells. ClpP mRNA and protein expression was efficiently silenced by three different siRNAs in MDA-MB-231 and ZR-75-1 cells (Figs. 3C–3F), and overexpression efficiency in MCF-7 and T47D cells was confirmed by RT-qPCR (Fig. S1A).

The suppressive effect of ClpP silencing on cancer cell growth was confirmed by the colony formation assay. The colony formation capabilities of the BC cell lines MDA-MB-231 and ZR-75-1 were markedly inhibited by si-ClpP-B (p < 0.01 and <0.001, respectively) (Figs. 4A–4E).

The transwell migration and invasion assay was performed to quantitatively assess cell metastasis and invasiveness. The results showed that cell migration and invasion were significantly reduced in cells transfected with si-ClpP-B, as compared with control cells, due to the down-regulation of MMP7 and vimentin, and the up-regulation of E-cadherin (Figs. 4F–4Q and Figs. 5O–5T). Conversely, the numbers of migratory and invasive MCF-7 and T47D cells were markedly increased by ClpP overexpression (Figs. S1B–S1E).

Flow cytometry was carried out to detect cell apoptosis. The percentages of apoptotic MDA-MB-231 and ZR-75-1 cells were increased by si-ClpP-B (p < 0.01 and <0.001, respectively), accompanied by increased levels of cleaved caspase-9, cleaved caspase-8 and cleaved poly (ADP-ribose) polymerase (PARP) (Figs. 5A–5N).

To confirm the specificity of ClpP siRNAs, the same functional experiments were performed with a second siRNA (si-ClpP-A). The results of the loss-of-function studies were consistent (Fig. S2).

These data revealed the anti-proliferative, anti-migration, anti-invasion and pro-apoptotic roles of silencing ClpP in BC cells.

The tumor suppressive effect of silencing ClpP is mediated by the Src/PI3K/Akt signaling pathway

Previous studies have reported that the PI3K/Akt signaling pathway is involved in cellular transformation, tumorigenesis, cancer progression, and proliferation of BC cells (Guerrero-Zotano, Mayer & Arteaga, 2016; Sharma et al., 2017). Further, ClpXP reported to mediate cell migration, invasion, and metastasis in vivo by increasing phosphorylation of the key cellular kinases Akt and Src (Seo et al., 2016). Here, western blot analysis was conducted to confirm whether the Src/PI3K/Akt pathway is involved in ClpP-induced BC progression. As shown in Fig. 6, silencing of ClpP inhibited the activation of Src, resulting in the inhibition of PI3K phosphorylation and the downstream signaling molecule Akt, which led to a series of changes in the activities of related molecules associated with cell proliferation, apoptosis, migration and invasion. These data confirmed the effect of ClpP on the proliferation and invasion capabilities of MDA-MB-231 and ZR-75-1 BC cells, as well as the induction of apoptosis.