Cristina Espinosa-Diez

MicroRNAs in Cancer: challenges and opportunities in early detection, disease monitoring, and therapeutic agents.

Purpose of ReviewThe goals of this review are to examine the usefulness of microRNAs (miRNAs) as diagnostic and prognostic biomarkers for cancer and to evaluate the applicability of miRNAs as cancer therapeutics.Recent FindingsExamination of miRNA milieu from body fluids offers a new alternative for quick, affordable, and easy analysis of disease status in patients. Blood-based exosomal miRNAs have increased stability and are an excellent choice for clinical cancer diagnostics and prognostics. Currently, there are many miRNA signatures associated with cancer and progression but there is no consensus among multiple serum and tumor sample studies. Off-target and immunological effects remain an obstacle for the use of miRNAs as novel chemotherapeutics in the clinic. Recent developments in nanotechnology and drug delivery systems which target the tumor microenvironment may provide an alternative therapeutic approach with decreased toxicity.SummaryThis review critically evaluates the literature investigating the use of miRNAs as biomarkers and their future as potential therapeutics.

MicroRNA-mediated regulation of glutathione and methionine metabolism and its relevance for liver disease.

The discovery of the microRNA (miRNA) family of small RNAs as fundamental regulators of post-transcriptional gene expression has fostered research on their importance in every area of biology and clinical medicine. In the particular area of liver metabolism and disease, miRNAs are gaining increasing importance. By focusing on two fundamental hepatic biosynthetic pathways, glutathione and methionine, we review recent advances on the comprehension of the role of miRNAs in liver pathophysiology and more specifically of models of hepatic cholestasis/fibrosis and hepatocellular carcinoma.

DNA damage dependent hypomethylation regulates the pro-angiogenic LncRNA MEG9

Changes in gene expression are key for the cells to adapt and response to intrinsic and extrinsic stimulus. It has been shown that genotoxic stress induces global hypomethylation as a result of decreased expression of DNA methyl transferases (DNMT). We hypothesized that DNA damage suppresses long non-coding RNA expression in the vasculature via DNA methylation leading to more robust DNA repair/survival or cellular senescence/death cell fate decisions. We show here that ionizing radiation reduces the expression of DNMTs in the vascular endothelium and this leads to increased expression of the anti-apoptotic lncRNA MEG9. MEG9 is a lncRNA from the DLK1-DIO3 ncRNA cluster. Loss-of-function studies using RNA gapmers indicate that MEG9 protects endothelial cells from DNA damage induced cell death. Consistent with this phenotype, knockdown of MEG9 decreases growth factor dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Mechanistically, we observed that MEG9 knockdown decreased the expression of cell survival genes including survivin and induced the expression of pro-apoptotic genes such as Bad/Bax. Taken together, our findings illustrate how DNA methylation at selective lncRNA loci can regulate their expression and drive endothelial cell fate decisions.

Differential regulation of microRNA-15a by radiation affects angiogenesis and tumor growth via modulation of acid sphingomyelinase

Activation of acid sphingomyelinase (SMPD1) and the generation of ceramide is a critical regulator of apoptosis in response to cellular stress including radiation. Endothelial SMPD1 has been shown to regulate tumor responses to radiation therapy. We show here that the SMPD1 gene is regulated by a microRNA (miR), miR-15a, in endothelial cells (ECs). Standard low dose radiation (2 Gy) upregulates miR-15a and decreases SMPD1 levels. In contrast, high dose radiation (10 Gy and above) decreases miR-15a and increases SMPD1. Ectopic expression of miR-15a decreases both mRNA and protein levels of SMPD1. Mimicking the effects of high dose radiation with a miR-15a inhibitor decreases cell proliferation and increases active Caspase-3 & 7. Mechanistically, inhibition of miR-15a increases inflammatory cytokines, such as IP10, activates caspase-1 inflammasome and increases Gasdermin D, an effector of pyroptosis. Importantly, both systemic and vascular-targeted delivery of miR-15a inhibitor decreases angiogenesis and tumor growth in a CT26 murine colorectal carcinoma model. Taken together, our findings highlight a novel role for miR mediated regulation of SMPD1 during radiation responses and establish proof-of-concept that this pathway can be targeted with a miR inhibitor.

NextGen VOICES: A postdoc's purpose

We gave young scientists this challenge: What is the purpose of a postdoc? Address this question by writing a job advertisement for the perfect postdoc position. We received responses from scientists around the world representing a variety of fields. Some wrote advertisements for their ideal postdoc

MicroRNA regulation of the MRN complex impacts DNA damage, cellular senescence, and angiogenic signaling

MicroRNAs (miRs) 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 a miR. We demonstrate that genotoxic stress-induced miR-494 inhibits the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Gain- and loss-of-function experiments show that miR-494 exacerbates DNA damage and drives endothelial senescence. Increase of miR-494 affects telomerase activity, activates p21, decreases pRb pathways, and diminishes angiogenic sprouting. Genetic and pharmacological disruption of the MRN pathway decreases VEGF signaling, phenocopies miR-494-induced senescence, and disrupts angiogenic sprouting. Vascular-targeted delivery of miR-494 decreases both growth factor-induced and tumor angiogenesis in mouse models. 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.

MicroRNA perturbation of the MRN complex buffers DNA damage response from VEGF signaling

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 factor -induced 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.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, 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. miR-99b diminishes VEGF signaling, transcriptional responses and proliferation. Disruption of the MRN complex induces CD44, a known driver of senescence and regulator 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.

MicroRNA-451a regulates colorectal cancer radiosensitivity

Colorectal cancer (CRC) is a leading cause of cancer-related death. The responses of CRC to standard of care adjuvant therapies such as radiation or chemotherapy are poorly understood. MicroRNAs (miRs) are small non-coding RNAs that affect gene expression programs in cells by downregulating specific mRNAs. In this study, we discovered a set of microRNAs upregulated rapidly in response to a single 2 Gy dose fraction of γ-radiation in a mouse colorectal carcinoma xenograft model. The most upregulated candidate in our signature, miR-451a inhibits tumor cell proliferation and attenuated surviving fraction in longer-term cultures. Conversely, inhibition of miR-451a increased proliferation, tumorsphere formation and surviving fraction of tumor cells. Using a bioinformatics approach, we identified four genes-CAB39, EMSY, MEX3C and EREG as targets of miR-451a. Transfection of miR-451a decreased both mRNA and protein levels of these targets. Importantly, we found miR-451a expression was decreased with tumor stage in a small subset of CRC patients. Finally, analysis of a TCGA colorectal cancer dataset reveals that the CAB39 and EMSY are upregulated at the protein level in a significant number of CRC patients and correlates with poorer overall survival. Taken together, our data indicates miR-451a influences the radiation sensitivity of colorectal carcinomas.

Abstract 1111: Reprogramming the breast cancer microenvironment using microRNAs that target DNA repair

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA 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.