Contributed equally
Developmental Immunology, German Cancer Research Center, Heidelberg, Germany
Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK
MicroRNA (miR)-106b~25 cluster regulates bypass of doxorubicin and γ-radiation induced senescence by downregulation of the E-cadherin transcriptional activator EP300. We asked whether upregulation of miR-106~25 cluster generates cells with a truly multidrug resistant (MDR) phenotype and whether this is due to upregulation of the ATP-binding cassette (ABC) transporter P-glycoprotein. We used minimally transformed mammary epithelial breast cancer cells (MTMECs) in which the miR-106b~25 cluster was experimentally upregulated by lentiviral transfection or in which hairpins targeting either EP300 or E-cadherin mRNAs have been expressed with lentiviruses. We find that overexpression of miR-106b~25 cluster led to the generation of MDR MTMECs (resistant to etoposide, colchicine and paclitaxel). Paclitaxel resistance was also studied after experimental downregulation of EP300 or E-cadherin. However none of these cells overexpressed P-glycoprotein or where able to efflux a fluorescent derivative of paclitaxel, making this phenotype drug-transporter independent. Paclitaxel treatment in MTMECs led to an increase in early apoptotic cells (Annexin V-positive), activation of caspase-9 and increase in the proportion of cells at the G2/M phase of the cell cycle. However, MTMEC overexpressing miR-106b~25 cluster, or with EP300 or E-cadherin downregulated, showed less activation of apoptosis, caspase-9 and caspase-3/-7 activities. Thus, miR-106b~25 cluster controls transporter-independent MDR by apoptosis evasion via downregulation of EP300.
Chemotherapy is widely used for cancer treatment. Unfortunately, in a high proportion of cases, cancer cells become insensitive to the cyctotoxic drugs and are able to proliferate and metastasize, normally with fatal consequences. How cancer cells acquire drug resistance remains to be fully understood, although the effector molecules and pathways involved in the process are relatively well characterized. Cancer cells can increase the activity of DNA repair enzymes, metabolize drugs or activate anti-apoptotic mechanisms (
MicroRNAs (miRs) are a class of 18- to 24-nucleotide single-stranded non-coding RNAs as negative regulators of gene expression by triggering translation repression through partial complementation to 3′-untranslated region (UTR) of target mRNAs (
The miR-106b~25 cluster, consisting of miR-106b, miR-93 and miR-25, is highly conserved in vertebrates, and is located in intron 13 of the minichromosome maintenance complex component 7 (
Here we report that breast cancer cells overexpressing miR-106b~25 cluster, or in which EP300 or E-cadherin have been downregulated by RNA interference, have a P-glycoprotein-independent (transporter independent) MDR phenotype that involves apoptosis evasion.
Minimally transformed mammary epithelial cells (MTMECs) overexpressing the miR-106b~25 cluster by lentiviral transfection (MTMEC-miR-106b~25) or expressing a short hairpin targeting EP300 or E-cadherin (MTMEC-shEP300 and MTMEC-shCDH1, respectively) mRNAs have been described (
MTMEC-derived cells were seeded, at least in duplicate, at a density of 3×105 cells in 25-cm2 culture flasks and exposed to a single dose of drug for 3 days. Cells were kept in culture for 21 days with drug-free medium changes every three days. Drug resistant clones were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet and counted.
Functional drug efflux assays were performed using 0.1
Cell surface P-glycoprotein (ABCB1) was determined by flow cytometry in a Becton Dickinson FACSDiva using the phycoerythrin-conjugated UIC2 antibody (Immunotech, Marseille, France) or the corresponding isotype control (Sigma-Aldrich) essentially as described (
For mRNA detection, total RNA (isolated using a miRCURY RNA isolation kit; Exiqon) was reverse transcribed with RNase H+ MMLV reverse transcriptase (iScript cDNA Synthesis kit) and real-time quantitative PCR was performed using SYBR-Green (Bioline) and
Apoptotic assessment was by detection of active caspase-9 and -3/-7 using Caspase-Glo assays (Promega) following the manufacturer's protocol. Caspase activity was normalized to cell density determined by sulphorhodamine B (Sigma-Aldrich) staining (
The drug concentration necessary to kill 50% of cells (IC50) was obtained after sulphorhodamine B (Sigma-Aldrich) staining (
Statistical evaluations were performed by Student's t-test for paired data, and data were considered significant at a p-value <0.05.
We have recently reported that experimental upregulation of the miR-106b~25 cluster in MTMEC cells leads to the acquisition of doxorubicin and γ-radiation resistance (
Doxorubicin, at low to moderate concentrations, and γ-irradiation trigger a senescent phenotype, very similar to the well-characterized replicative senescence, often termed drug (or therapy)-induced senescence (
Thus, upregulation of miR-106b~25 cluster confers cells the ability to generate resistance to a variety of structurally and mechanistically different drugs, a hallmark of MDR (
The three miRs in the miR-106b~25 cluster bind
The above, and our previously published data, indicate that downregulation of EP300 leads to the MDR phenotype.
Upregulation of ABCB1 (P-glycoprotein) is the main mechanism by which cells become multidrug resistant. To test whether the multidrug resistant phenotype of MTMEC-miR-106b~25 and MTMEC-shEP300 cells was due to ABCB1, we tested first
As several other transporters can be responsible for multi-drug resistance (
In summary, the multidrug resistance phenotype of MTMECs, either overexpressing miR-106b~25 or with EP300 downregulated, is not due to transporter activity.
As taxanes block cells in the G2/M phase of the cell cycle (
Apoptosis evasion is another mechanism by which cancer cells may become resistant to a variety of drugs such as doxorubicin, etoposide or paclitaxel (
As paclitaxel triggers activation of caspase-9 in MTMECs (
Thus, overexpression of miR-106b~25, or downregulation of EP300 (a direct target of the three miRs in the cluster), leads to a transporter-independent MDR phenotype involving apoptosis evasion.
We have previously determined that doxorubicin resistance in MTMECs due to overexpression of miR-106b~25 cluster, or to downregulation of EP300, can be mimicked by experimentally downregulating E-cadherin expression (
Here we report that the axis EP300→E-cadherin, which is controlled by the miR-106b~25 cluster, regulates paclitaxel resistance in breast cancer cells by apoptosis evasion. This pathway also determines the resistance to DNA damaging agents, such as doxorubicin or γ-radiation, bypassing therapy-induced senescence, hallmarks of MDR. This phenotype was independent of membrane pumps, but involved apoptosis evasion. Thus, transporter-independent MDR can by generated by modulation of the miR-106b~25 cluster→EP300→E-cadherin pathway.
Resistance to chemotherapeutics used in cancer therapy remains one of the main hurdles to overcome for the successful treatment of this disease. Although in many cases the initial response to chemotherapy is positive, in a high proportion of cases, and after a disease-free period, resistant cells give rise to secondary tumors normally at distant sites following metastasis, normally with fatal consequences (
There are several effectors of MDR, although the most common is upregulation of drug transporters such as ABCB1 (
Paclitaxel, as well as docetaxel, the other taxane currently used in the clinic, is a microtubule-stabilizing agent that interferes with spindle microtubule dynamics causing cell cycle arrest and apoptosis (
The MDR phenotype can be fully mimicked by experimental downregulation of E-cadherin in MTMECs. However, we cannot rule out the possibility that either the miRs in the miR-106b~25 cluster, or EP300, or both, act on downstream molecules that regulate themselves drug resistance. miRs act repressing the expression of hundred of targets and EP300, which as a transcriptional co-activator, can also affect expression of many genes. This offer the prospect of finding novel molecules and regulatory pathways controlling transporter-independent MDR.
3′-untranslated region
ATP-binding cassette
DMEM containing 0.1% bovine serum albumin
drug concentration necessary to kill 50% of cells
Dulbecco's modified Eagle's medium
epithelial-to-mesenchymal transition
microRNA
minimally transformed mammary epithelial breast cancer cells
multidrug resistant
We thank the China Scholarship Council (Y.H.), Commonwealth Scholarship Comission (M.A.), Cancer Research UK China Programme (Y.Z., C.C., E.Y.), Chinese National Natural Sciences Foundation (81402480 to Y.H.), the Science and Technology Foundation of Tianjin Municipal Health Bureau (2014KZ078 to Y.H.) for their support.
Overexpression of the miR-106b~25 cluster leads to the MDR phenotype. MTMECs overexpressing miR-106b~25 cluster are able to generate etoposide- (upper panels), colchicine- (middle panels) and paclitaxel- (lower panels) resistant clones. Cells were treated for 3 days with drugs and drug-resistant clones were stained with crystal violet after 3 weeks (right panels) and number of clones counted (left panels). MTMECs transfected with empty vector (MTMEC-ev) were used as controls. Both cells have been fully described previously (
Paclitaxel triggers apoptosis in MTMECs. (A) Detection of early apoptotic cells by Annexin V staining. MTMECs were treated with increasing paclitaxel concentrations for 24 h and stained with propidium iodide and Annexin V-FITC prior to flow cytometry. Early apoptotic cells (Annexin V-positive and propidium iodide-negative; lower right quadrants in the left panel) increased in a paclitaxel dose-dependent manner (right panel). (B) Paclitaxel triggers activation of caspase-9. MTMECs were treated with increasing paclitaxel concentrations and the activation of caspase-9 measured after 72 h as activation was not detectable after 24 h (data not shown). Data are normalized to cell density determined by sulphorhodamine B staining. Numerical data represent the average ± SD of at least three different experiments (*P<0.05). A representative flow cytometry plot is shown.
Downregulation of EP300 leads to paclitaxel resistance. (A) Drug sensitivity curves of MTMECs treated with paclitaxel for 72 h determined by sulphorhodamine B staining. The IC50 of control cells (MTMEC-ev) and those overexpressing miR-106b~25 cluster (MTMEC-miR-106b~25) or with EP300 downregulated (MTMEC-shEP300) is indicated in the abscissa. (B) Cells were treated for 3 days with paclitaxel and drug-resistant clones were stained with crystal violet after 3 weeks (upper panel) and number of clones counted (lower panel). Numerical data represent the average ± SD of at least three different experiments (*P<0.05). A representative picture of paclitaxel-resistant clones is shown.
ABC transporters are not responsible for the MDR phenotype of cells either overexpressing miR-106b~25 cluster or with EP300 downregulated. (A)
G2/M cell cycle arrest induced by paclitaxel is abolished in MTMEC-miR-106b~25 and MTMEC-shEP300 cells. Cell cycle analysis of MTMECs treated with paclitaxel for 48 h. Cells were stained with propidium iodide and gated according to their fluorescence to differentiate cell cycle phases (from left to right: G1, S, G2/M). Experiments were performed in triplicate and a representative plot is shown (upper panel). Histogram data (lower panel) represent the average of three independent experiments. For clarity error bars have been omitted (typical variation between experiments: ±10%).
Paclitaxel resistance in cells overexpressing miR-106b~25 cluster, or with downregulation of EP300, is due to apoptosis evasion. (A) Detection of apoptotic cells by Annexin V staining. MTMECs were treated with paclitaxel for 48 h and stained with propidium iodide and Annexin V-FITC prior to flow cytometry. Number of apoptotic cells (Annexin V-positive cells, both in early apoptosis, lower right quadrant, and late apoptosis, upper right quadrant) decreased in both MTMEC-miR-106b~25 and MTMEC-shEP300 cells. A representative dot plot is shown on the left. (B) Activation of both caspase-3/-7 and -9 was determined after paclitaxel treatment for 48 h. Data are normalized to cell density determined by sulphorhodamine B staining. Numerical data represent the average ± SD of at least three different experiments (*P<0.05).
Downregulation of E-cadherin leads to a MDR phenotype which is transporter-independent. (A) MTMEC-shCDH1 are able to generate paclitaxel-resistant clones. Cells were treated for 3 days with paclitaxel and drug-resistant clones were stained with crystal violet after 3 weeks (left panels) and number of clones counted (right panels). MTMECs transfected with empty vector (MTMEC-ev) were used as controls. (B) Paclitaxel IC50 of MTMECs treated with paclitaxel for 72 h was determined by sulphorhodamine B staining. (C) Flow cytometric analysis of ABCB1 expression using the phycoerythrin-conjugated UIC2 antibody (upper panel) and BODIPY-paclitaxel efflux (lower panel) in MTMEC-shCDH1 and control MTMEC-ev cells. (D) Activation of both caspase-3/-7 and -9 was determined after paclitaxel treatment for 48 h. Data are normalized to cell density determined by sulphorhodamine B staining. Numerical data represent the average ± SD of at least three different experiments (*P<0.05). Pictorial data show representative paclitaxel resistant clones and flow cytometry histograms.