Background
The key options in breast cancer treatment so far include surgery, chemotherapy, radiotherapy, and molecular targeted therapy [
1]. Out of these approaches, chemotherapy appears to be the most widely adopted. In particular, paclitaxel (TAX) was commonly used as one of the first-line chemotherapy drugs in breast cancer therapy and has shown remarkable efficacy in inhibiting tumor growth through mitotic arrest [
2‐
4]. Despite the excellent initial therapeutic efficacy, drug resistance of TAX develops in 90% of breast cancer patients during the disease progression [
5]. The molecular mechanism of such resistance remains elusive and has become a clinical issue that requires an immediate solution.
Metadherin (MTDH), also known as LYRIC, AEG-1, or 3D3, has been expressed in multiple tumor types as an oncogene and associated with aberrant proliferation and drug resistance of tumor cells [
6‐
10]. In particular, a recent study reported that MTDH gene promotes the cisplatin resistance of cervical cancer cells by activating the Erk/nuclear factor-kappa B (Erk/NF-κB) pathway and decreasing cleavage of caspase-3 [
11‐
14]. Based on these findings, we explored the relationship between MTDH gene expression and TAX resistance in breast cancer cell lines as well as the effect of MTDH knockdown on TAX therapeutic efficiency. For in vivo therapeutic study, we employed targeted nanocarriers to specifically co-deliver anti-MTDH small interfering RNA (siRNA) (for tumor-specific silence of MTDH gene) and TAX drug.
We first explored the role of MTDH gene in the TAX therapeutic efficiency toward breast cancer cells and then investigated whether a nanoparticle (NP)-based co-delivery method can be used to address TAX resistance issue in breast cancer treatment. We hypothesized that the two drugs-loaded NPs displayed onsite gene silencing in tumor tissues, which improved drug sensitivity of TAX with good tolerability. In addition, poly(lactic-co-glycolic acid) (PLGA) polymeric molecule is approved by the US Food and Drug Administration with high biosafety; this study thus presents a promising strategy for clinical practice.
Methods
Patients and clinicopathology characteristics
This is a retrospective study that was approved by the Fourth Hospital of Hebei Medical University in Shijiazhuang, China. In this study, we selected 44 cases with breast cancer, which were proven to be breast invasive ductal carcinoma by pathological diagnosis from March to December 2010. The median follow-up time was 84 months (range of 8–90 months). The primary endpoints were disease-free survival (DFS) and overall survival (OS). We detected the samples from carcinoma of 44 patients before neoadjuvant chemotherapy. Immunohistochemistry was used to detect MTDH expression in all tissues. All the patients were recommended to use combination chemotherapy of taxol and anthracycline. Clinical benefit was evaluated by the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and pathologic complete response (pCR) rates after neoadjuvant therapy. pCR was defined as ypT0 ypN0. All of above procedures were approved by the Fourth Hospital Ethics Committee of Hebei Medical University in Shijiazhuang, China (SCXK2009–0037).
Immunohistochemistry
All of the immunohistochemistry slides for MTDH were reviewed again by two independent pathologists. Immunohistochemistry staining of 4-μm sections of formalin-fixed paraffin-embedded tissue was rehydrated and incubated with anti-MTDH primary monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA) or phosphate-buffered saline (PBS) at 4 °C overnight, followed by sequential incubation with MaxVision™/horseradish peroxidase (HRP) and diaminobenzidine (DAB). Then slides were counterstained with hematoxylin, dehydrated, and mounted.
The levels of MTDH expression were evaluated on the basis of the staining intensity (SI) and percentage of positively stained tumor cells (PP). SI was defined as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). PP was graded according to the following criteria: 0 (no positive tumor cells), 1 (1%–20% positive tumor cells), 2 (21%–50% positive tumor cells), 3 (51%–70% positive tumor cells), and 4 (>70% positive tumor cells). The immunoreactive score (IRS) was calculated as follows: IRS = SI × PP. IRS of 0 means negative expression, IRS of 1–3 means weakly positive, IRS of 4–6 means moderately positive, and IRS of 8–12 means strong positive. Here, low expression was defined as an IRS of 3 or less. High expression was defined as an IRS of 4 and more [
15].
Cell lines and reagents
The human breast cancer cell lines MCF-7 and MDA-MB-435S were purchased from the American Type Culture Collection (Manassas, VA, USA) and propagated in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Biological Industries Israel Beit-Haemek Ltd., Kibbutz Beit-Haemek, Israel) and antibiotics. All cells were maintained in 5% CO2 at 37 °C. TAX was purchased from Yangzijiang Medicine Co. Ltd. (Jiangsu, China).
The plasmids containing MTDH gene or MTDH-short hairpin RNA (MTDH-shRNA) (5′-gcaattgggtagacgaagaaa-3′) were designed and amplified by transfecting into Escherichia coli. DH5α. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of MTDH mRNA and protein of MTDH-shRNA to verify the effect of transfection. Plasmids enveloped in lentivirus were incubated with MCF-7 cells for 6 h according to the MOI (multiplicity of infection) value and the virus titer and subsequently placed in fresh medium. Puromycin (0.4 μg/mL) was used to screen stable transfection cell lines.
RT-PCR analysis
Total RNA from treated cells was extracted with Trizol (Takara, Dalian, China) in accordance with the instructions of the manufacturer. Total RNA was used to synthesize cDNA by using PrimeScript RT reagent Kit (Takara). Then RT-PCR was carried out using Power Up SYBR Green Master Mix (Life Technologies, Thermo Fisher Scientific). The reaction was conducted using the following parameters: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s during 40 cycles. Internal control and primers for RT-PCR were obtained from the reference. RT-PCR was then employed to determine the change of MTDH mRNA in MCF-7–MTDH cell line and MCF-7–MTDH–shRNA cell line. The experiments were repeated for three times and data were analyzed using 2
−∆∆Ct. The data were normalized to the geometric mean of housekeeping gene
β-actin to control the variability in expression levels. RT-PCR primers were synthesized by SBS Genentech Co. Ltd. (Shanghai, China). The specific primers for MTDH and reference gene (β-actin) are as follows:
-
MTDH forward: 5′-AAATAGCCAGCCTATCAAGACTC-3′;
-
MTDH reverse: 5′-TTCAGACTTGGTCTGTGAAGGAG-3′.
-
β-actin forward, 5′-GCTACAGCTTCACCACCACAG-3′;
-
β-actin reverse, 5′-GGTCTTTACGGATGTCAACGTC-3′.
Western blot analysis
Cells were lysed and total proteins were separated by 10% SDS-PAGE and transferred (300 mA, 2 h) onto a PVDF membrane. After blotting with 5% nonfat milk, the membranes were incubated with primary antibodies (anti-MTDH 1:20000, anti-p65 1:5000, anti-p-p65 (S536) 1:1000, anti-IκBα1:1000, and β-actin 1:1000) at 4 °C overnight. Then the membranes were washed by TBS-T buffer and incubated with secondary HRP-labeled anti-rabbit antibody at room temperature for 1 h and washed with TBS-T buffer three times (10 min each time). The target proteins were visualized with a chemiluminescence system (Gene Company Ltd., Shanghai, China) and normalized to β-actin from the same membrane.
Cell apoptosis and cycle detection
Cell apoptosis was performed using an Annexin V-PE/7-AAD Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China). The experiments were carried out strictly in accordance with the instructions of the manufacturer. The cells were then analyzed by Beckman Coulter Cytomics FC 500 flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). The data were analyzed by EXPO32 ADC analysis software. Cell cycle analysis was performed by using the standard method with some modifications. In brief, cells were fixed with 75% ethanol at 4 °C overnight. The fixed cells were washed by PBS and suspended with 200 μL RNaseA at 37 °C for 10 min, and 250 μL PI (100 μg/mL) was added to stain the DNA of cells in the dark for 15 min. Cell cycle was analyzed with a Beckman Coulter Cytomics FC 500 flow cytometry, and the data were analyzed by Multicycle AV for Windows (version 295) software.
Cell viability assay
Cell viability was determined by a Cell Counting Kit-8 (CCK-8) assay. MCF-7, MCF-7-vector, MCF-7–MTDH, and MCF-7–MTDH–shRNA cells were seeded into 96-well plates at a density of 1 × 104/well (TAX 0 μg/mL) or 5 × 104/well (TAX 1 μg/mL) in 100 μL RPMI-1640 medium. After incubation in 5% CO2 at 37 °C overnight, the RPMI-1640 medium in each well was replaced with a different concentration of TAX (0 and 1 μg/mL) and further incubated for 0, 24, 48, and 72 h. Afterwards, 10 μL of CCK-8 was added to each well for another 2 h at 37 °C. The absorbance at 450 nm was read by the Microliate Reader (BioTek, Winooski, VT, USA). The inhibitory rate of cell growth was calculated on the basis of the following equation: cell growth inhibition rate = (1 − experimental OD450 / control OD450) × 100%. The experiments were repeated three times.
Tumorigenicity assay
MCF-7, MCF-7-vector, MCF-7–MTDH, and MCF-7–MTDH–shRNA cells (5 × 106 in 0.1 mL) were injected subcutaneously into 4-week-old female nude mice, respectively. TAX treatment was started at the third week after cell injection. The mice were randomly assigned to an untreated group and TAX treatment groups. The dose of TAX was 10 mg/kg and administered by intraperitoneal (IP) injection once a week for a total of four injections. Tumor volume was measured every three days (volume = 0.5 × length × width2, measured with a Vernier caliper). After the last treatment, the mice were sacrificed and the tumors were removed for weight analysis.
Preparation of NPs
For poly(etherimide)-poly(lactic-co-glycolic acid) (PEI-PLGA) NP synthesis, 20 mg was dissolved in 1 mL of methylene chloride and mixed with 0.2 mL deionized water. The mixture was emulsified through sonication by using probe sonicator at 25% power for 5 min. Then 2 mL of 2% poly(vinyl alcohol) (PVA) and 0.2 mL of hydrophobic TAX (with different ratios) dissolved in methylene chloride were added into the mixture. The solution was emulsified again at 30% power for 5 min, added dropwise into 10 mL of 0.6% PVA, and stirred for 3 min. The organic solvent was removed in a rotary evaporator under reduced pressure. The core particles containing TAX were centrifuged at 12,000 revolutions per minute (rpm) for 5 min and rinsed with deionized water. For loading siRNA, different ratios of TAX were added into the solution of core NPs and stirred at a rate of 200 rpm for 20 min. The core NPs absorbed by siRNA on their surface were centrifuged at 12,000 rpm for 5 min.
Morphological characterization of NPs
The NPs were negatively stained with uranyl acetate solution (2%) and deposited on a carbon-coated copper grid. The morphology was characterized with a transmission electron microscope (TEM) (JEM-200CX; Jeol Ltd., Tokyo, Japan). Size distribution (diameter in nanometers) and surface charge (zeta potential in millivolts) of NPs were determined by using a ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd., Malvern, UK) equipped with a He-Ne Laser beam at a wavelength of 633 nm and a fixed scattering angle of 90. Determinations were performed at 25 °C for samples appropriately diluted in distilled water.
Tumor-bearing nude mouse model
Six-week-old female BALB/c nude mice were purchased from Beijing Vital River Laboratories (Beijing, China). Human breast cancer cells (MCF-7, 2.0 × 106 cells in 50 mL PBS) mixed with 50 mL of Matrigel were transplanted into the mammary fat pads of the mice and allowed to grow to a tumor size of about 100 mm3 (volume = 0.5 × length × width2, measured with a Vernier caliper). The mice were then randomly divided into different experimental groups. All procedures were approved by the Committee on the Ethics of Animal Experiments of the Health Science Center of Peking University (Beijing, China).
Statistical analysis
Data analyses were performed with one-way analysis of variance (ANOVA) and the least significance difference (LSD) multiple comparisons test with PASW Statistics 23. Tumor volumes were compared by using a Kruskal–Wallis test followed by the Mann–Whitney test.
Discussion
MTDH was identified as an oncogene that functions in both drug resistance and metastasis [
16]. Upregulation of the MTDH gene could promote the proliferation of a variety of tumor cells, such as esophageal cancer, gastric cancer, glioma, and breast cancer [
17‐
20]. Previous study showed that overexpression of MTDH induces estrogen-independent growth of MCF-7 breast cancer cells and mediates tamoxifen resistance [
21]. Similarly, in our previous studies, overexpression of MTDH enhances the resistance of MDA-MB-231 cells to doxorubicin [
9]. In contrast, downregulation of MTDH could inhibit tumor cell growth, induce apoptosis, and increase the sensitivity of tumor cells to chemotherapeutic drugs. In gastric cancer, studies showed that knockdown of MTDH by siRNA in SGC790 cells could apparently inhibit cell proliferation by blocking cell cycle in G
0/G
1 phase [
18].
In our study cohort of 44 patients with breast cancer, we found that MTDH expression was negatively correlated with the probability of DFS and efficacy of TAX treatment, which should be further confirmed in a large population of patients with breast cancer. At cellular level study, we tested the cell proliferation of MCF-7–MTDH and MCF-7–MTDH–shRNA cells by CCK-8 assay and found that MTDH knockdown inhibits cell growth. The results of flow cytometry demonstrated that knockdown of MTDH resulted in an increase of G0/G1 phase cells and reduction of S and G2/M phase cells but that MTDH overexpression induced cell cycle arrest in S phase. Additionally, knockdown of MTDH inhibits the growth of xenograft tumor in vivo. Taken together, our results suggest that MTDH expression plays a crucial role in the MCF-7 breast cancer cell proliferation and is potentially useful for breast cancer treatment.
As one of the most important anticancer drugs, TAX has been widely used for chemotherapy in various malignant tumors for about 40 years [
22] and is the first-line chemotherapy drug in breast cancer therapy [
23]. By stabilizing the microtubule polymer and preventing microtubules from disassembly, TAX arrests the cell cycle in the G
2/M phase and induces cell apoptosis [
23‐
25]. However, the chemotherapy resistance is a major limitation of its effect and impacts the prognosis of patients with breast cancer. In the present work, we determined the sensitivity of wild type of MCF-7 (MCF-7–MTDH) and MTDH silencing cell (MCF-7–MTDH–shRNA) to TAX treatment. The results suggested that MCF-7–MTDH–shRNA was inhibited by TAX with a much higher rate than MCF-7–MTDH. We further examined the cell apoptosis rate and found that the apoptosis induced by TAX in MCF-7–MTDH–shRNA cells was higher than that in MCF-7–MTDH cells. Furthermore, the percentage of G
2/M phase in MCF-7–MTDH–shRNA treated with TAX was significantly higher than that in the control and MCF-7–MTDH groups. In in vivo experiments, compared with the MCF-7–MTDH and control groups, the volume of MCF-7–MTDH–shRNA xenograft tumors treated with TAX was significantly smaller. These data together suggest that overexpression of MTDH resisted TAX but that MTDH knockdown increased the sensitivity of MCF-7 cells to TAX treatment. In addition, MTDH plays a key role in the activation of diverse signaling pathways, including PI3K/Akt, NF-κB, and Wnt/β-catenin pathways [
3,
26]. The activation of NF-κB is critical to the resistance of tumor cells to cytotoxic agents and microtubule-disrupting agents [
27,
28]. We also examined the protein expression level of p65 and IκBα in various MCF-7 cells and showed that MTDH overexpression was correlated with chemoresistance to TAX but that MTDH knockdown increased the sensitivity of TAX by inhibiting the NF-κB/IκBα pathway. This implies that one can increase the effectiveness of TAX treatment on breast cancer by lowering MTDH expression in tumor cells.
Chemoresistance is currently the major cause for breast cancer treatment failure, especially for metastatic breast cancer. Numerous siRNAs have been demonstrated to be effective for in vivo tumor growth modulations [
29], but the delivery of siRNAs in vivo has been challenging for antitumor therapy because of their instability in physiological conditions, improper cellular distribution, and low bioactivity [
30]. Naked siRNA has a short half-life in the bloodstream because of rapid degradation by nucleases in plasma or excreted by kidney [
31]. Moreover, owing to high molecular weight, hydrophilic properties, and high density of charge, naked siRNA hardly penetrates across cell membranes [
32]. Using NPs, especially the biodegradable polymer NPs to load siRNA can realize controlled and targeted drug delivery with high efficacy and low side effects [
33,
34]. Also, the polymeric NPs readily realize the co-delivery of siRNA with hydrophobic or hydrophilic drugs [
35]. In order to reverse drug resistance and improve the utilization of drug effectively, researchers have developed multiple nanocarriers and different dosage forms, such as NP albumin-bound TAX [
36]. In this study, for tumor-specific MTDH knockdown, we constructed an amphiphilic PLGA-based copolymer NP for co-delivery of anti-MTDH siRNA and TAX into tumors. In vivo imaging results showed that the two drugs-loaded NPs (NP-TAX–siRNA) accumulated mainly in the tumor tissues, because of the passive targeting ability from the enhanced penetration and retention (EPR) effect of tumor vessels [
37,
38], and inhibited tumor growth dramatically, further confirming that MTDH silencing effectively enhances the TAX therapeutic efficiency. In addition, throughout the whole therapeutic experiment, neither weight loss nor tissue damage was observed in the NP-TAX–siRNA–treated mice, indicating the biosafety of NP-TAX–siRNA for tumor treatment.
Conclusions
In summary, we have revealed, for the first time, that overexpression of MTDH in breast cancer cells is related to TAX chemotherapeutic drug resistance. To achieve in vivo therapeutic assessment by tumor-specific knockdown of MTDH, we have devised a polymer-based nanocarrier to co-deliver anti-MTDH siRNA and TAX into tumor tissues. The designed NPs were composed of a cationic copolymer, which wrapped TAX in the inside and adsorbed the negatively charged siRNA on their surface. After systemic administration, the NPs had good tumor-targeting ability based on the EPR effect of tumor vasculatures and displayed effective antitumor activity without overt side effects. Based on our study, we provide a new strategy for reversing TAX resistance in breast cancer treatment, especially for those with high MTDH protein level.
Acknowledgments
We thank L.Z. Xu (Medical and Health Analysis Center of Peking University) for animal imaging as well as technical and methodological assistance. We thank Suping Li (CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China) for our nanoparticle experiment and her useful suggestions on the manuscript.