Colony Survival V0.4.5 Hack Working
Gifford Brickley
Copyright: 2013 Martin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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].
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.
In response to IR, miR-335 is downregulated by pCREB in an ATM dependent manner. MiR-335 downregulates CtIP and likely modulates the initiation of DNA end resection and repair. MiR-335 is also subject to epigenetic regulation by DNA methylation. The shaded box indicates that the miR-335-CtIP signaling axis is modulated by both irradiation and DNA methylation.
A miRNA microarray screen identified several miRNAs that appeared to have expression levels regulated in an ATM-dependent manner. We chose to focus on miR-335 as it appeared to be downregulated in response to IR in WT-LCLs but not in A-T LCLs. Real time quantitative PCR (RT-qPCR) confirmed that miR-335 was downregulated in all three WT-LCLs post-IR and that this downregulation was absent in the A-T LCLs tested (Figure 2A); a result also found by Smirnov and Cheung [33]. Downregulation of miR-335 post-IR was also true in another ATM proficient cell type, MCF7 cells. Treatment of cells with IR or doxorubicin, another potent DSB inducer, resulted in a similar downregulation of miR-335, indicating that this was likely a general response to DSBs (Figure 2B). The lack of miR-335 downregulation in A-T LCLs strongly suggested that ATM kinase activity is required for the IR-induced miR-335 downregulation observed in WT-LCLs and MCF7 cells. Pre-treatment of MCF7 cells with KU-55933, an ATM kinase specific inhibitor [34], led to the loss of IR-induced miR-335 downregulation (Figure 2C), confirming the requirement of ATM kinase activity for the differential expression patterns observed in WT and A-T LCLs.
Because ATM has not been reported to function as a transcription factor, it was unlikely that it acted directly to repress miR-335 transcription after DNA damage induced by IR. MiR-335 is located within the second (2nd) intron of the MEST gene suggesting that miR-335 is co-regulated via a common promoter with its host gene (Figure 2D) [35]. Post-IR damage to HeLa and HEK-293T cells showed downregulation of both miR-335 and MEST mRNA by RT-qPCR, suggesting that they are, indeed, co-transcribed under the same promoter (Figure 2E). To identify potential transcription regulatory elements of both MEST and miR-335, we analyzed the 2 kb DNA sequence upstream of the translation start site of MEST using the transcription factor search program, ConSite. We focused our search on transcription factors known to interact with ATM that might result in the downregulation of miR-335 levels. Two putative CREB binding sites were identified from this search (CACGTCGCGCTG and CGCGGCAACCAG) (Figure 2D). Previous studies have reported that ATM phosphorylates CREB in response to DNA damage and that this phosphorylation reduces its transcription activity [36]. Thus, CREB was postulated to be the likely mechanism for ATM-dependent downregulation of miR-335. We tested this using siRNA to knock down CREB protein expression and found that, indeed, the IR-induced downregulation of miR-335 was abolished in siCREB-transfected HeLa cells (Figure 2F). In addition, pre-IR basal levels of miR-335 were markedly decreased indicating that CREB drives a portion of the endogenous miR-335 expression observed in WT cells (Figure 2F). We used a chromatin immunoprecipitation (ChIP) assay to demonstrate that CREB binds to the MEST promoter region in non-irradiated HeLa cells while this binding disappeared post-IR (Figure 2G), suggesting that IR dissociates CREB from the MEST promoter, therefore, reducing its transcription effect.
The miRNA microarrays identified several patient-derived RS-LCLs with elevated levels of miR-335 before and after IR. To further support our working model of the proposed ATM-miR335-CtIP pathway and the impacts of miR-335 expression on the DDR, we chose to focus on two RS-LCLs previously described by our lab, RS7 [25] and RS73 [28], with constitutive miR-335 overexpression. MiR-335 overexpression was validated by RT-qPCR and miR-335 levels were elevated >10 fold when compared to WT and A-T LCL controls (Figure 5A). Significant reduction of CtIP protein expression was observed in RS7 (Figure 5B), similar to HeLa cells overexpressing miR-335 (Figure 3C). To further demonstrate the inverse relationship between miR-335 and CtIP protein levels in the naturally miR-335 overexpressing RS-LCLs, an antisense morpholino oligonucleotide (AMO-miR-335) was used to suppress miR-335 expression. AMO-miR-335 was designed to be complementary to miR-335 thereby inhibiting the processing of pre-miR-335 into mature transcripts and also to block the binding of mature transcripts to mRNA targets. Treatment of RS7 and RS73 LCLs with AMO-miR-335 resulted in increased CtIP protein levels, confirming that overexpression of miR-335 was responsible for reductions in CtIP in both RS7 and RS73 LCLs (Figure 5C).
(A) RT-qPCR indicating >10-fold increases in miR-335 expression in RS7 and RS73 LCLs when compared to WT and A-T LCL controls. Three different WT LCLs were used in RT-qPCR experiments and were used to normalize the expression values for A-T, RS7, and RS73 LCLs. (B) Immunoblotting of cytoplasmic and nuclear lysates isolated from RS7 cells with or without 10 Gy IR indicated reductions in CtIP protein levels. Two bands were noted, representing modified and unmodified CtIP. (C) Treatment of RS7, RS73 and MCF7 cells (also miR-335 overexpressing) with AMO-miR-335 increased nuclear CtIP protein levels compared to WT and AT cells. The quantification of CtIP is shown below the blot after normalization to the loading control.
In a previous study, RS7 cells demonstrated a RDS phenotype and a delay in DNA repair, similar to HeLa cells overexpressing miR-335 [25]. RS73 also displayed delays in DNA repair demonstrated by γ-H2AX foci kinetics and a G2/M checkpoint defect-a checkpoint that is also dependent on CtIP [28], [38]. In addition, RS73 LCLs were found to have a defect in BRCA1 foci formation [28], similar to miR-335 overexpressing HeLa cells, and subsequent studies of RS7 LCLs also showed reduced BRCA1 foci formation (Figure S6). Taken together, these data support an operational model in which miR-335 impacts the DDR through CtIP and BRCA1. To test this hypothesis, the constitutively miR-335 overexpressing RS7 and RS73 LCLs were treated with AMO-miR-335, which resulted in the partial restoration of BRCA1 foci (Figure 6A). The DDR defects observed in RS7 and RS73 suggest that miR-335 modulates the DDR, and when strongly overexpressed, reduces DNA repair. It also provides one possible explanation for the reduced colony survival observed in these RS-LCLs [22], [42]. To directly demonstrate the connection between miR-335 overexpression and radiosensitivity, we treated RS7 and RS73 cells with AMO-miR-335 and found that clonogenic survival after 1 Gy of IR significantly improved with AMO-miR-335 treatment but was not affected by AMO-Control (AMO-CTL) treatments (Figure 6B). In addition, MCF7 cells have high levels of miR-335 expression and treatment with AMO-miR-335 increased CtIP protein levels (Figure 5C). Pre-IR treatment of MCF7 cells with AMO-miR-335 increased the IR-induced formation of BRCA1 foci at 8 hours and also enhanced the clonogenic survival of cells at different doses of irradiation (Figure S7A and S7B).
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.