original article

© The American Society of Gene & Cell Therapy

Therapeutic Delivery of miR-200c Enhances Radiosensitivity in Lung Cancer Maria Angelica Cortez1, David Valdecanas1, Xiaochun Zhang1, Yanai Zhan2, Vikas Bhardwaj1, George A Calin3, Ritsuko Komaki4, Dipak K Giri5, Caio C Quini6, Tatiana Wolfe1, Heidi J Peltier7, Andreas G Bader7, John V Heymach8, Raymond E Meyn1 and James W Welsh4 1 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; 2Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; 3Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; 4Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; 5Sipaumdi Pathology Consultancy, Pearland, Texas, USA; 6Department of Physics and Biophysics, Sao Paulo State University ­(UNESP), Botucatu, Sao Paulo, Brazil; 7Mirna Therapeutics, Inc., Austin, Texas, USA; 8Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial–mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA doublestrand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future. Received 27 January 2014; accepted 16 April 2014; advance online publication 17 June 2014. doi:10.1038/mt.2014.79

INTRODUCTION Therapeutic resistance is the primary factor that limits the effectiveness of current therapies for solid tumors. Strategies for overcoming this resistance should readily translate into improved outcomes. This concept is particularly relevant for overcoming resistance to ionizing radiation, which is currently the only potentially curative nonsurgical approach for most solid tumors, including nonsmall cell lung cancer (NSCLC). Unfortunately, most patients with NSCLC present with nodal involvement, which precludes the use of tumoricidal radiation doses, which in

turn leads to resistance and recurrent disease. To improve outcomes in such cases, we and others are studying agents that target signaling pathways that mediate treatment resistance (e.g., c-Met and the epidermal growth factor inhibitor).1–5 The limitation of this approach is that targeting a single point in a complex pathway can itself prompt the development of resistance. Therapeutic delivery of synthetic microRNAs (miRNAs) that mimic endogenous tumor suppressor miRNAs has emerged as a promising approach for treating cancer.6–9 MiRNAs target multiple cellular processes and thus in theory can have broader effects beyond current approaches that are limited to targeting single aspects of a cellular pathway. MiRNAs are small, noncoding RNAs of ~22 nucleotides in length that participate in the regulation of most genes in the human genome.10 As a single miRNA can target hundreds of mRNAs and miRNAs are involved in virtually all biologic processes, aberrant miRNA expression is involved in the initiation of many diseases, including cancer. Genome-wide miRNA-expression profiles have shown that specific cancer types, including NSCLC, have unique miRNA signatures.11,12 The ability to inhibit oncogenic miRNAs or replace them with tumor suppressor miRNAs may complement traditional treatments such as chemotherapy and radiation. However, the role of miRNAs in mediating resistance to radiotherapy is poorly understood. Therefore, the ultimate goal of this work is to assess the potential applicability of miRNA delivery, in combination with radiation therapy, to treat NSCLC. Our focus has been on miR-200s, which are often suppressed in lung cancer and are key regulators of the epithelial-to-­ mesenchymal transition,13 a process linked to the development of tumor cell resistance to various types of therapy. The miR-200s consist of five members in two clusters; in humans, miR-200a, miR-200b, and miR-429 are located on chromosome 1, and miR200c and miR-141 are on chromosome 12.14 Interestingly, zinc finger E-box-binding homeobox (ZEB1) transcription factors have been shown to reciprocally repress members of the miR-200 family to control the balance between epithelial and mesenchymal states.15,16 Loss of miR-200s enhances cancer aggressiveness and metastasis, whereas replacement of miR-200s inhibits cell growth

Correspondence: James William Welsh, Department of Radiation Oncology, Unit 97, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030, USA. E-mail: [email protected]

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Therapeutic Delivery of miR-200c in Lung Cancer

RESULTS MiR-200c overexpression sensitizes lung cancer cells to radiation

in several types of tumors, including NSCLC.17 Therapeutic delivery of miR-200 has been noted to markedly reduce metastasis and angiogenesis and to induce vascular normalization in several types of cancer.18 Nonetheless, the potential benefit of systemically delivered miR-200s in combination with radiation remains unclear. As radiation is known to induce cell death by inducing reactive oxygen species (ROS) and p21,19,20 we postulated that miR200c could potentiate the therapeutic effects of radiation. In this report, we show that miR-200c overexpression increased cellular radiosensitivity by directly regulating the oxidative response genes PRDX2, SESN1, and GAPB/Nrf2 so as to inhibit DNA doublestrand breaks (DSBs) repair, increase levels of ROS, and upregulate p21. We also demonstrated the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer by using a xenograft mouse model of lung cancer. Collectively, these results suggest that miR-200c delivery combined with radiation therapy may represent a new therapeutic approach for NSCLC.

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To clarify how miR-200s affect radioresistence in NSCLC, we transiently transfected A549 cells with miR-200a, miR-200b, miR-200c, miR-141, or scrambled control miRNA mimics and analyzed clonogenic survival. We also tested miR-205, which shares targets with the miR-200 family. Cells were plated 48 hours after transfection and treated with 2, 4, or 6  Gy of radiation. Transfection of A549 cells with miR-200a, miR-200b, or miR-205 did not affect clonogenic survival (Figure 1a–c). However, A549 cells transfected with miR141 or miR-200c were significantly more sensitive to the cytotoxic effects of radiation than were cells transfected with a scrambled control (Figure 1d,e). The sensitizing enhancement ratio for miR-200c was 1.72 and for miR-141 was 1.33 in A549 cells. We focused our subsequent studies on miR-200c, which had the most potent effect on cell survival after irradiation compared with other miR-200s.

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Figure 1 MiR-200c overexpression enhances reproductive cell death after irradiation. (a–e) A549 cells were transiently transfected with m ­ iR-200a, -b, or -c, miR-141, or miR-205. Cells transfected with miR-200c and miR-141 were more sensitive to the cytotoxic effects of radiation (2, 4, or 6 Gy) than were cells transfected with a scrambled control. (f-h) A549, H460, and H1299 cells stably overexpressing miR-200c were more sensitive to the cytotoxic effects of radiation (2, 4, or 6 Gy) than were A549, H460 and H1299 cells stably expressing a scrambled control sequence. (i) Histone H2AX phosphorylation on serine-139 (y-H2AX) was up-regulated in A549 cells overexpressing miR-200c at 30 minutes and at 4, 16, and 24 hours after exposure to 4 Gy. Representative figure of y-H2AX detection at 24 hours after irradiation of 4 Gy. (j) y-H2AX was up-regulated in A549 cells overexpressing miR-200c at 4 and 24 hours after exposure to 4 Gy. MiR-200c overexpression led to downregulation of RAD51 at 4 and 24 hours after cells were irradiated at 4 Gy. A549 cell lysates were collected and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis for western blotting. Actin was used as an internal control.

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ZEB1 knockdown recapitulated miR-200coverexpression-induced responses to radiation treatment On the basis of studies establishing that ZEB1 negatively regulates the expression of miR-200s, including miR-200c,21 we next examined the effect of ZEB1 knockdown on the resistance and sensitivity to radiation in NSCLC cell lines. We chose three NSCLC cell lines with high ZEB1 expression (H460, H1299, and Calu-6) and used small hairpin RNA to establish three matched pairs of stable cell lines with and without ZEB1 knockdown (H460/shZEB1 and H460shCV (control vector), H1299/ shZEB1 and H1299/shCV, Calu6/shZEB1 and Calu6/shCV) (Figure 2a–c). Morphologic changes associated with the epithelial-to-mesenchymal transition phenotype were assessed with phase-contrast microscopy, and ZEB1 expression was assessed by western blotting. We tested radiosensitivity in terms of clonogenic survival by plating these stable cell lines and treating them with radiation at 2, 4, or 6  Gy. All three cell lines with stable ZEB1 knockdown were more sensitive  to the cytotoxic effects of radiation than were controls (Figure 2d–f). Sensitizing enhancement ratio values were 1.3, 1.2, and 1.2 for H460, H1299 and Calu6 ZEB1 knockdown, respectively. Next, in investigating

To confirm that miR-200c overexpression sensitized NSCLC cells to radiation, we evaluated the radiosensitivity of A549, H460, and H1299 cells made to stably overexpress miR-200c (confirmed by fluorescence-activated cell sorting and quantitative polymerase chain reaction (qPCR)) (Supplementary Figure S1) by using clonogenic survival analysis. Similar to our findings with transiently transfected cells, cells stably overexpressing miR-200c were significantly more sensitive to the cytotoxic effects of radiation than were controls (Figure 1f–h). Sensitizing enhancement ratio values for A549, H460, and H1299 cells stably overexpressing miR-200c were 1.49, 1.24, and 1.26, respectively. Given our findings that miR-200c modulated the radiosensitivity of NSCLC cells, we then investigated the effect of miR-200c on radiation-induced DNA DSBs and the kinetics of DNA repair. We detected γ-H2AX foci (indicative of DSBs) in A549 cells overexpressing miR-200c at 30 minutes and at 4, 16, and 24 hours after exposure to 4 Gy of radiation; we further found that the number of foci in those cells was five times higher than that in cells with scrambled control (Figure 1i). We also analyzed γ-H2AX and RAD51 protein levels and found that overexpression of miR200c led to upregulation of γ-H2AX and downregulation of RAD51, a protein involved in homologous recombination and DNA repair (Figure 1j).

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Figure 2 ZEB1 knockdown recapitulates miR-200c overexpression-induced responses to radiation treatment. (a-c) Specific shRNAs were used to establish three matched pairs of stable cell lines with and without ZEB1 knockdown (H460/shZEB1 and H460shCV, H1299/shZEB1 and H1299/ shCV, and Calu6/shZEB1 and Calu6/shCV). Morphologic changes associated with the epithelial-to-mesenchymal transition phenotype and ZEB1 expressions were assessed by phase contrast microscopy and western blotting, respectively. (d-f) ZEB1 knockdown cells were more sensitive to the cytotoxic effects of radiation (2, 4, or 6 Gy) than were cells stably expressing a scrambled control sequence. (g-i) Histone H2AX phosphorylation on serine 139 (γ-H2AX) was detected in ZEB1 knockdown cells at 30 minutes and at 2 and 4 hours after exposure to 4 Gy.

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levels. We analyzed ROS levels in A549 and H460 cells stably overexpressing miR-200c by using CellROX Deep Red Reagent (Life Technologies, Carlsbad, CA) and found higher ROS levels in those cells compared with scrambled-miRNA controls (Figure 3a,b). We next tested ROS levels in A549 and H460 cells stably overexpressing miR-200c after treatment with H2O2 (500  μmol/l) and irradiation (6  Gy). We found that the combination of miR-200c overexpression plus H2O2 produced higher ROS levels compared with scrambled-miRNA controls (Figure 3c,d). Similarly, treating A549 and H460 cells overexpressing miR-200c when analyzed 8  hours after irradiation also augmented intracellular ROS levels relative to cells with scrambled miRNA (Figure 3e,f). ROS levels return to control levels after 24 or 48 hours either after irradiation or treatment with H2O2 (data not shown). Next, because ZEB1 negatively

how ZEB1 knockdown affected radiation-induced DNA DSBs and the kinetics of DNA repair, we measured γ-H2AX foci in ZEB1 knockdown cell lines at 30 minutes and at 2 and 4 hours after exposure to 4 Gy of radiation and found that the numbers of foci in the knockdown cells were higher than in cells with control vector (Figures 2g–i). We also measured γ-H2AX foci in ZEB1 knockdown cell lines at 24 hours after exposure to 4 Gy of radiation and found no significant differences between ZEB1 knockdown cell lines compared to control (data not shown).

MiR-200c overexpression augments intracellular ROS levels and increases p21 expression After finding that miR-200c overexpression may enhance radiation-induced DSBs, we next tested the effect of miR-200c on the oxidative stress response by analyzing intracellular ROS

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Figure 3 MiR-200c overexpression augments intracellular ROS levels and increases p21 expression. (a,b) A549 and H460 cells overexpressing miR-200c had higher ROS levels than cells with a scrambled control. (c,d) miR-200c overexpression plus H2O2 (500 μmol/l) produced higher ROS levels than did miR-200c overexpression alone in A549 and H460 cells. (e,f) Treatment with radiation (XRT; 6 Gy) for 8 hours increased intracellular ROS levels in A549 and H460 overexpressing miR-200c relative to scrambled controls. (g) Western blotting showed that p21 levels were higher in A549 and H460 cells overexpressing miR-200c than scrambled controls. Vinculin was used as an internal control. (i) MiR-200c overexpression induces senescence in A549 cells. Senescence-associated beta-galactosidase (SA-B-gal) activity was significantly higher in cells overexpressing miR-200c than in control cells. Arrows indicate senescent cells. (h,j) miR-200c overexpression and ZEB1 knockdown significantly reduced proliferation in non-small cell lung cancer cell lines.

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MiR-200c directly regulates the oxidative stress– responsive genes PRDX2, GABP/Nrf2, and SESN1 Because miR-200c overexpression increased intracellular ROS levels, we next aimed to identify oxidative stress–responsive genes that are potentially regulated by this miRNA. We searched for potential targets by using 10 different programs and found that the 3′ untranslated regions (UTRs) of human PRDX2 contains evolutionarily conserved binding sites specific for miR200c. Previous studies have implicated PRDX2 in antioxidant defense.24–26 Therefore, we analyzed PRDX2 expression in A549 and H460 cells transfected with miR-200c by using qPCR and western blotting. Enforced overexpression of miR-200c suppressed the expression of PRDX2 mRNA and protein compared with a scrambled control (Figure 4a,b). To determine whether miR-200c interacts directly with the putative target gene PRDX2, we co-transfected 3′ UTR luciferase reporter vectors with miR200c precursors and used luciferase assays to confirm that miR200c regulates PRDX2 expression. Specifically, luciferase activity

regulates miR-200c, we measured ROS levels with a dichlorodihydrofluorescein diacetate (DCFDA) assay in ZEB1 knockdown H460, Calu-6, and H1299 stable cells at 24  hours after irradiation. In agreement with our previous results, ZEB1 knockdown increased intracellular ROS levels in all three cell lines compared with cells stably transfected with a control vector (Supplementary Figure S2). Next, because upregulation of p21 is associated with increased ROS levels,22 we tested if miR200c overexpression would increase p21 expression by using western blotting. As was true in other in vitro models,23 miR200c overexpression led to increased p21 expression in NSCLC cells (Figure 3g). Because p21 overexpression decreases proliferation and induces senescence, we next evaluated the effect of miR-200c overexpression on proliferation and senescence. As expected, miR-200c overexpression significantly reduced proliferation and increased senescence of NSCLC cells (Figure 3h,i). In agreement with these results, ZEB1 knockdown significantly reduced the proliferation of H1299 cells (Figure 3j).

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Figure 4 MiR-200c radiosensitizes lung cancer cells to radiation via PRDX2 regulation. (a,b) Overexpression of miR-200c in A549 and H460 cells decreased the expression of PRDX2 mRNA and protein levels compared with scrambled control. (c) Luciferase activity was reduced in cells transfected with miR-200c, PRDX2 3′ untranslated region constructs compared with a scrambled construct. Mutation of the miR-200c interaction sites rescued the luciferase activity, thus confirming that miR-200c directly interacts with the PRDX2 3′ untranslated regions s. (d) PRDX2 knockdown alone or in combination with H2O2 (500 μmol/l) or irradiation (XRT; 6 Gy) produced significantly higher ROS levels compared with negative controls. (e) Knockdown of PRDX2 significantly decreased proliferation rates in cells treated with H2O2 (500 μmol/l) or irradiation (XRT; 6 Gy). (f) PRDX2 silencing promoted p21 upregulation in H460 cells. (g) PRDX2 knockdown sensitized A549 and H460 cells to irradiation.

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was reduced in cells transfected with miR-200c and PRDX2 3′ UTR constructs compared with scrambled controls. Mutation of miR-200c interaction sites rescued the luciferase activity, thus confirming that miR-200c directly interacts with the PRDX2 3′ UTRs (Figure 4c). To confirm that miR-200c’s effect on ROS levels was due to PRDX2 downmodulation, we knocked down PRDX2 expression by using specific siRNAs and tested ROS levels with a DCFDA assay after treatment with H2O2 (500 μmol/l) or irradiation (6 Gy). We found that PRDX2 knockdown alone or H2O2 or radiation produced significantly higher ROS levels compared with a negative control (Figure 4d). We next tested the effect of PRDX2 knockdown on proliferation of H460 cells treated with H2O2 or radiation. Knockdown of PRDX2 significantly decreased proliferation rates in cells treated with H2O2 or radiation (Figure 4e). Because miR-200c overexpression increased both oxidative stress and p21 levels, we then tested the effect of PRDX2 knockdown on

p21 expression levels. We found that PRDX2 silencing promoted p21 upregulation in H460 cells (Figure 4f). Next, to confirm that miR-200c sensitizes NSCLC cells by downregulating PRDX2, we silenced this protein by using specific siRNA and analyzed cell survival after irradiation. We found that PRDX2 knockdown sensitized A549 and H460 cells to radiation treatment (Figure 4g). Sensitizing enhancement ratio values for A549 and H460 transfected with PRDX2 knockdown were 1.2 and 1.4, respectively. These findings suggest that miR-200c increased ROS and p21 levels via PRDX2 downregulation. We also validated two other oxidative response genes, GABPA/Nrf2 and SESN1, as being direct targets of miR-200c (Supplementary Figure S3a–e). In addition, knockdown of GABP/Nrf2 and SESN1 sensitized lung cancer cells to radiation (Supplementary Figure S3c,f). Sensitizing enhancement ratio values for A549 with GABP/Nrf2 and SESN1 knockdown were 1.4 and 1.3, respectively.

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Figure 5 Therapeutic delivery of miR-200c enhances radiosensitivity in a mouse model of lung cancer. (a) Systemic delivery of miR-200c-loaded liposomes (NOV340/miR-200c) leads to accumulation of miR-200c mimics in tumor and other tissues after subcutaneous injection in mice. Two mice per treatment group were sacrificed 24 hours after the third dose of the miRNA, and the tissues and transplanted tumors were harvested for total RNA extraction and evaluation of miR-200c levels. MiR-200c levels were higher in the NOV340/miR-200c-only and NOV340/miR-200c plus radiation (XRT) groups in tumor, liver, brain, and lung, and slightly higher in the whole blood in all three treatment groups compared with control. (b) Therapeutic delivery of miR-200c significantly sensitized lung cancer cells to radiation (XRT). The effect of NOV340/miR-200c on radioresponse of H1299 cells was measured by a tumor growth delay assay. The normalized tumor growth delay was 28.3 ± 6.6 days, and the enhancement factor was 1.5. (c) Illustration of our working hypothesis that miR-200c promotes radiosensitivity by downmodulating oxidative response genes increasing intracellular ROS levels and DNA damage, and decreasing proliferation via p21 upregulation. Table 1 NOV340/miR-200c and radioresponsiveness of H1299 cells implanted in mice Treatment conditiona

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28.3 ± 6.6

Six mice per treatment group. bTime (in days) required for tumors to grow from 8 to 12 mm minus time (in days) for tumors to grow from 8 to 12 mm in the untreated control. cTime (in days) required for tumors to grow from 8 to 12 mm in the combination treatment group minus time (in days) for tumors to grow from 8 to 12 mm in the NOV340/miR-200c-only control. dNormalized growth delay of combination treatment group divided by the absolute growth delay of the radiation-only control.

a

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Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer To examine the effect of miR-200c on radiosensitivity in vivo, we administered a liposomal nanoparticle loaded with miR-200c mimics, NOV340/miR-200c (Mirna Therapeutics, Austin, TX) in combination with fractionated irradiation to xenograft tumors in mice. Intramuscular (i.m.) tumors were created by inoculating 1 × 106 H1299 cells into the right leg of each mouse. When the tumors reached 8 mm in diameter, the mice were randomly assigned to one of four groups: control; NOV340/miR-200c-only; radiation; and NOV340/miR-200c plus radiation. The formulation was given as subcutaneous peritumoral injections at a dose of 5 mg/kg, and local irradiation was given to a total dose of 20 Gy, in 4-Gy fractions given over 5  days starting when tumors were 8  mm. For the combination therapy condition, NOV340/miR200c was given 1 hour before the radiation. We first investigated the accumulation of miR-200c mimics in tumor and other tissues after subcutaneous injection as follows. At 24 hours after the third dose of the miRNA, two mice per group were killed, and tissues were harvested for total RNA extraction and subsequent evaluation of miR-200c levels. Increased miR-200c levels were detected in the groups given NOV340/miR-200c or NOV340/miR-200c plus radiation in tumor, liver, brain, and lung (Figure 5a). A slight increase in miR-200c accumulation was noted in the whole blood in the NOV340/miR-200c-only, radiation-only, and NOV340/ miR-200c plus radiation groups compared with the control. Next, we analyzed the effect of the liposomal miR-200c formulation on radioresponse of implanted H1299 cells measured by tumor growth delay. For those experiments, the mice were killed when tumors reach 14–15  mm in diameter. Our in vivo results validated our previous in vitro observations in that miR-200c significantly (P  =  0.0026) sensitized lung cancer cells to radiation (Figure 5b). The combination of NOV340/miR-200c and radiation delayed tumor growth to a mean (±SE) of 41.9  ±  6.6  days compared with the control (11.1 ± 1.5 days), NOV340/miR-200conly (13.6 ± 1.6 days), or radiation-only (30.0 ± 3.0 days) groups. The normalized tumor growth delay was 28.3 ± 6.6 days, and the enhancement factor was 1.5 (Table 1).

DISCUSSION Our key findings in this study, designed to test a novel approach to rendering treatment-resistant lung cancer sensitive to therapy again, are as follows. First, both transient and stable transfection of lung cancer cell lines with miR-200c constructs radiosensitized those cell lines. These findings agree with those of others showing that miR-200c overexpression sensitized breast and lung cancer cells to radiation.27,28 Next, we found that miR-200c replacement led to upregulation of γ-H2AX and downregulation of RAD51, indicating that miR-200c overexpression suppressed DNA repair, after irradiation. The finding that miR-200c iinhibits DSBs repair in combination with radiation was shown by others in a study of breast cancer.28 Given evidence that ZEB1 negatively regulates miR-200c,21 we knocked down ZEB1 in three lung cancer cell lines and confirmed that ZEB1 knockdown enhanced radiationinduced cytotoxicity and also inhibited DNA repair. Next, as radiation-induced cytotoxicity is mediated primarily by the generation of ROS and ROS-driven oxidative stress, we investigated 1500

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the effect of miR-200c on the oxidative stress response and found that overexpression of miR-200c led to higher ROS levels and increased p21 expression in response to treatment with H2O2, radiation, or both. In line with our findings, Mateescu et al29 demonstrated that miR-141 and miR-200a target p38α to modulate the oxidative stress response in ovarian cancer and that the expression of miR-200s could be induced by ROS. Magenta et al23 also found that miR-200c overexpression induced cell growth arrest, apoptosis, and senescence through ZEB1 downmodulation; these phenomena were also induced by H2O2 and were partially rescued by miR-200c inhibition. Our findings complement these previous reports by demonstrating that miR-200c could not only promote cell growth arrest and apoptosis through ZEB1 but also through increasing intracellular ROS levels. Next, we sought genes that are involved in the oxidative stress response and have conserved binding sites in their 3′ UTRs for miR-200c and identified three such genes: PRDX2, GABPA/Nrf2, and SESN1. PRDX2, an efficient redox protein that neutralizes H2O2, has a critical role in antioxidant defense in cancer cells.24–26,30 PRDX2 expression in breast cancer cells facilitates the metastasis of those cells to the lung, suggesting that PRDX2 may be useful for stratifying patients with a new diagnosis of breast cancer for specific therapy based on ROS-inducing drugs.31 PRDX2 also has an important role in angiogenesis in that it protects the vascular endothelial growth factor receptor (VEGFR)-2 from oxidation.32 We further found that miR-200c overexpression decreased PRDX2 mRNA and protein levels, and miR-200c inhibition increased PRDX2 expression. We further confirmed that the effect of miR-200c on ROS levels, p21 expression, and radiosensitivity in these cell lines was due to PRDX2 regulation. In line with these findings, others have shown that silencing PRDX2 expression sensitized glioma cells to radiation.33 We also confirmed that two other oxidative response genes, SENS1 and GABPA/Nrf2, are direct targets of miR-200c. SESN1 is a stress-inducible protein that negatively regulates ROS levels in several cancer cell lines under oxidative stress.25 SESN1, along with sulfiredoxins, participates in regeneration of overoxidized forms of peroxiredoxins, including PRDX2, and thus regulates the redox balance in cells.24,25 Interestingly, others have shown that silencing SESN1 in human fibroblasts inhibited cell proliferation and accelerated cell senescence triggered by ROS accumulation,25 and cells overexpressing SESN1 were more resistant to oxidative stress and hypoxia.25,34 GABP/Nrf2 regulates PRDX5, a mitochondrial PRDX that regulates ROS produced by mitochondria.35,36 GABP/Nrf2 also regulates Yes-associated protein (YAP), which regulates several genes involved in antioxidant functions such as SOD2, SOD3, PRDX1, and NQO1.37 Interestingly, p21 expression has been shown to be increased in Gabpa−/− mouse embryonic fibroblasts,38 suggesting another potential mechanism by which miR-200c could increase p21 expression. We further found that knockdown of GABP/Nrf2 and SESN1 sensitized lung cancer cells to radiation, implicating these three genes in the ability of miR-200c to sensitize lung cancer cells to radiation. Others have found that miR-200c can sensitize cells to radiation via regulation of TANK-binding kinase 1 (TBK1)28 and, surprisingly, the VEGF-VEGFR2 pathway.27 Finally, we confirmed that miR-200c enhanced radiosensitivity in an in vivo model of lung cancer, as demonstrated by assays www.moleculartherapy.org  vol. 22 no. 8 aug. 2014

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of tumor growth delay in mice implanted with H1299 lung cancer cells. These findings are in agreement with our in vitro results demonstrating that miR-200c overexpression significantly sensitize lung cancer cells to radiation and is consistent with our hypothesis that miR-200c promotes radiosensitivity by downmodulating oxidative response genes and inhibiting DNA repair (Figure 5c). In conclusion, we demonstrated that miR-200c increases radiosensitivity in lung cancer cells by regulating the oxidative stress response via direct regulation of PRDX2, GAPB/Nrf2, and SESN1 and by inhibiting repair of radiation-induced DSBs. Our in vivo experiments support the therapeutic potential of systemic delivery of miR-200c in combination with therapies known to increase oxidative stress, such as radiation.

MATERIALS AND METHODS Cell lines and irradiation. The established lung cancer cell lines A549, H460, H1299, Calu6, and HCC1833 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. Cells were irradiated at room temperature with a Mark I 137Cs irradiator (JL Shepherd & Associates, San Fernando, CA) at a dose rate of 3.5 Gy/minute. Establishment of stable miR-200c–expressing and stable ZEB1 knockdown cells. Vectors for the premicroRNA expression constructs Lenti-miR-200c

(PMIRH200cPA-1) and pCDH-CMV-MCS-EF1-copGFP (from System Biosciences, Mountain View, CA) and pRS-shZEB1 and pRS-control (from OriGene, Rockville, MD) were transiently transfected with a pPACKH1 HIV Lentivector Packaging Kit (System Biosciences) into 293TN cells by using Lipofectamine 2000 and Plus reagents (both from Life Technologies). Scrambled pCDH-CMV-MCS-EF1-copGFP and pRS-control were used as controls. Viral supernatant was collected 3  days after transfection and mixed with PEG-it Virus Precipitation Solution (System Biosciences) overnight at 4 °C. NSCLC cells were infected and incubated with the viral particles supplemented with transdux (System Biosciences) overnight at 37 °C. Fluorescence-activated cell sorting was used to sort populations expressing green fluorescence protein (GFP) and evaluate infection efficiency (as the percentage of copGFP-positive cells, which was >85%). Successful establishment of NSCLC overexpressing miR-200c cells was verified by qPCR (Supplementary Figure S1). Successful establishment of ZEB1 knockdown cells was verified by western blotting (Figure 2a–c).

Clonogenic survival assay. NSCLC cells transfected with miR-200c, siPRDX2, siGABP/Nrf2, siSESN1, and their respective controls were seeded in triplicate in 60-mm dishes and allowed to stabilize overnight. The next day, the cells were irradiated to a dose of 0, 2, 4, or 6 Gy, counted, seeded in 60-mm dishes, and incubated for 12 days to allow macroscopic colony formation. Colonies were fixed and stained for 5 minutes with 0.5% crystal violet (Sigma-Aldrich, St Louis, MO) in methanol. The number of colonies formed in each treatment group was counted, with a cutoff of 50 viable cells per colony. Survival was calculated relative to that of unirradiated cells (survival = (plating efficiency of treated cells)/(plating efficiency of control cells) where plating efficiency = (number of colonies formed by treated cells)/(number of colonies formed by untreated cells)). Detection of γ-H2AX. For this assay, 200,000 cells were plated on cover-

slips, placed in 35-mm dishes, and allowed to attach overnight. Cells were then irradiated (4 Gy), incubated for 4–24 hours, and then fixed with 1% paraformaldehyde for 10 minutes, followed by 70% ethanol for 10 minutes at room temperature. The cells were then treated with 0.1% NP40 in phosphate-buffered saline (PBS) for 20 minutes, washed in PBS four times, and then blocked with 5% bovine serum albumin in PBS for 30 minutes. The cells were then incubated with anti-γ-H2AX antibody (Millipore, Billerica,

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MA) in 5% bovine serum albumin in PBS overnight. The next day, the cells were incubated with fluorescein-isothiocyanate-labeled secondary antibody at a dilution of 1:300 in 5% bovine serum albumin in PBS for 30 minutes. Cells then were incubated in the dark with 4 4′,6-diamidino2-phenylindole dihydrochloride (1 mg/ml) in PBS for 5  minutes, and coverslips were mounted on a slide with an antifade solution (Molecular Probes; Invitrogen, Carlsbad, CA). Slides were examined with a fluorescence microscope (Leica, Buffalo Grove, IL), and images were captured by a charge-coupled device camera and imported into the Advanced Spot Image analysis software package. For each treatment condition, the numbers of γ-H2AX foci were determined in at least 50 cells. Identifying potential microRNA targets. Potential miR-200c targets were

identified by using the target prediction databases miRNA body map (http://www.mirnabodymap.org/; Ghent University, Belgium)39 and miRwalk.40 These databases compare predicted targets from mirBase release 14, TargetScan 5.1, miRDB 3.0, MicroCosm v5, DIANA 3.0, TarBase v.5c, PITA catalog v6, RNA22 (August 2007), and miRecords v2. Transfection. Pre-microRNAs miR-200c, siRNA against PRDX2, GABP/ Nrf2, and SESN1, and their respective negative controls (scrambled oligos) (Life Technologies) were reverse-transfected into lung cancer cell lines with Lipofectamine 2000 (Life Technologies) at a final concentration of 100 nmol/l. Quantitative polymerase chain reaction. Total RNA was isolated from

cells with Triazol (Life Technologies) for miRNA analysis according to the manufacturer’s protocol. To analyze expression of mature microRNA, total RNA was retrotranscribed with miRNA-specific primers with a TaqMan MicroRNA Reverse Transcription kit (Life Technologies), followed by qPCR with Taqman MicroRNA assays according to the manufacturer’s protocol. For studies of PRDX2, GABP/Nrf2 and SESN1 expression, mRNA was retrotranscribed by using a Superscript III kit (Life Technologies) and analyzed by qPCR using SYBR Green (Life Technologies) with specific primers (Supplementary Table S1) according to the manufacturer’s protocol. The comparative Ct method was used to calculate the relative abundance of miRNA and mRNAs compared with U6 and B2M expression, respectively.

Protein extraction and western blot analysis. Total protein was extracted

by using NP40 lysis buffer (0.5% NP40, 250  mmol/l NaCl, 50  mmol/l HEPES, 5 mmol/l ethylenediaminetetraacetic acid, and 0.5 mmol/l egtazic acid) supplemented with protease inhibitors cocktails (Sigma-Aldrich). Lysates were centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected for experiments. Protein lysates (40 μg) were resolved on denaturing gels with 4–20% sodium dodecyl sulfate-polyacrylamide and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with the following antibodies: primary antibodies, anti-PRDX2 (Lab Frontier, Seoul, Korea), antiGABP/Nrf2 (Pierce-Thermo Fisher Scientific, Rockford, IL), anti-SESN1 (Novus Biologicals, Littleton, CO), anti-ZEB1, anti-p21, anti-RAD51 (Cell Signaling Technologies, Beverly, MA), anti-H2AX (Millipore), anti-actin, anti-vinculin (Sigma), and secondary antibody labeled by horseradish peroxidase (Amersham GE Healthcare). The secondary antibody was visualized by using a chemiluminescent reagent Pierce ECL kit (Thermo Fisher Scientific, Waltham, MA).

Vector construction. The 3′ UTR wild-type vectors for PRDX2 (gene ID: 7001), GABP/Nrf2 (gene ID: 2551), and SESN1 (gene ID: 27244) were purchased from SwitchGear Genomics (Carlsbad, CA). These vectors were used to generate mutant vectors (6-bp deletion) by using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using the primers shown in Supplementary Table S2. Luciferase assay. A549 cells were plated in 96-well dishes at 4 × 104 cells/

well. Cells were transfected with miR-200c or scrambled constructs

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(100  nmol/l) with PRDX2-3′ UTR, GABP/Nfr2-3′ UTR, or SESN1-3′ UTR constructs (wt or mut); at 48 hours after transfection, cells were incubated for 15 minutes with 20 µl/well of 1× Passive Lysis Buffer (Promega, Madison, WI). Firefly and renilla luciferase activities were measured sequentially by using dual-luciferase assays (Promega) with a Fluostar Optima plate reader (BMG Lab Technologies GmbH, Durham, NC). Three independent experiments were performed, and values are shown as mean ± standard error of the mean (SEM). Intracellular ROS assay. ROS levels were determined with CellROX Deep

Red Reagent (Life Technologies) and 2′,7′-dichlorofluorescein diacetate (DCFDA)-Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, Cambridge, MA) according to the manufacturer’s recommendations. For cells that are GFP-positive (green), such as A549 and H460 miR200c-overexpressing lines, we used CellROX Deep Red Reagent to measure ROS. In other models that do not express GFP, we used the DCFDA assay. Fluorescence intensity was detected at different intervals by using a FLUOstar Optima fluorescence plate reader (BMG Lab Technologies) with excitation/emission of 640/665 nm (Deep Red) or 485/530 nm (DCFDA). Cells were treated with H2O2 for 6 hours or radiation for 8 hours before ROS assay. Three independent experiments were performed. Values are shown as mean ± SEM. Senescence assay. Senescence was quantified by the expression of betagalactosidase (SA-beta-Gal) activity according to the manufacturer’s recommendations (Abcam). Cells overexpressing miR-200c or scrambled sequences were plated at 500,000 cells/well in 6-well plates for 72  hours before staining. Representative images (100×) were taken from diverse areas of cell culture by phase-contrast microscopy to assess the number of positive cells. Proliferation assay. Numbers of viable cells were analyzed based on ­ uantification of ATP by the CellTiter-Glo Assay (Promega) according q to the manufacturer’s recommendations and read with a Fluostar Optima plate reader (BMG Lab Technologies). Three independent experiments were performed. Values are shown as mean ± SEM. In vivo tumor model and administration of the miR-200c/liposome complexes NOV340/miR-200c. The mice used in this study were bred

and maintained in our own specific pathogen-free mouse colony. Male nude (nu/nu) mice were used for H1299 xenograft studies. They were housed five per cage in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the US Department of Agriculture and Department of Health and Human Services. The human H1299 tumor cell line (CRL-5803; ATCC) was originally derived from a human lung carcinoma. Before tumor cell injection, tumor cell suspensions were prepared from cells grown in monolayers in vitro. H1299 tumors were generated by intramuscular injection of 1 × 106 cells in a volume of 20 µl into the right hind leg of 40 male nude Nu/Nu SPF mice aged 3–4 months. When tumors grew to 8 mm (range 7.8–8.3 mm) in diameter, mice were assigned to the following groups (10 mice each): Control (no treatment), NOV340/ miR-200c (five doses of 5 mg/kg each given SQ on Monday, Wednesday, and Friday, radiation-only (4 Gy once per day for 5 days, total 20 Gy), or NOV340/-miR-200c (five 5 mg/kg doses as described above) + radiation. The miRNA was given 1–2 hours before radiation. NOV340/miR-200c is a liposomal nanoparticle that contains a synthetic, double-stranded mimic of the tumor suppressor miRNA miR-200c (Mirna Therapeutics).41 The liposome is composed of amphoteric lipids that change their net charge depending on the pH of the environment, such that is becomes cationic at low pH and anionic at higher pH.42 At neutral pH 7.4, NOV340/miR200c forms a particle with an average diameter of 120 nm and an average ­zeta-potential of −18  mV. Mice were irradiated with a 137Cesium device (dose rate 4 Gy/minute) as follows. Mice were immobilized in a jig, and

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tumors were centered in a 3-cm-diameter circular field. All mice were checked 2–3 times a week after irradiation to measure tumor diameter. For the growth-control assays, mice were killed when tumors reached 14–15 mm in diameter. Tumor growth delay was defined as the time for tumors to grow from 8 to 12 mm in diameter for treated mice as compared to tumor growth in untreated mice. Biodistribution of systemically delivered miR-200c mimics. At 24 hours after the third SQ dose of the microRNA, two mice per group were killed and the tumor, liver, brain, lung, and whole blood were harvested for total RNA extraction and subsequent evaluation of miR-200c tissue levels. To detect miRNAs, total RNA from flash-frozen mouse tissues (tumor, liver, lung, and brain) and mouse whole blood was isolated using the mirVANA PARIS RNA isolation kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Either 10 ng of total RNA per tissue or a 1:10 dilution of whole blood total RNA eluent was converted to cDNA by using MMLV-RT (Invitrogen) incubated as follows: 16 °C for 15 minutes; 42 °C for 15 minutes; and 85 °C for 5 minutes. TaqMan miRNA assays were used to analyze the expression of hsa-miR-200c and hsa-miR-24 oligonucleotides (Applied Biosystems, Foster City, California). After the cDNA synthesis, qPCR was done with an ABI Prism 7900HT SDS (Applied Biosystems) using Platinum Taq Polymerase (Invitrogen) under the following cycling conditions: 95 °C for 1 minute (initial denature); then 50 cycles of 95 °C for 5 seconds, and 60 °C for 30 seconds. Statistical analysis. Statistical comparisons were made using Student’s t tests. P < 0.05 were considered to represent statistically significant differences. All statistical analyses and graphing were done with Graph Pad software (GraphPad Prism, San Diego, CA) and Excel (Microsoft, Redmond, WA). The enhancement in sensitization to radiation was calculated for all clonogenic assays based on the Linear–Quadratic (L–Q) model for biological effect.9 The statistic coefficients of determination (R-squared) calculated for each L–Q fitted curve were always better than 0.996. From the L–Q curves obtained for control or drug-treated samples, the classic radiosensitization enhancement factor was calculated as the ratio of radiation doses required to reduce the surviving populations to 10%. OriginLab version 8.1 (OriginLab, Northampton, MA) was used for mathematical calculations.

SUPPLEMENTARY MATERIAL Figure S1. Establishment of A549, H460, and H1299 cells stably overexpressing miR-200c. Figure S2.  ROS levels in ZEB-knockdown stable cell lines. Figure S3. MiR-200c regulates other oxidative responsive genes ­sensitizing lung cancer cells to radiation. Table S1.  Primers designed for gene expression analysis by quantitative polymerase chain reaction. Table S2.  Primers designed for site-directed mutagenesis.

ACKNOWLEDGMENTS The authors thank Christine F. Wogan, MS, ELS, of MD Anderson’s Division of Radiation Oncology, for editorial contributions. Financial support was provided by Lung Cancer Research Foundation grant. This work was made possible through the generosity of the family of M. Adnan Hamed and the Orr Family Foundation to MD Anderson Cancer Center’s Thoracic Radiation Oncology program. Other financial support was provided by the National Cancer Institute (K12 11111246; PI Robert Bast Jr, Subproject # 9276; P01CA06294; R01s CA155196 and CA160398; P50 CA070907; and P30 CA016672), Department of Defense (BATTLE award W81XWH-06-1-0303, PROSPECT award W81XWH-07-1-03060); and research support from the Wiegand Foundation. CCQ was supported by FAPESP (process number 2013/20842-6). H.J.P. and A.G.B. were supported by a commercialization grant from the Cancer Prevention Research Institute of Texas (CPRIT). H.J.P. and A.G.B. are employees of Mirna Therapeutics which develops miRNA-based therapies. All other authors declare no competing financial interests.

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References

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Molecular Therapy  vol. 22 no. 8 aug. 2014

Therapeutic Delivery of miR-200c in Lung Cancer

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