MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab
- Kai-Liang Zhang†1, 2, 8,
- Xuan Zhou†3,
- Lei Han1, 8,
- Lu-Yue Chen1, 8,
- Ling-Chao Chen4, 8,
- Zhen-Dong Shi1, 8,
- Ming Yang5,
- Yu Ren6,
- Jing-Xuan Yang2,
- Thomas S Frank2,
- Chuan-Bao Zhang7, 8,
- Jun-Xia Zhang1, 8,
- Pei-Yu Pu1, 8,
- Jian-Ning Zhang1, 8,
- Tao Jiang7, 8,
- Eric J Wagner9Email author,
- Min Li2Email author and
- Chun-Sheng Kang1, 8Email author
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 29 December 2013
Accepted: 11 March 2014
Published: 20 March 2014
Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown.
miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy.
In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the β-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation.
miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy.
Glioblastoma is the most common and fatal primary brain tumor in adults . The survival time varies depending on the patient’s genetic background [2, 3]. PTEN mutation and EGFR amplification are key prognostic factors in patients with anaplastic astrocytoma and in older patients with glioblastoma multiforme . Molecular therapies targeting EGFR have been developed in recent years, such as gefitinib, but many patients do not respond well to EGFR inhibitors, including those with non-small-cell lung cancer or glioblastoma . This is exemplified by the EGFR pathway’s contribution to radiation or chemo resistance in glioma .
MicroRNAs target the 3’ UTRs of oncogenes and tumor suppressor genes therefore contributing to the tumorigenesis of various human cancers . We previously identified a group of microRNAs (miR-21, miR-23b, miR-27b and miR-524-5p) that regulate proliferation, invasion and apoptosis in glioma [8–11]. Additionally, we demonstrated that the expression profile of miR-566 as well as that of four other miRNAs (miR-181d, miR-518b, miR-524-5p and miR-1227) correlated with the prognosis of glioblastoma patients . The function of miR-181d, miR-518, and miR-1227 have been reported in glioma or in other cancer types [13–15], however, there are no reports about miR-566 function till now.
Numerous studies have demonstrated that miRNAs contribute to chemotherapy resistance [16–18], most likely by regulating pro-survival pathways involved in drug resistance. Accumulating evidence suggests that microRNAs can regulate EGFR signaling, correlate with EGFR expression and influence gefitinib’s efficacy. For example, a study of lung cancer suggested that miRNA-128b directly regulated EGFR and loss of heterozygosity (LOH) was frequent in tumor samples, correlating significantly with the clinical response and survival following gefitinib treatment . Furthermore, miR-21 repressed p53-mediated apoptosis in response to chemotherapeutic agents, such as doxorubicin and other DNA damage-inducing agents, thereby contributing to drug resistance in glioblastoma cells . In this study, we focused on the function of miR-566 in EGFR signaling. We hypothesized that miR-566 could regulate the EGFR pathway and influence the sensitivity of glioma cells to anti-EGFR therapy.
miR-566 is over-expressed in glioma cell lines and activates EGFR/Akt signaling
Inhibition of miR-566 inhibits proliferation and invasiveness of glioma cells
Having confirmed that a miR-566 inhibitor could deactivate the EGFR pathway and inhibit the proliferative and invasive behavior of glioma cells, we then demonstrated whether the functions of miR-566 were mainly through the EGFR pathway. U87 glioma cells were first infected with lenti-AS-566 and then EGF was introduced to activate EGFR signaling. Results showed that EGF partially reversed the effects of lenti-AS-566 (Additional file 1: Figure S2).
VHL is a direct target of miR-566
miR-566 regulates the EGFR pathway through the VHL/β-catenin and VHL/HIF-1α axis
miR-566 regulates the formation of the β-catenin/HIF-1α complex and sensitizes glioma cells to nimotuzumab therapy
Synergistic activity of miR-566 inhibition and nimotuzumab in glioma cells and xenograft model
To our knowledge, there are no reports on the function of miR-566 in human cancers including glioma. In the present study, we confirmed that miR-566 was upregulated in human glioma cells, and repressing miR-566 could inactivate the EGFR pathway largely by targeting VHL. Further studies demonstrated that miR-566 regulated the VHL/β-catenin and VHL/HIF-1α axis in the transcription of EGFR. In addition, miR-566 is responsible for the formation of a β-catenin/HIF-1α complex. Finally, we confirmed that miR-566 inhibition could be synergistic with nimotuzumab therapy.
The EGFR pathway is activated in glioma and other human cancers, including lung, breast and colorectal [25–28]. The FDA has approved two types of anti-EGFR agents: low molecular weight tyrosine kinase inhibitors (TKIs) and mAbs that inhibit the EGFR extracellular domain. Clinically used anti-EGFR drugs include gefitinib, erlotinib, lapatinib, cetuximab and panitumumab [29, 30]. TKIs are effective therapies in human non-small cell lung cancer [31, 32]. However, TKIs such as gefitinib and erlotinib have had limited clinical success in treating glioblastoma . Moreover, all patients treated with cetuximab or panitumumab for colorectal cancer suffered from acute and subacute cutaneous side effects . Nimotuzumab is an anti-EGFR mAb developed at the Center of Molecular Immunology in Havana, Cuba. The clinical trials of nimotuzumab demonstrated that severe cutaneous adverse events were extremely rare. Furthermore, grade 3 and 4 acneiform eruptions commonly associated with other anti-EGFR mAbs were absent . Glioblastoma patients could benefit from nimotuzumab therapy, but the molecular expression profiles of GBM patients differ from one another. Personalized and combination therapy are needed.
MiRNAs are small, non-coding RNAs that can function as oncogenes or tumor suppressors by inhibiting the expression of numerous target genes. EGFR signaling can also be regulated by numerous miRNAs. For example, miR-7 is down-regulated in human glioblastoma and directly inhibits EGFR expression by targeting its 3’ UTR. In addition, miR-7 suppresses Akt pathway activation independent of its EGFR inhibition . We previously demonstrated that miR-21 is upregulated in glioma cells and that blocking its expression inactivates EGFR/Akt signaling in a PTEN-independent manner . In the present study, for the first time, we confirmed that miR-566 is upregulated in human glioma cells. In vitro and in vivo studies demonstrated that miR-566 inhibition deactivated EGFR/Akt signaling and slowed the proliferation of glioma cells.
Studies have demonstrated that miRNAs influence the response to chemotherapies for ovarian cancer, pancreatic cancer, bladder cancer and glioblastoma [37–40]. In a study conducted by Liana Adam, miR-200 expression regulated the epithelial-to-mesenchymal transition in bladder cancer cells and reversed EGFR therapy resistance . In a study by Masahiro Seike, miR-21 was up-regulated in the lung adenocarcinoma cell line H3255, which contains an EGFR mutation and is hypersensitive to EGFR TKI AG1478. The inhibition of miR-21 enhanced AG1478-induced apoptotic activity in these lung cancer cells, which showed intermediate sensitivity to AG1478. Another study demonstrated that epidermal growth factor (EGF) and MET receptors modulated the expression of miR-30b, miR-30c, miR-221 and miR-222. These microRNAs are also responsible for gefitinib-induced apoptosis and the epithelial-mesenchymal transition of NSCLC cells in vitro and in vivo by inhibiting the expression of the genes encoding BCL2-like 11 (BIM), apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ϵ (PKC-ϵ) and sarcoma viral oncogene homolog (SRC) . Our previous data demonstrated that miR-21 is involved in the regulation of anti-EGFR therapy .
Because miR-566 can regulate EGFR signaling, we wondered whether it could sensitize glioma to the effects of nimotuzumab in vitro and in vivo and its underlying mechanism. We identified VHL as a potential functional target of miR-566. A 3’ UTR luciferase assay was performed to determine whether miR-566 binds to the 3’ UTR of the VHL gene. The relative luciferase level for the VHL gene was significantly higher in lenti-AS-566-infected glioma cells than in lenti-NC-infected controls, and Western blot analysis confirmed these findings. The results demonstrated that the expression of the VHL protein is significantly upregulated in lenti-AS-566 infected cells. These results suggest that VHL is a direct target of miR-566. Furthermore, we confirmed that miR-566 regulated the formation of a β-catenin/HIF-1α complex. Both β-catenin and HIF-1α are important transcription factors for EGFR. Finally, studies demonstrated that the proliferation and invasion of glioma cells are attenuated when co-treated with lenti-AS-566 and nimotuzumab. The same results were confirmed in nude mice treated with lenti-AS-566 and nimotuzumab.
In conclusion, this is the first report to demonstrate that miR-566 expression is significantly increased in glioma cells. miR-566 modulated the EGFR pathway through direct targeting of VHL. We have identified the survival-related miRNA miR-566 as a regulator that influences the response to anti-EGFR therapy. Our study could have important implications for glioblastoma patients in the development of novel therapeutics.
Materials and methods
Cell culture and chemical reagents
The human glioma cell lines U87, LN229, SNB19, LN308 and U251 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human astrocytes (Invitrogen, Carlsbad, CA) were derived from human brain tissues. The human glioma cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Waltham, MA). Astrocytes were cultured in GIBCO Astrocyte Medium supplemented with N-2, FBS and EGF. Cells were cultured in a humidified 10% CO2 atmosphere at 37˚C. LiCl (Acros Organics, New Jersey, USA) and CoCl2 (Sinopharm Chemical, Shanghai, China) were diluted in phosphate-buffered saline (PBS).
Lentiviral infection, gene transfection and qRT-PCR
Lentiviruses containing a miR-566 inhibitor segment (lenti-AS-566) or negative control (lenti-NC) segment were obtained from Genepharma (Shanghai, China). The human glioma cell lines U87 and LN229 were infected with the viral suspension. pcDNA3 and pcDNA3-VHL plasmids were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Cells were harvested 48 h after infection or transfection, and RNA and protein extractions were performed. TRIzol (Invitrogen, Carlsbad, California) was used to isolate total RNA. To detect miR-566, stem-loop reverse transcription-polymerase chain reaction (RT-PCR) was performed with a one-step RNA PCR kit (Takara, Otsu, Shiga, Japan) according to the manufacturer’s instructions. Real-time PCR was performed by SYBR green detection with a forward primer for the mature miRNA sequence and a universal adaptor reverse primer. For the analysis of EGFR, AKT1, AKT2 and AKT3 messenger RNA (mRNA) expression, complementary DNA (cDNA) synthesis was performed using random primers under standard conditions. mRNA expression was quantified using the ΔΔCt method. GAPDH served as the internal control. All miRNA expression data were normalized to a U6 small nuclear RNA from the same sample. All reactions were performed in triplicate.
Plasmid construction and 3’ UTR analysis
The VHL expression plasmid pcDNA3-VHL was kindly provided by Professor Jinquan Cheng (H. Lee Moffitt Cancer Center and Research Institute, Florida). Glioma cells were transfected with 100 ng TOP-FLASH or FOP-FLASH plasmid (Millipore, Billerica, Massachusetts). The cells were then treated with lenti-AS-566 or VHL plasmid with or without LiCl. At 24 h after transfection, cell lysates were prepared with Dual Luciferase Lysis Buffer (Promega, Agora, Fitchburg Center, Fitchburg, Wisconsin), and luciferase activity was measured with a microplate reader (Mithras LB940; Berthold Technologies GmbH, Bad Wildbad, Germany). The transfection efficiency was normalized using Renilla luciferase activity. Experiments were performed at least 3 times; representative data from a single experiment are shown.
The putative miR-566 binding site of the VHL 3’ UTR was inserted into the pGL3-control vector (Promega, Agora, Fitchburg Center, Fitchburg, Wisconsin) at the Xba I site. For the VHL mutant reporter, the seed region of the VHL 3’ UTR was deleted to remove all nucleotides with complementarity to miR-566. For 3’ UTR luciferase assays, glioma cells were co-treated with lenti-NC or lenti-AS-566. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Agora, Fitchburg Center, Fitchburg, Wisconsin) 48 h after transfection.
Protein extraction, immunoblotting and immunoprecipitation
Glioma cells were washed in PBS and lysed with ice-cold RIPA buffer (Pierce, Brebieres, France) containing the protease inhibitor PMSF (Sigma, St. Louis, MO). Protein quantification was performed with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA). A DUALXtract Cytoplasmic and Nuclear Protein Extraction Kit (Dualsystems Biotech, Schlieren, Switzerland) was used to isolate cytoplasmic and nuclear proteins from cultured glioma cells.
The protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Roche, Basel, Switzerland). Membranes were immunostained with specific antibodies according to standard protocols. Antibody-labeled protein bands on the membranes were detected with a G:BOX F3 (Syngene, Cambridge, UK).
For immunoprecipitation, cells were lysed with IP lysis buffer (Pierce, Rockford, USA). The cell lysates were then subjected to immunoprecipitation with 1–5 mg of antibodies and Protein A/G agarose beads (Pierce, Rockford, USA) overnight at 4°C with constant agitation. Control samples were incubated with agarose beads after immunoprecipitation with a control immunoglobulin. The immunoprecipitated complexes were then washed with wash buffer. The proteins were eluted, boiled and subjected to SDS-PAGE analysis.
For immunofluorescence analysis, glioma cells were seeded on poly-L-Lysine treated coverslips (BD, USA). The cells were then infected with lenti-566 or lenti-NC with or without LiCl or CoCl2. After 48 h, the cells were fixed in cold methanol for 2 min. The cells were then washed 3 times in PBS and incubated in blocking buffer for 30 min at room temperature. Next, the cells were washed in PBS and incubated overnight at 4°C with β-catenin and HIF-1α primary antibodies (Cell Signaling Technology). The cells were again washed in PBS, followed by incubation with a fluorescent secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI solution for 5 min. Confocal images of the cells were acquired on a confocal microscope (FV500) with a 40 × water immersion lens and a 1.20 numerical aperture using FluoView software (Olympus, Japan).
Colony formation, invasion, cell cycle distribution and apoptosis analysis
For the colony formation assay, 2,000 glioma cells treated with lenti-AS-566, lenti-NC, VHL plasmid or nimotuzumab were plated in complete growth media in a fresh 6-well plate and allowed to grow until visible colonies formed. Cold methanol was used to fix the cell colonies, and colonies were stained with 0.1% crystal violet for 15 min, washed, air dried, photographed and counted.
Corning transwell insert chambers (Corning, New York) and BD Matrigel Invasion Chambers (BD Biosciences, Bedford, MA) were used for the cell invasion experiment. The prepared cells were added to the chamber and incubated for 24 h at 37˚C. Cells that invaded the lower chamber through the membrane were fixed with 20% methanol and stained with 0.1% crystal violet, imaged and counted.
The cell cycle was analyzed by flow cytometry. Pretreated U87 and LN229 cells were washed with PBS, trypsinized, fixed in 70% ethanol, washed and incubated in phosphate-buffered saline containing propidium iodide and RNase A (Sigma, St. Louis, MO) for 30 min at 37˚C. The cell cycle distributions were determined using a DNA stain (4’,6-diamidino-2-phenylindole). The data are the mean ± SD of 3 independent experiments.
Forty-eight hours after transfection, cells were harvested, washed, resuspended in staining buffer and examined using an Annexin V FITC Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China). The apoptotic distribution of the cells in each sample was then determined using fluorescence-activated cell sorting. Annexin V-positive cells were regarded as apoptotic cells.
Athymic mice (4 weeks of age) were intracranially implanted with 5 × 105 U87 cells (pretreated with lentivirus containing the miR-566 inhibitor segment or negative control segment) under the direction of a stereotactic instrument. Four days after cell implantation, mice were injected intraperitoneally with nimotuzumab or control PBS every other day. Bioluminescence imaging was used to detect intracranial tumor growth. Mice were anesthetized, injected with D-luciferin (Promega, Agora, Fitchburg Center, Fitchburg, Wisconsin) at 50 mg/mL intraperitoneally and imaged with the IVIS Imaging System (Caliper Life Sciences) for 10–120 s. To quantify bioluminescence, identical circular regions of interest were drawn around the entire head of each animal, and the integrated flux of photons (photons per second) in each region of interest was determined by using the Living Images software package (Caliper Life Sciences). Data were normalized to the bioluminescence at the initiation of treatment for each animal. The error bars shown in the figures indicate SDs. All protocols involving animals were performed in accordance with an approved Institutional Animal Care and Use Committee protocol.
This work was supported partially by the National High Technology Research and Development Program 863 (2012AA02A508), the China National Natural Scientific Fund (81172406, 81101916 and 81001128), the Natural Science Foundation of Tianjin Municipal Science and Technology Commission (12JCZDJC24300 and 12ZCDZSY17300), China Scholarship Council (CSC), and the Dr. Marnie Rose Foundation.
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