https://orcid.org/0000-0001-9210-3901
Abstract
Chemotheraputic drug resistance is a critical factor associated with the poor survival in advanced/metastatic pancreatic cancer (PC) patients.
Human pancreatic cell lines Capan-1 and BXPC-3 were cultured with different concentrations of erlotinib (0, 10, 50, and 100 μm) for 48 h. The relative cell viability and apoptosis was detected using MTT assays and flow cytometry apoptosis analysis, respectively. Transfection of pcDNA-EphA2, si-EphA2 and miR-124 mimic/inhibitor was used to modulate the intracellular level of EphA2 and miR-124. The interaction between miR-124 and the 3′UTR of EphA2 was explored using dual luciferase reporter assay.
Compared with BXPC-3 cells, Capan-1 cells showed resistance to differential concentration treatment of erlotinib. The expression of EphA-2 was significantly increased and the expression of miR-124 was significantly decreased in Capan-1 cells. Overexpressing EphA2 induced resistance of BXPC-3 cells to erlotinib treatment. And EphA2 was identified as a novel target gene for miR-124. MiR-124 overexpression was able to sensitize the response of Capan-1 cells to erlotinib through inhibiting EphA2. Furthermore, both miR-124 overexpression and EphA2 inhibition sensitized Capan-1 cells to erlotinib in xenograft model.
Our study demonstrated that EphA2 rescued by miR-124 downregulation conferred the erlotinib resistance of PC cell Capan-1 with K-RAS mutation.
Introduction
Pancreatic cancer (PC) is a highly malignant digestive tract tumour that has a poor prognosis.[1] PC cells are often overexpressed epidermal growth factor receptor (EGFR) and its endogenous ligand, therefore, important progress has been made in the EGFR targeting strategies of advanced PC.[2] Erlotinib, a small molecule EGFR-tyrosine kinase inhibitor (EGFR-TKI), can effectively improve the clinical efficacy of gemcitabine-based chemotherapies in the treatment of advanced PC, as shown by improving overall survival and clinical benefit response.[3,4] However, the clinical application of erlotinib is limited by the resistance of PC cells to erlotinib as evidenced by the findings that erlotinib in combination with capecitabine led to a 10% objective response rate.[5] Overcoming erlotinib resistance has become a critical issue for improving the outcome of PC chemotherapy.
K-RAS protooncogene is one of the genes with high mutation frequency of >90% in PC,[6] which is closely related to the development of PC and confers a unfavourable prognosis in PC patients.[7] The somatic mutation in K-RAS was confirmed to promote the malignant transformation of tumour cells and resistance to anticancer agents.[8] Studies have reported that the PC cells lines carrying mutated K-RAS showed high resistance to gemcitabine.[9] Recent evidence also suggested that mutations of K-RAS predicted an insensitivity of PC cells to erlotinib. K-RAS-mutated PC cells lines MiaPaCa-2, Panc-1 and AsPc-1 displayed the intense resistance to erlotinib.[10] To date, the K-RAS mutation-mediated resistance to erlotinib in PC is still poorly understood.
Erythropoietin-producing hepatocellular receptor 2 (EphA2), belonging to the family of receptor tyrosine kinases (RTKs), has a crucial role in promoting cell adhesion, proliferation and metastasis through interacting with its specific ligand, EphrinA1, thereby inducing tumour metastatic progression and angiogenesis.[11] Recent studies showed that EphA2 was required for erlotinib resistant of non-small cell lung cancer, and pharmacological inhibition of EphA2 significantly decreased the resistance of lung cancer cells to erlotinib in mice, indicating that EphA2 may be a promising target for overcoming erlotinib resistance.[12] High EphA2 expression was also found in PC, and the serum soluble EphA2 fragment level was potentially used as a diagnostic marker for PC.[13] In light of the fact that EphA2 knockout could effectively attenuate the invasion and metastasis of PC,[14] we're interested in that whether EphA2 had correlation with erlotinib resistance in PC.
The altered microRNA (miRNA) expression was a frequent event in many cancer types and involved in tumour initiation, progression and metastasis.[15] MiR-124 was significantly downregulated in PC tissues that facilitates the progression and metastasis of PC.[16] MiR-124 overexpression has been found to increase the gemcitabine-induced cell apoptosis in PC cells, including PANC-1 and MIAPaCa-2 cells.[17] Lastly, miR-124 decreased the clonogenic growth of mesenchymal K-RAS mutant non-small cell lung cancer (NSCLC) cell lines.[18] Preliminary association studies found that a miR-124-binding site was presented in the 3′-UTR of EphA2 gene (microrna.org), suggesting that EphA2 was a potential target gene of miR-124. It was therefore inferred that miR-124 induced the expression inhibition of EphA2 that was beneficial to attenuating K-RAS mutation-mediated resistance to erlotinib in PC.
Methods and materials
Cell culture and treatment
Human pancreatic ductal adenocarcinoma cell lines, Capan-1 cells (mutation in KRAS) and BXPC-3 cells (wild type KRAS) were obtained from the American Type Culture Collection (ATTC). Capan-1 cells were cultured in Iscove's Modified DMEM (IMDM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (HyClone, Logan, UT, USA). BXPC-3 cells were grown in RPMI containing 10% FBS (Gibco) and 1% penicillin-streptomycin (HyClone). All cells were cultured at 37 °C with 5% CO2. For erlotinib treatment, Capan-1 or BXPC-3 cells were cultured with different concentrations of erlotinib (0, 10, 50 and 100 μm) for 48 h.
MTT assays
After erlotinib treatment and/or cell transfection, the cell viability was detected using MTT assays. Capan-1 cells or BXPC-3 cells (6 × 103 cells/well) were cultured in 96-well plates and then incubated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) solution (10 μl/well, 5 mg/ml) (Sigma, St Louis, MO, USA) for 4 h as per the manufacturer's guidelines. The medium in each well was then replaced with DMSO (100 μl/well). The absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad, Foster, CA, USA).
Flow cytometry apoptosis analysis
The treated cells were harvested and washed twice with PBS for apoptosis analysis using fluorescein isothiocyanate (FITC) Annexin V apoptotis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocols. Briefly, the cells (1 × 106 cells) were suspended in 100 μl Annexin V binding buffer and then stained with 5 μl stains containing FITC conjugated Annexin V and propidium iodide (PI) from the detection kit at room temperature for 30 min in the dark. The cells were analyzed acquiring 10 000 events by Becton–Dickinson FACSVerse™ flow cytometry (Becton Dickinson, Heidelberg, Germany) with excitation at 488 nm and emission at 530 nm.
Western blot analysis
The cells were lysed with RIPA cell lysis buffer according to the manufacturer's protocols, and the protein concentration was determined using the Bradford protein assay (Thermo Scientific, Waltham, MA, USA). The protein samples were run with SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The proteins on PVDF membranes were blocked using 5% skim milk in TBST for 2 h. The proteins were incubated with the primary antibodies against EphA2 (1 : 500; Abcam, Cambridge, Cambridgeshire, UK), Bcl-2 (1 : 500; Sigma), Bax (1 : 2000; Sigma), c-Raf (1 : 1000), p-c-Raf (1 : 2000), p-AKT (1 : 1000), AKT (1 : 1000), p-mTOR (1 : 5000), mTOR (1 : 1000) and β-actin (1 : 1000; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. The primary antibodies against c-Raf, p-c-Raf, p-AKT, AKT, p-mTOR and mTOR were obtained from Cell Signaling Technology (Danvers, MA, USA). The proteins were then incubated with the horseradish peroxidase-conjugated secondary antibody and visualized using a chemiluminescent detection system (Pierce, Rockford, IL, USA). In addition, band density was analysed using ImageJ software.
Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted using TRIzol (Invitrogen, Waltham, MA, USA) and quantified using a NanoDrop 2000 (Thermo Scientific). For detection of EphA2 mRNA level, total RNA was reversely transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Dalian, China). For detection miR-124 expression, the reverse transcription was performed with the specific primer for miR-124 (5-GTCGTATCCATGCGGGTCTGGTGTCTTCATACGACCAACAAA-3). The qRT-PCR was performed using SYBR Green qPCR Kit (Invitrogen) with housekeeping gene U6 as the internal reference for miR-124 and GAPDH as the internal reference for EphA2 mRNA. The specific primers for qRT-PCR were as follows:
miR-124, (F) 5-AGGCCUCUCUCUCCGUGUUCAC-3, (R) 5-CAGCCCCATTCTTGGCATTCAC-3;
U6, (F) 5-GCTTCGGCAGCACATATACTAAAAT-3, (R) 5-CGCTTCACGAATTTGCGTGTCAT-3;
EphA2, (F) 5-ACCAGGCTGTACTCAAGTTTAC-3, (R) 5-GCCTTCAGCGTCCCTTTAT-3;
GAPDH, (F) 5-TGCACCACCAACTGCTTAGC-3, (R) 5-GGCATGCACTGTGGTCATGAG-3. All primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China).
Cell transfection
Transfection of pcDNA-EphA2, si-EphA2 and miR-124 mimic/inhibitor was used to modulate the intracellular level of EphA2 and miR-124. The transfection was performed using Lipofectamine 2000 transfection reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's protocols. MiR-124 mimic (5-CCGUAAGUGGCGCACGGAAU-3), inhibitor (5-GGCAUUCACCGCGUGCCUUA-3) and their corresponding controls were obtained from Guangzhou RiboBio Co., Ltd (Guangzhou, China). The working concentration of miR-124 mimic was 50 nm, and the working concentration of miR-124 inhibitor was 80 nm. The expression plasmid of EphA2 (pcDNA-EphA2) and siRNA targeting EphA2 (si-EphA2) were obtained from Shanghai GenePharma Co., Ltd (Shanghai, China).
Dual luciferase assays
According to the putative miR-124-binding site in the 3′UTR of EphA2 (WT), the 3′UTR of EphA2 with miR-124-binding site mutation was also designed (Figure 4a left). The 3′UTR of EphA2 with or without miR-124-binding site mutation were amplified and cloned into the pmirGLO Dual-Luciferase Expression Vector (Promega, Madison, WI, USA), constructing the reporter vectors, pmirGLO-EphA2-3′UTR-wt (WT) and pmirGLO-EphA2-3′UTR-Mut (Mut). The pmirGLO-EphA2-3′UTR-wt and pmirGLO-EphA2-3′UTR-Mut were cotransfected respectively into 293T cells with miR-124 mimic, inhibitor or the corresponding control. After 48 h, the relative luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega).
Studies in mice
BALB/C mice (18–22 g, 6–8 weeks) were purchased from Zhejiang University. All protocols in mouse experiments were followed the NIH Guidelines for the Care and Use of Laboratory Animals. This study was approved by the Ethics Committee of Zhejiang Provincial People's Hospital. Twenty-four BALB/C mice were randomly divided into six groups (n = 6 per group): Lenti-vector+PBS, Lenti-vector+erlotinib, Lenti-miR-124 + PBS, Lenti-miR-124 + erlotinib, Lenti-sh-EphA2 + PBS, Lenti-sh-EphA2 + erlotinib. Capan-1 cells were transfected with Lentivirus expression vector containing miR-124 (Lenti-miR-124), lentivirus expression vector interfering EphA2 (Lenti-sh-EphA2) or the control, Lenti-vector. For the mice in Lenti-vector+PBS, Lenti-vector+erlotinib groups, Capan-1 cells transfected with Lenti-vector (1 × 106 cells in 200 μl PBS) were subcutaneously injected into the mice. After 7 days, the mice were administered intragastrically with erlotinib (50 mg/kg/day) or PBS for 21 days. For the mice in Lenti-miR-124 + PBS, Lenti-sh-EphA2 + PBS, Lenti-miR-124 + erlotinib, Lenti-sh-EphA2 + erlotinib groups, the mice received Capan-1 cells (1 × 106 cells in 200 μl PBS) transfected with Lenti-miR-124 or Lenti-sh-EphA2. After 7 days, the mice were administered intragastrically with erlotinib (50 mg/kg/day) or the same dose of PBS for 21 days. The tumour volume was measured before the first administration, and was monitored every 3 days for 24 days. All mice were killed after the last measurement to collect the tumour tissues for weighing and qRT-PCR analysis.
Statistical analysis
All data from at least three independent experiments were expressed as mean ± standard deviation (SD) and analysed using Graphpad Prism 6 (Graphpad Software Inc., La Jolla, CA, USA) with Student's t-test and one-way ANOVA. For multiple testing, one-way ANOVA was followed by the Bonferroni test when variances were homogeneous, and Kruskal–Wallis test was performed to compare the variables of multiple-groups when variances were not homogeneous. P < 0.05 was considered as statistically significant difference.
Results
Capan-1 cells (mutation in KRAS) had resistance to erlotinib treatment compared with BXPC-3 cells (wild type KRAS)
Both Capan-1 cells and BXPC-3 cells are pancreatic ductal adenocarcinoma cells, and the major difference between the two is that Capan-1 cells have mutation in KRAS while BXPC-3 cells have wild type KRAS. It was observed that the relative viability of BXPC-3 cells was decreased by erlotinib treatment in a dose-dependent manner (Figure 1a). After 48 h of treatment, 10 μm erlotinib significantly reduced BXPC-3 cells viability. Meanwhile, compared with the untreated cells, 10 μm and 50 μm erlotinib had no obvious influence on the viability of Capan-1 cells, and 100 μm erlotinib reduced the cell viability with significant difference (Figure 1a). Moreover, apoptosis was significantly increased in the erlotinib-treated BXPC-3 cells, and the apoptosis of the erlotinib-treated Capan-1 cells was also a slightly increased, but not significant (Figure 1b). The ratio of Bax to Bcl-2 is an important marker for apoptotic status. Western blot analysis revealed that erlotinib treatment up-regulated Bax protein and down-regulated Bcl-2 in BXPC-3 cells, about three times increase in the ratio of Bax to Bcl-2 (Figure 1c). The application of erlotinib at 100 μm increased the ratio of Bax to Bcl-2 in Capan-1 cells by 60%. Compared with BXPC-3 cells, Capan-1 cells were shown to be relatively insensitive to erlotinib.
Capan-1 cells (mutation in KRAS) had resistance to erlotinib treatment compared with BXPC-3 cells (wild type KRAS). Capan-1 or BXPC-3 cells were cultured with different concentrations of erlotinib (0, 10, 50 and 100 μm) for 48 h. (a) The relative viability of Capan-1 and BXPC-3 cells was detected using MTT assays. (b) The flow cytometry apoptosis analysis was performed to evaluate the apoptosis. (c) The protein levels of Bcl-2 and Bax were detected using western blot analysis. Compared with the cells treated with erlotinib (0 μm) group, *P < 0.05. n = 3.
EphA2 induced resistance of BXPC-3 cells to erlotinib
As shown in Figure 2, EphA2 mRNA (Figure 2a) and protein (Figure 2b) expressions were increased in Capan-1 cells compared with BXPC-3 cells. We next investigated the role of EphA2 in the resistance of pancreatic ductal adenocarcinoma cells to erlotinib. Transfection of pcDNA-EphA2 significantly elevated the levels of EphA2 mRNA and protein in BXPC-3 cells (Figure 3a and 3b). Interestingly, EphA2 expression in the pcDNA-EphA2-transfected cells was significantly higher than that in the pcDNA-EphA2-transfected cells treated with 50 μm erlotinib (Figure 3a and 3b). Furthermore, EphA2 overexpression not only significantly increased the viability and decreased apoptosis of BXPC-3 cells, but also attenuated the negative effect of erlotinib (50 μm) on BXPC-3 cells (Figure 3c and 3d). It was further observed that EphA2 overexpression with or without added erlotinib (50 μm) resulted in decreased ratio of Bax to Bcl-2 in BXPC-3 cells (Figure 3e).
EphA2 expression was increased in Capan-1 cells compared with BXPC-3 cells. (a) EphA2 mRNA expression in Capan-1 and BXPC-3 cells was detected using qRT-PCR analysis. (b) EphA2 protein level was determined using western blot analysis. Compared with BXPC-3 cells, *P < 0.05. n = 3.
EphA2 induced resistance of BXPC-3 cells to erlotinib. BXPC-3 cells were transfected with pcDNA-EphA2 or its control, pcDNA and then treated with erlotinib (50 μm) for 48 h. (a) EphA2 mRNA and (b) protein level was determined using qRT-PCR and western blot analysis, respectively. (c) The relative viability of Capan-1 and BXPC-3 cells was detected using MTT assays. (d) The apoptosis was evaluated using flow cytometry apoptosis analysis. (e) The protein levels of Bcl-2 and Bax were detected using western blot analysis. *P < 0.05. n = 3.
EphA2 was negatively regulated by miR-124 in Capan-1 cells
According to the putative miR-124-binding site in the 3′UTR of EphA2 (WT), the 3′UTR of EphA2 with miR-124-binding site mutation (Mut) was also designed (Figure 4a, left). Dual luciferase assay was performed to identify the interaction between the 3′UTR of EphA2 and miR-124. The relative luciferase activity was negatively regulated by miR-124 in the cells transfected with the reporter vectors containing the 3′UTR of EphA2; meanwhile, miR-124 had no discernible effect on the relative luciferase activity of the cells transfected with the reporter vectors containing the 3′UTR mutation of EphA2 (Figure 4a, right). The transfection efficiency of miR-124 mimic and inhibitor in Capan-1 cells was verified using qRT-PCR analysis (Figure 4b). And miR-124 overexpression significantly reduced the expression of EphA2, but miR-124 inhibition significantly increased EphA2 expression in Capan-1 cells (Figure 4c and 4d). Given the obvious differences in EphA2 expression between Capan-1 cells and BXPC-3 cells (Figure 2), we were also interested in the expression profile of miR-124 in Capan-1 cells and BXPC-3 cells. Results showed that miR-124 expression was markedly lower in Capan-1 cells than that in BXPC-3 cells (Figure 4e).
EphA2 was negatively regulated by miR-124 in Capan-1 cells. (a) The interaction between the 3′UTR of EphA2 and miR-124. According to the putative miR-124-binding site in the 3′UTR of EphA2 (WT), the 3′UTR of EphA2 with miR-124-binding site mutation was also designed (left). Dual luciferase assay was performed to identify the interaction between the 3′UTR of EphA2 and miR-124. The reporter vectors, pmirGLO-EphA2-3′UTR-wt (WT) and pmirGLO-EphA2-3′UTR-Mut (Mut), were constructed and cotransfected, respectively, into 293T cells with miR-124 mimic or inhibitor. (b) Capan-1 cells were transfected with miR-124 mimic, inhibitor or the corresponding controls. After 48 h, the expression of miR-124 was determined using qRT-PCR analysis. (c) EphA2 mRNA and (d) protein level was determined using qRT-PCR and western blot analysis, respectively. (e) The expression of miR-124 in Capan-1 and BXPC-3 cells. *P < 0.05. n = 3.
MiR-124 sensitized Capan-1 cells to erlotinib through inhibiting EphA2
In the next series of experiments, we determined the role of miR-124 in the resistance of Capan-1 cells to erlotinib. MiR-124 overexpression significantly decreased the expression of EphA2 mRNA in Capan-1 cells with or without exogenous erlotinib (Figure 5a). Moreover, miR-124 overexpression down-regulated the levels of EphA2 protein and phosphorylation levels of its downstream signalling molecules, including p-c-Raf, p-AKT and p-mTOR (Figure 5b). Most importantly, miR-124 overexpression magnified the negative effects of erlotinib on Capan-1 cells, as shown by the decreased cell viability and increased apoptosis (Figure 5c and 5d). The ratio of Bax to Bcl-2 in Capan-1 cells treated with erlotinib was also increased by miR-124 overexpression. The above data suggested that miR-124 sensitized Capan-1 cells to erlotinib. To define whether EphA2 was involved in the mechanism of miR-124 control the resistance of Capan-1 cells to erlotinib, EphA2 expression was significantly down-regulated in Capan-1 cells by means of transfection of siRNA targeting EphA2, which was also accompanied by a significant decrease in the levels of p-c-Raf, p-AKT and p-mTOR (Figure 5f and 5g). Knockdown of EphA2 had the similar effect on the resistance of Capan-1 cells to erlotinib as miR-124 overexpression, reducing cell viability (Figure 5h), promoting apoptosis (Figure 5i) and increasing the ratio of Bax to Bcl-2 (Figure 5j) under exogenous erlotinib (50 μm, 48 h) stimulation.
MiR-124 sensitized Capan-1 cells to erlotinib through inhibiting EphA2. Capan-1 cells were transfected with miR-124 mimic or its control, pre-NC and then treated with erlotinib (50 μm) for 48 h. Detection of (a) EphA2 mRNA and (b) the protein levels of EphA2, c-Raf, p-c-Raf, p-AKT, AKT, p-mTOR and mTOR. (c) The relative viability of Capan-1 and BXPC-3 cells was detected using MTT assays. (d) The apoptosis was evaluated using flow cytometry apoptosis analysis. (e) The protein levels of Bcl-2 and Bax were detected using western blot analysis. Capan-1 cells were transfected with si-EphA2 or its control, si-NC and then treated with erlotinib (50 μm) for 48 h. Detection of (f) EphA2 mRNA and (g) the protein levels of EphA2, c-Raf, p-c-Raf, p-AKT, AKT, p-mTOR and mTOR. (h) The relative viability of Capan-1 and BXPC-3 cells was detected using MTT assays. (i) The apoptosis was evaluated using flow cytometry apoptosis analysis. (j) The protein levels of Bcl-2 and Bax were detected using western blot analysis. *P < 0.05. n = 3.
Both miR-124 overexpression and EphA2 inhibition sensitized Capan-1 cells to erlotinib in mice
The above data were also further confirmed in the nude mice model. Capan-1 cells transfected with Lenti-miR-124, Lenti-sh-EphA2 or Lenti-vector (1 × 106 cells in 200 μl PBS) were subcutaneously injected into the nude mice (n = 6 per group). The mice received erlotinib (50 mg/kg/day) or PBS by intragastric administration for 21 days as described in Sec 2 section. The mice subcutaneously injected with the Lenti-miR-124/sh-EphA2/vector-transfected Capan-1 cells and treated with PBS were used as control. The tumour volume and weight did not differ significantly between Lenti-vector+PBS and Lenti-vector+erlotinib groups (Figure 6a and 6b). However, compared with Lenti-vector+PBS group, both Lenti-miR-124 and Lenti-sh-EphA2 decreased the tumour volume and weight of subcutaneous xenografts in nude mice without erlotinib therapy (*P < 0.05) (Figure 6a and 6b). Moreover, upon the treatment of erlotinib, the tumour volume and weight were significantly reduced in the mice with either miR-124 overexpression or EphA2 inhibition compared with Lenti-vector+erlotinib group (#P < 0.05) (Figure 6a and 6b). Importantly, the mice in Lenti-miR-124 + erlotinib group had smaller tumour volume and weight than that of the mice in Lenti-miR-124 + PBS group (&P < 0.05) (Figure 6a and 6b). A similar difference was also observed between Lenti-sh-EphA2 + erlotinib and Lenti-sh-EphA2 + PBS groups (%P < 0.05) (Figure 6a and 6b). Results of qRT-PCR analysis showed that Lenti-miR-124 significantly increased miR-124 level of tumour tissues, and EphA2 was significantly decreased by Lenti-miR-124 and Lenti-sh-EphA2 (Figure 6c and 6d). These findings suggested that in addition to inhibit tumour growth, miR-124 overexpression and EphA2 inhibition could sensitize Capan-1 cells to erlotinib in mice.
Both miR-124 overexpression and EphA2 inhibition sensitized Capan-1 cells to erlotinib in mice. Twenty-four BALB/C mice were randomly divided into four groups (n = 6 per group): Lenti-vector+PBS, Lenti-vector+erlotinib, Lenti-miR-124 + PBS, Lenti-miR-124 + erlotinib, Lenti-sh-EphA2 + PBS, Lenti-sh-EphA2 + erlotinib. Capan-1 cells were transfected with Lentivirus expression vector containing miR-124 (Lenti-miR-124), lentivirus expression vector interfering EphA2 (Lenti-sh-EphA2) or the control, Lenti-vector. For the mice in Lenti-vector+PBS, Lenti-vector+erlotinib groups, Capan-1 cells transfected with Lenti-vector (1 × 106 cells in 200 μl PBS) were subcutaneously injected into the mice. After 7 days, the mice were administered intragastrically with erlotinib (50 mg/kg/day) or PBS for 21 days. For the mice in Lenti-miR-124 + PBS, Lenti-sh-EphA2 + PBS, Lenti-miR-124 + erlotinib, Lenti-sh-EphA2 + erlotinib groups, the mice received Capan-1 cells (1 × 106 cells in 200 μl PBS) transfected with Lenti-miR-124 or Lenti-sh-EphA2. After 7 days, the mice were administered intragastrically with erlotinib (50 mg/kg/day) or the same dose of PBS for 21 days. (a) The tumour volume was measured before the first administration, and was monitored every 3 days for 24 days. (b) The tumour weight was also measured. The expressions of (c) miR-124 and (d) EphA2 mRNA in tumour tissues were determined using qRT-PCR analysis. *P < 0.05 vs Lenti-vector+PBS; #P < 0.05 vs Lenti-vector+erlotinib; &P < 0.05 vs Lenti-miR-124 + PBS; %P < 0.05 vs. Lenti-sh-EphA2 + PBS.
Discussion
The present study determined that EphA2 was up-regulated in K-RAS mutant PC cells Capan-1 and involved in the K-RAS mutation-mediated resistance to erlotinib in PC. Activating mutations in K-RAS was an important genomic event for the initiation and progression of PC.[19] Additionally, growing evidence has demonstrated that K-RAS mutations resulted in the resistance to anticancer agents, which also is the critical factor for chemotherapy failure. K-RAS mutation was confirmed to render gemcitabine resistance of PC cells.[20] Erlotinib is a small molecule EGFR-TKI, and its integration with gemcitabine-based chemotherapy has a statistically significant survival benefit over gemcitabine alone for treatment of advanced PC in a double-blind, international phase III trial.[21] Unfortunately, the resistance of PC cells to erlotinib was developed and sharply decreased the clinical outcomes of advanced/metastatic PC patients.[5] K-RAS-mutated PC cells lines MiaPaCa-2, Panc-1 and AsPc-1 were reported to display the intense resistance to erlotinib.[10] This study also revealed that K-RAS mutant PC cells Capan-1 had resistance to erlotinib treatment compared with BXPC-3 cells with the wild-type K-RAS.
EphA2 has been implicated to contribute to induce tumour metastatic progression and angiogenesis. And high expression of EphA2 was found in PC compared with healthy controls.[22] Here, it was observed that EphA2 expression was significantly higher in Capan-1 cells than that in BXPC-3 cells, which might suggest a certain role of EphA2 in the resistance of PC to erlotinib. Importantly, EphA2 is capable of increasing the migratory capacity and drug resistance of tumour cells. EphA2 has previously been demonstrated to induce erlotinib resistant of non-small cell lung cancer.[12] In our study, BXPC-3 cells had some degree of sensitivity to erlotinib, which was consistent with the study of Naomi and his team.[10] Interestingly, the reconstruction of EphA2 expression in BXPC-3 cells obviously induced the resistance of BXPC-3 cells to erlotinib. Moreover, EphA2 inhibition sensitized Capan-1 cells to erlotinib both in vitro and in vivo studies. The above evidence demonstrated the functional involvement of EphA2 alteration in erlotinib resistance of K-RAS-mutated PC cells Capan-1.
Accumulating evidence has indicated that the dysregulation of miRNA was a frequent event in many cancer types, including PC.[23] MiRNA has recently been shown to mediate resistance to anticancer agents in PC. For instance, miRNA-210 was down-regulated in gemcitabine-resistant PC cells, and miRNA-210 overexpression enhanced the sensitivity of PC cells to gemcitabine.[24] MiR-21, as an oncogene, was highly expressed in PC and induced the growth, migration, invasion and 5-fluorouracil resistance of PC cells.[25] MiR-124 was also an important biomarker for PC, and the down-regulation of miR-124 was significantly associated with the poor prognosis of PC patients.[16] It was subsequently proved that miR-124 overexpression was conducive to recover the gemcitabine-induced cell apoptosis in PC cells, PANC-1 and MIAPaCa-2.[17] In the current study, the expression profile of miR-124 in Capan-1 cells and BXPC-3 cells was first analysed, and results showed that miR-124 expression was markedly lower in Capan-1 cells than that in BXPC-3 cells. We also confirmed that EphA2 was a novel target gene of miR-124 and miR-124 sensitized Capan-1 cells to erlotinib through inhibiting EphA2. PI3K/Akt/mTOR and canonical MAPK pathways have been confirmed to be important for the resistance to erlotinib of PC cells.[26,27] Moreover, both Akt/mTOR and c-Raf/MEK/ERK pathways were the downstream signalling of EphA2.[28] In the current study, both miR-124 overexpression and EphA2 knockdown could down-regulate the levels of p-c-Raf, p-AKT and p-mTOR in vitro, reinforcing the existence of miR-124/EphA2 axis in resistance to erlotinib in Capan-1 cells. However, further studies will be needed to identify whether and how EphA2 regulates the erlotinib resistance of PC cell Capan-1 through activating Akt and c-Raf signalling.
In conclusion, our data indicated that EphA2 rescued by miR-124 downregulation conferred the erlotinib resistance of PC cell Capan-1 with K-RAS mutation. Although further investigations are needed to clarify the role of EphA2 in the erlotinib resistance of the other K-RAS-mutated PC cell lines, this study shed a light on the connections between EphA2 and the erlotinib resistance of PC cell Capan-1. The identification of key regulators and mechanisms in erlotinib resistance may eventually contribute to improve the clinical treatment for PC.
Declaration
Funding
This study was supported by Natural Science Foundation of Zhejiang Province (Grant No. LQ17H160016) and Zhejiang Provincial Medical and Healthy Science and Technology Projects (Grant No. 2015KYA029).
Conflicts of interest
The Authors have no conflicts of interest to disclose.
References
Author notes
These authors contributed equally to this work.
