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. 2013 Dec 31;8(3):458–468. doi: 10.1016/j.molonc.2013.12.011

DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity‡

Marijn TM van Jaarsveld 1,†, Maikel D Wouters 2,†, Antonius WM Boersma 1, Marcel Smid 1, Wilfred FJ van IJcken 3, Ron HJ Mathijssen 1, Jan HJ Hoeijmakers 2, John WM Martens 1, Steven van Laere 4, Erik AC Wiemer 1,✉, Joris Pothof 2,✉
PMCID: PMC4685152  NIHMSID: NIHMS560411  PMID: 24462518

Abstract

The DNA damage response (DDR) is activated upon DNA damage and prevents accumulation of mutations and chromosomal rearrangements, both driving carcinogenesis. Tumor cells often have defects in the DDR, which in combination with continuous cell proliferation are exploited by genotoxic cancer therapies. Most cancers, overcome initial sensitivity and develop drug resistance, e.g. by modulation of the DDR. Not much is known, however, about DNA damage responsive microRNAs in cancer therapy resistance. Therefore, we mapped temporal microRNA expression changes in primary breast epithelial cells upon low and high dose exposure to the DNA damaging agents ionizing radiation and cisplatin. A third of all DDR microRNAs commonly regulated across all treatments was also misexpressed in breast cancer, indicating a DDR defect. We repeated this approach in primary lung epithelial cells and non‐small cell lung cancer samples and found that more than 40% of all DDR microRNAs was deregulated in non‐small cell lung cancer. Strikingly, the microRNA response upon genotoxic stress in primary breast and lung epithelial cells was markedly different, although the biological outcome of DNA damage signaling (cell death/senescence or survival) was similar. Several DDR microRNAs deregulated in cancer modulated sensitivity to anti‐cancer agents. In addition we were able to distinguish between microRNAs that induced resistance by potentially inducing quiescence (miR‐296‐5p and miR‐382) or enhancing DNA repair or increased DNA damage tolerance (miR‐21). In conclusion, we provide evidence that DNA damage responsive microRNAs are frequently misexpressed in human cancer and can modulate chemotherapy sensitivity.

Keywords: DNA damage response, MicroRNAs, Cancer, Chemotherapy, Therapy resistance

Highlights

  • MicroRNA profiling revealed a general DNA damage‐induced microRNA response.

  • However, this response differed between primary breast and lung epithelial cells.

  • At least 30% of these general responsive microRNAs were misexpressed in cancer.

  • These DNA damage responsive microRNAs modulated sensitivity to genotoxic treatments.

  • MicroRNA‐21 might mediate an improved DNA repair or an increased damage tolerance.

1. Introduction

The DNA damage response (DDR) maintains genome stability by protecting the genome against mutations and chromosomal rearrangements, the driving events of carcinogenesis. The DDR comprises DNA repair, cell cycle arrest to allow the cell time to repair the damage and, if DNA damage is beyond repair, induction of apoptosis or cellular senescence (permanent cell cycle arrest) (Hoeijmakers, 2009). The DDR forms an active barrier against tumorigenesis. Consequently, most, if not all, pre‐malignant cells obtain at least one defect in the DDR, which allows them to progress into more malignant states (Bartkova et al., 2005; Gorgoulis et al., 2005; Hanahan and Weinberg, 2011). Defects in the DDR combined with increased proliferation of tumor cells are exploited by DNA damaging cancer therapy to selectively target tumor cells. For instance, ionizing radiation (IR), alkylating agents, anthracyclines and platinum‐containing compounds induce DNA damage and are used to treat a wide‐range of cancers. Therapy resistance, which can be either innate or acquired, is a major impediment for the successful treatment of cancer. Cellular resistance to genotoxic agents can be achieved by impaired delivery, uptake or retention of drugs (Gottesman, 2002). In addition, cancer cells modulate therapy sensitivity by altering DDR pathways involved in DNA repair (Bouwman and Jonkers, 2012; Lord and Ashworth, 2012) apoptosis and senescence (Gordon and Nelson, 2012; Igney and Krammer, 2002). Therefore, studying the DDR and its regulation can result in new leads to combat therapy resistance (Bartek and Lukas, 2007).

MicroRNAs (miRNAs) are an important class of regulatory factors. These small non‐coding RNA molecules regulate gene expression by translational repression and/or mRNA degradation (Bartel, 2009). Most, if not all, tumors display deregulated miRNA expression compared to normal tissue (Farazi et al., 2011). Moreover, emerging evidence indicates important roles for miRNAs in the DDR and in anti‐cancer therapy (Wiemer, 2011; Wouters et al., 2011). Based on these data, we hypothesized that miRNAs involved in the DDR might be altered in human cancer due to DDR defects, which in turn could modulate chemotherapy sensitivity. We used a large‐scale miRNA genomics approach to screen miRNA expression in both primary cell lines exposed to genotoxic stress and human tumors to identify these relationships.

2. Materials & methods

2.1. Cell culture and reagents

Human Mammary Epithelial progenitor Cells (HMEpC) and Human Small Airway Epithelial progenitor Cells (HSAEpC) were obtained from Promocell (Germany) and cultured according to the supplier's instructions. Cells were expanded up to population doubling 5, after which they were aliquotted and frozen in culture medium (Promocell) containing 10% DMSO and 5% Human Serum Albumin (HSA, Octalbine). All experiments were carried out with cells cultured from a freshly thawed vial that were expanded till population doubling 10. The HMEpC/HSAEpC retained their proliferation characteristics up to 16 population doublings. Cancer cell lines H226 (CRL‐5826), H460 (HTB‐177), MDA‐MB‐231 (HTB‐26), MDA‐MB‐157 (HTB‐24) and SK‐BR‐3 (HTB‐30) were obtained from the ATCC. Cisplatin, doxorubicin and paclitaxel were obtained from Pharmachemie, the Netherlands.

2.2. Clonal survival assay

Clonal survival assays were performed after exposure of cells to a continuous treatment of cisplatin or a pulse of IR. A total of 1*104 primary epithelial cells, or 250–1250 lung and breast cancer cells were seeded in 3 cm dishes and cultured at 3% O2, conditions that approximate tissue oxygen levels. After 16 h, cells were treated with IR or cisplatin. Untreated cells formed 100–200 colonies after 7 (cancer cell lines) or 10 days (epithelial cell lines). Colonies were fixed with 50% methanol, 7% acetic acid and stained with 0.1% Coomassie‐blue before counting.

2.3. Western blotting

15‐20 μg of total protein was subjected to SDS‐PAGE/Western blotting. Specific proteins were detected with antibodies against PARP1 (SA‐249; Biomol) and p53 (SC‐126, Santa Cruz). Goat‐anti‐mouse conjugated to HRP (Santa Cruz, SC‐2005) was used as secondary antibody.

2.4. FACS

Human primary epithelial cells were incubated for 15 min with 1 μM Bromodeoxyuridine (BRDU), trypsinized and washed with PBS before fixation with 70% ethanol. After proteolytic treatment with pepsine and HCl, cells were incubated with FITC‐conjugated antibody against BRDU (BD Biosciences, #3447583) and finally propidium iodide (PI) (1 μg/mL, Sigma), after which the cell cycle profile was analyzed.

To analyze IL6/IL8 levels, medium supernatant was collected at the indicated times and incubated with human IL6/IL8 capture beads (Cytometric Bead Array (CBA) flex set, BD Biosciences), according to manufacturer's instructions. These beads have unique fluorescence intensities, allowing simultaneous detection of IL6/IL8.

2.5. MiRNA profiling

MiRNA RT‐qPCR expression analysis using microfluidic cards (A&B TLDA arrays, Applied Biosystems) was performed on 8 normal breast tissue samples and 84 breast tumor tissue samples as described before (Van der Auwera et al., 2010). The represented breast cancer subtypes are listed in Supplemental Table 1.

MiRNA microarray analysis was performed on (i) Untreated and cisplatin/IR treated HMEpC and HSAEpC cells (4 replicates for each condition), (ii) 18 lung tumor tissue samples and 14 adjacent ‘normal’ lung tissue samples (the represented non‐small cell lung cancer (NSCLC) subtypes are listed in Supplemental Table 1), and (iii) 52 breast cancer cell lines (Riaz et al., 2013) and 12 lung cancer cell lines. Total RNA was isolated with RNA Bee (BioConnect, the Netherlands). One μg RNA was hybridized with an LNA™‐based probeset (Exiqon) version 10 (all cell lines) or version 7 (lung tissue), spotted in duplicate on Nexterion E slides (Pothof et al., 2009). Version 10 probeset contains 1344 probes capable of detecting 725 human miRNAs, version 7 contains 576 probes of which 328 recognize human miRNAs. Slides were scanned and quantified with Imagene software. After background subtraction, expression values were quantile normalized. Low expressed miRNAs and obvious outliers were removed from probeset version 10, while low expressed miRNAs from probeset 7 were removed after statistical analysis. The resulting datasets consisted of (i) 696 (ii) 328 and (iii) 425 human miRNAs.

2.6. Transfections

Breast and lung cancer cell lines (50% confluent) were transfected with miRNA mimics, scrambled control mimic or fluorescent transfection control mimic (Dharmacon, miRIDIAN mimics; 50 nM final concentration) using Dharmafect 1 according to supplier's protocol. After 48 h cells were exposed to anticancer drugs for 24 h after which cell viability was tested by MTT assay (Carmichael et al., 1987). For clonal survival assays, cells were cultured at 3% O2 to approximate tissue oxygen levels. Cells were seeded in 6 well plates at 80% confluency, and transfected two times at 6 h intervals with the appropriate miRNA mimics using RNAiMax (Invitrogen) according to the supplier's protocol. After the second transfection overnight (16 h), cells were trypsinized and seeded as described above for clonal assays or seeded on glass slides. The latter cells were treated the following day with cisplatin and fixed for immune fluorescence staining (Rademakers et al., 2003) with 53BP1 antibody (H‐300; Santa Cruz Biotechnology) and DAPI staining.

2.7. Statistical analysis

Significant Analysis of Micro‐Arrays (SAM) statistical analysis was carried out to identify differentially expressed miRNAs in the array datasets (Tusher et al., 2001). For HMEpC and HSAEpC cells, SAM analysis was performed to compare miRNA expression levels in treated versus untreated cells for each time point. Differentially expressed miRNAs that showed a fold change <1.5 fold were excluded, resulting in datasets of 122 HMEpC and 123 HSAEpC regulated miRNAs. SAM was also performed to compare breast and lung tumors with their corresponding normal tissue. A flow chart depicting the various statistical and experimental steps is presented in Supplemental Figure 1. Statistics and heatmap generation have been performed using TM4 microarray software suite (Saeed et al., 2003). Correlation between miRNA expression levels and drug sensitivity in the NCI60 panel was assessed using Pearson correlation statistics and Benjamini‐Hochberg's FDR was used to control for multiple hypothesis testing (Supplementary Table 2). Paired sample T‐tests were performed on MTT data of minimally three independent experiments, to test whether differences between miRNA mimic or scrambled mimic transfected cells were significant (Figure 5).

Figure 5.

Figure 5

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MiRNA overexpression modulates the response to DNA damaging agents in breast and lung cancer cell lines. MiRNAs were overexpressed in cell lines that had a low basal expression of the respective miRNA. For miR‐193a‐3p and miR‐296‐5p breast cancer cell line MDA‐MB‐157 was selected (A, upper left panel). For miR‐183 breast cancer cell line MDA‐MB‐231 was used (A, upper middle panel). For let‐7a breast cancer cell line SK‐BR‐3 was used (A, upper right panel). For miR‐16 and miR‐21 lung cancer cell line H460 was selected (A, lower left panel) and for miR‐382 lung cancer cell line H226 (A, lower middle panel) was used. A: Drugs were added 48 h after transfection with a scrambled control or a miRNA mimic. An MTT assay was carried out after 24 h of continuous exposure (for dose: see Supplemental Table 4). The viability of drug treated cells is depicted relative to the viability of untreated cells which is arbitrarily set at 100%. Shown is a representative experiment (average values ± S.D., n = 3). Paired sample t‐tests were used to assess whether differences between independent experiments were consistent. Asterisks indicate statistical significance but only in those experiments in which the difference between viability of control mimic and miRNA mimic transfectants was consistent in three independent experiments, *p < 0.05, **p < 0.01. B: Clonogenic survival assay of cells transfected with selected miRNAs. Colonies were counted one week after treatment with a pulse of IR or continuous exposure of cisplatin. The amount of colonies under untreated conditions was set at 100%. For each miRNA transfection the same cell‐lines were used as in A. A representative experiment is shown (n = 2) and depicted are average values ± S.D. (n = 3). C: 53BP1 immune fluorescence staining of H460 cells transfected with miR‐21 and treated for 8 h with 5 μM cisplatin. DAPI stains the nuclei. A representative experiment is shown (n = 2). 53BP1 foci were quantified in >200 nuclei for both conditions. The graph depicts the average amount of foci per nucleus normalized to the control.

3. Results

3.1. Characterization of the DDR in primary epithelial mammary cells

To identify miRNAs that are regulated upon DNA damage (DDR miRNAs) and misexpressed in human tumors we focused on normal breast tissue and breast cancer. Because established breast cancer cell lines likely have defects in the DDR, most notably loss of p53 expression, and therefore might not – or aberrantly – express specific DDR miRNAs upon genotoxic stress treatment, we used primary human mammary epithelial progenitor cells (HMEpCs) to monitor DDR miRNAs. HMEpCs were characterized by their ability to form mammospheres, which is associated with stemness (Dontu et al., 2003) and displayed cellular senescence after approximately 16 population doublings. Treatment with cisplatin resulted in p53 upregulation (Supplemental Figure 2A), indicating that HMEpCs are wild‐type primary cells that exhibit a normal p53 response.

The severity of DNA damage determines the outcome of DDR signaling, i.e. repair and survival or apoptosis/senescence. Since there is evidence in the literature that all these branches within the DDR can be defective in cancer, conditions were established to identify miRNAs that are regulated upon DNA damage in general, independent of biological outcome. Therefore, we used two genotoxic therapeutic agents (cisplatin and IR) for which we determined a dose that allows for cellular recovery after DNA damage and a dose that induces primarily cell death or senescence. To define the conditions for recovery we used a clonal survival assay, which determines the capacity of individual cells to recover and form colonies after DNA damage. We chose 0.25 μM cisplatin or 1.5 Gy IR, both resulting in a 50% reduction of HMEpC colony formation (Supplemental Figure 2B), thus 50% of all initially damaged cells can still grow out into a colony within 10 days. As expected, these conditions activate the DDR and induce cell cycle arrest (Supplemental Figure 2B).

Subsequently, using an MTT assay, we established a higher dose at which the cells undergo apoptosis after cisplatin treatment. Treatment with 15 μM cisplatin for 48 h led to a 50% reduction of HMEpC viability (Supplemental Figure 3A), which was accompanied by decreased cellular PARP1 levels (Supplemental Figure 3B), a measure for apoptosis. Note that at this dose all cells will eventually undergo apoptosis. In contrast to cisplatin, a high dose of IR did not lead to apoptosis but to cellular senescence, which does not affect cellular viability as measured by the MTT assay (Supplemental Figure 3C) (Bunnak et al., 1994; Coppe et al., 2008; Sabin and Anderson, 2011). Therefore, we determined the dose at which HMEpCs display a total, permanent stop of cell division, which was at 6 Gy or higher (Supplemental Figure 3D). Cellular senescence is associated with the secretion of cytokines in the medium, such as IL‐6 and IL‐8 (Coppe et al., 2008). In line with this, we found that IR enhanced the secretion of IL‐6 and IL‐8 (Supplemental Figure 3E). Thus, we show that HMEpCs have at least a p53 proficient DDR, which can result in cell cycle arrest, apoptosis and senescence depending on the type of DNA damage and we have determined doses to be used in our profiling studies.

3.2. Regulation of DDR miRNAs and their differential expression in breast cancer

In order to characterize the miRNA response to DNA damage, HMEpCs were treated with the low and high dose of cisplatin and IR. Based on miRNA kinetics after DNA damage treatment (Pothof et al., 2009; Zhang et al., 2011), we isolated total RNA at 6 h, 12 h, and 24 h after start of treatment for each genotoxic treatment and dose. MiRNA profiling was performed using miRNA arrays containing Locked Nucleic Acid‐based capture probes against 725 human miRNAs. Reproducibility between biological replicates (n = 4) was analyzed before normalization, which demonstrates that the correlation between replicates was higher than between non‐replicates (Supplemental Figure 4). After normalization, we identified condition‐specific regulation of miRNAs dependent on type of genotoxic stress (Supplemental Figure 5A) and dose (Supplemental Figure 5B/C) or miRNAs that exhibited an opposite regulation after IR and cisplatin treatment (Supplemental Figure 5D). As explained, we focused on general miRNA responders to DNA damage, i.e. significantly regulated miRNAs across all genotoxic conditions per time point. We identified several general DDR miRNAs and most were regulated at 6 h and 12 h after treatment (Figure 1), which is in agreement with published miRNA expression kinetics after DNA damage (Pothof et al., 2009; Zhang et al., 2011). In conclusion, we have identified several miRNAs that can be characterized as general responders to genotoxic cancer treatments in HMEpCs.

Figure 1.

Figure 1

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MiRNAs regulated in four DNA damaging conditions. HMEpC cells were treated with the indicated doses of cisplatin (μM) and IR (Gy). Each miRNA was identified by SAM statistics, but significant miRNAs with a fold‐change lower than 1.5 fold were excluded. Shown are standard heatmaps, which depict the average fold change. To calculate the average fold change, we divided the average expression value after damage (n = 4) by the average control value (n = 4) at the same time point. To give the best representation of differences in expression, we chose arbitrary colors to depict fold changes between −1.75 en +1.75. A white color indicates a fold change of 1.00.

We next examined whether these general genotoxic stress‐responsive miRNAs were differentially expressed as part of a defective DDR in breast tumors compared to normal breast tissue. To that end, we used a miRNA expression dataset consisting of 84 breast tumor samples and 8 normal breast tissue samples (Van der Auwera et al., 2010) in which 439 human miRNAs were found expressed by RT‐PCR. Subsequently, we selected only those miRNAs present on both the micro‐array and RT‐PCR platforms and found that 31% of our general responder miRNA set (10 out of 32) was differentially expressed between normal breast tissue and cancer (Figure 2). The fact that almost one third of general DNA damage responsive miRNAs in wild‐type HMEpCs are deregulated in human breast cancer indicates that these tumors have a defect in the DDR.

Figure 2.

Figure 2

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Overlap between general miRNA responders to DNA damage in primary breast epithelial cells and miRNAs deregulated in breast tumors. A total of 412 miRNAs were detected in both datasets, of which 121 miRNAs were differentially expressed in breast tumors compared to healthy tissue and 32 miRNAs were similarly regulated in all DNA damaging conditions in HMEpC. Expression values of the 10 overlapping miRNAs in healthy tissue and breast tumor samples are depicted in the heatmap. Fold changes indicate the difference in expression compared to the average of normal breast tissue samples.

3.3. DNA damage responsive miRNA expression in epithelial small airway progenitor cells and non‐small cell lung cancer

To test whether this approach could also be applied to other epithelial cells, tissues and corresponding tumors, we used primary human small airway epithelial progenitor cells (HSAEpCs), healthy lung tissue and non‐small cell lung cancer (NSCLC) samples. First, we established treatment doses of cisplatin and IR as described for HMEpCs. Since HSAEpCs did not form colonies, we used an adapted clonal survival assay in which we counted the number of surviving cells. Similar to HMEpCs treatment 0.25 μM cisplatin and 1.5 Gy IR resulted in a 50% cell number reduction (Supplemental Figure 2C). These doses were sufficient to induce cell cycle arrest (Supplemental Figure 2C). A high dose of 40 μM cisplatin, which was >2 fold higher than HMEpCs, resulted in 50% reduction in viability as seen in an MTT assay (Supplemental Figure 3A) and was accompanied by p53 activation and apoptosis activation as seen by reduction of PARP1 levels (Supplemental Figure 2A/3B). Together, this indicates that HSAEpCs have a wild‐type p53 response. IR at a dose of 6 Gy completely abolished cell division in the HSAEpC (Supplemental Figure 3D) and induced IL‐6 and IL‐8 secretion indicative of cellular senescence (Supplemental Figure 3E).

Using these established doses, we performed miRNA profiling in HSAEpCs identical as described for HMEpCs. Most miRNAs were regulated 6 h and 12 h after all 4 treatments (Figure 3), which was similar to HMEpCs. We also revealed miRNAs in HSAEpC which were treatment or dose‐specific (Supplemental Figure 5E/F) and were different from miRNAs regulated under similar conditions in HMEpCs. In line with the p53 activation observed upon DNA damage, p53 target miRNA miR‐34b was found upregulated in both HMEpCs and HSAEpCs (Supplemental Figure 6A). We identified several miRNAs that showed similar expression patterns in both HSAEpC and HMEpC after DNA damage (Supplemental Figure 6B), however, most differentially expressed miRNAs characterized as general DNA damage responders were specific for either HSAEpC or HMEpC (Supplemental Figure 6A). These observations indicate that similar types of DNA damage do induce overlapping but also clearly different miRNA expression alterations in distinct epithelial cell types, although the biological outcome is identical, suggesting that cell type or cellular origin is intrinsically a major determinant in the miRNA response to DNA damage.

Figure 3.

Figure 3

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MiRNAs regulated in four DNA damaging conditions. HSAEpC cells were treated with the indicated doses of cisplatin and IR. Each miRNA was identified by SAM statistics, but significant miRNAs with a fold change lower than 1.5 fold were excluded. Shown are standard heatmaps, which depict the average fold change. To calculate the average fold change, we divided the average expression value after damage (n = 4) by the average control value (n = 4) at the same time point. To give the best representation of differences in expression, we selected arbitrary colors to depict fold changes between −1.75 en +1.75. A white color indicates a fold change of 1.00.

To determine whether general DNA damage responsive miRNAs in HSAEpCs were misexpressed in human NSCLC, we used the miRNA expression profiles of 14 normal lung and 18 NSCLC samples and selected those present on both platforms. Next, we searched for miRNAs overlapping between general DNA damage responders in HSAEpCs and the set of 42 miRNAs differentially expressed in NSCLC and identified a 43% overlap (6 out of 14) (Figure 4). These findings, together with the HMEpC data, suggest that deregulation of DDR miRNAs in breast and lung tumors are indicative of DDR defects.

Figure 4.

Figure 4

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Overlap between general miRNA responders to DNA damage in primary lung epithelial cells and miRNAs deregulated in NSCLC. A total of 158 miRNAs were detected in both datasets, of which 42 miRNAs were differentially expressed in lung tumors compared to healthy tissue and 14 miRNAs were similarly regulated in all DNA damaging conditions in HSAEpC. Expression values of the 6 overlapping miRNAs in healthy tissue and lung tumor samples are depicted in the heatmap. Fold changes indicate the difference in expression compared to the average of normal lung tissue samples.

3.4. DNA damage responsive miRNAs alter sensitivity towards DNA damaging treatments

It is conceivable that DDR miRNAs that are deregulated in cancer modulate the response of tumors to genotoxic anti‐cancer therapy. To test this hypothesis, we first correlated the expression levels of our 10 general DNA damage responsive miRNAs that were misexpressed in breast cancer with the response to chemotherapeutics using the data available for the NCI60 panel (Blower et al., 2008), a well‐defined set of cancer cell lines for which the sensitivity for multiple chemotherapeutics is known. We have assessed the response to cisplatin and doxorubicin, which induces double strand DNA breaks, and to paclitaxel as a control to monitor general stress sensitivity. Paclitaxel is not genotoxic (Danesi et al., 2010), but affects microtubule dynamics, thereby inhibiting mitosis, and also stimulates reactive oxygen species production (Alexandre et al., 2007). In this analysis, we found that expression levels of 3 out of 9 (33%) miRNAs that could be analyzed showed a correlation (p < 0.05) with cisplatin resistance. This percentage was higher than all other analyzed breast tumor miRNAs that were not generally regulated after DNA damage (21%) (Supplemental Table 2). Notably 44% of DDR miRNAs correlated with doxorubicin resistance compared to 20% of the non‐DDR tumor miRNAs. Furthermore, we found that 33% of DDR miRNAs associated with paclitaxel resistance versus 10% of non‐DDR miRNAs, indicating that these miRNAs control general stress resistance. In addition, these analyses provide evidence that several DDR miRNAs deregulated in cancer are associated with anti‐cancer therapy sensitivity.

In order to demonstrate that general DDR miRNAs misexpressed in breast cancer affect viability of breast cancer cells upon exposure to chemotherapeutic agents we overexpressed a selection of overlapping miRNAs (miR‐296‐5p, miR‐193a‐3p, miR‐183, let‐7a) in breast cancer cell lines that have a low basal expression of these specific miRNAs (Supplemental Table 3). MiRNA overexpressing cells were treated with an IC50 dose of cisplatin, doxorubicin or paclitaxel based on the sensitivity of mock‐transfected cells (Supplemental Table 4). After 24 h of continuous exposure we measured the viability of exposed cells compared to mock‐treated cells by MTT assay. MiR‐296‐5p and miR‐193a‐3p overexpression consistently induced resistance to cisplatin, whereas miR‐183 overexpression induced sensitivity. MiR‐296‐5p overexpression also led to resistance for doxorubicin and paclitaxel, while let‐7a overexpression did not modulate chemotherapy sensitivity (Figure 5A). Next, we examined whether overexpression of general DDR miRNAs from HSAEpCs deregulated in NSCLC (miR‐16, miR‐21, and miR‐382) in lung cancer cells can modulate chemotherapy sensitivity. MiR‐382 and miR‐21 overexpression had no effect or resulted in resistance, while miR‐16 promoted sensitivity to cisplatin and doxorubicin (Figure 5A). Thus, we demonstrate that several general DDR miRNAs deregulated in human cancer are capable of modulating chemotherapy sensitivity.

Drug tolerant cells (including cisplatin resistant tumor cells) may temporarily acquire a quiescent phenotype and later recover cell proliferation and cause tumor relapse (Sharma et al., 2010). Cells that undergo quiescence in response to genotoxic stress treatment are detected by the MTT assay as viable, but will not (or very slowly) grow out into colonies. Resistance associated with colony outgrowth is a sign of enhanced DNA repair or increased drug clearance. Therefore, we performed clonal survival assays to determine whether resistance in the MTT assay is associated with colony outgrowth. We omitted miR‐193a‐3p in our analysis, because its overexpression already abolished colony formation in non‐genotoxic treated cells. We observed that only miR‐21 overexpression led to increased cisplatin resistance as indicated by increased colony formation. In contrast, miR‐296‐5p and miR‐382 transfected cells, which appear resistant to cisplatin in the MTT assay, were as sensitive as control cells (Figure 5B). In addition, miR‐21 also enhanced resistance to IR. Together, this suggests that miR‐296‐5p and miR‐382 induce drug resistance by undergoing quiescence whereas miR‐21 induces drug resistance via improved repair, drug clearance or enhanced DNA damage tolerance. In line with this, increased miR‐21 levels resulted in a decreased number of 53BP1 foci (Figure 5C), which is a marker for unrepaired double strand DNA breaks (DSBs) (Rodier et al., 2011), suggesting (in combination with the enhanced colony formation) that its mode of action involves improved repair or drug clearance. In toto, our data indicate that DDR miRNAs are partially cell type specific, are frequently found deregulated in human cancer and can modulate sensitivity towards genotoxic therapeutic anti‐cancer treatments.

4. Discussion

In this study, we used primary wild‐type epithelial cells from breast and lung to delineate DDR miRNAs, reasoning that intrinsic DDR defects in cancer cell lines might not give rise to a wild‐type DDR miRNA expression profile, which is required to distinguish deregulated DDR miRNAs in human cancer. We have demonstrated that miRNA expression is altered in primary epithelial cells derived from breast and lung tissue in response to DNA damage in a cell type specific manner. Subsequently, we have shown that these DDR miRNAs are frequently deregulated in breast cancer or NSCLC and can modulate the sensitivity to genotoxic, but also non‐genotoxic, anti‐cancer drugs.

In separate studies, it has been established that miRNAs play an important role in the DDR (Pothof et al., 2009; Wouters et al., 2011) and exposure to DNA damage results in specific miRNA expression changes (Pothof et al., 2009; Simone et al., 2009; Suzuki et al., 2009; Zhang et al., 2011). In addition, most, if not all, human tumors exhibit defects in the DDR (Bartkova et al., 2005; Gorgoulis et al., 2005; Hanahan and Weinberg, 2011) and deregulated miRNA expression (Farazi et al., 2011; Garzon et al., 2009). Finally, multiple reports point towards an important role for miRNAs in chemotherapy sensitivity (Allen and Weiss, 2010; Giovannetti et al., 2012; Haenisch and Cascorbi, 2012; van Jaarsveld et al., 2013; Wiemer, 2011). The careful setup of this study allowed us to combine all these observations to provide evidence for our hypothesis that miRNAs under control by the DDR might be altered in human cancer due to DDR defects, which in turn could modulate chemotherapy sensitivity. Some of these DDR miRNAs, e.g. miR‐16 and miR‐21, were already known to be regulated upon DNA damage and to play a role in therapy resistance (Li et al., 2009; Shi et al., 2010; Xia et al., 2008). Additional miRNAs identified by us have also been associated with the DDR or drug resistance (miR‐183, miR‐193a‐3p) (Dai et al., 2011; Ma et al., 2012), which confirms our approach to identify putative modulators of drug resistance.

MiR‐21 was found overexpressed in our cancer datasets and has been reported to be a frequently upregulated oncogenic miRNA in various solid cancers (Volinia et al., 2006). Of all miRNAs conferring drug resistance, miR‐21 overexpression unexpectedly led to increased colony formation after both IR and cisplatin and to decreased numbers of 53BP1 foci. DSBs as induced by IR or caused by cisplatin‐induced DNA crosslinks regressing into DSBs during DNA replication, need to be repaired to complete S‐phase and continue cell division. Thus a large increase in colony outgrowth as seen when miR‐21 is overexpressed is most logically explained by improved DNA repair, enhanced drug clearance or by increased DNA damage tolerance. The observation that miR‐21 overexpression also increases colony outgrowth after IR, a direct DNA damaging insult that is not inhibited by drug clearance mechanisms, suggests that miR‐21 facilitates DNA damage repair and/or tolerance. MiR‐21‐mediated induction of drug resistance has been shown before and linked to cell survival pathway induction (Giovannetti et al., 2010, 2006, 2007, 2010). We propose that besides activation of described cellular survival pathways, miR‐21 also enhances DNA repair assisting in drug tolerance.

We focused on general miRNA responders upon genotoxic stress, i.e. cisplatin, a DNA crosslinking agent generating DSBs during DNA replication and IR, which also generates DSBs. Therefore, these general miRNA responders could also be regarded DSB responsive miRNAs. Additional comparative studies are required to determine whether these miRNAs are in fact general genotoxic or DSB specific responsive miRNAs. There are several lines of evidence that DSB repair is defective in breast cancer and NSCLC (Ashworth, 2008; Postel‐Vinay et al., 2012; Tseng et al., 2009) and therefore cisplatin and IR are likely well‐chosen genotoxic anti‐cancer treatments for our study. Depending on the specific DDR defect in other tumor categories it can be envisaged however, that different genotoxic agents inducing bulky or alkylated adducts, should be used in an approach as taken here. Systematic miRNA profiling of tumors before, during and after genotoxic chemotherapy or treatment (IR) and of circulating miRNA in the peripheral blood may – in conjunction with objective clinical response data and knowledge of their involvement in therapy resistance – reveal tumor and/or serum biomarkers that predict whether a tumor is responsive for a given anti‐cancer therapy. In addition, the therapeutic targeting of these miRNAs – either by overexpression or inhibition ‐ could sensitize tumors to therapy and enhance the progression free and overall survival.

Surprisingly, we observed that different miRNAs were regulated after identical genotoxic treatment in HMEpC and HSAEpC. While both cell lines were treated at equitoxic doses, which in both cell lines led to identical biological outcomes (repair/recovery; apoptosis/senescence), the miRNA response was markedly different. If we assume that these DDR miRNAs in each of the examined cell lines contribute to the observed cellular responses, it would mean that a distinct set of cell‐type specific miRNAs drives a part of the DDR in each of these cell types. Importantly, our findings suggest that at least part of the DDR is different in the two epithelial cell lines at the level of miRNA expression, although the biological outcome upon similar genotoxic insult is similar. This has implications for the approach taken here. Not only should one use primary wild‐type cells in screens aiming at identifying miRNAs in cancer etiology and behaviour, but also cell types that resemble tumor origin as close as possible. In conclusion, we have shown that DDR miRNAs are frequently deregulated in human cancer and can modulate the cellular response to (genotoxic) anti‐cancer treatments. Moreover, these miRNAs could be promising targets to sensitize tumors to therapy.

Author contributions

M.v.J and M.D.W designed and performed experiments, analyzed all data and wrote manuscript; A.B generated miRNA array data of lung cancer samples; W.v.IJ spotted miRNA arrays; M.S was involved in statistical analyses; R.M, J.H and J.M commented on manuscript; J.M provided breast cancer cell lines; S.v.L provided miRNA RT qPCR data of breast cancer samples; E.W and J.P conceived study, designed and supervised experiments and wrote manuscript. All authors have read and agreed with the manuscript.

Author information

Micro‐array data is available through the public repository database ArrayExpress at accession code: GSE47526. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to J.P (j.pothof@erasmusmc.nl) or E.W (e.wiemer@erasmusmc.nl).

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Acknowledgements

We thank Sabine van Steenbergen‐Langeveld for performing measurements of IL6/IL8 in medium supernatant and Christel Kockx for printing microarray slides and providing technical assistance with the miRNA array hybridization.

Supplementary data 1.

1.1.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2013.12.011

van Jaarsveld Marijn T.M., Wouters Maikel D., Boersma Antonius W.M., Smid Marcel, van IJcken Wilfred F.J., Mathijssen Ron H.J., Hoeijmakers Jan H.J., Martens John W.M., van Laere Steven, Wiemer Erik A.C. and Pothof Joris, (2014), DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity, Molecular Oncology, 8, doi: 10.1016/j.molonc.2013.12.011.

‡

This project was supported by a Dutch Cancer Society grant (EMCR 2007‐3794) and mRACE grant (J.P, E.W, M.v.J and M.D.W), a ZonMW Veni grant (016‐076‐069) (J.P) and grants from DDResponse, the National Institute of Health (NIH)/National Institute of Ageing (NIA) (AG0171242‐10), NIEHS (1UO1 ES011044), the Royal Academy of Arts and Sciences of the Netherlands (academia professorship) and a European Research Council Advanced Grant (DamAge 233424) (J.H). The authors declare no potential conflicts of interest.

Contributor Information

Erik A.C. Wiemer, Email: e.wiemer@erasmusmc.nl

Joris Pothof, Email: j.pothof@erasmusmc.nl.

References

  1. Alexandre, J. , Hu, Y. , Lu, W. , Pelicano, H. , Huang, P. , 2007. Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Res.. 67, 3512–3517. [DOI] [PubMed] [Google Scholar]
  2. Allen, K.E. , Weiss, G.J. , 2010. Resistance may not be futile: microRNA biomarkers for chemoresistance and potential therapeutics. Mol. Cancer Ther.. 9, 3126–3136. [DOI] [PubMed] [Google Scholar]
  3. Ashworth, A. , 2008. A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. J. Clin. Oncol.. 26, 3785–3790. [DOI] [PubMed] [Google Scholar]
  4. Bartek, J. , Lukas, J. , 2007. DNA damage checkpoints: from initiation to recovery or adaptation. Curr. Opin. Cell Biol.. 19, 238–245. [DOI] [PubMed] [Google Scholar]
  5. Bartel, D.P. , 2009. MicroRNAs: target recognition and regulatory functions. Cell. 136, 215–233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bartkova, J. , Horejsi, Z. , Koed, K. , Kramer, A. , Tort, F. , Zieger, K. , Guldberg, P. , Sehested, M. , Nesland, J.M. , Lukas, C. , Orntoft, T. , Lukas, J. , Bartek, J. , 2005. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature. 434, 864–870. [DOI] [PubMed] [Google Scholar]
  7. Blower, P.E. , Chung, J.H. , Verducci, J.S. , Lin, S. , Park, J.K. , Dai, Z. , Liu, C.G. , Schmittgen, T.D. , Reinhold, W.C. , Croce, C.M. , Weinstein, J.N. , Sadee, W. , 2008. MicroRNAs modulate the chemosensitivity of tumor cells. Mol. Cancer Ther.. 7, 1–9. [DOI] [PubMed] [Google Scholar]
  8. Bouwman, P. , Jonkers, J. , 2012. The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat. Rev. Cancer. 12, 587–598. [DOI] [PubMed] [Google Scholar]
  9. Bunnak, J. , Takigami, M. , Ito, H. , Shinozawa, T. , 1994. Gamma irradiation effects on cultured cells: investigated by the MTT method. J. Radiat. Res.. 35, 205–212. [DOI] [PubMed] [Google Scholar]
  10. Carmichael, J. , DeGraff, W.G. , Gazdar, A.F. , Minna, J.D. , Mitchell, J.B. , 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res.. 47, 936–942. [PubMed] [Google Scholar]
  11. Coppe, J.P. , Patil, C.K. , Rodier, F. , Sun, Y. , Munoz, D.P. , Goldstein, J. , Nelson, P.S. , Desprez, P.Y. , Campisi, J. , 2008. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol.. 6, 2853–2868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dai, Y. , Xie, C.H. , Neis, J.P. , Fan, C.Y. , Vural, E. , Spring, P.M. , 2011. MicroRNA expression profiles of head and neck squamous cell carcinoma with docetaxel-induced multidrug resistance. Head Neck. 33, 786–791. [DOI] [PubMed] [Google Scholar]
  13. Danesi, C.C. , Bellagamba, B.C. , Dihl, R.R. , de Andrade, H.H. , Cunha, K.S. , Spano, M.A. , Reguly, M.L. , Lehmann, M. , 2010. Mutagenic evaluation of combined paclitaxel and cisplatin treatment in somatic cells of Drosophila melanogaster . Mutat. Res.. 696, 139–143. [DOI] [PubMed] [Google Scholar]
  14. Dontu, G. , Abdallah, W.M. , Foley, J.M. , Jackson, K.W. , Clarke, M.F. , Kawamura, M.J. , Wicha, M.S. , 2003. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev.. 17, 1253–1270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Farazi, T.A. , Spitzer, J.I. , Morozov, P. , Tuschl, T. , 2011. miRNAs in human cancer. J. Pathol.. 223, 102–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garzon, R. , Calin, G.A. , Croce, C.M. , 2009. MicroRNAs in cancer. Annu. Rev. Med.. 60, 167–179. [DOI] [PubMed] [Google Scholar]
  17. Giovannetti, E. , Erozenci, A. , Smit, J. , Danesi, R. , Peters, G.J. , 2012. Molecular mechanisms underlying the role of microRNAs (miRNAs) in anticancer drug resistance and implications for clinical practice. Crit. Rev. Oncol. Hematol.. 81, 103–122. [DOI] [PubMed] [Google Scholar]
  18. Giovannetti, E. , Funel, N. , Peters, G.J. , Del Chiaro, M. , Erozenci, L.A. , Vasile, E. , Leon, L.G. , Pollina, L.E. , Groen, A. , Falcone, A. , Danesi, R. , Campani, D. , Verheul, H.M. , Boggi, U. , 2010. MicroRNA-21 in pancreatic cancer: correlation with clinical outcome and pharmacologic aspects underlying its role in the modulation of gemcitabine activity. Cancer Res.. 70, 4528–4538. [DOI] [PubMed] [Google Scholar]
  19. Gordon, R.R. , Nelson, P.S. , 2012. Cellular senescence and cancer chemotherapy resistance. Drug Resist. Updat.. 15, 123–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gorgoulis, V.G. , Vassiliou, L.V. , Karakaidos, P. , Zacharatos, P. , Kotsinas, A. , Liloglou, T. , Venere, M. , Ditullio, R.A. , Kastrinakis, N.G. , Levy, B. , Kletsas, D. , Yoneta, A. , Herlyn, M. , Kittas, C. , Halazonetis, T.D. , 2005. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature. 434, 907–913. [DOI] [PubMed] [Google Scholar]
  21. Gottesman, M.M. , 2002. Mechanisms of cancer drug resistance. Annu. Rev. Med.. 53, 615–627. [DOI] [PubMed] [Google Scholar]
  22. Haenisch, S. , Cascorbi, I. , 2012. miRNAs as mediators of drug resistance. Epigenomics. 4, 369–381. [DOI] [PubMed] [Google Scholar]
  23. Hanahan, D. , Weinberg, R.A. , 2011. Hallmarks of cancer: the next generation. Cell. 144, 646–674. [DOI] [PubMed] [Google Scholar]
  24. Hoeijmakers, J.H. , 2009. DNA damage, aging, and cancer. N. Engl. J. Med.. 361, 1475–1485. [DOI] [PubMed] [Google Scholar]
  25. Igney, F.H. , Krammer, P.H. , 2002. Death and anti-death: tumour resistance to apoptosis. Nat. Rev. Cancer. 2, 277–288. [DOI] [PubMed] [Google Scholar]
  26. Li, Y. , Li, W. , Yang, Y. , Lu, Y. , He, C. , Hu, G. , Liu, H. , Chen, J. , He, J. , Yu, H. , 2009. MicroRNA-21 targets LRRFIP1 and contributes to VM-26 resistance in glioblastoma multiforme. Brain Res.. 1286, 13–18. [DOI] [PubMed] [Google Scholar]
  27. Lord, C.J. , Ashworth, A. , 2012. The DNA damage response and cancer therapy. Nature. 481, 287–294. [DOI] [PubMed] [Google Scholar]
  28. Ma, K. , He, Y. , Zhang, H. , Fei, Q. , Niu, D. , Wang, D. , Ding, X. , Xu, H. , Chen, X. , Zhu, J. , 2012. DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression. J. Biol. Chem.. 287, 5639–5649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meng, F. , Henson, R. , Lang, M. , Wehbe, H. , Maheshwari, S. , Mendell, J.T. , Jiang, J. , Schmittgen, T.D. , Patel, T. , 2006. Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology. 130, 2113–2129. [DOI] [PubMed] [Google Scholar]
  30. Meng, F. , Henson, R. , Wehbe-Janek, H. , Ghoshal, K. , Jacob, S.T. , Patel, T. , 2007. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology. 133, 647–658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Postel-Vinay, S. , Vanhecke, E. , Olaussen, K.A. , Lord, C.J. , Ashworth, A. , Soria, J.C. , 2012. The potential of exploiting DNA-repair defects for optimizing lung cancer treatment. Nat. Rev. Clin. Oncol.. 9, 144–155. [DOI] [PubMed] [Google Scholar]
  32. Pothof, J. , Verkaik, N.S. , van, I.W. , Wiemer, E.A. , Ta, V.T. , van der Horst, G.T. , Jaspers, N.G. , van Gent, D.C. , Hoeijmakers, J.H. , Persengiev, S.P. , 2009. MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. Embo J.. 28, 2090–2099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rademakers, S. , Volker, M. , Hoogstraten, D. , Nigg, A.L. , Mone, M.J. , Van Zeeland, A.A. , Hoeijmakers, J.H. , Houtsmuller, A.B. , Vermeulen, W. , 2003. Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions. Mol. Cell Biol.. 23, 5755–5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Riaz, M. , van Jaarsveld, M.T. , Hollestelle, A. , Prager-van der Smissen, W.J. , Heine, A.A. , Boersma, A.W. , Liu, J. , Helmijr, J. , Ozturk, B. , Smid, M. , Wiemer, E.A. , Foekens, J.A. , Martens, J.W. , 2013. MicroRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs. Breast Cancer Res.. 15, R33 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rodier, F. , Munoz, D.P. , Teachenor, R. , Chu, V. , Le, O. , Bhaumik, D. , Coppe, J.P. , Campeau, E. , Beausejour, C.M. , Kim, S.H. , Davalos, A.R. , Campisi, J. , 2011. DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion. J. Cell Sci.. 124, 68–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sabin, R.J. , Anderson, R.M. , 2011. Cellular senescence – its role in cancer and the response to ionizing radiation. Genome Integr.. 2, 7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saeed, A.I. , Sharov, V. , White, J. , Li, J. , Liang, W. , Bhagabati, N. , Braisted, J. , Klapa, M. , Currier, T. , Thiagarajan, M. , Sturn, A. , Snuffin, M. , Rezantsev, A. , Popov, D. , Ryltsov, A. , Kostukovich, E. , Borisovsky, I. , Liu, Z. , Vinsavich, A. , Trush, V. , Quackenbush, J. , 2003. TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 34, 374–378. [DOI] [PubMed] [Google Scholar]
  38. Sharma, S.V. , Lee, D.Y. , Li, B. , Quinlan, M.P. , Takahashi, F. , Maheswaran, S. , McDermott, U. , Azizian, N. , Zou, L. , Fischbach, M.A. , Wong, K.K. , Brandstetter, K. , Wittner, B. , Ramaswamy, S. , Classon, M. , Settleman, J. , 2010. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell. 141, 69–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shi, L. , Chen, J. , Yang, J. , Pan, T. , Zhang, S. , Wang, Z. , 2010. MiR-21 protected human glioblastoma U87MG cells from chemotherapeutic drug temozolomide induced apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 activity. Brain Res.. 1352, 255–264. [DOI] [PubMed] [Google Scholar]
  40. Simone, N.L. , Soule, B.P. , Ly, D. , Saleh, A.D. , Savage, J.E. , Degraff, W. , Cook, J. , Harris, C.C. , Gius, D. , Mitchell, J.B. , 2009. Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS ONE. 4, e6377 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Suzuki, H.I. , Yamagata, K. , Sugimoto, K. , Iwamoto, T. , Kato, S. , Miyazono, K. , 2009. Modulation of microRNA processing by p53. Nature. 460, 529–533. [DOI] [PubMed] [Google Scholar]
  42. Tseng, R.C. , Hsieh, F.J. , Shih, C.M. , Hsu, H.S. , Chen, C.Y. , Wang, Y.C. , 2009. Lung cancer susceptibility and prognosis associated with polymorphisms in the nonhomologous end-joining pathway genes: a multiple genotype-phenotype study. Cancer. 115, 2939–2948. [DOI] [PubMed] [Google Scholar]
  43. Tusher, V.G. , Tibshirani, R. , Chu, G. , 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. U S A. 98, 5116–5121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Van der Auwera, I. , Limame, R. , van Dam, P. , Vermeulen, P.B. , Dirix, L.Y. , Van Laere, S.J. , 2010. Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype. Br. J. Cancer. 103, 532–541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van Jaarsveld, M.T. , Helleman, J. , Boersma, A.W. , van Kuijk, P.F. , van Ijcken, W.F. , Despierre, E. , Vergote, I. , Mathijssen, R.H. , Berns, E.M. , Verweij, J. , Pothof, J. , Wiemer, E.A. , 2013. miR-141 regulates KEAP1 and modulates cisplatin sensitivity in ovarian cancer cells. Oncogene. 32, 4284–4293. [DOI] [PubMed] [Google Scholar]
  46. Volinia, S. , Calin, G.A. , Liu, C.G. , Ambs, S. , Cimmino, A. , Petrocca, F. , Visone, R. , Iorio, M. , Roldo, C. , Ferracin, M. , Prueitt, R.L. , Yanaihara, N. , Lanza, G. , Scarpa, A. , Vecchione, A. , Negrini, M. , Harris, C.C. , Croce, C.M. , 2006. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. U S A. 103, 2257–2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wiemer, E.A.C. , 2011. Role of MicroRNAs in anticancer drug resistance. In Cho W.C.S., MicroRNAs in Cancer Translational Research. Springer Verlag; 449–483. [Google Scholar]
  48. Wouters, M.D. , van Gent, D.C. , Hoeijmakers, J.H. , Pothof, J. , 2011. MicroRNAs, the DNA damage response and cancer. Mutat. Res.. 717, 54–66. [DOI] [PubMed] [Google Scholar]
  49. Xia, L. , Zhang, D. , Du, R. , Pan, Y. , Zhao, L. , Sun, S. , Hong, L. , Liu, J. , Fan, D. , 2008. miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int. J. Cancer. 123, 372–379. [DOI] [PubMed] [Google Scholar]
  50. Zhang, X. , Wan, G. , Berger, F.G. , He, X. , Lu, X. , 2011. The ATM kinase induces microRNA biogenesis in the DNA damage response. Mol. Cell. 41, 371–383. [DOI] [PMC free article] [PubMed] [Google Scholar]

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