Published OnlineFirst January 27, 2015; DOI: 10.1158/0008-5472.CAN-14-2783 Cancer Molecular and Cellular Pathobiology Research
MicroRNA-22 Suppresses DNA Repair and Promotes Genomic Instability through Targeting of MDC1 Jung-Hee Lee1,2, Seon-Joo Park1,3, Seo-Yeon Jeong1, Min-Ji Kim1, Semo Jun1,4, Hyun-Seo Lee1, In-Youb Chang1,5, Sung-Chul Lim6, Sang Pil Yoon7, Jeongsik Yong8, and Ho Jin You1,4
Abstract
MDC1 is critical component of the DNA damage response overexpression of constitutively active Akt1, homologous recom- (DDR) machinery and orchestrates the ensuring assembly of the bination was inhibited by miR-22–mediated MDC1 repression. DDR protein at the DNA damage sites, and therefore loss of In addition, during replicative senescence and stress-induced MDC1 results in genomic instability and tumorigenicity. How- premature senescence, MDC1 was downregulated by upregulat- ever, the molecular mechanisms controlling MDC1 expression are ing miR-22 and thereby accumulating DNA damage. Our results currently unknown. Here, we show that miR-22 inhibits MDC1 demonstrate a central role of miR-22 in the physiologic regulation translation via direct binding to its 30 untranslated region, leading of MDC1-dependent DDR and suggest a molecular mechanism to impaired DNA damage repair and genomic instability. We for how aberrant Akt1 activation and senescence lead to increased demonstrated that activated Akt1 and senescence hinder DDR genomic instability, fostering an environment that promotes function of MDC1 by upregulating endogenous miR-22. After tumorigenesis. Cancer Res; 75(7); 1–13. 2015 AACR.
Introduction DNA double-strand breaks (DSB) activate the DDR by trigger- ing the kinase activity of ataxia telangiectasia mutated (ATM), Repeated exposure to both exogenous and endogenous insults thereby initiating a signaling cascade in which the histone variant challenges the integrity of cellular genomic material. Eukaryotes H2AX (g-H2AX), located at DSB sites, becomes phosphorylated, have evolved a system called the DNA damage response (DDR), and other DDR factors, including the adaptor protein mediator of which allows cells to sense DNA damage and orchestrate the DNA damage checkpoint 1 (MDC1), are recruited. MDC1 ampli- appropriate cell-cycle checkpoints and DNA repair mechanisms fies the ATM signaling activity, leading to a higher percentage of (1). The failure to respond to DNA damage is a characteristic phosphorylated H2AX proteins and contributing to the recruit- associated with genomic instability and with the onset of diseases, ment and retention of additional DDR factors at the sites of DNA including neurodegenerative diseases, immune deficiency, can- damage (3). Thus, MDC1 has been termed a master regulator, cer, and premature aging (2). modulating the specific chromatin microenvironment required to maintain genomic stability. MDC1 knockout mice show chromo- somal instability, defective DNA repair, and radiation sensitivity 1 DNA Damage Response Network Center,Chosun University School of (4). Furthermore, loss of MDC1 is associated with an increased Medicine, Gwangju, Republic of Korea. 2Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju, occurrence of tumors in mice (5), and reduction or lack of MDC1 Republic of Korea. 3Division of Natural Medical Sciences, Chosun has been observed in breast and lung carcinoma cells in humans 4 University School of Medicine, Gwangju, Republic of Korea. Depart- (6). Therefore, cellular levels of MDC1 appear to impact genomic ment of Pharmacology, Chosun University School of Medicine, Gwangju, Republic of Korea. 5Department of Anatomy, Chosun Uni- instability and tumorigenicity directly. Although posttranslational versity School of Medicine, Gwangju, Republic of Korea. 6Department modification via small ubiquitin-like modifiers affects the stability of Pathology,Chosun University School of Medicine, Gwangju, Repub- of MDC1 and its function in DDR (7–9), little is known about how lic of Korea. 7Department of Anatomy, School of Medicine, Jeju National University, Jeju-Do, Republic of Korea. 8Department of Bio- the expression of MDC1 is regulated and which pathophysiologic chemistry, Molecular Biology and Biophysics, University of Minnesota conditions are associated with this regulation. Twin Cities, Minneapolis, Minnesota. miRNAs are small noncoding RNAs that suppress protein 0 Note: Supplementary data for this article are available at Cancer Research synthesis, usually by interacting with the 3 -untranslated region Online (http://cancerres.aacrjournals.org/). (30-UTR) of target mRNAs (10). Several lines of evidence suggest Corresponding Authors: Ho Jin You, Chosun University Medical School, 375 that miRNAs negatively regulate the expression of DDR proteins Seosuk-dong, Gwang-ju 501-759, Republic of Korea. Phone: 82-62-230-6337; (11–14). Therefore, miRNAs may play an important role in the Fax: 82-62-233-3720; E-mail: [email protected]; Jung-Hee Lee, Phone: 82-62- regulation of DDR and may contribute to the maintenance of 230-6399; Fax: 82-62-230-6586; E-mail: [email protected]; and Jeongsik genomic integrity. Yong, Phone: 612-626-2420; Fax: 612-625-2163; E-mail: [email protected] To investigate the possibility that some specific miRNA might doi: 10.1158/0008-5472.CAN-14-2783 directly regulate MDC1 expression and its function, we screened 2015 American Association for Cancer Research. for miRNAs that could potentially regulate MDC1 expression and
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A B 1.5
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0.9
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0.3 α-Tubulin 0.0 Normalized luciferase luciferase Normalized activity
C HeLa U2OS HEK293T D miR-22 –+ –+ –+
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BRCA1 0.9 0.9 0.9
α-Tubulin 0.6 ∗∗ 0.6 ∗∗ 0.6 ∗∗
30 ∗∗ 15 150 Relative MDC1 0.3 0.3 0.3 ∗∗ ∗∗ mRNA expression 20 10 100 0 0 0 miR-22–+ –+ –+
10 5 50 expression
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miR-Ctrl E F miR-22 1.6 ∗∗ ns
1.2
0.8
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Normalized luciferase Normalized activity 0 Vector MDC1 MDC1 3′-UTR-wt 3′-UTR-mt
Figure 1. miR-22 directly affects MDC1 expression. A, HEK293T cells were cotransfected with the MDC1 30-UTR luciferase reporter vector along with the candidate miRNAs, which were predicted by at least five bioinformatics algorithms, or the miRNA-negative control (miR-Ctrl). Results are shown as mean SD (n ¼ 3). B, the levels of MDC1 protein were measured using Western blotting in HEK293T cells transfected with the indicated miRNAs. C, indicated cells were transfected with control miRNA or miR-22. The levels of indicated proteins were determined using Western blotting (top). miR-22 levels in the indicated cells were determined using real-time qPCR analysis (bottom). Results are shown as mean SD (n ¼ 3). , P < 0.01. D, expression of MDC1 mRNA in miR-22–transfected HeLa, U2OS, and HEK293T cells was quantitated using real-time qPCR. Results are shown as mean SD (n ¼ 3). , P < 0.01. E, a schematic representation of MDC1 30-UTR. Red, the seed sequence of miR-22. F, MDC1 30-UTR-wt and MDC1 30-UTR-mt were cotransfected with miR-22 in HEK293T cells. Luciferase activity was measured 24 hours after the transfection. Data represent mean SD; n ¼ 3; ns, not significant; , P < 0.01.
fi fi identi ed miR-22 as an miRNA that could speci cally suppress Materials and Methods MDC1 expression. We show that miR-22–mediated downregula- tion of MDC1 induces impaired DDR activation and genomic Antibodies instability. Further, we demonstrated that this new pathway plays a Polyclonal MDC1 antibody (R2) was raised in rabbit against a crucial role in the regulation of DNA repair in sustained activation glutathione S-transferase fusion protein containing the BRCT of Akt1 and senescence and may represent a new therapeutic target. domain of MDC1 (residues 1882-2089). Anti-MDC1 polyclonal
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miR-22 Impedes DDR Function of MDC1
MDC1 cDNA – – + A miR-22 ++– B MDC1
HA-MDC1 MDC1 cDNA – – + α-Tubulin miR-22 –++ 1.2 ∗∗ miR-Ctrl+control vector MDC1 ∗∗ ∗∗ ∗∗ miR-22+control vector 80 0.9 miR-22+MDC1 cDNA ∗∗ 60 ∗∗ γ-H2AX -H2AX 0.6 ∗∗ γ 40 ∗∗ ∗∗ ∗∗ (%)staining 20
merge Relative 0.3 ∗∗ ∗∗ 0
Relative BrdUrd incoporation (%) incoporation Relative BrdUrd MDC1 cDNA – – + 0.0 DAPI miR-22 –++ 0 2 5 10 20 50 Gy 2 hours after-IR treatment
C MDC1 cDNA – – + D MDC1 cDNA –– + E MDC1 cDNA –– + miR-22 –+ + miR-22 –+ + miR-22 –+ + MDC1 MDC1 MDC1
HA-MDC1 HA-MDC1 HA-MDC1
α-Tubulin α-Tubulin α-Tubulin
miR-22
–– + miR-control Control vector MDC1 cDNA MDC1 cDNA miR-Ctrl+control vector miR-22 –+ + miR-22+control vector Comet miR-22+MDC1 cDNA tail ∗∗ 100
P = 0.0008 P = 0.0001 15 ∗∗ ∗∗ ∗∗ ∗∗ 75 ∗∗ ∗∗ 10 60 10
45 ∗∗ 30 5 Relative cell survival (%)survival Relative cell 1 15 02510 Gy Comet tail moment (%) Comet tail 2 hours after-IR treatment
0 Number of chromosomal breaks 0 MDC1 cDNA – – + Control-miR Control vector MDC1 cDNA miR-22 –++ miR-22 F
Figure 2. miR-22–mediated MDC1 downregulation leads to impaired DNA damage response. A, BrdUrd incorporation was measured using a colorimetric assay after indicated doses of IR, using U2OS cells transfected with indicated combinations of miRNAs and HA-MDC1 constructs. Results are shown as mean SD (n ¼ 3). , P < 0.01. B and C, miR-22–expressing U2OS cells were transfected with miR-22–insensitive MDC1 and irradiated with 10 Gy of IR. Cells were then analyzed by g-H2AX and MDC1 staining 16 hours after IR (B) and by comet assay 3 hours after IR (C). Representative images and quantification of unrepaired DSBs are shown. DAPI was used for nuclear staining. Results are shown as mean SD (n ¼ 3). , P < 0.01. D, representative images and quantification of chromosome breaks indicated cells exposed to IR. Arrows, chromosome breaks. (Continued on the following page.)
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antibody (Ab11170; Abcam) was used for immunohistochemis- MDC1 30-UTR luciferase reporter that was lacking predicted try. The following antibodies were used in this study: anti-53BP1 seed region for the miR-22. As shown in Fig. 1F, deletion of the (BD), anti-BRCA1 (Santa Cruz Biotechnology), anti–g-H2AX miR-22 binding site in the seed region of the MDC1 30-UTR (Upstate), anti-Akt (Cell Signaling Technology), anti-phospho abrogated the suppressive ability of miR-22. These results Akt1 (Ser473) (Cell Signaling Technology), and anti–a-Tubulin together demonstrate that miR-22 represses MDC1 expression (Santa Cruz Biotechnology). by directly targeting its 30-UTR. To determine whether the miR-22 could affect the recruitment DR-GFP assay (homologous recombination assay) of MDC1 and its downstream targets to sites of DNA damage, we U2OS-DR-GFP cells were transfected with control or CA-Akt1 introduced miR-22 into U2OS cells and subjected them to ion- vector using lipofectamine 2000, and sequentially transfected izing radiation (IR), and examined MDC1, 53BP1, and BRCA2 with miR-22 inhibitor, and then infected with I-SceI–carrying foci formation by immunofluorescence. We observed that miR-22 adenovirus at an estimated multiplicity of infection of 10. After 72 greatly reduced the percentage of cells expressing IR-induced hours, GFP-positive cells were measured by FACS (FACSCalibur; MDC1 foci (Supplementary Fig. S2A). In addition, formation of BD Biosciences). The acquired data were analyzed using Cell- both 53BP1 and BRCA1 foci was also decreased in miR-22– Quest Pro software (BD Biosciences). overexpressing U2OS cells as compared with control cells (Sup- plementary Fig. S2B and S2C). Because miR-22 did not affect Clonogenic survival assay 53BP1 and BRCA1 protein levels (Fig. 1C), these results suggest 2 After treatment with irradiation, 5 10 cells were imme- that miR-22 inhibits IR-induced MDC1 foci formation, which in diately seeded on 60-mm dish in triplicate and grown for 2 to 3 turn suppresses the recruitment of 53BP1 and BRCA1 to sites of weeks at 37 C to allow colonies to form. Colonies were stained DNA damage. with 2% methylene blue/50% ethanol and were counted. The fraction of surviving cells was calculated as the ratio for the miR-22 affects DDR function of MDC1 and induces genomic fi plating ef ciency of treated cells over untreated cells. Cell instability survival results are reported as the mean value SD for three MDC1 plays an important role in DSB repair (15,16) and independent experiments. checkpoint activation (17–19). Therefore, we examined the Additional materials and methods can be found in Supple- effect of miR-22 on intra-S phase cell-cycle checkpoint. We mentary Information. found that the radiation-resistant DNA synthesis was induced in U2OS cells by overexpressing miR-22 (Fig. 2A, empty Results squares). Further, overexpression of miR-22 in U2OS cells had MDC1 is a direct target of miR-22 significantly more residual DSBs than control cells, as evi- To search for miRNAs that regulate MDC1 expression, we denced by the increase in signal intensity of g-H2AX staining carried out a comprehensive bioinformatics analysis to generate and by the increase in comet tail moments (Fig. 2B and C, a selective miRNA library that could then be used for screening. middle columns; Supplementary Fig. S3A and S3B). We then From this analysis, a total of 8 miRNAs were identified as candi- analyzed metaphase spreads of control and miR-22–expressing dates (Supplementary Table S1), and each was reversely screened U2OS cells after IR exposure. The results showed that over- for the effect on MDC1 expression by using a luciferase assay. The expression of miR-22 into U2OS cells could significantly results of the luciferase assays revealed that overexpression of miR- increase the number of chromosome breaks as compared with 22 led to remarkably lower luciferase activity compared with control cells (Fig. 2D, middle column). scrambled control miRNA (Fig. 1A). Consistent with these results, To determine whether the effect of miR-22 on radiation-resis- when overexpressed, only miR-22 significantly reduced endoge- tant DNA synthesis and DSB repair was mediated via its effect on nous MDC1 protein expression in HEK293T cells (Fig. 1B). The MDC1, we cotransfected U2OS cells with miR-22 and an miR-22– MDC1 expression level decreased as the concentrate of transfected insensitive MDC1 expression plasmid. Our results showed that miR-22 or its premature hairpin (pre–miR-22) was increased the nontargetable MDC1 fully rescued the radiation-resistant (Supplementary Fig. S1). miR-22–mediated repression of MDC1 DNA synthesis (Fig. 2A, black squares), DSB repair defect was not restricted to HEK293T cells, as we also observed specific (Fig. 2B and C, third columns; Supplementary Fig. S3), and repression in HeLa and U2OS cells (Fig. 1C). Using real-time chromosome breaks (Fig. 2D, third column). This evidence shows quantitative PCR (qPCR), we found that MDC1 mRNA was that miR-22 inhibits DDR through the downregulation of MDC1. reduced more than 50% when miR-22 was overexpressed (Fig. We then determined whether the suppressed DSB repair by 1D), indicating that miR-22 posttranscriptionally downregulates miR-22 leads to increased cellular sensitivity to IR. We found that MDC1, probably by promoting both mRNA decay and inhibiting the survival fractions of colonies in miR-22–expressing cells translation. following IR were significantly reduced relative to those of control We next analyzed putative miR-22 target site using TargetS- cells (Fig. 2E, empty squares). Of notice, overexpressing miR-22– can algorithm (MIT; release 6.2) and found that bases 317 to insensitive MDC1 significantly reduced the extent of miR-22– 339 in the MDC1 30-UTRarecomplementarytothetargetsites mediated sensitization to IR in miR-22–expressing cells (Fig. 2E, of miR-22 (Fig. 1E). To determine whether miR-22 binds to this black squares), further supporting working model that miR-22 site to repress MDC1 expression, we constructed a mutant modulates the DSB repair through MDC1.
(Continued.) Results are shown as mean SD (n ¼ 3). E, cell viabilities of indicated U2OS cells after indicated doses of IR were examined by the clonogenic survival assay. Results are shown as mean SD (n ¼ 3). , P < 0.01. F, array CGH profiles of clones derived from GM00637 cells transfected with control miRNA or miR-22. Chromosomal regions above or below the red dotted line indicate amplifications or deletions of genomic positions, respectively.
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A U2OS HCT116 B 2.4 3 ∗∗ ∗∗ U2OS HCT116
1.6 2 1.2 1.2
0.9 0.9 expression 0.8 1 Relative miR-22 0.6 ∗∗ 0.6 0 0 ∗∗ CA-Akt1 –+ –+
Relative MDC1 0.3 0.3 Akt1 mRNA expression 0 0 pAkt1 Vector CA-Akt1 Vector CA-Akt1
MDC1
α-Tubulin
U2OS HCT116 C U2OS HCT116 D ∗∗ ∗∗ ∗∗ ∗∗ 1.6 2 Anti–miR-22 – – + ––+ CA-Akt1 –++ –++ 1.6 1.2 MDC1 1.2 0.8 pAkt1 0.8 Relative MDC1
mRNA expression 0.4 Akt1 0.4
α-Tubulin 0 0 Anti–miR-22 – – + ––+ CA-Akt1 –++ –++ Vector E CA-Akt1 F CA-Akt1+anti–miR-22 1.5 ns ns ∗∗ ∗∗ 1.2
0.9 High pAkt1
0.6
0.3 High MDC1 Low pAkt1 Low MDC1 Low Normalized luciferase luciferase activity Normalized 0 MDC1 MDC1 3′-UTR-wt 3′-UTR-mt G 15
10 MDC1 5
0 0 2 4 6 8 10 pAkt1
Figure 3. Activated Akt1 downregulates MDC1 through upregulation of miR-22. A, Western blot analysis of MDC1 expression using cell extracts from control vector– or CA-Akt1–transfected U2OS or HCT116 cells. miR-22 expression was quantitated using real-time qPCR. Results are shown as mean SD (n ¼ 3). , P < 0.01. B, the level of MDC1 mRNA in U2OS and HCT116 cells transfected with CA-Akt1 was measured using real-time qPCR. Results are shown as mean SD (n ¼ 3). , P < 0.01. C and D, CA-Akt1–expressing cells were transfected with anti–miR-22. Two days after the transfection, the levels of MDC1 protein (C) and mRNA (D) were measured using Western blotting or real-time qPCR, respectively. Results are shown as mean SD (n ¼ 3). , P < 0.01. E, luciferase assay with reporter constructs either wild or mutated 30-UTR of MDC1 in indicated plasmid-transfected HEK293T cells. Data represent mean SD; n ¼ 3; ns, not significant; , P < 0.01. F, immunohistochemistry analysis for phospho-Akt1 (pAkt1) and MDC1 using a prostate tumor tissue array. Hematoxylin counterstain (blue) was included for nuclei staining. Scale bar, 25 mm. G, a scatter plot showing the negative correlation between pAkt1 and MDC1 expression in the prostate cancer tissue microarray. The P value and Pearson correlation coefficient (r) were calculated.
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A U2OS HCT116 B
IR 0 0.5 1 3 6 h 0 0.5 1 3 6 h U2OS HCT116 MDC1 Anti–miR-22 –– + –– + CA-Akt1 –+ + –+ + DAPI MDC1 Vector γ-H2AX DAPI
DAPI γ-H2AX
MDC1 DAPI
DAPI CA-Akt1 ∗∗ ∗∗ γ-H2AX ∗∗ ∗∗ 80 75
DAPI 60 60 45 40 80 Vector 80 Vector 30 CA-Akt 1 CA-Akt1 % of Cells 20 % of Cells 60 60 15
with MDC1 with MDC1 > 5 foci 0 with MDC1 > 5 foci 0 40 40 Anti–miR-22 –– + –– + CA-Akt1 –+ + –+ + 20 20 ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ 0 0 % of Cells with MDC1 MDC1 >5 with foci of Cells % % of Cells with MDC1 MDC1 >5 fociwith of Cells % 00.513 6 h 00.51 3 6 h ∗∗ ∗∗ I-Scel endonuclease 3 C restriction site D 2.5 SceGFP iGFP 2 + I-Scel 1.5 DSB 1
HR 0.5 GFP-expressing cells (%) GFP-expressing cells wtGFP iGFP 0 Anti–miR-22 ––+ CA-Akt1 –++
∗∗ ∗∗ E U2OS HCT116 F 4.5 MDC1 cDNA ––+ ––+ CA-Akt1 –++ –++ 3 MDC1
pAkt1 1.5
Akt1 (%) cells GFP-expressing 0 MDC1 cDNA ––+ α-Tubulin CA-Akt1 –++
Figure 4. Activated Akt1 suppresses HR by downregulation of MDC1. A, U2OS and HCT116 cells transfected with control vector or CA-Akt1 were irradiated with 10 Gy and fixed for immunofluorescence staining of MDC1 and g-H2AX at the indicated time points. Results are shown as mean SD (n ¼ 3). , P < 0.01. B, representative images (top) and quantification (bottom) of IR (10 Gy)-induced MDC1 and g-H2AX foci in cells cotransfected with anti–miR-22 and CA-Akt1 or with CA-Akt1 alone. Results are shown as mean SD (n ¼ 3). , P < 0.01. C, a schematic showing the assay for the fluorescence- based measurement of HR-mediated DSB repair. D, U2OS DR-GFP cells were cotransfected with anti-miR22 and CA-Akt1 or with CA-Akt1 alone, and the percentage of cells expressing GFP was measured using flow cytometry. Results are shown as mean SD (n ¼ 3). , P < 0.01. E, MDC1 expression was reconstituted by transfecting miR-22–insensitive MDC1 into U2OS or HCT116 cells with high Akt1 activity. F, HR assay indicated that overexpression of miR- 22–insensitive MDC1 significantly increases the HR efficiency in CA-Akt1–expressing U2OS DR-GFP cells. Results are shown as mean SD (n ¼ 3). , P < 0.01.
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miR-22 Impedes DDR Function of MDC1
A MRC-5 IMR-90 Senescence Senescence
Young Replication H2O2 Young Replication H2O2
SA-β-gal
Morphology
80 3.5 75 3
60 2.8 60 2 2.1 45 40 1.4 30 1 20 0.7 15 miR-22 expression miR-22 expression 0 0 0 miR-22 expression
-gal–positive cell (%) SA- β -gal–positive cell (%) SA- β -gal–positive cell 0 Y R-S H-S Y R-S H-S Y R-S H-S Y R-S H-S
MRC5 IMR-90 B Y R-S Y H-S C 1.2 1.2 MDC1 MRC-5 0.9 0.9 α-Tubulin
0.6 0.6 MDC1 IMR-90 0.3 0.3 α-Tubulin MDC1 mRNA expression mRNA MDC1 MDC1 mRNA expression MDC1 mRNA 0 0 Y R-S H-S Y R-S H-S D E Young cells Presenescent cells Ant-control Anti-control Anti–miR-22 MRC-5 IMR-90 IR
MDC1 MRC-5 MRC-5 MDC1 α-Tubulin
Ant–miR-22 DAPI
Young Senescence Young Senescence IMR MDC1 - 2.5 3 90
2 DAPI 2 1.5 MRC-5 IMR-90
1 75 75 1
0.5 60 60 Relative miR-22 expression Relative miR-22 expression Relative miR-22 expression 0 0 45 45 Anti–miR-22 H2O2 30 30
15 15 % of Cells with MDC1 MDC1 % of Cells with foci >5 0 MDC1 % of Cells with foci >5 0 Ant–miR-22 Young Senescence Young Senescence
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To assess the subsequent genomic effects resulting from the The Pearson correlation analysis demonstrated a statistically numerous chromosomal breaks in miR-22–expressing cells, we significant inverse relationship between pAkt1 and MDC1 performed array comparative genomic hybridization (array CGH) levels (Fig. 3G). To further investigate the role of miR-22 on using human fibroblast GM00637 cells. From this analysis, we pAkt-mediated suppression of MDC1 levels in prostate cancer, conclude that there was a high frequency of chromosomal abnor- we measured the miR-22, p-Akt, and MDC1 levels of fresh malities in miR-22–expressing cells, including clonal amplifica- human prostate cancer tissues. We observed that miR-22 levels tions and deletions in discrete regions (Fig. 2F; Supplementary were significantly higher in prostate cancer samples with high Fig. S4). Taken all together, these results provide evidence that pAktandlowMDC1expressionthaninthosewithlowpAkt miR-22–mediated downregulation of MDC1 results in defects in and high MDC1 expression (Supplementary Fig. S5A and S5B). DSB repair and allows cells to bypass an intra–S-phase checkpoint These results further strengthen the notion that activated causing a decrease in chromosome integrity. Akt1 downregulates MDC1 protein through upregulation of miR-22. Akt1 downregulates MDC1 expression through upregulation of miR-22 Akt1 inhibits homologous recombination through Endogenous miR-22 expression appears to be dependent on miR-22–mediated MDC1 suppression particular abnormal pathologic contexts, including high Akt1 We next examined the effect of activated Akt1 on the DDR activity (20) and replicative-induced senescence (21). Akt1 is function of MDC1 by monitoring the recruitment of MDC1 to frequently activated in many tumor types (22), and its overstim- sites of DNA damage after IR. As shown in Fig. 4A, CA-Akt1 greatly ulation leads to a strong repression of homologous recombina- reduced the percentage of cells with IR-induced MDC1 foci. tion (HR), causing genomic instability (23–25). Thus, miR-22– However, transfection of the anti–miR-22 could completely res- mediated downregulation of MDC1 is hypothesized to abrogate cue the inhibitory effect of CA-Akt1 on MDC1 foci formation (Fig. HR in Akt1-activated cancer cells. To determine whether activated 4B), suggesting that inhibition of MDC1 foci by CA-Akt1 is due to Akt1 negatively regulates MDC1 through the induction of miR-22, upregulation of miR-22. U2OS and HCT116 cells were transfected with constitutively Because it has been demonstrated that MDC1 plays an impor- active Akt1 (CA-Akt1). As expected, overexpression of CA-Akt1 tant role in the HR (15), we tested whether activated Akt1 inhibits in either U2OS or HCT116 cells led to increased miR-22 expres- HR via suppressing MDC1 expression. To this end, we assayed for sion (Fig. 3A, top panel). Intriguingly, in comparison with control HR-mediated repair of an I-SceI–induced DSBs, in U2OS cells, cells, CA-Akt1–transfected cells exhibited diminished levels of using a recombination substrate DR-GFP (27). When DSBs are MDC1 protein (Fig. 3A, bottom panel) and mRNA (Fig. 3B). To repaired by HR, GFP is expressed and levels can be quantitated demonstrate whether activated Akt1 controls MDC1 expression using flow cytometry (Fig. 4C). Consistent with the role of through miR-22, we cotransfected U2OS and HCT116 cells with activated Akt1 in HR (23–25), cells overexpressing CA-Akt1 had the CA-Akt1 expression vector and a miR-22 antisense oligonu- significantly reduced HR efficiency (Fig. 4D, middle bar). Intrigu- cleotide (anti–miR-22). We found that inhibition of endogenous ingly, anti–miR-22 treatment enhanced the recovery of HR activity miR-22 led to an increase in MDC1 protein and mRNA in CA- in CA-Akt1–overexpressing U2OS DR-GFP cells (Fig. 4D, third Akt1–overexpressing cells (Fig. 3C and D). Moreover, CA-Akt1 bar). Moreover, the efficiency of HR significantly improved with significantly inhibited MDC1 30-UTR luciferase activity relative to miR-22–insensitive MDC1 transfection (Fig. 4E and F). Together, control (Fig. 3E). Under these experimental conditions, CA-Akt1– these results suggest that induction of miR-22 and subsequent induced decrease of luciferase activity was significantly attenuated downregulation of MDC1 are responsible for the repression of HR when anti–miR-22 was introduced, suggesting that CA-Akt1 neg- in high Akt1-activated cells. atively regulates MDC1 via miR-22. It has been reported that miR-22 is frequently overexpressed Repression of MDC1 by miR-22 in senescent cells in prostate tumor tissues, and its expression is positively Given that our data support a role for miR-22 in abrogation of correlated with the level of phosphorylated Akt1 (pAkt1; MDC1 expression and its DDR function and recent studies have ref. 26). Using this system, we asked whether Akt1 activity shown that miR-22 family is upregulated during replicative senes- directly affects MDC1 levels in vivo. To this end, we examined cence of human fibroblasts (21), we next sought to assess whether levels of the pAkt1 and MDC1 in prostate tumor tissue array by upregulation of miR-22 could downregulate MDC1 expression immunohistochemistry. We observed an increase in the level of during both replicative senescence and premature stress-induced Ser473 phosphorylation of Akt1 in the prostate tumor speci- senescence. Senescence was induced in both human embryonic mens, which was the region with the low MDC1 expression lung fi broblasts MRC-5 and IMR-90 cells by either serial passaging (Fig. 3F, left panel). On the other hand, specimens with weak or by treatment with either hydrogen peroxide (H2O2) or busulfan pAkt signals had strong MDC1 staining (Fig. 3F, right panel). (BU). As expected, the level of miR-22 was markedly higher in
Figure 5. Cellular senescence leads to miR-22–mediated MDC1 deficiency. A, senescence in MRC-5 and IMR-90 cells was induced by either serial passage (R-S) or through treatment with H2O2 (H-S). Representative images for cell morphology and SA-b-gal activity in young (Y) and senescent cells are shown. The histograms (bottom) show the percentage of SA-b-gal–positive cells (left) and miR-22 levels (right). Results are shown as mean SD (n ¼ 3). , P < 0.01. B and C, the level of MDC1 protein (B) and mRNA (C) in young (Y), replicative senescent (R-S), and H2O2-induced premature senescent (H-S) cells. Results are shown as mean SD (n ¼ 3). , P < 0.01. D, rescue of MDC1 expression level by transfecting anti–miR-22 into H2O2-induced premature senescent cells. Results are shown as mean SD (n ¼ 3). , P < 0.01. E, MDC1 foci formation in young and H2O2-induced premature senescent cells, with and without IR treatment and with and without anti–miR-22. Results are shown as mean SD (n ¼ 3). , P < 0.01.
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replicatively senescent cells than in young cells. Like replicative Inverse correlation between MDC1 and miR-22 expression in senescent cells, H2O2- and BU-induced prematurely senescent aged human and mouse tissues cells also showed much higher miR-22 expression compared with We next sought to assess whether miR-22 expression inversely control young cells (Fig. 5A; Supplementary Fig. S6A). Impor- correlates with the MDC1 expression in aged human and mouse tantly, all of these senescent cells showed significantly reduced tissues. To test this, we measured miR-22 and MDC1 expression MDC1 protein and mRNA (Fig. 5B and C; Supplementary Fig. S6B levels in lung and colon tissues from young and old mice using and S6C). Thus, downregulation of MDC1 is considered as a real-time qPCR and Western blotting. For both lung and colon general phenomenon in cells undergoing replicative senescence or tissues, the older mice had higher levels of miR-22 (Fig. 7A) stress-induced premature senescence. and lower levels of both MDC1 mRNA and protein expression The DDR function of MDC1 during senescence was then (Fig. 7B) than younger mice. Based on our previous observations, examined by immunofluorescence for IR-induced MDC1 foci it is very likely that the decreased MDC1 levels in lung and colon formation. When young cells were exposed to IR, clearly visible tissues in older mice were a direct result of the high levels of miR- MDC1 foci formed (Supplementary Fig. S7A and S7B). In con- 22. We were able to show the same effect in human cells using trast, induction of MDC1 foci formation was significantly reduced peripheral blood mononuclear cells from young (n ¼ 7) and old in replicatively senescent and H2O2-induced prematurely senes- (n ¼ 11) human donors (Fig. 7C). Furthermore, the same was true cent cells. To confirm the role of miR-22 in this process, we for human tissue samples. When colon biopsies from young (17 transfected anti–miR-22 into H2O2-induced prematurely senes- and 24 years old) and old (64 and 68 years old) donors were cent cells and showed that MDC1 levels (Fig. 5D) and IR-induced subjected to immunohistochemical analysis, staining for MDC1 MDC1 foci formation (Fig. 5E) were restored. was much darker in younger colon tissue than the older tissue, again showing that MDC1 abundance decreases with aging (Fig. miR-22 inhibits DNA repair by downregulating MDC1 7D). Thus, miR-22–mediated downregulation of MDC1 expres- expression in senescent cells sion is a common mechanism in aging cells and is utilized in We then determined whether miR-22 inhibits MDC1 expres- diverse and widespread tissue types in mammals. sion contributing to repressing DSB repair in senescent cells. To test this, we introduced either anti–miR-22 or miR-22–insensitive MDC1 into senescent cells and examined the effect on DSB repair. Discussion Young cells that were exposed to IR repaired the majority of DSBs Akt1 is a serine/threonine kinase, which is a key downstream within 6 hours (Supplementary Fig. S8). In contrast, senescent target of the signaling pathway mediated by PI3K, and plays a cells still had numerous unrepaired DSBs 6 hours after IR pivotal role in the regulation of diverse cellular process, including exposure, but this effect was reversed if anti–miR-22 was cell growth, proliferation, and survival (32). Aberrant activation present (Fig. 6A). The DSB repair defect in senescent cells was of the PI3K/Akt1 pathway is a common event in a wide range of also fully rescued by overexpressing miR-22–insensitive MDC1 human cancers (22). Activated Akt1 stimulates NHEJ repair, (Fig. 6B; Supplementary Fig. S9), suggesting that miR-22 acts to which contributes to chemo- or radioresistance in some tumor regulate DSB repair in senescent cells by modulation expression cells with constitutive Akt1 activation (33–35). In contrast, acti- of MDC1. vation of Akt1 inhibits HR due to suppression of the DDR under Several studies have proposed that miR-22 is a tumor suppres- pathologic circumstances (23–25, 36). Because defective HR can sor because it induces senescence-like phenotypes in cancer lead to genome instability and predisposition to cancer (37), the cells and it triggers both growth suppression and apoptosis inhibition of HR by the activation of Akt1 may contribute to (21, 28–31). However, our data highlight an oncogenic function tumorigenesis. However, the precise mechanism by which acti- of miR-22, in which it inhibits DSB repair and consequently vated Akt1 exerts its influence on HR needs to be elucidated. In the increases the risk of genomic instability, a hallmark of cancer present study, we detected a decline in HR in cells expressing cells. Thus, we examined whether DNA damage accumulation constitutively active Akt1. This decline in HR was associated with accompanied miR-22–induced cellular senescence in cancer cells. the upregulation of miR-22, which caused the loss of MDC1 To this end, human breast cancer cells (MCF7 and MDA-MB-231) function. These data show that CA-Akt1 can reduce the efficiency and human cervical cancer cells (si-Ha) were transiently trans- of HR following DSBs, which correlates with the decreased fected with the miR-22, exposed to IR, and then stained cells recruitment of MDC1 to sites of DNA breaks. These results also for foci of g-H2AX, a marker of unrepaired DSBs. Consistent showed that both the inhibition of miR-22 and overexpression of with a previous report (21), introduction of miR-22 into cancer MDC1 completely restored the function of MDC1 in the DDR and cells caused a senescence-like phenotype, as observed by the HR in cancer cells with high Akt1 activity. This scenario clarifies increased senescence-associated b-galactosidase (SA-b-gal) how elevated Akt1 activity might increase genomic instability and activity (Supplementary Fig. S10). Notably, miR-22–induced foster an environment for cancer development. Remarkably, this senescent cells showed a dramatic reduction in MDC1 expres- inverse correlation between pAkt1 and MDC1 expression occurs sion (Fig. 6C, middle lanes in each panel) and a significant in vivo in human prostate tumor tissues. Thus, miR-22–mediated increase in the number of unrepaired DSBs as compared with MDC1 downregulation could be an underlying mechanism control cells (Fig. 6D, middle row in each panel). However, behind Akt1-mediated oncogenesis and anti–miR-22 may be a when miR-22–induced senescent cells were transfected with new potential therapeutic agent for Akt1-induced tumorigenesis. miR-22–insensitive MDC1 (Fig. 6C, third lanes in each panel), During cellular senescence or organismal aging, mammalian the suppression of the DSBs repair was almost abolished (Fig. cells accumulate mutations in their genome and often end up 6D, bottom rows in each panel). These results suggest that the with abnormal chromosomal rearrangements (38). These miR-22–mediated decrease in MDC1 plays a critical role in the mutations and genomic rearrangements arise from aberrant accumulation of DSBs in senescent cells. DSB repair (39–42). However, the molecular mechanism for
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A Young Senescence B Young Senescence Anti-ctrl Anti-ctrl Anti–miR-22 Mock Mock MDC1 cDNA MRC5 MRC5 IMR-90 IMR-90
MRC-5 IMR-90 MRC-5 IMR-90 15 12 12 20
12 9 9 15 9 6 6 10 6 3 3 5 3 Comet tail moment moment Comet tail(%) Comet tail moment moment (%) Comet tail 0 0 0 0 Ant–miR-22 MDC1 cDNA Young Senescence Young Senescence Young Senescence Young Senescence
C MCF7 MDA-MB-231 Si-Ha D MDC1 cDNA miR-22
HA-MDC1 MCF7 MDA-MB-231 Si-Ha γ γ γ miR-22 MDC1 cDNA MDC1 -H2AX DAPI -H2AX DAPI -H2AX DAPI
α-Tubulin
1.2 1.5 1.8
0.9 1.2 1.2 0.9 0.6 0.6 0.6
protein level protein level 0.3 Relative MDC1 0.3
0 0 0 MDC1 cDNA 75 80 80 miR-22 60 60 60
γ -H2AX 45 3.5 8 15 40 40 30
2.8 12 (%)staining 6 20 20 Relative 15 2.1 9 4 0 0 0 1.4 6 MDC1 cDNA
expression 2 0.7 3 miR-22 Relative miR-22 0 0 0 MDC1 cDNA miR-22
Figure 6. miR-22–mediated downregulation of MDC1 suppresses DNA repair in senescent cells. A, transfection of H2O2-induced premature senescent cells with anti–miR-22 increased DSB repair upon IR exposure, as measured by the comet assay. Results are shown as mean SD (n ¼ 3). , P < 0.01. B, comet assay revealed that overexpression of miR-22–insensitive MDC1 rescued DSB repair in H2O2-induced premature senescent. Results are shown as mean SD (n ¼ 3). , P < 0.01. C, the level of MDC1 protein was measured in MCF7, MDA-MB-231, and Si-Ha cells transfected with miR-22 or together with miR-22 and HA-MDC1. Middle, quantitation of Western blot analysis. Bottom, miR-22 levels. Results are shown as mean SD (n ¼ 3). , P < 0.01. D, miR-22–induced senescent cancer cells are defective in DSB repair as shown by increased g-H2AX staining. miR-22–insensitive MDC1 expression decreased g-H2AX staining. Results are shown as mean SD (n ¼ 3). , P < 0.01.
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miR-22 Impedes DDR Function of MDC1
A B Lung Colon Young Aging Young Aging Lung Colon MDC1 2 2.5 α-Tubulin 2 1.5 1.2 1.2 1.5 1 0.9 0.9 1 0.6 0.6 0.5 0.5 0.3 0.3 Relative miR-22 expression Relative miR-22 expression 0 0 Young Aging Young Aging 0.0 0 Relative MDC1 mRNA level Young Aging Young Aging
P = 0.0024 P = 0.045 1.5 C 2.0
Figure 7. 1.5 Inverse correlations between miR-22 1.0 and MDC1 expression in aging tissues. A, expression of miR-22 was 1.0 measured using lung and colon tissues collected from young 0.5 (6 months) and old (23 months) 0.5 mice. Results are shown as