Background
Breast cancer (BC) is the most frequently diagnosed cancer in women worldwide and continues to be one of the leading causes of cancer-related deaths [
1,
2]. The standard treatment for BC mainly depends on the molecular subtype of the tumour. Triple-negative breast cancer (TNBC), which lacks oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounts for approximately 15–20% of all BCs. As a highly aggressive and heterogeneous tumour, it contributes significantly to tumorigenesis and resistance to chemotherapy and is thus associated with an increased risk of disease recurrence and death [
3]. Moreover, improper interventions, in terms of both timing and methods, lead to earlier relapse and a worse outcome [
4]. Hence, there is a desire to explore more effective strategies.
Over the last decade, advances in molecular translational research have heralded major breakthroughs in the understanding, diagnosis and management of breast cancer. Cell cycle progression undertakes a crucial role in cell proliferation, the aberration of which has been acknowledged as a hallmark of cancer [
5,
6]. CDK4 and CDK6, cyclin-dependent kinases that function in the form of cyclin D1-bound compounds at a cell cycle checkpoint, can promote G1-S phase transition in the cell cycle. A major target of CDK4 and CDK6 during cell cycle progression is the tumour suppressor retinoblastoma (RB) protein. When RB is phosphorylated, its growth-suppressive properties are inactivated, releasing E2Fs. Amplification and overactivation of the CDK4/6-cyclin D1-RB-E2F pathway have been observed in various malignancies, including BC [
7‐
12]. Selective CDK4/6 inhibitors “turn off” these kinases and dephosphorylate RB, resulting in the blockade of cell cycle progression in mid-G1 phase and preventing the proliferation of cancer cells [
13].
Although many pre-clinical and clinical trials based on different TNBC subtypes have been conducted, no explicit targets have yet been identified [
14,
15]. Clinical studies have confirmed the efficacy of cisplatin (CDDP), given through either single or combined administration, for TNBC. However, CDDP is highly toxic to the blood and nervous system and has limited survival benefits, so it is not routinely applied in chemotherapy regimens [
16,
17]. Palbociclib (PD0332991, PD), a selective CDK4/6 inhibitor, has been approved by the Food and Drug Administration (FDA) as a first-line endocrine-based therapy for postmenopausal women with hormone-receptor-positive (HR +), HER2-negative advanced or metastatic breast cancer [
14,
18‐
20]. Therefore, we wondered whether PD alone or in combination with CDDP could be applied as a novel treatment for TNBC. To answer this question we performed the current study to discover the effect of PD alone or combined with CDDP on TNBC cells and the corresponding mechanism.
Methods
Cell culture and treatments
The TNBC cell line MDA-MB-231 was kindly donated by Professor Erwei Song, University of Sun Yat-Sen in 11/2018. The TNBC cell lines MDA-MB-468 and HCC1937 were purchased from GeneChem Company (Shanghai, China) in 01/2019. All cells were free of mycoplasma contamination, and their identify was authenticated by short tandem repeat (STR) DNA profiling by Shanghai Biowing Applied Biotechnology Co., Ltd on 31/10/2019. All cells used in the experiments were within 30 passages after thawing. MDA-MB-231 cells were cultured in DMEM with 10% FBS (Gibco, Grand Island, NY, USA). MDA-MB-468 cells were cultured in RPMI-1640 medium with 10% FBS (Gibco). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. In vitro, cells were treated with 500 nM PD (PD-0332991, SelleckChem, Houston, TX, USA) or vehicle (PBS, BOSTER, Wuhan, China). MDA-MB-231 cells were treated with 50 μM CDDP, and MDA-MB-468 cells were treated with 1 μM CDDP (SelleckChem).
Apoptosis analysis
Cells (5 × 104/well) were seeded in triplicate in 10% RPMI-1640 medium/DMEM-FBS (complete medium) in 6-well plates and treated with PD and CDDP at the indicated concentrations separately or combined. After being treated for the defined duration, the cells were washed, resuspended in binding buffer, and stained with Annexin V-FITC/PI according to the manufacturer’s instructions (BD Biosciences). The apoptotic cell populations were analysed using flow cytometry (Beckman Coulter, CA). All assays were independently performed three times.
Cell cycle analysis
For cell cycle analysis, cells were harvested, washed with PBS, fixed in pre-chilled 70% ethanol, and kept overnight at − 20 °C. The fixed cells were then collected, washed, and resuspended in PBS. The cells were incubated with 1 mg/mL RNase and 50 µg/mL propidium iodide (PI) in the dark for 30 min at 37 °C and subjected to flow cytometry (Beckman Coulter, CA). The cell cycle results were analysed using FlowJo version 7.6.1. All assays were independently performed in triplicate.
Assessment of cell viability
The viability of the cells was assessed with Cell Counting Kit-8 reagent (CCK8, Dojindo, Tokyo, Japan). A total of 5000 to 10,000 cells per well, depending on the growth characteristics of each cell line were seeded in 96-well plates in triplicate. After adhering overnight, the designated drugs were added at different concentrations and/or sequences to the wells. After the defined duration, the supernatants were removed, and 100 μl of CCK8 solution (1:10 dilution) was added to the cells. After 2 h of incubation at 37 °C in the dark, the optical density (OD) at 450 nm was measured with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Each experiment was performed three times. The half maximal inhibitory concentration (IC50) was determined from dose–response curves generated by GraphPad Prism version 6.0. The combination index (CI) values for the combined treatment regimens with PD and CDDP were calculated with CompuSyn version 1.0 [
21].
Cells (500–1000/well) were seeded in 6-well plates and treated with the indicated drugs. After combination treatments, the medium was replenished every 3 days until cells in the control wells reached 80–100% confluence. The cell monolayers were then fixed and stained with a solution of 4% paraformaldehyde and 0.5% crystal violet for 30 min at room temperature, washed with water, and dried. Colonies containing 50 or more cells were visually identified and counted. Assays were performed with three independently treated cell populations.
Tumour xenograft studies
This study was approved by the Ethics Committees of Tongji Hospital and performed in accordance with the Guide for the Care and Treatment of Laboratory Animals of Tongji Hospital. Four-week-old female BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and quarantined alone for one week before the experiment. For MDA-MB-231 cell line xenograft models, 7 × 10
6 cells were suspended in 100 μl of PBS plus 50 μl of Matrigel (BD Biosciences, MA, USA) and subcutaneously injected into the left axilla. One week later, mice bearing engrafted tumours of 50 mm
3 were randomized to receive oral treatment with 150 mg/kg PD (n = 4), the intraperitoneal injection of 5 mg/kg CDDP (n = 4), PD-CDDP treatment (n = 4) or vehicle (PBS) treatment (n = 4) according to the dosing schedule provided in Fig.
4a. The perpendicular tumour diameters were measured with callipers. Tumour volumes were calculated as (length × width
2)/2 every 3 days. Tumours were weighed when the mice were euthanized by cervical dislocation. All mice were sacrificed when the tumour burden of the vehicle group was equal to 1000 mm
3. Tumours were fixed in 4% paraformaldehyde for paraffin embedding and used for immunohistochemical staining.
Immunohistochemistry
Tissue sections were incubated with antibody against Ki-67 (#9027, 1:400) (Cell Signaling Technology) overnight at 4 °C and stained with 3,3′-diaminobenzidine (DAB). Densitometry analysis was performed using ImageJ version 1.48v.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, California, USA). RB expression was measured in triplicate using SYBR Green qPCR Mix (Toyobo, Shanghai, China) according to the manufacturer’s instructions. Primer sequences were as follows: RB Forward: 5′-CTCTCGTCAGGCTTGAGTTTG-3′, RB Reverse: 5′-GACATCTCATCTAGGTCAACTGC-3′; GAPDH Forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′, and GAPDH Reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′. The comparative Ct method was used to calculate the relative mRNA expression, and GAPDH was used as an internal control.
Cell transfection
Plasmids containing small hairpin RNA (shRNA) targeting RB and negative control shRNA (shNC), RB-overexpressing plasmid and empty plasmid were purchased from RiboBio (Guangzhou, China). ShRNA was transfected into MDA-MB-231 cells, and RB-overexpressing plasmid and empty plasmid were transfected into MDA-MB-468 cells with X-tremeGENE HP DNA transfection reagent (Roche, CHE) according to the manufacturer’s instructions. The shRNA sequences were as follows: shNC, 5′-TTCTCCGAACGTGTCACGT-3′, shRB, 5′-CGGCTAAATACACTTTGTGAA -3’.
Immunofluorescence assay
Breast cancer cells were subjected to indirect immunofluorescence staining with γH2AX (Ser139, #9718, 1:400) and then labelled with FITC goat anti-rabbit IgG (#AS007, 1:200). Nuclei were stained with DAPI (Life Technology). Fluorescence images were acquired using an inverted fluorescence microscope (Olympus). ImageJ version 1.48v was utilized for foci measurement and image analysis.
Western blot analysis
Cell lysates were separated by SDS-PAGE, and the proteins were then transferred to PVDF membranes. The proteins were detected using antibodies against the following: RB (ab181616, 1:2000), cyclin D1 (ab40754, 1:1000), E2F1 (ab179445, 1:1000) (Abcam, Cambridge, UK), phospho-RB (p-RB) (S780) (#8180, 1:1000), PARP/cleaved PARP (#9542, 1:1000) (Cell Signaling Technology), and β-Actin (AC026, 1:100,000) (ABclonal, Boston, USA). Specific bands were visualized by ECL (Advansta, USA) and detected with an imaging system (Bio-Rad, USA).
Statistical analysis
Statistical significance was determined and means and standard deviations were calculated by GraphPad Prism version 6.0. All analyses were performed in triplicate, and P < 0.05 was used to indicate statistical significance. Data are expressed as the mean ± SD or mean. The significance of differences between two groups was analysed by two-tailed Student’s t-test. The Chou-Talalay method was performed to calculate the CI.
Discussion
In this study, we explored the effect of various combinations of PD and CDDP in the treatment of TNBC. We initially established three common drug regimens and discovered that compared with CDDP treatment alone, the simply combined or sequential use of PD and CDDP was no more effective in MDA-MB-231 cells. Therefore, we performed a deeper investigation of the drug regimens and found synergism in the PD-CDDP group. Based on the effect of PD on the cell cycle, when PD was used for 48 h and then withdrawn for 72 h, its effect in blocking the cell cycle was weakened, and some cells entered S and G2 phase. Therefore, it is reasonable to speculate that when PD is used for 48 h and then withdrawn for 48 h, most cells are synchronized in the cell cycle and ready to enter S phase, in which cells are more sensitive to CDDP, thereby achieving the synergistic effect observed in the PD-CDDP group. In contrast, tumour cells in the CDDP-PD group were at various stages of the cell cycle, which show quite different sensitivities to CDDP. Sequential use of PD following CDDP may induce the recovery of partial cells with DNA damage due to cell cycle extension, so CDDP-PD treatment ultimately produced an effect antagonistic to that of CDDP alone or the same effect as CDDP alone.
Combination therapy that relies on complementary mechanisms of antitumour activity has increasingly become a trend in cancer treatment [
24,
25]. Currently, an increasing number of targeted therapies, such as CDK inhibitors, combined with conventional chemotherapy regimens have been applied to improve the antitumour effects of the individual therapies and inhibit tumour resistance. For example, pre-treatment with PD could sensitize myeloma cells to bortezomib-induced apoptosis [
26]. Another study found that simultaneous combination treatment consisting of abemaciclib with paclitaxel or CDDP could achieve better efficacy than chemotherapy alone [
27]. In contrast with the above findings, Patrick et al. demonstrated that simultaneous combination treatment of carboplatin with PD decreased antitumour activity compared with carboplatin treatment alone in Rb-proficient mice and that the coadministration of PD with carboplatin had no effect on tumour growth in vivo [
28]. The above results indicate that care is required when combination treatments consisting of CDK inhibitors and chemotherapeutic drugs are designed. Different tumour types, the use of different chemotherapeutic agents, and even the use of different time points, as shown in our study, may have totally different effects. Improper strategies may be ineffective or even have results opposite of the intended results.
In addition, we demonstrate that the synergistic effect of PD with CDDP-mediated cytotoxicity occurs in an RB-dependent manner. In RB-knockdown or RB-deficient cell lines, PD could not induce cell cycle arrest, and PD-CDDP could not enhance the cytotoxic effect of CDDP, but the overexpression of RB restored the sensitivity of RB-deficient cells to PD. Some studies and our study have demonstrated that RB can act as a marker to select patients suffering from cancer who are likely to benefit from PD treatment, and the loss of RB function may be the main cause of primary and secondary drug resistance to PD [
29‐
31].
Overall, this is the first study to investigate the inhibition of TNBC cells by a combination strategy consisting of PD and CDDP under a specific drug regimen. However, there are several limitations in this study. We used only one RB-proficient cell line in this study, so we cannot rule out that the observed synergistic effect is specific to only MDA-MB-231 cells. Furthermore, the additional validation of CDDP-resistant cell lines in this study would have increased the clinical relevance of the study.
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