Rather than targeting tumour cells directly, elements of the tumour microenvironment can be modulated to sensitize tumours to the effects of therapy. Here we report a unique mechanism by which ectopic microRNA-103 can manipulate tumour-associated endothelial cells to enhance tumour cell death. Using gain-and-loss of function approaches, we show that miR-103 exacerbates DNA damage and inhibits angiogenesis in vitro and in vivo. Local, systemic or vascular-targeted delivery of miR-103 in tumour-bearing mice decreased angiogenesis and tumour growth. Mechanistically, miR-103 regulation of its target gene TREX1 in endothelial cells governs the secretion of pro-inflammatory cytokines into the tumour microenvironment. Our data suggest that this inflammatory milieu may potentiate tumour cell death by supporting immune activation and inducing tumour expression of Fas and TRAIL receptors. Our findings reveal miR-mediated crosstalk between vasculature and tumour cells that can be exploited to improve the efficacy of chemotherapy and radiation.
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.
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