Biology subjects

Ruhl, R.

Publications and source records attributed to Ruhl, R..

6 recordsLinked to original sources

bioRxiv2022

Tumor cell intrinsic RIG-I activation is sufficient to drive immune mediated tumor rejection

Targeting cytosolic nucleic acid sensors is a potent approach to drive type I interferon responses and anti-tumor immunity. Recent evidence suggests that activation of retinoic acid inducible gene-I (RIG-I) using synthetic hairpin RNA agonists decreases tumor progression in multiple preclinical models. However, the role of tumor cell intrinsic RIG-I in shaping tumor cell fates and the host immune microenvironment remains unclear. Here, we show that RIG-I expression is correlated with better overall survival and a distinct immune gene signature in specific human cancers including colorectal cancer. Activation of RIG-I in breast and colorectal cancer cells is sufficient to drive tumor cell death in vitro and significantly delay tumor growth in vivo in multiple preclinical models. Importantly, the efficacy of tumor cell RIG-I activation is lost in immune deficient mice suggesting the requirement of immune responses for this effect. We observe that tumor cell intrinsic RIG-I activation elicits a robust cellular and molecular immune response. We show that tumor cell RIG-I activation also leads to induction of specific immune checkpoints including PD-L1. Using a publicly available database, we found that RIG-I expression serves as an excellent prognostic marker for responders to checkpoint immunotherapy, particularly PD-L1/PD-1 across cancers. Finally, combination of tumor cell intrinsic RIG-I activation with anti-PD-L1 led to a synergistic decrease in tumor growth in a colorectal tumor model. Our findings suggest that tumor cell intrinsic RIG-I can be targeted to enhance anti-tumor immune responses and highlights a potential strategy for anti-cancer vaccines that can invigorate the immune system.

cancer biology↗

bioRxiv2022

DNA damage-induced lncRNA MEG9 impacts angiogenesis

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.

cell biology↗

bioRxiv2018

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.

cell biology↗

bioRxiv2018

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.

cancer biology↗

bioRxiv2017

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 {gamma}-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.

cancer biology↗

bioRxiv2017

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.

cancer biology↗