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Title: ATM–Dependent MiR-335 Targets CtIP and Modulates the DNA Damage Response}
Article:
Abstract:
ATM plays a critical role in cellular responses to DNA double-strand breaks( DSBs). We describe a new ATM–mediated DSB–induced DNA damage response pathway involving microRNA( miRNA): irradiation( IR)-induced DSBs activate ATM, which leads to the downregulation of miR-335, a miRNA that targets CtIP, which is an important trigger of DNA end resection in homologous recombination repair( HRR). We demonstrate that CREB is responsible for a large portion of miR-335 expression by binding to the promoter region of miR-335. CREB binding is greatly reduced after IR, corroborating with previous studies that IR-activated ATM phosphorylates CREB to reduce its transcription activity. Overexpression of miR-335 in HeLa cells resulted in reduced CtIP levels and post-IR colony survival and BRCA1 foci formation. Further, in two patient-derived lymphoblastoid cell lines with decreased post-IR colony survival, a “radiosensitive” phenotype, we demonstrated elevated miR-335 expression, reduced CtIP levels, and reduced BRCA1 foci formation. Colony survival, BRCA1 foci, and CtIP levels were partially rescued by miRNA antisense AMO-miR-335 treatment. Taken together, these findings strongly suggest that an ATM–dependent CREB–miR-335–CtIP axis influences the selection of HRR for repair of certain DSB lesions.
Introduction:
The DNA damage response( DDR) plays an essential role in deciding cell fate after DNA double strand breaks( DSBs) by arresting the cell cycle to allow evaluation of DNA integrity and signaling for repair or apoptosis[1]. In this way, the DDR maintains genomic stability and is an indispensable defense mechanism against cell death or tumor development[2],[3]. The molecular mechanisms of DSB-induced DDR have been extensively characterized and post-translational modifications of proteins, such as by phosphorylation, ubiquitinylation, sumoylation and acetylation, play a crucial role[4]. MiRNAs have recently emerged as endogenous gene regulators but their role in the DDR remains largely unexplored. MiRNAs downregulate protein expression by mRNA cleavage or translation repression[5], suggesting that miRNAs may be a new class of cellular regulators, targeting the protein components of the DDR pathways[6]. ATM( Ataxia-Telangiectasia Mutated) kinase plays a hierarchical role in the DSB-induced DDR[7],[8]. ATM coordinates many cellular processes of the ionizing radiation induced DDR starting with the phosphorylation of specific serines or threonines on downstream protein substrates[8],[9]. Three mechanisms have been reported to regulate ATM expression: 1) promoter methylation[10],[11], 2) transcription activators[12] and 3) miRNA interactions with mRNA[13]–[15]. Such modulation of ATM protein levels can result in the radiosensitization of cells[11],[16]. Classically, radiosensitization has been associated with mutations in the ATM gene that lead to loss of the protein and failure to activate downstream substrates. Recently, we have shown that ATM protein levels are reduced by miR-421 overexpression and that this reduction in ATM levels is sufficient to induce a cellular phenotype similar to that found in A-T patients[13]. The miR-421-mediated ATM downregulation was recently identified as a major factor associated with clinical radiosensitivity in a patient with squamous cell carcinoma[17]. Interestingly, the ATM/miRNA interaction is bidirectional. In addition to phosphorylating downstream protein targets, ATM also regulates the DDR by modulating the biogenesis of a subset of miRNAs by phosphorylating KH-type splicing regulatory protein( KSRP) which increases mature transcripts of a number of miRNAs[18]. Similarly, the breast cancer susceptibility gene, BRCA1, has been shown to modulate miRNA biogenesis[19]. BRCA1 plays an important role in avoiding tumor genesis likely through the maintenance of genomic stability by its role in cell cycle regulation and localization to DSBs thereby facilitating DNA repair[20]–[22]. Thus, ATM and BRCA1 highlight an intriguing connection between miRNA and the DDR. We have extensively profiled over 100 cell lines derived from patients with A-T-like phenotypes, e. g. reduced clonogenic survival levels characteristic of radiosensitive patients, but of primarily unknown etiology[23],[24]. These ‘radiosensitive’ lymphoblastoid cell lines( RS-LCLs) have been used to interrogate some of the mechanisms of DNA repair[25]–[28]. To complement the methodologies used in previous studies, we performed miRNA microarray measurements on RS-LCLs to identify miRNAs that associate with radiosensitivity and, thus, with DDR defects. In the present study we focus on one of these miRNAs, miR-335, which has been associated with increased metastatic and re-initiation potential of breast cancer when downregulated[29],[30]. MiRNA-335 is located within the mesoderm specific transcript homolog( MEST) gene on chromosome 7 which is a maternally imprinted gene[31]. We describe a new pathway triggered by ATM-dependent downregulation of miR-335 expression. We show that CREB( cAMP responsive element binding protein 1) binds to the MEST promoter and regulates miR-335 in an irradiation( IR)-dependent manner. Further, we demonstrate that this new pathway modulates the DDR via miR-335 suppression of CtIP( CtBP interacting protein/RBBP8), a protein that plays an important role in DNA end resection leading to the recruitment of BRCA1 to DSBs during homologous recombination repair( HRR)( Figure 1)[32]. We demonstrate dysfunction of these DDR mechanisms in two patient-derived RS-LCLs with constitutive miR-335 overexpression and rescue their radiosensitive cellular phenotypes by suppressing miR-335.
Discussion:
We have identified a novel ATM-mediated DDR pathway involving a CREB–miR-335–CtIP axis. The proposed pathway impacts upon BRCA1 foci formation and BRCA1 retention at DSBs, cell cycle checkpoint regulation, DNA repair, and cell survival. This regulatory pathway is responsive to DSBs and our working model places ATM kinase, and the inferred subsequent phosphorylation of CREB, as a novel down-regulating mechanism of MEST/miR-335 which supplements the suppression of maternally-derived transcripts by promoter hypermethylation( Figure 1)[29],[30],[36]. The methylation status of the MEST/miR-335 promoter region remained largely unchanged after IR, indicating that methylation does not play a significant role in the IR-induced modulation of miR-335( Figure S1B and S1C). The ChIP assay showed that CREB binds directly to the promoter region of MEST/miR-335( Figure 2G) and CREB siRNA significantly reduced basal miR-335 expression( Figure 2F), suggesting that CREB is responsible for a substantial portion of the basal miR-335 expression( Figure 2E). This made it technically difficult to fully assess the IR-induced response in cells where CREB was already knocked down. Previous studies indicate that ATM phosphorylates CREB after IR and this phosphorylation reduces the transcription activity of CREB[36]. Our data demonstrating the removal of CREB from the MEST/miR-335 promoter region by IR suggests that CREB is likely responsible for the IR-induced and ATM-dependent downregulation of miR-335. Tavazoie et al. discovered that miR-335 expression levels were significantly lower in some metastatic breast cancers and that overexpression of miR-335 suppressed tumor metastasis[29]. Interestingly, the MCF7 breast cancer cell line used in our study strongly expresses miR-335 and has reduced BRCA1 foci levels. BRCA1 mutations are associated with a large increase in the risk of breast cancer and our data suggest that miR-335 overexpression may be another mechanism by which the functions of this cancer-associated gene can be modulated. Our results also indicate that miR-335 overexpressing cells are more sensitive to IR-induced damage and that the significantly lower miR-335 expression found in the study by Tavazoie et al. may potentially render breast cancer cells more resistant to IR. This dual role of miR-335 might further explain why some metastatic breast cancers are more resistant to radiotherapy or chemotherapy. These studies suggest that miR-335 is a promising candidate as a radiosensitizer for breast cancer radiotherapy or chemotherapy if miRNAs could be used to target relevant genes. For example, overexpression of miR-335 should cause tumor cells to become more sensitive to IR or DSB-inducing agents, as observed in this study, and continued overexpression should suppress tumor metastasis and tumor re-initiation. Previous in vitro studies have described CtIP functions in the DDR as promoting DSB end-resection via the MRN complex[32], as well as helping to select the DNA repair mechanism activated after DSBs by promoting HRR via BRCA1 recruitment[37]. Thus, reductions in CtIP protein expression could have far-reaching consequences in the DDR, such as the effects on RDS, BRCA1 foci formation, and other DNA repair defects observed when miR-335 was overexpressed in HeLa or was endogenously overexpressed in RS7 and RS73 LCLs. These cells had reduced CtIP protein levels due to miR-335 overexpression and a cellular phenotype consistent with defects in the CtIP-BRCA1-mediated DDR[25],[28]. Transfection of CtIP to miR-335 overexpressing HeLa cells rescued the BRCA1 foci formation and clonogenic survival in these cells, further solidifying the notion that miR-335 modulates DDR through CtIP( Figure 4C and 4D). Interestingly, miR-335 overexpression also changed cell cycle distribution before IR( Figure S4). Given that CtIP has been previously shown to function in cell cycle regulation[22],[41],[43], it will be interesting to learn whether the miR-335-CtIP-regulated cell cycle affects the choice of DNA repair between non-homologous end joining and HRR. Our working model suggests that the downregulation of miR-335 by ATM–CREB in WT-LCLs could result in an increase in CtIP protein expression after IR-induced damage and subsequently enhance the DDR. However, significant changes in CtIP protein levels after IR were not observed in WT-LCLs, using the methods in this study. This suggests that, in WT-LCLs, the ATM–CREB–miR-335–CtIP axis plays a more subtle role in the DDR and most likely functions to ‘fine tune’ DNA repair. In contrast, RS7, RS73 and MCF7 cells illustrated the substantial influence that miR-335 can have on the DDR when grossly dis-regulated, such as by strong miR-335 overexpression. RS7 and RS73 have defects in post-IR DNA repair and cell cycle regulation and both, similarly, do not efficiently form or retain BRCA1 foci at the sites of DNA damage[25],[28]. Interestingly, both BRCA1 foci defects and reduced colony survival were responsive to suppression of miR-335 levels by AMO-miR-335. The partial abrogation of the defective DDR cellular phenotypes in these cells and in MCF7 cells( e. g. BRCA1 foci) suggests that miR-335 overexpression is responsible for many of the DDR defects observed in these cells and is an example of the substantial impact miR-335 can have upon the DDR. Human CtIP mutations have been implicated in Seckel and Jawad syndromes and cells from these patients present with hypersensitivity to DNA damage[44]. The present study suggests that miR-335 may be an additional mechanism by which CtIP can be disrupted in disease phenotypes and could present with similar clinical features( i. e., phenocopies) while lacking CtIP mutations.
ATM ( Ataxia-Telangiectasia Mutated ) serine/threonine kinase plays a critical role in coordinating the cellular response to DNA double-strand breaks ( DSBs ) , such as cell cycle checkpoint , DNA repair , and apoptosis . miRNAs have been reported to be involved in many cellular processes , and their role in DSB–triggered DNA damage response ( DDR ) is just being elucidated . Here we describe a novel DSB response mechanism whereby ATM–dependent miR-335 downregulates CtIP . CtIP is a multifunctional protein that is crucial for DNA homologous recombination repair; and we show , for the first time , that this protein is subject to miRNA downregulation . CtIP downregulation was verified in cell lines from radiosensitive patients , and we demonstrate that this pathway contributes to radiosensitization and DNA repair defects in BRCA1 foci formation and cell cycle checkpoint . Given that miR-335 is also a tumor metastasis suppressor , our finding suggests that miR-335 overexpression could not only increase tumor sensitivity to radiation or chemical therapy but also inhibit tumor metastasis or re-initiation of tumor growth .