Loss-of-function mutations of retinoblastoma family (Rb) proteins drive tumorigenesis by overcoming barriers to cellular proliferation. Consequently, factors modulating Rb function are of great clinical import. Here, we show that miR-335 is differentially expressed in human cancer cells and that it tightly regulates the expression of Rb1 (pRb/p105) by specifically targeting a conserved sequence motif in its 3′ untranslated region. We found that by altering Rb1 (pRb/p105) levels, miR-335 activates the p53 tumor suppressor pathway to limit cell proliferation and neoplastic cell transformation. DNA damage elicited an increase in miR-335 expression in a p53-dependent manner. miR-335 and p53 cooperated in a positive feedback loop to drive cell cycle arrest. Together, these results indicate that miR-335 helps control proliferation by balancing the activities of the Rb and p53 tumor suppressor pathways. Further, they establish that miR-335 activation plays an important role in the induction of p53-dependent cell cycle arrest after DNA damage. Cancer Res; 70(17); 6925–33. ©2010 AACR.
In mammals, a major tumor suppressor pathway is centerd on the family of retinoblastoma (Rb) proteins, consisting of Rb1 (pRb/p105), Rbl1 (p107), and Rbl2 (p130; refs. 1–3). Rb family proteins control cell cycle genes through interaction with the E2F family of transcription factors, as well as by direct recruitment of chromatin regulators to promoters (4, 5). However, although the Rb pathway is essential for cell cycle control and tumor suppression, information on microRNAs (miRNA) controlling the expression of Rb family proteins at the posttranscriptional level is limited. MiRNAs are 19- to 25-nucleotide noncoding RNAs that are critically involved in the regulation of gene expression by directing posttranscriptional gene repression. miRNAs can alter the expression of entire sets of genes and have been shown to act as targets and effectors in the aberrant control of tumor suppressors and oncogenes in human malignancies (6–10). The discovery of a cluster of six miRNAs (miR-290 cluster) targeting Rbl2 in mice suggested the existence of a miRNA network controlling Rb pathway activity (11, 12). Interestingly, whereas the miR-290 cluster lacks conservation in humans, an additional miRNA, miR-335, was predicted to target mammalian Rb1 in a conserved manner (11, 13). Recently, miR-335 was found to be upregulated in a set of human multiple myeloma cell lines and to inhibit metastatic cell invasion of breast cancer cells, indicating that miR-335 is implicated in human cancer (9, 14).
Here, we analyzed the role of miR-335 in the context of cell proliferation and show that miR-335 tightly controls the expression of human and mouse Rb1 by targeting its 3′ untranslated region (UTR) with high efficiency. High conservation of miR-335 and its Rb1 target sequence in placental mammals as well as differential expression in human cancer cell lines indicates an important role in cell cycle control. The impairment of the Rb pathway by miR-335 is paralleled by a significant upregulation of p53 levels resulting in reduced proliferation. Reducing p53 levels in the context of miR-335 overexpression is sufficient to drive proliferation and increased transformation. We further show that miR-335 is significantly upregulated on DNA damage in a p53-dependent manner and show that miR-335 mediates efficient cell cycle arrest on DNA damage in a positive feedback loop with p53.
We conclude that miR-335 controls cell proliferation in a p53-dependent manner, thus connecting the activities of the Rb and p53 tumor suppressor pathways.
Materials and Methods
Cell lines and cell cultivation
All cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and have not been cultured in the laboratory for longer than 6 months. ATCC authentication testing procedure includes Mycoplasma test, bacteria or fungi contamination, cellular contamination, cytochrome c oxidase subunit I testing, short tandem repeat analysis, cytogenetic analyses (G-banding, fluorescence in situ hybridization), flow cytometry, and immunohistochemistry. H1299 cells were cultured in RPMI medium, 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) penicillin/streptomycin. All other cell lines used in this study were maintained in DMEM, 10% (v/v) FBS, and 1% (v/v) penicillin/streptomycin. Cell growth was measured in triplicate after plating 5 × 104 cells. Cell number was counted at the indicated time points using a hemocytometer. Stable transfections of plasmids were performed using Fugene6 (Roche Applied Science); selection was done using blasticidin (10 μg/mL; U2OS-miR control and U20S-miR-335). Cells were treated with etoposide (5 μmol/L), actinomycin D (5 nmol/L), nocodazole (50 ng/mL), and 5-aza-cytidine (5 μmol/L) for the indicated duration.
Transient transfections of miRNAs or siRNA oligos
U2OS, H1299, MG-63, NIH 3T3, and C2C12 cells were transiently transfected with hsa_miR-335 mimic (C-300708-05, Thermo Scientific Dharmacon), antagomiR-335 hairpin miR-IDIAN inhibitor (IH-300708-07, Thermo Scientific Dharmacon), or a mix of all miR-290 cluster mimics (mmu_miR-290, mmu_miR-291-3b, mmu_miR-292-3b, mmu_miR-293, mmu_miR-294, and mmu_miR-295; Thermo Scientific Dharmacon) according to the manufacturer's suggestions. MiR-IDIAN miRNA mimic negative control miRNA (CN-00100-01-05, Thermo Scientific Dharmacon) was used as a negative control. Mimic miRNAs resemble siRNA molecules that recapitulate miRNA function. Total RNA and protein were prepared 72 hours after transfection. U2OS cells were transiently transfected with a siRNA against Rb1 (Thermo Scientific Dharmacon; sense sequence: GGAAGGACAUGUGAACUUAUU, antisense sequence: UAAGUUCACAUGUCCUUUCUU) or siRNA control siGENOME Non-Targeting siRNA #1 (D-001210-01-20, Thermo Scientific Dharmacon) according to the manufacturer's suggestions.
Analysis for miRNA expression
Total RNA was extracted and purified using TRIzol reagent (Gibco/BRL) and reverse transcribed using the miR-335 and RNU48 TaqMan MicroRNA Assay system (Applied Biosystems). The human snoRNA RNU48 was used as a reference. The stem-loop real-time PCR (RT-PCR) was performed according to conditions suggested by Applied Biosystems. Quantitative miRNA expression data were analyzed using a StepOnePlus Sequence Detection System (Applied Biosystems).
Western blot analysis and antibodies
Western blot analysis was performed according to standard procedures using the following primary antibodies: purified mouse anti-human Rb1 (BD Pharmingen), monoclonal anti-p53 (DO-1; Santa Cruz Biotechnology), mouse monoclonal anti-p53 (1C12; Cell Signaling Technology), rabbit polyclonal anti-p14ARF (Abcam), and anti-actin (Sigma). Secondary antibodies coupled to horseradish peroxidase were obtained from Sigma (antirabbit IgG peroxidase conjugate A-6154; antimouse IgG peroxidase conjugate A-4416).
Immunofluorescence analysis and 5-bromo-2′-deoxyruridine incorporation
Immunofluorescence and 5-bromo-2′-deoxyruridine (BrdUrd) incorporation were performed as previously described (15). Slides were analyzed on a Leica DM 4000B fluorescence microscope.
Determination of luciferase and cell cycle profile
Luciferase activity was determined using the Dual Luciferase kit (Promega) and was performed as previously described (16). Cell cycle profile was analyzed using a fluorescence-activated cell sorting (FACS) flow cytometer (FACSCalibur, Becton Dickinson).
miR-335 as a novel regulator of the tumor suppressor Rb1
In a recent study, miR-335 was predicted to target mouse Rb1; however, biological evidence remained unaddressed (11). Here, we show that miR-335 is identical in a series of placental mammalian species (Fig. 1A; Supplementary Fig. S1A). Importantly, classic sequence features known to boost target recognition, such as the seed region (nucleotides 2–10), additional base pairing at nucleotides 13 and 16, and the AU-rich sequence composition near the end of the miR-335 seed region, are highly conserved in the Rb1 3′UTR of placental mammals (ref. 17; Fig. 1A). We show by quantitative stem-loop RT-PCR that miR-335 is widely expressed in cell lines derived from human tumors (Fig. 1B). Of notice, miR-335 levels were close to the limit of detection in the U2OS and MG-63 osteosarcoma cell lines (Fig. 1B). Due to the fact that U2OS cells carry Rb1 and p53 wild-type alleles, we used U2OS cells as a gain-of-function model system to study the role of miR-335 in controlling the Rb pathway.
Transient transfection of U2OS cells with miR-335 efficiently reduced Rb1 protein levels, as detected by Western blotting and immunofluorescence analysis (Fig. 1C; Supplementary Fig. S1B). Control miRNA or the pooled mouse miR-290 cluster did not significantly affect Rb1 protein levels (Fig. 1C, left). Importantly, cotransfection of miR-335 with miR-335 antagomir molecules (antagomiR-335) rescues Rb1 protein levels (Fig. 1C, right). In addition, miR-335 efficiently reduced Rb1 protein levels in NIH 3T3 embryonic fibroblasts and C2C12 myoblast cells, indicating that target recognition by miR-335 is conserved also in mouse cells (Supplementary Fig. S1C and D). Next, we aimed to show that miR-335 directly controls human Rb1 expression by targeting the Rb1 3′UTR. Transient overexpression of miR-335 efficiently reduced the expression of a luciferase reporter fused to a region of the human Rb1 3′UTR containing the predicted miR-335 targeting region (position 358–380; Fig. 1A), but did not have an effect when the target region was deleted (Fig. 1D). We conclude that miR-335 efficiently controls Rb1 expression by directly targeting a sequence motif in the 3′UTR of Rb1 in a conserved manner.
miR-335 impairs proliferation and transformation
Retinoblastoma proteins play a major role in controlling cell cycle (5). Although loss of Rb1 is expected to be associated with increased cell proliferation, we found that U2OS cells display significantly reduced cell proliferation rates when transiently transfected with miR-335 (Fig. 2A, left; Supplementary Fig. S2A). Cell cycle profiles revealed that transfection of miR-335 causes an accumulation of cells in G0-G1 phase and reduced cell numbers in S phase (Fig. 2A, bottom). Consistent with this, we found that the incorporation of BrdUrd into the DNA of replicating cells was significantly reduced in these cells (Fig. 2A, right; Supplementary Fig. S2B). Importantly, transient overexpression of the Rb1 cDNA lacking its 3′UTR rescues impaired proliferation, BrdUrd incorporation, and cell cycle regulation in the context of ectopically increased miR-335 levels (Fig. 2B). We conclude that reduced cell proliferation and delayed S-phase entry are a direct consequence of targeting Rb1 by miR-335. Soft-agar colony formation assays are the most commonly used in vitro assays to determine anchorage-independent growth, a hallmark of cell transformation. We generated U2OS cells stably overexpressing miR-335 (Fig. 2C, left) and show that elevated miR-335 levels are associated with a significant impairment of colony formation (Fig. 2C, right).
Taken together, our results indicate that increased miR-335 levels activate a cellular response that compensates the growth advantage arising from impaired Rb pathway activity, resulting in reduced proliferation and neoplastic transformation in vitro.
miR-335 controls proliferation in a p53-dependent manner
Recently, inactivation of the Rb pathway in the developing human retina was shown to lead to activation of the ARF-MDM2-MDMX-p53 tumor surveillance pathway, thereby limiting proliferation (18). To test whether miR-335 can induce activation of the p53 pathway, we studied the Rb1 and p53 tumor suppressor pathways in U2OS cells in the context of altered miR-335 levels.
We found that the transient transfection of U2OS cells with miR-335 is associated with a concomitant 5-fold increase in p53 protein levels (Fig. 3A). In functional analogy, siRNA-mediated knockdown of Rb1 in U2OS cells was found to drive a marked increase of p53 protein levels whereas the transient overexpression of Rb1 readily reduced the expression of p53 (Supplementary Fig. S3A and B), thus confirming a functional link between the Rb and p53 tumor suppressor pathways in our model system. Importantly, reporter constructs containing a luciferase reporter under the control of promoters of the p53 target genes Mdm2, PG13, PIG3, p21, and Bax (16) were efficiently upregulated in U2OS cells with increased miR-335 levels (Fig. 3B). In line with this, reducing Rb1 expression in NIH 3T3 mouse embryonic fibroblasts and C2C12 cells using miR-335 is linked with a concomitant increase in p53 protein levels (Supplementary Fig. S1C and D). We conclude that limiting Rb levels by miR-335 results in a compensatory activation of the p53 pathway, aimed to protect from loss of Rb-dependent cell cycle regulation and uncontrolled proliferation of human and mouse cells.
We next addressed whether the activation of the p53 is responsible for reduced proliferation in the context of miR-335 overexpression. We reduced p53 levels in U2OS cells by stably expressing shRNAs directed against p53 (U2OS sh p53; Supplementary Fig. S3C) and used these cell lines to constitutively overexpress miR-335 or a control miRNA. miR-335 overexpression was confirmed by quantitative stem-loop RT-PCR (data not shown). As expected, stable overexpression of miR-335 in U2OS sh Control cells resulted in significantly reduced proliferation when compared with U2OS sh Control cells that stably express a control miRNA (Fig. 3C, left). Importantly, stable expression of miR-335 in U2OS cells with reduced p53 levels (U2OS sh p53) and transient transfection of miR-335 into the p53-deficient osteosarcoma cell line MG-63 significantly accelerates cell proliferation (Fig. 3C, right; Supplementary Fig. S3D). Consistent with this, we found that reduced soft-agar colony formation of sh Control U2OS cells overexpressing miR-335 (U2OS sh Control; Fig. 3D, left) was compensated by reducing p53 levels using stable shRNAi (U2OS sh p53; Fig. 3D, right). We conclude that reducing Rb1 protein by miR-335 overexpression activates the p53 tumor suppressor pathway to limit cell proliferation and in vitro transformation.
miR-335 drives hyperproliferation in the absence of p53
Next, we wished to confirm that cell proliferation is controlled by miR-335 in a p53-dependent manner. The human, p53-deficient non–small cell lung carcinoma cell line H1299 efficiently expresses miR-335, allowing the use of this cell line in miR-335 gain-of-function and loss-of-function experiments (Fig. 1B). Transient transfection of H1299 cells with miR-335 resulted in a significant reduction of Rb1 protein levels, accelerated cell proliferation, and neoplastic cell transformation due to the absence of the p53 tumor suppressor pathway (Fig. 4A and B). These findings are in line with the results obtained with MG-63 cells (Supplementary Fig. S3D). Importantly, we show that reducing endogenous miR-335 levels in H1299 cells by stable overexpression of antagomiR-335 results in a significant reduction of cell proliferation and impaired colony formation in soft-agar assays (Fig. 4C). Elevated Rb1 protein levels in antagomiR-335–expressing H1299 cells were confirmed by Western blotting (Fig. 4D). Together, our results show that miR-335 induces hyperproliferation and increases transformation in vitro in the absence of the p53 tumor suppressor pathway. We propose that miR-335 has an important role in balancing the Rb and p53 tumor suppressor pathways, affecting the control of proliferation and immortalization.
miR-335 and p53 cooperate in a positive feedback loop to drive DNA damage–mediated cell cycle arrest
Cellular insults such as DNA damage or oncogenic stress lead to the activation of the p53 pathway to induce cell cycle arrest or apoptosis (19, 20). To address whether miR-335 plays a role in the induction of p53-dependent cell cycle arrest, we measured miR-335 expression levels after induction of DNA damage in U2OS cells. Treatment with the DNA-damaging agents etoposide or actinomycin D causes cell cycle arrest in G1 or G2-M phase that is paralleled by an efficient increase in p53 protein levels and miR-335 expression (Fig. 5A; Supplementary Fig. S4A). In contrast, the microtubule-damaging agent nocodazole causes only a slight upregulation of p53 without significantly affecting miR-335 expression (Fig. 5A; Supplementary Fig. S4A). Importantly, actinomycin D or etoposide treatment of U2OS cells with reduced p53 levels (U2OS sh p53) and p53-deficient MG-63 cells does not result in increased miR-335 levels (Fig. 5B; Supplementary Fig. S4B and C). In addition, transient transfection of MG-63 cells with p53 causes an efficient increase in miR-335 levels that is paralleled by upregulation of the MEST (mesoderm specific transcript) gene that encodes miR-335 in intron 2 (Supplementary Fig. S5A). Together, these data indicate that miR-335 levels are increased during DNA damage by upregulation of MEST in a p53-dependent manner. Importantly, MEST luciferase reporter assays in MG-63 cells transiently transfected with p53 confirmed the presence of a p53-responsive promoter element at the transcriptional start site of the MEST-002 transcript (Supplementary Fig. S5B and C). To further investigate the role of p53 in the activation of MEST transcription, we induced DNA damage in U2OS cells and performed chromatin immunoprecipitation experiments. As expected, we found an efficient enrichment of p53 at the p21 promoter in etoposide-treated cells (Supplementary Fig. S5B and D). However, we were not able to detect p53 enrichment at consensus p53 binding sites in the MEST locus of cells after the induction of DNA damage (Supplementary Fig. S5B and D). Our results indicate that p53 controls miR-335 levels, but uses an indirect mechanism to control the transcription of the miR-335 encoding the MEST gene.
Downregulation of Rb1 by miR-335 mediates cell cycle arrest by activating the p53 tumor suppressor pathway (Fig. 3A and B). To test whether the upregulation of miR-335 is required for DNA damage–mediated cell cycle arrest, we assessed cell cycle profiles and BrdUrd incorporation of U2OS cells stably overexpressing miR-335 or antagomiR-335 after treatment with etoposide. As expected, overexpression of miR-335 results in a significantly delayed S-phase entry and reduced BrdUrd incorporation in untreated U2OS cells (Fig. 5C and D, top; Supplementary Fig. S6). AntagomiR-335 does not affect cell cycle regulation due to the fact that miR-335 levels are close to the detection limit in U2OS cells (Fig. 5C and D, top; Supplementary Fig. S6). In contrast, antagonizing the upregulation of miR-335 in etoposide-treated U2OS by stable overexpression of antagomiR-335 causes a significant 50% increase in the number of cells in S phase, reduced cell number in G1 phase, and a higher number of BrdUrd-positive cells, when compared with control cells or miR-335 overexpressing U2OS cells (Fig. 5C and D, bottom; Supplementary Fig. S6). Of notice, ectopic expression of miR-335 does not improve etoposide induced cell cycle arrest, suggesting that the activation of the miR-335 pathway by DNA damage has already reached saturation in our model system. Together, our data show the existence of an important connection between miR-335 and the p53 tumor suppressor pathway during DNA damage response. DNA damage activates the p53 pathway, driving the upregulation of miR-335. miR-335 represses Rb1 at the posttranscriptional level, enforcing the activation of the p53 tumor suppressor pathway. Together, these data indicate that miR-335 and p53 operate in a positive feedback loop to ensure efficient cell cycle arrest upon DNA damage (Fig. 6).
The retinoblastoma and p53 pathways represent the major tumor suppressor pathways in mammals and are crucial for the control of cell proliferation and the response to cellular insults (19–21). Rb is frequently mutated or expressed at low levels in several tumors such as retinoblastoma, osteosarcoma, as well as small lung, prostate, bladder, and breast carcinomas (22–26). Thus, miRNAs controlling Rb protein levels are of high clinical interest. Here, we show that miR-335 efficiently controls Rb1 (pRb/p105) protein levels by directly targeting a conserved region in the 3′UTR of Rb1 in placental mammals. Our data show that miR-335–mediated reduction of Rb1 protein results in the activation of the p53 pathway in human and mouse cells, impairing cell proliferation and neoplastic transformation in vitro. In line with this, impairing the p53 pathway is sufficient to drive hyperproliferation and increased transformation in the context of ectopically increased miR-335 levels. We conclude that miR-335 has an important role in balancing the p53 and Rb tumor suppressor pathways.
In normal cells, the Rb and p53 pathways integrate stimuli such as DNA damage and other stress signals, leading to cell cycle arrest and senescence. Activation of the p53 pathway represents the primary defence system against DNA double-strand breaks (27, 28). In this pathway, ARF inhibits HDM2 activity, leading to p53 stabilization and transcriptional activation of p21-mediated cell cycle arrest. Activation of the Rb pathway was postulated to occur as a secondary event to the activation of p53 by involving p21 (29, 30). The inhibition of cyclin-dependent kinases (CDK) by p21 and p16 keeps pRb in the hypophosphorylated form, thereby preventing E2F from transcribing S-phase genes (21). Several studies suggest that the p53 and Rb pathways are functionally connected: high E2F activity on Rb loss of function can lead to the activation of ARF expression and activation of p53; in addition, p21 inhibits CDKs, leading to hypophosphorylation of pRb (18, 31, 32). In our study, we show that activation of the p53 pathway during DNA damage results in transcriptional activation of the pri-miR-335 transcript MEST-002, leading to high miR-335 levels. Importantly, antagonizing the upregulation of miR-335 levels significantly impairs cell cycle arrest on DNA damage. The fact that miR-335–mediated reduction of Rb1 drives the activation of the p53 pathway suggests that one function of miR-335 upregulation during DNA damage is to reduce the Rb pathway and to enforce p53-mediated cell cycle arrest. Recent reports indicated that the outcomes of activation of the p53 and Rb pathways are distinct: Cells that activate the p53 pathway can resume growth after inactivation of p53, whereas cells that fully engage the Rb pathway for several days cannot reenter into cell cycle presumably due to heterochromatic silencing of E2F target genes and the formation of senescence-associated heterochromatic foci (33–35). Based on our results, we propose that miR-335 antagonizes the establishment of an irreversible growth arrest by Rb through promoting p53-mediated cell cycle arrest. The current model of Rb and p53 interaction proposes that high E2F activity can activate the p53 pathway. However, in our U2OS model system, the p16INK4a and p14ARF promoters are silenced by DNA methylation (36). We were speculating that miR-335 could interfere with the transcriptional silencing of p14ARF or modify E2F activity. However, ectopically increased miR-335 levels do not increase ARF protein levels or E2F activity in U2OS cells (Supplementary Fig. S7A and B). Instead, we show that miR-335 overexpression increases p53 at the protein level but does not significantly affect steady-state p53 RNA levels (Fig. 3A; Supplementary Fig. S7C). This suggests that miR-335/Rb1 uses an alternative pathway to increase p53 stability in U2OS cells. It is interesting to speculate that reducing Rb1 levels by miR-335 could drive increased rDNA transcription leading to ribosomal stress, which was shown to activate and stabilize p53 (19, 37, 38). In addition to its central role in cell cycle regulation and tumor suppression, pRb acts as a transcriptional regulator that controls the expression of DNA methyltransferases and transcription factors controlling development. This suggests that miR-335 could be an important factor in tuning the activity of Rb1 during organismal development.
Our data identify miR-335 as a potent regulator of Rb1 at the posttranscriptional level connecting the Rb1 and p53 tumor suppressor pathway activities. Beside a critical role in the execution of a p53-mediated cell cycle arrest during DNA damage response, we also want to propose that miR-335 could be a relevant factor that could increase cancer susceptibility under conditions with impaired p53 tumor suppressor activity by unleashing cells from the control of the Rb tumor suppressor pathway.
Future work will be aimed toward the dissection of the diverse roles of miR-335 balancing the Rb and p53 pathways in tumor suppression, development, and DNA damage response.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Andrea Bisso (Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB); Trieste, Italy) for providing the U2OS sh Control and sh p53 cell lines. M. Scarola performed all experiments shown in the article. S. Schoeftner generated cell lines and constructs. R. Benetti designed the experiments together with C. Schneider and M. Scarola. R. Benetti prepared the manuscript figures and wrote the paper together with S. Schoeftner.
Grant Support: Italian Association for Cancer Research (Associazione Italiana per la Ricerca sul Cancro) grant Rif 42/08, 6352 (R. Benetti).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received January 14, 2010.
- Revision received June 14, 2010.
- Accepted June 22, 2010.
- ©2010 American Association for Cancer Research.