The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer

    The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer John K. Chan, Kevin Blansit, Tuyen Kiet, Alexander Sherman, Gabriel Wong, Christine Earle, Lilly Y.W. Bourguignon PII: DOI: Reference:

S0090-8258(14)00094-8 doi: 10.1016/j.ygyno.2014.01.034 YGYNO 975362

To appear in:

Gynecologic Oncology

Received date: Accepted date:

19 September 2013 18 January 2014

Please cite this article as: Chan John K., Blansit Kevin, Kiet Tuyen, Sherman Alexander, Wong Gabriel, Earle Christine, Bourguignon Lilly Y.W., The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer, Gynecologic Oncology (2014), doi: 10.1016/j.ygyno.2014.01.034

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ACCEPTED MANUSCRIPT The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer John K. Chan1, Kevin Blansit1, Tuyen Kiet1, Alexander Sherman1, Gabriel Wong2, Christine Earle2, Lilly YW Bourguignon2 1

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Division of Gynecologic Oncology Department of Obstetrics, Gynecology & Reproductive Sciences University of California, San Francisco School of Medicine UCSF Helen Diller Family Comprehensive Cancer Center 1600 Divisadero Street, Box 1702 San Francisco, CA 94143 2

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Department of Medicine University of California, San Francisco School of Medicine Veterans Affairs Medical Center, Box 111N2 San Francisco, CA

Running Title: MiR-21 promotes drug resistance in ovarian cancer Financial Support: This work is supported by the John Kerner Fund, the Veterans Administration Merit Review Awards (RR & D-1I01 RX000601 and BLR & D-5I01 BX000628), United States Public Health grants (R01 CA66163) and DOD grant. LYWB is a VA Senior Research Career Scientist. Correspondence To: John K. Chan University of California, San Francisco School of Medicine UCSF Helen Diller Family Comprehensive Cancer Center 1600 Divisadero Street, Box 1702 San Francisco, CA 94143-1702 Telephone: 415-885-7561 Fax: 415-885-3586 Electronic mail: [email protected] Conflict of Interest Statement: The authors declare that there are no conflicts of interest. Text (excluding references): 3,251 Number of Figures: 6

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ACCEPTED MANUSCRIPT Abstract Background: MicroRNAs have been implicated in tumorigenesis, drug resistance, and

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prognosis in cancer. We investigated the role of microRNA-21 (miR-21) in regulating ovarian

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cancer drug resistance.

Methods: We used parental and cisplatin resistant ovarian cell lines to demonstrate the role of

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miR-21 in drug resistance and investigated the gene targets of miR-21. Fresh tumor specimens

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were used to validate our in vitro findings.

Results: Cisplatin resistant ovarian cells were four-fold more resistant compared to the parental

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cell line. MiR-21 was overexpressed in the resistant cell line on microRNA microarray, which was subsequently validated with qRT-PCR. Using anti-microRNA inhibitors, we demonstrated

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that miR-21 attenuation reversed the drug resistant phenotype in both the resistant and parental cell lines. The inhibition of miR-21 induced apoptosis based on annexin V-FITC immunostaining.

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Using western blot analysis, miR-21 knockdown enhanced the expression of tumor suppressor PDCD4, and attenuated apoptosis inhibitor c-IAP2. Using 101 specimens from advanced ovarian cancer patients enrolled in The Cancer Genome Atlas, we found that women with tumors that overexpressed miR-21 were associated with a shorter progression-free survival. Conclusion: Our data suggest that miR-21 regulates drug resistance via apoptosis and cellular survival pathways. Targeting miR-21 may have clinical utility in the treatment of resistant ovarian cancer. Keywords: miR-21; ovarian cancer; chemosensitivity; chemoresistance; microRNA Introduction In the United States, ovarian cancer is the most lethal gynecologic malignancy associated with 15,500 deaths in 2012 [1]. Although ovarian cancer patients have a good initial response to

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ACCEPTED MANUSCRIPT chemotherapy, the majority will recur and succumb to their disease due to drug resistance. Novel therapies are needed to target drug resistant ovarian cancer.

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MicroRNAs (miRs) are small non-coding nucleotides that post-transcriptionally regulate

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gene expression. These molecules may serve either as oncogenes or tumor suppressors to influence tumorigenesis and prognosis in ovarian cancer [2]. The role of microRNAs in drug

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resistance has received considerable attention. Investigators have shown microRNAs may

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modulate response to chemotherapy, and that inhibition of these microRNAs may reverse ovarian cancer drug resistance [3-10]. More specifically, prior studies have demonstrated that

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microRNAs regulate drug resistance via MDR1 expression, and that the attenuation of microRNAs increased sensitivity to cisplatin [11, 12].

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MiR-21 has been implicated in the tumorigenesis, drug resistance, and prognosis of breast, head/neck and ovarian cancers [13-15]. In breast and head/neck cancers, the inhibition of miR-21

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attenuated cell proliferation and tumor growth by augmenting apoptosis [15, 16]. Furthermore, Nam et al showed that dysregulation of miR-21 affects ovarian cancer prognosis, while others have shown that miR-21 was associated with ovarian cancer proliferation and cell migration via the PTEN tumor suppressor pathway [17, 18]. While prior studies have identified the association of miR-21 and prognosis, few have investigated the role miR-21 regulating ovarian cancer drug resistance. In addition, most reports used cell line data without validation from fresh tumor specimens. In this current report, we explored the role of miR-21 in regulating drug resistance and identified the downstream effectors of this microRNA. Methods Cell culture

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ACCEPTED MANUSCRIPT Parental A2780, cisplatin-resistant A2780-CP (Sigma-Aldrich, St. Louis, MO), or SKOV3 cells (ATCC, Manassas, VA) cells were cultured according to manufacturer’s guidelines,

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with anti-mycotic/anti-biotic supplemented and 10% fetal bovine serum (FBS).

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Transfection with anti-miR-21 inhibitor

Cells were transfected with 25nM or 50nM anti-miR-21 miRIDIAN Hairpin Inhibitor

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(Thermo Scientific Dharmacon, Lafayette, CO) using Lipofectamine 2000 transfection reagent

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(Invitrogen, Grand Island, NY) according to manufacture guidelines. 25 nM of negative control

additionally used in these experiments. MicroRNA microarray

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#2 (Thermo Scientific Dharmacon) and miR-21 mimic (Thermo Scientific Dharmacon) were

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To investigate differential microRNA expression between the parental and resistant cell line, microRNA microarray was used to assay RNA isolated by TRIzol (Invitrogen). The Exiqon

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microarray platform contained over 1,700 capture probes and covered all microRNAs annotated in miRBase 11.0 (The Gladstone Institute, San Francisco, Ca). qRT-PCR

MicroRNA overexpression was validated by qRT-PCR using RACE/SYBR Green method as previously described [19]. Total RNA from transfected cells (50nM of inhibitor or negative control #2) was isolated using TRIzol, polyadenlyated with E. coli Poly(A) Polymerase (New England Biolabs, Ipswich, MA), and reverse transcribed with SuperScript III First-Strand Synthesis System (Invitrogen) using 3’ RACE adapter (5’-GCG AGC ACA GAA TTA ATA CGA CTC ACT ATA GGT TTT TTT TTT TTV N-3’). qRT-PCR was performed with SYBR Green PCR Master Mix on ABI PRISM 7900HT Sequence Detection System (Invitrogen) using the following primers: 3’ RACE outer universal primer (5’-GCG AGC ACA GAA TTA ATA

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ACCEPTED MANUSCRIPT CGA CT-3’), and miR-21 specific primer (5’-TAG CTT ATC AGA CTG ATG TTG A-3’). The ΔCt values were normalized using endogenous RPL, and reported as fold expression (5’-ATG

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GCG AGG ATG GCA AGA AAA-3’ / 5’-GCT CAC TCC ATT GAT ACC TCT GA-3’).

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Cell Survival Assays

MTT assays (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and

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luminescent ATP growth based assays (Promega, Madison, Wi) were used to evaluate cisplatin

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drug resistance in the respective cell lines. A2780, A2780-CP, and SKOV3 cells were transfected with 25nM of anti-miR-21 inhibitor, miR-21 mimic, or negative control. Transfected cells were

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seeded in triplicate with 3,000 cells/well into 96-well plates to incubate overnight in complete media supplemented with 10% FBS. Serial dilutions of cisplatin (Sigma-Aldrich) from 0μM to

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20μM in serum-free antibiotic-free media were used to determine IC50. Final volume was 100μL per well, and cells were incubated for 72 hours. Cell survival was normalized to the absorbance

linear model.

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of 100%, and IC50 values of cisplatin were estimated from dose-response curves using a non-

Annexin V-FITC Apoptosis Staining Annexin V-FITC Apoptosis Detection Kit was used to detect apoptotic cells (eBioscience, San Diego, CA). A2780 and A2780-CP cells were transfected with 25 nM of anti-miR-21 inhibitor or negative control #2 as described above. Transfected cells were seeded in triplicate with 20,000 cells per cell into 24-well plates, then incubated overnight in complete media with 10% FBS. 1 µM of cisplatin suspended in DMSO was added to complete media and 3% FBS overnight. Binding reagent (1:50 dilution) and annexin V-FITC (1:200 dilution) were added 5 minutes before bright field and fluorescence images were captured. Western Blot of PDCD4, c-IAP2, Caspase 3, and Caspase 7

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ACCEPTED MANUSCRIPT Protein extract from lysed cells was acquired using RIPA lysis and stored at -20°C prior to use. BCA protein assay kit (Thermo Scientific Dharmacon) was used according to

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manufacturer protocol to measure protein concentration of lysates from transfected cells. 20µg of protein were loaded with sample and loading buffers in a 15-well NUPAGE™ 4-10% B/T gel

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and run at 200V for 30-45 minutes. BenchMark™ pre-stained ladder (Invitrogen) was loaded in

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the gel. The proteins were transferred onto BIO-RAD™ Trans-Blot (Hercules, CA)

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nitrocellulose membrane with NUPAGE Transfer Buffer (Invitrogen) and 10% methanol. 100V constant voltage was applied on ice for 1-2 hours.

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To evaluate the protein levels of tumor suppressor PDCD4 (Programmed Cell Death Protein 4) and survival protein c-IAP2 (Inhibitors of the Apoptosis Family of Proteins),

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immunoblot experiments were performed. Prior to incubating with monoclonal antibodies, the membrane was blocked with 5% bovine serum albumin in Tween-20 Tris-buffered saline

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(TTBS). We then immunoblotted using the following primary antibodies at 4°C overnight at a dilution of 1:1,000: mouse anti-PDCD4 (Abnova, Walnut, CA), mouse anti-c-IAP2 (BD Pharmigen, San Diego, CA), mouse anti-caspase 3 (Abcam, Cambridge, Massachusetts), mouse anti-capsase 7 (Abcam, Cambridge, Massachusetts), or mouse anti-α-actinin (Santa Cruz Biotech, Santa Cruz, Ca) as a loading control. Secondary goat anti-mouse IgG-Peroxidase conjugated (1:10,000 to 1:20,000 dilution) was used to visualize bands using Super Signal West Pico Chemiluminescent reagent (Thermo Scientific) at room temperature for 1 hour. Membrane was washed with TTBS for 5 minutes, three times, prior to adding chemiluminescent substrate. The Cancer Genome Atlas To validate the association of miR-21 with drug resistance in ovarian cancer patients’ specimens, we extracted clinical and microRNA expression data from recurrent patients enrolled

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ACCEPTED MANUSCRIPT in The Cancer Genome Atlas (TCGA) (). Patient microRNA expression profiles were measured with Agilent Gene Expression Array, as

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described [20]. Level 3 data was extracted from the unc.edu__H-microRNA_8x15Kv2__mirna_

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expression_analysis.txt files from TCGA data portal. Using matching patient barcodes, clinicalpathologic information from clinical_patient_all_OV.txt file was merged with microRNA

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expression for correlative studies. Progression-free survival was defined as the end of treatment

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from cisplatin to initiation of another chemotherapy regimen. Computational methods and statistical analyses

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Targetscan 5.1 (http://targetscan.org) was used to evaluate potential targets of miR-21. Ttest and Pearson correlation calculations were performed with R (R Foundation for Statistical

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Computing, Vienna, Austria). Kaplan Meier log-rank estimate and Cox Proportional Hazard analyses were performed with SPSS 17.0 software (IBM, Armonk, New York)

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Results Overexpression of MiR-21

After 72 hours exposure to cisplatin, the A2780-CP cells were 4-fold more resistant than parental A2780 cells based on MTT proliferation assay (IC50 15.9 vs. 4.0 μM; p=0.02) (Figure 1). In our DNA microarray, we identified a 2.1 fold increase in miR-21 expression in the resistant vs. parental cell lines. Other microRNAs that were differentially expressed included: miR-7d (2.1 fold), miR-10b (2.4), miR-29a (2.5), miR-98 (2.3), Let-7a (3.4), miR-10a (3.6), miR-422a (4.3), miR-27b (4.8), miR-93 (5.8), and miR-190 (6.8). Building on our prior work with breast cancer, we explored the pathways and downstream targets in drug resistant ovarian cancer [8]. Using qRT-PCR, we showed that miR-

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ACCEPTED MANUSCRIPT 21 was overexpressed in our resistant A2780-CP cells by 2.4 fold compared to parental cells (3.0 vs. 1.3; p<0.01) (Figure 3).

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Investigation of MiR-21 Mechanism

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To investigate miR-21’s regulation the drug resistant phenotype, we transfected anti-miR21 inhibitors into the parental and resistant cell lines. Using qRT-PCR, we validated the

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inhibition of miR-21 (1.3 vs. 0.3, p<0.01; 3.0 vs. 0.4, p<0.01) (Figure 3). In parental and resistant

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cell lines, the inhibition of miR-21 enhanced the sensitivity of these cells to cisplatin by 45% and 31%, respectively (p=0.05; p=0.02) (Figures 2A and 2B). In another series of experiments, we

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found that miR-21 mimic transfection resulted in enhanced survival of parental A2780 and SKOV3 cells (190%, p=0.02; and 181%, p=0.03 respectively) (Supplemental Figure 1A and B).

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Using the apoptosis marker annexin V-FITC, we showed that the attenuation of miR-21 increased the number of immunopositive cells by 28% and 17%, in the parental and resistant cell

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lines respectively (p<0.01; p=0.05) (Figure 4). This data suggest that miR-21 may regulate drug resistance via augmented apoptotic pathways. Previous studies have shown that tumor suppressor protein, PDCD4 (Programmed Cell Death Protein 4) serves as one of the target proteins for miR-21 [21, 22]. Using Targetscan, we demonstrated that PDCD4 is a potential target of miR-21 (context score -0.44). In this study, we found that anti-miR-21 inhibitor enhanced PDCD4, caspase 3, and caspase 7 expression in the resistant and parental cell lines. Furthermore, we demonstrated that the inhibition of miR-21 attenuated the expression of the apoptotic inhibitor, c-IAP2 in both cell lines (Figure 5, Supplemental Figure 3). Validation Using TCGA Tumor Specimens

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ACCEPTED MANUSCRIPT To validate our in vitro studies using immortalized cell lines, we evaluated survival of patients enrolled in The Cancer Genome Atlas (TCGA). Of 434 fresh tumors submitted to the

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TCGA, we extracted genomic and clinical data from all stage III recurrent ovarian cancer

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patients. Of these 101 women, the median age was 63 years (range: 38-87). The majority of patients (94%) underwent primary cytoreductive surgery followed by adjuvant platinum-based

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chemotherapy. Using the microRNA expression data, patients with tumors overexpressing miR-

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21 (above 75th percentile of expression) had a decreased progression-free survival (PFS) compared to those with normal expression (6-month PFS of 33% vs. 65%) with corresponding

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median PFS of 4.3 months vs. 8.6 months (p=0.03) (Figure 6). Upon further analysis using data from tumors of TCGA, we found a correlation between miR-21 and PDCD4 expression in

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advanced stage, treatment–resistant tumors (rho= -0.23, p=0.03) (Supplemental Figure 2A). Additionally, the drug resistant tumors that overexpressed miR-21 also had a higher expression

Discussion

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of c-IAP2 (rho= 0.34, p<0.01) (Supplemental Figure 2B).

Although most advanced ovarian cancer patients initially respond to chemotherapy, the majority will recur and succumb to their disease. As such, research towards defining the mechanisms and overcoming drug resistance may improve the outcomes of women with this aggressive cancer. MicroRNAs are small non-coding nucleotides that regulate genes post-transcriptionally. Prior studies have shown that microRNAs may be implicated in cancer tumorigenesis and prognosis [13, 14]. The role of microRNAs in cancer drug resistance has received significant attention. Previous studies have shown that miR-21 is associated with drug resistance in breast, pancreatic and head/neck cancers [9, 10, 15, 23]. In ovarian cancer, miR-21 is upregulated and

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ACCEPTED MANUSCRIPT associated with metastasis and poor prognosis [17, 18, 24]. Researchers have shown that inhibition of miR-21 resulted in decreased cell invasion and proliferation [25].

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In this report, we showed that the inhibition of miR-21 enhanced the sensitivity of ovarian

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cancer cells to cisplatin. These findings are consistent with those of other solid tumors such as breast, colorectal and head/neck cancer. Prior research from our group and others has

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demonstrated that miR-21 inhibition resulted in enhanced chemosensitivity in breast cancer,

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head/neck and colorectal cancer [8, 15]. Although we were able to enhance the sensitivity to chemotherapy in both sensitive and resistant cell lines, the effect of the inhibitors was attenuated

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in the resistant cells. These findings may be explained by higher level of miR-21 observed in the resistant cell lines as demonstrated in our qRT-PCR experiments (Figure 3). Moreover, we were

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able to decrease the sensitivity of ovarian cancer cells to cisplatin by transfecting miR-21 mimic (Supplemental Figure 1A). Using an additional ovarian cancer cell line, we validated these

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results using SKOV3 ovarian cells (Supplemental Figure 1A). In another series of experiments, we attempted to target miR-21 with higher concentrations of inhibitor, but were unable to observe an incremental dose response. It is possible that this can be attributed to other factors related to drug resistance such as drug transporters and cell survival signals [26, 27]. Apoptosis has been demonstrated to be an important contributor to the development of drug resistance. Attenuation of the apoptotic pathway has been shown to increase proliferation and promote drug resistance using in vitro models [26, 28, 29]. In the clinical setting, patients with tumors that overexpressed the apoptotic inhibitor survivin were more likely to develop drug resistant cancers with a poorer prognosis [30, 31]. Lou et al also demonstrated that miR-21 downregulation promotes apoptosis in OVCAR3 cells [25]. However, these studies did not

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ACCEPTED MANUSCRIPT evaluate miR-21 and its role in drug resistant ovarian cancer. Our study demonstrated that the inhibition of miR-21 resulted in the reversal of the cisplatin resistant phenotype.

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Although miR-21 overexpression has been shown to promote metastasis and proliferation,

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there is limited information on the role of miR-21 in drug resistance. Prior data from our laboratory and others have shown that survival proteins may play a significant role in drug

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resistance in breast and other cancers [8, 32, 33]. MiR-21 has been shown to be an oncogene in

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breast cancer, head/neck cancer and glioblastomas, by upregulating PDCD4 [8, 15, 34]. Based on data from this current report, we hypothesize that miR-21 overexpression downregulates the

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tumor suppressor gene PDCD4, leading to an increase in the inhibitor of apoptosis protein cIAP2 and subsequent chemotherapy resistance.

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Previous studies have also demonstrated that downregulation of PDCD4 results in drug resistance via enhanced expression of c-IAP2 and MDR1 [8]. c-IAP2 has been implicated in the

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inhibition of caspase activity, a key player in programmed cell death [35, 36]. In multiple myeloma, investigators showed that c-IAP2 and other IAP proteins in association with NF-ΚB signal transduction play a significant role in drug resistance [37]. Others have found that c-IAP2 increases apoptosis and enhances sensitivity of cancer cells to chemotherapy [35, 38]. Therefore, these molecules may serve as therapeutic targets for resistant tumors in ovarian and other cancers. Given these aforementioned findings from our immortalized cell lines, it is important to validate these results with fresh tumor specimens from patients. In our analysis of 101 recurrent and advanced stage ovarian cancer patients enrolled in The Cancer Genome Atlas (TCGA), we demonstrated that upregulation of miR-21 was associated with shorter progression-free survival. Other investigators have shown that the dysregulation of other microRNAs including let-7b, miR-200a, and miR-27a are associated with drug resistance and poorer survival in ovarian cancer

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ACCEPTED MANUSCRIPT [7, 17]. In addition, Lou et al showed the clinical association of miR-21 and higher grade and stage of disease [14]. TCGA contains genomic data from high quality biospecimens of primary,

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newly diagnosed, untreated, serous cancers from 15 academic centers. In addition, all tumors are

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matched with non-tumor tissue controls from the same patient. The data from these human specimens allowed us to confirm our laboratory findings in the clinical setting.

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The interpretation of our results needs to be considered with the following limitations.

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Our analysis only evaluated one immortalized cell line and its resistance to cisplatin. Although platinum containing agents remains the primary adjuvant therapy for ovarian cancer, the

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examination of other cytotoxic agents such as paclitaxel and doxorubicin should also be studied. In fact, preliminary data from our group also suggested that miR-21 may play a role in taxane

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resistance and the inhibition of miR-21 increases sensitivity to paclitaxel [39]. Although our current study only focused on miR-21, it is well recognized that microRNAs function in a

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complex network with other microRNAs in the modulation of drug resistance. In our microRNA microarray analysis of both cell lines and human specimens, we identified a panel of microRNAs that may predict surgical, chemotherapy response and survival outcomes [40]. As such, further studies are needed to elucidate the functional significance of microRNA profiles rather than distinct microRNAs involved in ovarian cancer drug resistance. Furthermore, in our microRNA analysis of human specimens, we were only able to demonstrate a median progression-free survival difference of four months in those with high vs. normal miR-21 expression using clinical data from the TCGA. Although our survival analysis was statistically significant, the marginal difference may not be clinically relevant. Moreover, we were unable to demonstrate an overall survival difference based on miR-21 expression. These findings may be due to the fact that these patients were heavily pretreated for their recurrent

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ACCEPTED MANUSCRIPT disease. Although we described the clinical outcomes data based on PFS, this analysis was not well defined in the TCGA. There is a lack of detailed information on more reliable

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measurements of recurrence or progression based on RECIST and tumor markers such as CA-

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125. In addition, this study only utilized a single tissue cohort, without validation from an independent cohort of patients. Nevertheless, it is important to analyze fresh tumor specimens

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with clinical outcomes to confirm the findings of our laboratory studies.

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This is one of the first reports to investigate the role of miR-21 overexpression in ovarian cancer drug resistance. Our study demonstrates that downregulation of miR-21 results in

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apoptosis via modulation of PDCD4 and c-IAP2. These results were also subsequently validated in patients’ tumors with clinical information. Taken together, our findings suggest that miR-21

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inhibition and its ability to promote apoptosis and cisplatin-induced cell death may be used as an

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important target in designing novel therapies toward reversing ovarian cancer drug resistance.

Conflict of Interest Statement:

The authors declare that there are no conflicts of interest.

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Sultan A KT, Chan J, Wang Y, Francisco B. . Significance of MicroRNAs in determining

taxane resistance in ovarian cancer. Gynecologic Oncology. Sherman A FK, Korkola J, Levine D, Olshen A, Chan J. Differential microRNA

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Figure 1. MTT assay A2780 vs. A2780-CP after treatment with cisplatin Figure 2. Reversal of cisplatin resistance after transfection with anti-miR-21 inhibitor in A.

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Figure 3. MiR-21 overexpression in A2780-CP cells compared to A2780 cells

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Figure 6. Progression-free survival of recurrent ovarian cancer patients based on miR-21 expression

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Highlights - We demonstrated that miR-21 is overexpressed in cisplatin resistant ovarian cancer cell line - The inhibition of miR-21 reversed drug resistance in vitro - Overexpression of miR-21 is associated with worse patient survival

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