Background
Ovarian cancer is the most common malignant tumor of the female reproductive tract. It was reported to occur in 22,240 women and caused 14,080 deaths in 2017 in the world [
1]. Around 30 different types of ovarian cancer are defined based on the cell origins, such as epithelial ovarian cancer (EOC), germ cell ovarian cancer, and stromal cell ovarian cancer. Thereinto, EOC is the most common type and accounts for approximately 90% of ovarian malignancy [
2].
Chemoresistance is a major challenge for current ovarian cancer therapy. The mechanism of EOC chemoresistance is not completely uncovered. Studies from different groups indicated that epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) were closely associated with the chemoresistance, metastasis and tumor relapse in EOC patients [
3]. EMT is a biological process in which the phenotype of epithelial cells is transformed into a mesenchymal phenotype by specific procedures [
4]. During EMT, cells downregulate the epithelial cellular adhesion markers and gain mesenchymal properties by increasing the expression of mesenchymal markers [
5,
6]. Recently, a close association between EMT-like cancer cells and chemotherapeutic resistance has been revealed [
7,
8]. However, the role of EMT in EOC chemoresistance is not fully elucidated.
On the other hand, mounting evidence suggests that CSCs are the culprits of therapeutic resistance and cancer metastasis, leading to the tumor relapse and death in patients [
9]. CSCs are capable of self-renewal, recapitulating the heterogeneity of the tumors, and differentiating into a new tumor bulk in the appropriate microenvironment. K Abubaker, et al. [
10] found that surviving ovarian cancer cells in mice xenograft after chemotherapy were enriched with CSC phenotypes [85]. Furthermore, IA Silva, et al. [
11] reported that ALDH
+CD133
+ CSC-like cells within human primary ovarian tumors displayed enhanced chemoresistant phenotypes as compared to the parental cells. Also, the presence of these cells in primary ovarian tumors was associated with a shorter disease-free survival and overall survival in patients [
11]. These studies suggest that CSCs are closely associated with ovarian cancer chemoresistance.
Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is an important intracellular pathway in regulating cell cycle, quiescence, and proliferation. Various somatic mutations in phosphatase and tensin homolog (PTEN), Akt1, and mTOR that induce enhanced PI3K/Akt/mTOR signaling have been found in ovarian cancer [
12‐
16]. The hyperactivation of PI3K/Akt/mTOR signaling was implicated in cancer metastasis and chemoresistance [
17‐
19]. It was reported that PI3K regulated G1 cell cycle and apoptosis in ovarian cancer by activating Akt/mTOR/p70S6K1 signaling [
20], and inhibition of PI3K can disrupt ovarian cancer cell proliferation and trigger cell death. More importantly, the inhibition of PI3K/Akt/mTOR signaling was found to re-sensitize chemoresistant ovarian cancer cells to chemotherapeutic drugs [
21,
22]. These findings establish PI3K/Akt/mTOR signaling as an attractive therapeutic target for ovarian cancer treatment. However, the correlation between PI3K/Akt/mTOR signaling and EMT or CSC in EOC chemoresistance remains unclear.
In the current study, we found that the acquisition of EMT and enhancement of CSC marker expression in chemoresistant EOC cells were associated with the activation of PI3K/Akt/mTOR signaling. More importantly, BEZ235 in combination with cisplatin can significantly reduce the expressions of EMT and CSC markers and restore the sensitivity of chemoresistant EOC cells to cisplatin. These results may provide further evidence for better understanding the mechanism underlying EOC chemoresistance and evaluating the effect of PI3K inhibition in EOC chemosensitization.
Methods
Antibodies and reagents
Antibodies used for this study are summarized in Additional file
3: Table S1. Cisplatin, carboplatin, paclitaxel were obtained from Sigma-Aldrich (NSW, Australia), while BEZ235 (dactolisib) was obtained from Jomar Life Research (Melbourne, VIC, Australia). BEZ235 stock solution was prepared at 1 mg/mL in N-methyl-2-pyrrolidone + polyethylene glycol 300 (1:9, v/v), while other drug stock solutions were prepared in dimethyl sulfoxide (DMSO, Sigma–Aldrich). All working solutions were diluted using cell culture medium with the final concentration of organic solvent lower than 0.1%.
Cell culture
A2780 and A2780-cis EOC cell lines were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). The development of A2780-cis cell line is described in
https://www.sigmaaldrich.com/catalog/product/sigma/cb_93112517. IGROV1 cell line was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA), while IGROV1-cis EOC cell line was kindly provided by Prof. Jan H.M. Schellens (Netherlands Cancer Institute, AMS, Netherland). The development of IGROV1-cis cell line is described in a previous publication [
23]. All cell culture reagents were purchased from ThermoFisher Scientific (Melbourne, VIC, Australia). All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin, and 50 μg/mL streptomycin. 1 μM and 3.3 μM cisplatin were added to the medium every 2–3 passages for A2780-cis and IGROV1-cis cell lines, respectively. All cell lines were maintained in a humidified incubator at 37 °C and 5% CO
2.
Proliferation assay
1 × 103 cells were seeded in 96-well plates and cultured for consecutive 7 days at 37 °C and 5% CO2. The cell number was detected using CyQUANT® Cell Proliferation Assay Kit (Life Technologies, Scoresby, VIC, Australia) according to the protocol. The fluorescence intensity was measured using a microplate reader (Bio-Tec, Hercules, CA, USA) with excitation at ~ 485 nm and emission at ~ 530 nm.
Cell viability detection
Cell viability detection was performed using MTT assay. Briefly, 8 × 103 cells were seeded in 96-well plates for 24 h and then treated with a range of concentrations of drugs. After 48-h incubation, the cell viability was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/mL) and Bio-Tec microplate reader at 562 nm wavelength. The cell viability curves were generated by Prism 7 (GraphPad, San Diego, CA, USA) and IC50 values were calculated.
Matrigel invasion assay
The invasion ability of A2780, A2780-cis, IGROV1 and IGROV1-cis cells was evaluated by Matrigel Transwell® plates (BD Bioscience, NSW, Australia) according to manufacturer’s specification. The invasion ability was calculated as follows: invasion ratio = [(average number of cells invading through matrigel insert) / (average number of cells migrating through control insert)] × 100%.
Wound-healing assay
Cell migration ability was evaluated by wound-healing assay. Briefly, 3 × 105 cells were seeded in 6-well plates for 24 h. Monolayer cells were scraped using a sterile 200 μL tip, which caused a straight scratch (wound). The wounded monolayer cells were washed to remove cell debris and then replenished with fresh cell culture medium. Representative images were obtained after 0 h, 48 h, 96 h at 40x magnification using a light microscope.
Cell cycle analysis
Cell cycle analysis was conducted using flow cytometry. Briefly, the sample containing trypsinized adherent, and floating cells with 1 μL FxCycle Violet stain was analyzed at 405-nm excitation using FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and emission was collected with a 450/50 band-pass filter. The results further were analyzed using FlowJo V10 software (Tree Star Inc., Ashland, OR, USA) to determine cell cycle distribution.
Immunofluorescence
Cells grown on glass cover-slips (1 × 10
5 cells) were fixed by methanol for 10 mins at RT, incubated with various primary antibodies (Additional file
3: Table S1) and then Alexa Fluro-488 goat anti-rabbit IgG, Alexa Fluro-488 goat anti-mouse IgG, or Alexa Fluro-488 donkey anti-goat IgG. Propidium iodide (PI) (0.2 mg/L) was used for nuclei staining. The staining results were visualized using FV300/FV500 laser scanning confocal microscope (Olympus, Tokyo, Japan). The mean intensity of immunostaining from three randomly selected fields was scored as 0 (negative, < 25%), 1 (weak, 25–50%), 2 (moderate, 50–70%), and 3 (strong, > 75%) by two observers (J.B. and Y.L.).
Quantitative real-time PCR (qRT-PCR)
Primers for EMT (E-cadherin, N-cadherin, Vimentin), CSC (CD44v6, CD117, ALDH1A1, Snail) markers, and GAPDH (control) were synthesized by Invitrogen (Mulgrave, VIC, Australia). Samples were analyzed as previously described [
24]. The values were obtained as the threshold cycle for each gene and normalized to the reference gene (GAPDH). The 2
-ΔΔCT method was used to calculate relative changes for each gene in different cell lines.
Western blot
Protein expressions were examined using western blot as previously described [
25]. The membrane proteins were incubated with different primary antibodies (Additional file
3: Table S1), followed by incubation with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using ImageQuant LAS4000 system (GE Healthcare, USA).
1.5 × 103 cells were seeded in 10-cm dishes for 48 h at 37 °C and 5% CO2 and then treated with vehicle control, ½ IC50 BEZ235, ½ IC50 cisplatin, or combination treatment of ½ IC50 BEZ235 and ½ IC50 cisplatin, respectively. After 2-day treatment, the drug-containing medium was replaced with fresh media and all cells were incubated for an additional 10 days until colonies were large enough to be clearly distinguished. The colonies, defined as groups of > 50 cells, were counted manually with the aid of INT2 inverted microscope (Olympus, Tokyo, Japan).
Apoptosis detection
Cell apoptosis was detected using AO/EB assay. 2 × 105 A2780-cis or IGROV1-cis cells were treated with vehicle control, ½ IC50 BEZ235, ½ IC50 cisplatin, or combination treatment of ½ IC50 BEZ235 and ½ IC50 cisplatin for 24 h, respectively. Cells were then stained with AO/EB (Sigma-Aldrich) according to manufacturer’s protocol and examined by FV300/FV500 laser scanning confocal microscope (Olympus, Tokyo, Japan) at 200x magnification. Apoptotic cells were characterized by nuclear condensation and fragmentation.
Reactive oxygen species (ROS) assay
2 × 105 A2780-cis and IGROV1-cis cells were treated with vehicle control, ½ IC50 BEZ235, ½ IC50 cisplatin, or combination treatment of ½ IC50 BEZ235 and ½ IC50 cisplatin, respectively. After treatments for 48 h, the cells were detached, centrifuged, and re-suspended in 1× PBS. CellROX® Green Reagent (Life Technologies, Scoresby, VIC, Australia) was added to the cells to a final concentration of 5 μM and incubated at 37 °C for 30 mins according to the manufacturer’s protocol. The fluorescence was detected by FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA, USA) with excitation at ~ 485 nm and emission at ~ 520 nm.
Statistical analysis
All experiments were repeated in triplicate. The data were expressed as mean ± standard deviation (SD) for continuous variables while frequencies (%) for categorical variables. Students’ t test and one-way ANOVA with post-hoc Tukey’s test were used to compare the data from different groups. P < 0.05 was considered statistically significant.
Discussion
Chemoresistance is a major clinical challenge for ovarian cancer therapy. The mechanisms underlying EOC chemoresistance remain elusive. Hence, elucidating the mechanisms underlying EOC chemoresistance might be expected to improve the poor prognosis. In this study, using the paired chemoresistant EOC cell lines, we demonstrate the role and association of PI3K/Akt/mTOR signalling, EMT, and CSC in EOC chemoresistance. A summary of our findings is shown in Fig.
5.
Our results demonstrate that cisplatin-resistant EOC cells are cross-resistant to chemotherapeutic drugs, including cisplatin, carboplatin, and paclitaxel, suggesting that common resistant mechanisms might exist within EOC cells. In addition, we also found that EOC cells possessing stem-cell or EMT characteristics were enriched, and the PI3K/Akt/mTOR pathway was activated in two cisplatin-resistant EOC lines. Furthermore, the dual inhibitor, BEZ235, was found to inhibit the cell viability of cisplatin-resistant EOC cells, reverse EMT, repress stem-cell-associated gene expression, and re-sensitize chemoresistant EOC cells to cisplatin.
In the first stage, we compared the characteristics of two pairs of EOC cell lines, the chemoresistant cell lines (A2780-cis and IGROV1-cis) and the corresponding chemosensitive cell lines (A2780 and IGROV1). The cisplatin-resistant cells showed multidrug-resistance, which might be developed by several mechanisms, such as decreased drug uptake, increased drug efflux, activation of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced apoptosis [
26].
In this study, the decreased E-cadherin and increased N-cadherin and Vimentin were found in cisplatin-resistant EOC cells compared with parental cells, which is consistent with the findings from other groups in EOC and lung cancer [
27]. The increased expressions of EMT-related transcription factors, including Snail, Slug, Twist2, and Zeb2, at the gene level in A2780-cis cells further support our observation [
28]. B Davidson, et al. [
29] recently found that EMT markers-Vimentin and Zeb1 can be used as biomarkers for predicting poor response to chemotherapy in metastatic serous ovarian carcinoma effusions, further confirming the significance of EMT in EOC chemoresistance. Increasing findings indicate that EMT and CSC have a close link and share the same markers and properties [
30]. Our results suggest that the acquisition of EMT in EOC cisplatin resistance is accompanied by an enhanced CSC marker expression, further proving the close association of EMT with CSCs.
The CSC theory offers a potential explanation for the relapse and resistance that occur in many tumors including ovarian cancer after therapy [
31]. CSCs play very important roles in cancer metastasis and chemoresistance [
32]. Targeting CSCs is promising and may overcome chemoresistance and lead to the cure of EOC [
3]. In the current study, we found that two cisplatin-resistant EOC cell lines consistently showed increased ovarian cancer stem cell markers, indicating that CSCs may have an association with chemoresistance. Furthermore, the lower cell proliferation rate (quiescent status), increased G0/G1 phase, increased invasion and migration capability, were also observed in cisplatin-resistant cell lines, which are recognized characteristics maintained by CSCs [
33‐
36].
Accumulating evidence demonstrates that PI3K/Akt/mTOR signalling, EMT, and CSCs play important roles in EOC progression, metastasis, and chemoresistance [
3]. In this study, we found this pathway was activated in two cisplatin-resistant cell lines. Therefore, important questions arise as to whether there is a relationship between the activation of PI3K/Akt/mTOR signalling and EMT or enhanced CSC marker expression, and whether targeting this pathway could be effective to overcome EOC chemoresistance.
To deeply study the involvement of PI3K/Akt/mTOR signaling with acquisition of EMT and enhanced CSC marker expression in EOC chemoresistance, a dual inhibitor of PI3K/Akt/mTOR pathway, BEZ235, was selected for advancing our investigation. BEZ235, which targets both pan class I PI3K and mTOR, has been reported to inhibit cancer cell growth in vitro and in vivo [
37,
38]. This dual inhibitor has been used in a Phase I/II clinical trial for advanced solid tumors and may be useful for EOC clinical trials in the future [
39,
40]. In this study, an interesting finding is that the combination treatment or BEZ235 alone can reverse EMT and greatly reduce the expressions of CSC markers in cisplatin-resistant EOC cells compared with control or cisplatin alone, suggesting that the inhibition of PI3K/Akt/mTOR signaling can suppress EMT and CSC marker expression. Another interesting finding is that cisplatin can enhance CSC marker expression. This result is in line with the enhanced CSC marker expression found in EOC-cis cell lines as compared to the corresponding cisplatin-sensitive parental cells. Our results demonstrate that the inhibition of PI3K/Akt/mTOR pathway by BEZ235 confers a strong preferentially inhibitory effect on EMT and CSC. Also, more apoptosis was induced by combination therapy compared to single BEZ235 or cisplatin treatment, indicating that BEZ235 can increase chemosensitivity in cisplatin-resistant cells through inhibiting PI3K/Akt/mTOR signaling. Of note, BEZ235 has been demonstrated to be effective in targeting cells with CSC characteristics [
19]. Dubrovska et al. found that the inhibition of PI3K/mTOR activity by BEZ235 led to a decrease in the population of CD133
+/CD44
+ prostate cancer progenitor cells in a mouse xenograft model [
41]. It is well known that conventional chemotherapy drugs effectively target processes that cancer cells need to grow and divide, such as the ability to replicate DNA. CSCs are relatively resistant to chemotherapy mostly because of the quiescence and thus can be enriched by chemotherapy, which is evidenced by our data and findings from other researchers [
10,
42]. These results support BEZ235 as a good candidate for CSC treatment.
Furthermore, we found that ROS and cell death caused by combination therapy were more significant than BEZ235 or cisplatin treatment alone in EOC-cis cell lines, indicating that inhibited oxidative stress is another possible mechanism underlying EOC cisplatin resistance and apoptosis [
43]. ROS has an important implication in cancer chemoresistance and was reported to be associated with a developing cancer stemness [
44,
45]. These studies further support our findings, i.e. increased ROS level and weakened CSC marker expression after combination treatment. It will be interesting to investigate the exact mechanisms of how ROS gets involved in EOC chemoresistance in the following study.
Finally, further investigation in high grade serous ovarian cancer is needed as an extension of this study to confirm the crosslink of PI3K/Akt/mTOR signaling with EMT and CSC. Findings from transgenic mice may be helpful for better understanding the relative importance of this pathway and underlying mechanisms in regulating EMT and stemness of ovarian cancer. Besides, cells sorted based on certain ovarian CSC surface markers may reveal the exact role of PI3K/Akt/mTOR signaling in regulating different ovarian CSCs at a single-cell level.
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