Anastasov et al. Radiation Oncology 2012, 7:206 http://www.ro-journal.com/content/7/1/206
RESEARCH
Open Access
Radiation resistance due to high expression of miR-21 and G2/M checkpoint arrest in breast cancer cells Nataša Anastasov1*, Ines Höfig1, Iria Gonzalez Vasconcellos1, Kristina Rappl2, Herbert Braselmann3, Natalie Ludyga2, Gert Auer4, Michaela Aubele2 and Michael J Atkinson1,5
Abstract Background: There is evidence that the extent of the G2/M arrest following irradiation is correlated with tumour cell survival and hence therapeutic success. We studied the regulation of cellular response to radiation treatment by miR-21-mediated modulation of cell cycle progression in breast cancer cells and analysed miR-21 expression in breast cancer tissue samples with long-term follow up. Methods: The miR-21 expression levels were quantified (qRT-PCR) in a panel of 86 cases of invasive breast carcinomas in relation to metastasis free survival. The cellular radiosensitivity of human breast cancer cells after irradiation was determined comparing two cell lines (T47D and MDA-MB-361) by cell proliferation and colony forming assays. The influence of miR-21 overexpression or downregulation on cell cycle progression and G2/M checkpoint arrest after irradiation was assessed by flow cytometric analysis. Results: The expression of miR-21 was transiently increased 8 hours after irradiation in the radioresistant T47D cells and significantly changed with lower extent in radiosensitive MDA-MB-361 cells. Anti-miR-21 treated breast cancer cells failed to exhibit the DNA damage-G2 checkpoint increase after irradiation. Apoptotic activity was significantly enhanced from 7% to 27% in T47D cells and from 18% to 30% in MDA-MB-361 cells 24 hours after 5 Gy irradiation. Additionally, we characterized expression of miR-21 in invasive breast carcinomas. In comparison to non-cancerous adjacent breast tissue, tumours samples had increased miR-21 expression that inversely correlated with the distant metastases-free survival of patients (p = 0.029). Conclusions: Our data indicate that miR-21 expression in breast cancer cells contributes to radiation resistance by compromising cell cycle progression. These data point to the potential of combining radiotherapy with an anti-miR-21 as a potent G2/M check point inhibitor in overcoming radiation resistance of tumours. Keywords: MiR-21, Breast cancer, Radiation resistance, G2/M checkpoint arrest
Background MicroRNAs (miRNAs) are functional small nucleic acids that regulate the stability and translational efficiency of target messenger RNAs [1]. Altered expression of miRNAs has been demonstrated in several human cancers where miRNA 'signatures' are found to be informative for tumour classification and clinical outcome [2,3]. Although several miRNAs are upregulated in specific * Correspondence:
[email protected] 1 Institute of Radiation Biology, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Muenchen, Germany Full list of author information is available at the end of the article
tumour types [4], a global reduction of miRNA abundance is the more common trait in human cancers. This results in considerable influence on the transformed phenotype [5]. In human breast cancer the deregulation of miRNA expression was first demonstrated by Iorio et al. [6], who suggested a possible role for miRNAs as robust biomarkers for breast cancer diagnosis and prognosis. Recent studies [7] provide evidence that miRNAs are involved in many of the cellular regulatory processes, including activation of different signaling pathways and induction of apoptosis [8,9].
© 2012 Anastasov et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Anastasov et al. Radiation Oncology 2012, 7:206 http://www.ro-journal.com/content/7/1/206
The heterogeneity of human cancers requires the use of multiple therapeutic modalities, including radiation therapy. However, the development of radioresistance presents a problem over prolonged courses of treatment [7,10]. Only a few studies have described the effect of radiation on miRNA expression profiles [7,11,12]. There are indications that radiation sensitivity may be manipulated by influencing the expression of a single miRNA species [11,13,14]. However, little is known of the underlying mechanisms. A more detailed knowledge about radiation influences on miRNA expression in tumour cells is important for improving the effectiveness and reducing the side effects of radiotherapy. Overexpression of miR-21 has been previously reported [15-17]. Patients with high tumour miR-21 expression have a worse clinical outcome than those with low tumour miR-21 expression [18]. A possible explanation was provided by a genome wide search for miR-21 targets. This suggested a functional link between miR-21 and the p53 tumour suppressor pathway [17,19], where p53-induced proteins provoke apoptosis in response to DNA damage after irradiation in cancer. One possibility to improve therapeutic strategy is the modulation of cell cycle progression. The fact that the radiation-induced G2-phase block is a universal event in tumour cells renders the G2/M checkpoint as target for improved efficacy of radiation therapy [20]. Most of the cancer cells have mutations in genes involved in the G1 checkpoint such as p53, Rb, p16, MDM2 and cyclin D1 [21,22]. Interestingly the G2 checkpoint is usually retained in the cancer cells with impaired G1 checkpoint. Therefore if the G2 checkpoint is selectively disrupted the cancer cells with impaired G1 checkpoint would become more sensitive to the DNA-damaging treatment compared with normal cells because normal cells still retain G1 checkpoint intact [21]. In this study, we characterized expression of miR-21 in 86 invasive mammary carcinomas, supporting poor prognostic effects with high miR-21 expression. Additionally, we identify changes in miR-21 levels and cellular response regulation after irradiation in breast cancer cells. Furthermore, we give evidence that modulating the miR21 expression level would be an important milestone in efficient breast cancer radiation therapy treatment.
Methods Growth and maintenance of cell lines
The breast cancer cell line MDA–MB–361 was cultured in DMEM (Dulbecco Modified Eagles medium) with 20% FCS, (Invitrogen, Carlsbad, CA) and T47D was maintained in RPMI 1640 (Roswell Park Memorial Institute medium) supplemented with 10% FCS and human insulin (10 μg/ml). The cell cultures were maintained in a water humidified 37°C incubator with 5% CO2.
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Ionizing radiation treatment
Irradiation of cell cultures containing 1 × 106 log phase cells was performed with a Cs-137 irradiator (HWM D2000, Siemens, Germany) at a dose rate of 0.95 Gy/min. Doses of 2.5 Gy; 5.0 Gy or 7.5 Gy were administered at room temperature and control cells were sham irradiated. The exposed and sham irradiated cells were subsequently incubated at 37°C and harvested after indicated time points for RNA and protein isolation. The experiment was repeated for each dose in triplicate. Lentivirus production and infection of breast cancer cell lines
Replication-defective lentiviral particles were produced by transient co-transfection of HEK293T cells in a 10 cm petri dish with 16 μg, 8 μg and 4 μg of packaging plasmids pMDLg/pRRE, pRSV. Rev and pMD2.G (a kind gift from D. Trono, École polytechnique fédérale de Lausanne) and 8 μg of lentiviral transduction vector pGreenPuro (pGP; System Biosciences, California) using Lipofectamine 2000 (Life Technologies, California) according to the manufacturer’s instructions. The pGP vector (named EV – empty virus in results section) was used as the backbone for miR-21 overexpression and miR-21 downregulation (anti-miR-21) by specific miRNA oligo cloning (pmiRZIP-21 - Cat. Nr. MZIP21PA-1-GVO-SB; Biocat, Heidelberg, Germany). The virus particles were harvested 48 hours after transfection, cleared and concentrated as previously described [23]. According to virus titer determination virus productions ranged between 108 and 109 TU/ml (TU - Transduction Units). Viral infection of breast cancer cells was performed using protocols previously described [24]. Briefly, 2 × 105 cells per well were infected with 4 × 105 TU/ml (defined as 2 MOI – multiplicity of infection) and three days after infection GFP expression was monitored. After infection 5 × 105 cells were irradiated for indicated time points. Microscopic analysis was done 48 hours post irradiation (HBO 50/AC and AxioCam MRC, Carl Zeiss AG, Germany). RNA isolation for miRNA expression analysis
Paraffin-embedded tissue was microdissected with a sterile needle from 5 μm thick sections using a stereo microscope (Stemi 2000, Zeiss, Germany). A consecutive H&E-stained section was used for guidance. Tumour cell material (containing at least >80% tumour cells) was collected from all cases. Additionally, histologically normal ductal epithelium material was collected from five cases as control tissue. Total RNA was isolated from microdissected tissues as previously described [25]. After digestion in lysis buffer and 500 μg/ml proteinase K the RNA was purified by phenol/chloroform extraction, ethanol precipitated, and dissolved in 20 μl RNase-free water.
Anastasov et al. Radiation Oncology 2012, 7:206 http://www.ro-journal.com/content/7/1/206
Five microlitres (100 ng) of RNA were reversetranscribed using MultiScribeTM reverse transcriptase (Applied Biosystems; Foster City, CA, USA) [26]. Further processing and evaluation of the results was performed according to the manufacturer’s instructions. Total RNA was isolated from each of the breast cancer cell lines (MDA-MB-361 and T47D) after irradiation. Cells were pelleted by centrifugation at 1500 rpm for 5 min, and washed with 1 ml Dulbecco’s phosphate-buffered saline (PBS) without MgCl2 and CaCl2 (Invitrogen, Carlsbad, CA, USA). Small RNAs (
