MicroRNAs contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by two distinct microRNAs. We demonstrate that genotoxic stress-induced miR-494 and miR-99b inhibit the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Functionally, gain and loss of function experiments show that miR-494 and miR-99b affect telomerase activity, activate p21 and Rb pathways and diminish angiogenic sprouting in vitro and in vivo.Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating endothelial senescence and proliferation. Vascular-targeted delivery of miR-494 decreases both growth factorinduced and tumor angiogenesis in mouse models. Mechanistically, disruption of the MRN complex induced CD44, a known driver of senescence and regulator of VEGF signaling in addition to suppressing IL-13 a stimulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence and highlight the potential of targeting DNA repair to disrupt pathological angiogenesis.
Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.
Endothelial cells are highly responsive to environmental changes that allow them to adapt to intrinsic and extrinsic stimuli and switch their transcriptome accordingly to go back to vascular homeostasis. Our previous data demonstrated that small non-coding-RNAs respond quickly to genotoxic stressors and determined endothelial cell fate and DNA damage response. To further understand the contribution of non-coding-RNAs, we profiled differentially expressed long non-coding RNAs in response to genotoxic stress and compared them to pro-angiogenic growth factor signaling. We identified the Maternally expressed gene 9 (MEG9) as a cytoprotective lncRNA in the endothelium. Gain and Loss-of-function studies indicate that MEG9 prevents endothelial cells from cell death, suggesting that MEG9 responses to genotoxic stress can be an adaptive and protective mechanism. Consistent with this phenotype, the knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Deletion of the MEG9 ortholog, Mirg, in mice results in increased vascular leak in Matrigel plugs and a sex and age-dependent decrease in platelets. Mechanistically, we observed that both MEG9 knockdown in vitro and Mirg-deleted mice in vivo activated common pathways, including apoptosis, clotting, and inflammation. Indeed, the proinflammatory adhesion molecule ICAM1 was significantly increased in human and mouse endothelial cells in a MEG9-dependent manner, supporting the increased vascular permeability observed on MEG9 deficient cells. Taken together, our findings illustrate how genotoxic stress responses through dynamic modulation of lncRNAs, such as MEG9, trigger adaptive mechanisms to maintain endothelial function, while loss of these molecules contributes to maladaptive responses and endothelial cell dysfunction.
The tumor microenvironment (TME) plays a critical role in orchestrating immune infiltration, tumor progression and response to therapeutics. Therefore, strategies to manipulate the TME including cytokine gene therapy, antibodies to reverse macrophage polarization etc are under active investigation. We have identified an alternative approach to target the TME by disrupting DNA repair in the tumor endothelial cells (ECs). We discovered a seven-microRNA (miR) signature induced in ECs in in vitro and in vivo by oxidative stress and DNA damage. The top miR candidate in this signature, miR-103 altered the TME by inducing DNA damage in ECs, eliciting type I interferons, upregulating immune costimulatory receptors and decreasing PD-L1 expressing tumor associated macrophages and granulocytes. Moreover, miR-103 treatment had a paracrine effect on the tumors and upregulated Fas and TRAIL receptors. Mechanistically, these functions of miR-103 were largely due to its downregulation of the three prime exonuclease TREX1. Local, systemic or vascular targeted delivery of miR-103 decreased both angiogenesis and tumor burden in multiple mouse tumor models. Complementary to the role of miR-103, two additional miRs in the miR signature, miRs 494 and 99b each induced senescence in the vasculature by downregulating the Mre11a-Rad50-NBN (MRN) complex. Ectopic expression of miRs 494 or 99b decreased telomerase activity, increase p21 levels with a concomitant decrease in pRb levels. Vascular targeted delivery of miR-494 decreased angiogenesis in vivo whereas systemic delivery decreased tumor growth. Interestingly, both miR mimics and the MRN knockdowns induced the transcription of a number of senescence associated genes including CD44. Taken together these data suggest that miRs 494 and 99b targeting of the MRN complex induces senescence. The MRN complex interacts with the ATM kinase, histone H2AX and TREX1 suggesting that the miRs we identified disrupt critical nodes of a DNA Damage Response (DDR) network. Our findings reveal a complex, miR mediated cross talk between EC DNA damage pathways, the TME and tumor cells. These interactions can be exploited for designing therapies that synergize with tumor cell killing to enhance host anti-tumor responses. Citation Format: Cristina Espinosa-Diez, RaeAnna Wilson, Rebecca Ruhl, Nathan Kanner, Namita Chatterjee, Clay Hudson, Sudarshan Anand, Shushan Rana. Reprogramming the tumor microenvironment by targeting endothelial DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1819. doi:10.1158/1538-7445.AM2017-1819
Preclinical and clinical studies have revealed that tumor endothelium is abnormal, resistant to genotoxic stress, and as such, functions as a key determinant of therapeutic responses to radiation and chemotherapy. While it is well established that radiation and chemotherapy cause DNA damage in tumor vasculature, the molecular mechanisms leading to subsequent cell cycle arrest, apoptosis or senescence in vascular cells are poorly understood. Therefore, identifying and understanding factor(s) that mediate DNA damage responses in tumor endothelial cells will provide potential targets for sensitizing tumor vasculature to radiation and other DNA damaging agents and improve their therapeutic efficacy in cancer. Recent data indicates that microRNAs (miRs) are potent regulators of DNA damage responses in the tumor microenvironment. miRs are short 20-22 nucleotide (nt) RNA molecules that regulate gene expression by binding to partially complementary sites in target mRNAs. Since miRs mediate several physiological processes in endothelial cells, we hypothesized that miRs regulate endothelial (EC) DNA damage responses. We used an expression screen to identify miRs induced by radiation, cisplatin or hydrogen peroxide in human ECs and identified seven specific miRs unique to intrinsic EC apoptosis pathways regulated by genotoxic stress. In vitro gain-of-function assays show that three of them, miR-21, miR-99b and miR-494 lead to endothelial senescence by impairing telomerase function and inhibit sprouting angiogenesis in vitro, in a 3D assay. Strikingly, we observed that these three miRs each target every member of the MRN (Mre11a-Rad50 and NBS1) complex, a critical part of the cellular DNA repair machinery. MRN complex plays a vital role in DNA ds break repair, replication, and telomere maintenance. Pulldown of a mutant RNA Induced Silencing Complex (RISC) from cells transfected the miR mimics enriched for the MRN mRNAs suggesting direct miRNA-MRN complex mRNA binding. Consistent with these results, knockdown of the MRN complex recapitulated the effects of the miRs, reproducing the senescence phenotype, angiogenesis inhibition and also impaired telomerase activity. Since MRE-11a is upregulated in human breast cancer patients, we asked if there was any differential expression of miR-494 in either the tumor ECs or tumor cells. Interestingly, ISH of a breast cancer tissue array revealed a significant reduction in tumor miR-494 levels compared with the adjacent normal tissue. Furthermore, ectopic expression of miR-494 diminished breast cancer cell proliferation in 2D and 3D. Our observations indicate that miR-494 behaves as a tumor suppressor microRNA by targeting the MRN complex, inducing senescence, cell cycle arrest and decreases angiogenesis. Therefore, we propose that restoration of these miRs targeting the MRN complex in breast cancer is likely to synergize with DNA damaging agents and decrease tumor burden. Citation Format: Cristina Espinosa-Diez, RaeAnna Wilson, Nathan Kanner, Rebecca Ruhl, Christina M. Hipfinger, Sudarshan Anand. Reprogramming the breast cancer microenvironment using microRNAs that target DNA repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1111.
Radiation therapy is a big part of standard of care in oncology. However, in the long- term patients treated with radiation are at higher risk of suffering cardiovascular events. It is well described that the genotoxic stress induced by Ionizing radiation in normal cardiovascular tissue triggers genetic and epigenetic changes. In addition to coding mRNAs, non-coding RNAs are also highly regulated by changes in methylation and transcription, leading to a tight regulatory response to damage. Genotoxic stress induces global hypomethylation due to decreased expression of DNA methyltransferases (DNMT). We observed that DNMT1, DNMT3A, and DNMT3b are downregulated in response to radiation treatment, in a dose-response manner in human endothelial cells. We have also observed that methylation changes produced by radiation affect a specific ncRNA cluster, DLK1-DIO3. Previous results from our lab indicate that ncRNAs from this cluster are highly responsive to different genotoxic agents, including radiation. Given the essential role of microRNAs and Long non- coding RNAs (lncRNAs) from the DLK1-DIO3 cluster in cardiovascular development and aging, we are interested in understanding how the epigenetic changes induced by radiotherapy affect to lncRNAs expression, and how they influence cardiovascular health in the long term after treatment. We have identified that a specific lncRNA from the DLK1-DIO3 cluster, MEG9, increases in ECs after exposure to ionizing radiation. Interestingly, knockdown of the individual DNMTs enzymes indicates a significant upregulation of MEG9 when DNMT3b is inhibited, more so after radiation treatment. To explore the role of this lncRNA, we performed loss-of-function studies. MEG9 inhibition not only diminished proliferation but also increased apoptosis through caspase 3/7 activation. Consistent with this phenotype, knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Taken together, our findings illustrate how DNA methylation at particular lncRNA loci can regulate their expression and drive endothelial cell fate decisions. Our work illustrates how epigenetic changes may affect the long- term cardiovascular function of cancer patients submitted to radiation therapy.
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